PAH-modulating compositions and methods

The disclosure provides, e.g., compositions, systems, and methods for targeting, editing, modifying, or manipulating a host cell's genome at one or more locations in a DNA sequence in a cell, tissue, or subject. Gene modifying systems for treating phenylketonuria (PKU) are described.

1. 11. A template RNA comprising, from 5′ to 3′: a) a gRNA spacer that is complementary to a first portion of a human PAH gene, wherein the gRNA spacer comprises an RNA sequence of nucleotides 1 to 20 of SEQ ID NO: 37,221; b) a gRNA scaffold that binds a SpyCas9 domain; c) a heterologous object sequence comprising a mutation region to correct a mutation in a second portion of the human PAH gene, wherein the heterologous object sequence comprises a nucleotide sequence of nucleotides 97 to 105 of SEQ ID NO: 37,221; and d) a primer binding site (PBS) sequence comprising at least 3 bases with 100% identity to a third portion of the human PAH gene, wherein the PBS sequence comprises a nucleotide sequence of nucleotides 106 to 116 of SEQ ID NO: 37,221.
2. 22. The template RNA of claim 1, wherein the mutation to be corrected in the human PAH gene is R408W.
3. 33. The template RNA of claim 1, wherein the gRNA spacer has a length of 20 nucleotides.
4. 44. The template RNA of claim 1, wherein the heterologous object sequence has a length of 9-16 nucleotides.
5. 55. The template RNA of claim 1, wherein the heterologous object sequence comprises, from 5′ to 3′, a post-edit homology region, a mutation region, and a pre-edit homology region.
6. 66. The template RNA of claim 1, wherein the heterologous object sequence consists of an RNA sequence of GGGCCGAGG.
7. 77. The template RNA of claim 1, wherein the PBS sequence has a length of 11-12 nucleotides.
8. 88. The template RNA of claim 1, wherein the PBS sequence consists of an RNA sequence of 106 to 116 of SEO ID NO: 37,221.
9. 99. The template RNA of claim 1, wherein the gRNA scaffold comprises an RNA sequence having at least 90% identity to nucleotides 21 to 96 of SEQ ID NO: 37,221.

CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of PCT/US2022/076058, filed Sep. 7, 2022, which claims the benefit of U.S. Provisional Application No. 63/241,897, filed Sep. 8, 2021, U.S. Provisional Application No. 63/303,927, filed Jan. 27, 2022, and U.S. Provisional Application No. 63/367,025, filed Jun. 24, 2022. The contents of the aforementioned applications are hereby incorporated by reference in their entirety.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in XML, format compliant with WIPO Standard ST.26 and is hereby incorporated by reference in its entirety. Said XML copy, created on Sep. 15, 2023, is named V2065-702520FT_SL.xml and is 51,647,577 bytes in size.
BACKGROUND
Integration of a nucleic acid of interest into a genome occurs at low frequency and with little site specificity, in the absence of a specialized protein to promote the insertion event. Some existing approaches, like CRISPR/Cas9, are more suited for small edits that rely on host repair pathways, and are less effective at integrating longer sequences. Other existing approaches, like Cre/loxP, require a first step of inserting a loxP site into the genome and then a second step of inserting a sequence of interest into the loxP site. There is a need in the art for improved compositions (e.g., proteins and nucleic acids) and methods for inserting, altering, or deleting sequences of interest in a genome.
PKU is an inherited disorder involving an autosomal recessive inborn error of metabolism caused by a deficiency in the hepatic enzyme PAH. PAH catalyzes the hydroxylation of phenylalanine to tyrosine, the rate-limiting step in phenylalanine metabolism. The reaction is dependent on tetrahydrobiopterin (BH4), as a cofactor, molecular oxygen, and iron. Loss-of-function mutations in one, or both, copies of the PAH gene lead to a non-functional, or less efficient enzyme. This ultimately results in phenotypically severe forms of PKU where phenylalanine in the blood can accumulate to toxic concentrations, with impaired levels of plasma tyrosine. Additionally, the deficiency prevents normal synthesis of downstream products, including dopamine, norepinephrine, and melanin.
The PAH genomic sequence and its flanking regions span about 171 kb, containing 13 exons. Study of pathogenic allelic variants have identified more than 500 different disease-causing mutations in the PAH gene (Mitchell, et al. Genet Med. 2011; 13:697-707). Of these mutations, approximately 62% have been characterized as missense, 13% deletions, 11% splice, 6% silent, 5% nonsense, 2% insertion, and <1% deletion or duplication of exons. The identification of several PAH mutations have been described for their effects on enzymatic activity using enzyme kinetics and crystallographic studies. Mutations affecting the catalytic binding mode, including Y138F, S23A, and Y377F, were observed with reduced propensity for tetramer formation (Flydal, et al. PNAS. 2019; 116(23):11229-34). Other residues that interact with BH4 in the precatalytic conformation (amino acids 245-255, 286, 322, and 325) also interact with BH4 in the catalytic conformation, and, in addition, these sites are actually associated with severe destabilization of PAH.
Naturally occurring N-terminal PAH mutations have been determined to be distributed in a nonrandom pattern, clustering within residues 46-48 (GAL motif) and 65-69 (IESRP motif (SEQ ID NO: 37634)), both motifs highly conserved in pyruvate dehydrogenase (PDH) (Gjetting, et al. Am./J. Hum. Genet. 2001; 68:1353-60). Structure-function studies demonstrated that mutations in these regions drastically reduced phenylalanine binding. Most missense mutations identified in PKU to date result in phenotypic outcomes associated with misfolding of the PAH enzyme, increased protein turnover, and loss of enzymatic function. Residues in exons 7-9 and in interdomain regions within the subunit appear to play an important structural role and constitute hotspots for destabilization. Additionally, using recombinant forms of hPAH, mutations in BH4 responsive domains, including R408W and Y414C showed residual activity, but had perturbed allostery suggesting altered protein conformation (Gersting, et al. Hum. Genet. 2008; 83:5-17). Mutation analyses and structure-function analyses have identified a robust genotype-phenotype mapping for PAH's role in PKU; however, outside of lifetime symptom management strategies, there has not been a successful cure.
Dietary therapy of phenylalanine (Phe) remains to be the mainstay treatment for PKU since its introduction in 1953. In the 1970s, tetrahydrobiopterin (BH4) and neurotransmitter precursor (L-dopa/carbidopa and 5-hydroxytryptophan) combination therapy showed promise in modulating PKU. Since its institution as a therapy, synthetics such as sapropterin have been formulated for as small molecule isomers of BH4. Although, this form of therapy is generally only useful in patients with mild subsets of PAH-deficient PKU. It is thought that the therapy responsiveness is associated with mutations in the PAH gene resulting in some residual enzyme activity. At high blood concentrations, Phe in the blood will compete with other large neutral amino acids (LNAAs) for transport across the blood-brain barrier. LNAA supplementation has been shown to reduce cerebral Phe concentrations despite the observed increase in plasma Phe levels. Likewise, dietary supplementation with glycomacropeptides (GMP) has been observed to significantly reduce ureagenesis, improved protein retention, and Phe utilization. Although, these strategies do little to address the increased blood levels of Phe or the genotypic drivers.
Modern non-dietary approaches include the development of PAH-based fusion proteins and enzyme substitution therapies. Enzyme substitution therapies can include administration of phenylalanine ammonia-lyase (PAL) to a patient. PAL is an enzyme which catalyzes the conversion of Phe to transcinnamic acid and insignificant amounts of ammonia. Early studies using PAL administered in enteric-coated gelatin capsules to PKU patients, showed reductions in Phe levels; however, repeated dosing in vivo resulted in mounting of immune responses. Although, these approaches are not practical from a clinical perspective as several intravenous injections would be required due to the limited half-life of circulating enzymes. Gene therapy has shown some promise, for example using viral vectors, in rescuing PAH functionality. However, the efficacy of this strategy is hampered by the very low gene transfer rate and transient transgene expression. Accordingly, there is a need for new and more effective treatments for targeting PAH in PKU.
SUMMARY OF THE INVENTION
This disclosure relates to novel compositions, systems, and methods for altering a genome at one or more locations in a host cell, tissue, or subject, in vivo or in vitro. The disclosure provides gene modifying systems that are capable of modulating (e.g., inserting, altering, or deleting sequences of interest) phenylalanine hydroxylase (PAH) activity and methods of treating phenylketonuria (PKU) by administering one or more such systems to alter a genomic sequence, such as to correct mutations, within the PAH gene on the human chromosome 12q23.2 involved as a genetic driver in PKU.
In one aspect, the disclosure relates to a system for modifying DNA to correct a human PAH gene mutation causing PKU comprising (a) a nucleic acid encoding a gene modifying polypeptide capable of target primed reverse transcription, the polypeptide comprising (i) a reverse transcriptase domain and (ii) a Cas9 nickase that binds DNA and has endonuclease activity, and (b) a template RNA comprising (i) a gRNA spacer that is complementary to a first portion of the human PAH gene, (ii) a gRNA scaffold that binds the polypeptide, (iii) a heterologous object sequence comprising a mutation region to correct the mutation, and (iv) a primer binding site (PBS) sequence comprising at least 3, 4, 5, 6, 7, or 8 bases of 100% homology to a target DNA strand at the 3′ end of the template RNA. In some embodiments, the PAH gene may comprise a R408W mutation. In some embodiments, the PAH gene may comprise a R261Q mutation. In some embodiments, the PAH gene may comprise a R243Q mutation. In some embodiments, the PAH gene may comprise a IVS10-11G>A mutation. The template RNA sequence may comprise a sequence described herein, e.g., in Table 1A, 1B, 1C, 1D, 3A, 3B, 3C, 3D, 4A, 4B, 4C, 4D, 5A-5F, 8A-8D, E3, E3A, BB, E5, E5A, E6, or E6A.
The gRNA spacer may comprise at least 15 bases of 100% homology to the target DNA at the 5′ end of the template RNA. The template RNA may further comprise a PBS sequence comprising at least 5 bases of at least 80% homology to the target DNA strand. The template RNA may comprise one or more chemical modifications.
The domains of the gene modifying polypeptide may be joined by a peptide linker. The polypeptide may comprise one or more peptide linkers. The gene modifying polypeptide may further comprise a nuclear localization signal. The polypeptide may comprise more than one nuclear localization signal, e.g., multiple adjacent nuclear localization signals or one or more nuclear localization signals in different regions of the polypeptide, e.g., one or more nuclear localization signals in the N-terminus of the polypeptide and one or more nuclear localization signals in the C-terminus of the polypeptide. The nucleic acid encoding the gene modifying polypeptide may encode one or more intein domains.
Introduction of the system into a target cell may result in insertion of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, or 1000 base pairs of exogenous DNA. Introduction of the system into a target cell may result in deletion, wherein the deletion is less than 2, 3, 4, 5, 10, 50, or 100 base pairs of genomic DNA upstream or downstream of the insertion. Introduction of the system into a target cell may result in substitution, e.g., substitution of 1, 2, or 3 nucleotides, e.g., consecutive nucleotides.
The heterologous object sequence may be at least 5, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, or 700 base pairs.
In one aspect, the disclosure relates to a pharmaceutical composition comprising the system described above and a pharmaceutically acceptable excipient or carrier, wherein the pharmaceutically acceptable excipient or carrier is selected from the group consisting of a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle. In one aspect, the disclosure relates to a pharmaceutical composition comprising the system described above and multiple pharmaceutically acceptable excipients or carriers, wherein the pharmaceutically acceptable excipients or carriers are selected from the group consisting of a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle, e.g., where the system described above is delivered by two distinct excipients or carriers, e.g., two lipid nanoparticles, two viral vectors, or one lipid nanoparticle and one viral vector. The viral vector may be an adeno-associated virus (AAV).
In one aspect, the disclosure relates to a host cell (e.g., a mammalian cell, e.g., a human cell) comprising the system described above.
In one aspect, the disclosure relates to a method of correcting a mutation in the human PAH gene in a cell, tissue or subject, the method comprising administering the system described above to the cell, tissue or subject, wherein optionally the correction of the mutant PAH gene comprises an amino acid substitution of W408R, Q261R, and/or Q243R (reversing the pathogenic substitution which is R408W, R261Q, or R243Q). In another aspect, the correction of the mutant PAH gene comprises a nucleic acid substitution of IVS10-11A>G (reversing the pathogenic substitution which is IVS10-11G>A). The system may be introduced in vivo, in vitro, ex vivo, or in situ. The nucleic acid of (a) may be integrated into the genome of the host cell. In some embodiments, the nucleic acid of (a) is not integrated into the genome of the host cell. In some embodiments, the heterologous object sequence is inserted at only one target site in the host cell genome. The heterologous object sequence may be inserted at two or more target sites in the host cell genome, e.g., at the same corresponding site in two homologous chromosomes or at two different sites on the same or different chromosomes. The heterologous object sequence may encode a mammalian polypeptide, or a fragment or a variant thereof. The components of the system may be delivered on 1, 2, 3, 4, or more distinct nucleic acid molecules. The system may be introduced into a host cell by electroporation or by using at least one vehicle selected from a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle.
Features of the compositions or methods can include one or more of the following enumerated embodiments.
Enumerated Embodiments


    
    
        1. A template RNA comprising, e.g., from 5′ to 3′:
        
            (i) a gRNA spacer that is complementary to a first portion of the human PAH gene, wherein the gRNA spacer has a sequence comprising the core nucleotides of a gRNA spacer sequence of Table 1A, Table 1B, Table 1C, or Table 1D, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the gRNA spacer (e.g., comprises one or more flanking nucleotides that are adjacent to the core nucleotides), or wherein the gRNA spacer has a sequence of a spacer chosen from Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A;
            (ii) a gRNA scaffold that binds a gene modifying polypeptide (e.g., binds the Cas domain of the gene modifying polypeptide),
            (iii) a heterologous object sequence comprising a mutation region to introduce a mutation into (e.g., to correct a mutation in) a second portion of the human PAH gene (wherein optionally the heterologous object sequence comprises, from 5′ to 3′, a post-edit homology region, a mutation region, and a pre-edit homology region), and
            (iv) a primer binding site (PBS) sequence comprising at least 3, 4, 5, 6, 7, or 8 bases with 100% identity to a third portion of the human PAH gene.
        
        
        2. The template RNA of embodiment 1, wherein the heterologous object sequence comprises the core nucleotides of an RT template sequence from Table 3A, Table 3B, Table 3C, or Table 3D, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the RT template sequence, or wherein the heterologous object sequence comprises a sequence of an RT template sequence from Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A.
        3. The template RNA of embodiment 1, wherein the heterologous object sequence comprises the core nucleotides of the RT template sequence of Table 3A, Table 3B, Table 3C, or Table 3D that corresponds to the gRNA spacer sequence, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the RT template sequence (e.g., comprises one or more flanking nucleotides that are adjacent to the core nucleotides), or wherein the heterologous object sequence comprises a sequence of an RT template sequence from Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, E5A, E6, or E6A.
        4. The template RNA according to any one of embodiments 1-3 wherein the PBS sequence has a sequence comprising the core nucleotides of the PBS sequence from the same row of Table 3A, Table 3B, Table 3C, or Table 3D as the RT template sequence, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 5′ end of the flanking nucleotides of the PBS sequence (e.g., comprises one or more flanking nucleotides that are adjacent to the core nucleotides).
        5. The template RNA according to any one of embodiments 1-3, wherein the PBS sequence has a sequence comprising the core nucleotides of a PBS sequence of Table 3A, Table 3B, Table 3C, or Table 3D that corresponds to the RT template sequence, or a sequence having 1, 2, or 3 substitutions thereto, the gRNA spacer sequence, or both, and optionally comprises one or more consecutive nucleotides starting with the 5′ end of the flanking nucleotides of the PBS sequence, or wherein the PBS sequence comprises a PBS sequence from Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A that corresponds to the RT template sequence, or a sequence having 1, 2, or 3 substitutions thereto, the gRNA spacer sequence, or both.
        6. The template RNA according to any of embodiments 1-5, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold of Table 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
        7. The template RNA according to any of embodiments 1-5, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold of Table 12 that corresponds to the RT template sequence, the gRNA spacer sequence, or both, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
        8. A template RNA comprising, e.g., from 5′ to 3′:
        
            (i) a gRNA spacer that is complementary to a first portion of the human PAH gene,
            (ii) a gRNA scaffold that binds a gene modifying polypeptide (e.g., binds the Cas domain of the gene modifying polypeptide),
            (iii) a heterologous object sequence comprising a mutation region to introduce a mutation into a second portion of the human PAH gene, wherein the heterologous object sequence comprises the core nucleotides of an RT template sequence of Table 3A, Table 3B, Table 3C, or Table 3D, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the RT template sequence, or wherein the heterologous object sequence comprises an RT template sequence of Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A; and
            (iv) a PBS sequence comprising at least 3, 4, 5, 6, 7, or 8 bases of 100% identity to a third portion of the human PAH gene.
        
        
        9. The template RNA of embodiment 8, wherein the gRNA spacer comprises the core nucleotides of a gRNA spacer sequence of Table 1A, Table 1B, Table 1C, or Table 1D, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the gRNA spacer sequence, or wherein the gRNA spacer comprises a gRNA spacer sequence of Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A.
        10. The template RNA of any one of embodiments 1-9, wherein the gRNA spacer comprises ACCTCAATCCTTTGGGTGTA (SEQ ID NO: 16355), or a sequence having 1, 2, or 3 substitutions thereto.
        11. The template RNA of any one of embodiments 1-9, wherein the gRNA spacer comprises CCTCAATCCTTTGGGTGTAT (SEQ ID NO: 16332), or a sequence having 1, 2, or 3 substitutions thereto.
        12. The template RNA of any one of embodiments 1-9, wherein the gRNA spacer comprises TGGGTCGTAGCGAACTGAGA (SEQ ID NO: 16102), or a sequence having 1, 2, or 3 substitutions thereto.
        13. The template RNA of any one of embodiments 1-9, wherein the gRNA spacer comprises GGGTCGTAGCGAACTGAGAA (SEQ ID NO: 16084), or a sequence having 1, 2, or 3 substitutions thereto.
        14. The template RNA of any one of embodiments 1-9, wherein the gRNA spacer comprises TAGCGAACTGAGAAGGGCCA (SEQ ID NO: 16011), or a sequence having 1, 2, or 3 substitutions thereto.
        15. The template RNA of any one of embodiments 1-9, wherein the gRNA spacer comprises ACTTTGCTGCCACAATACCT (SEQ ID NO: 16032), or a sequence having 1, 2, or 3 substitutions thereto.
        16. The template RNA of embodiment 8, wherein the heterologous object sequence comprises the core nucleotides of the gRNA spacer sequence of Table 1A, Table 1B, Table 1C, or Table 1D that corresponds to the RT template sequence, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the gRNA spacer sequence, or wherein the heterologous object sequence comprises the nucleotides of the gRNA spacer sequence of Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A that corresponds to the RT template sequence, or a sequence having 1, 2, or 3 substitutions thereto.
        17. The template RNA according to any one of embodiments 8-16, wherein the PBS sequence has a sequence comprising the core nucleotides of the PBS sequence from the same row of Table 3A, Table 3B, Table 3C, or Table 3D as the RT template sequence, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 5′ end of the flanking nucleotides of the PBS sequence.
        18. The template RNA according to any one of embodiments 8-16, wherein the PBS sequence has a sequence comprising the core nucleotides of a PBS sequence of Table 3A, Table 3B, Table 3C, or Table 3D that corresponds to the RT template sequence, or a sequence having 1, 2, or 3 substitutions thereto, the gRNA spacer sequence, or both, and optionally comprises one or more consecutive nucleotides starting with the 5′ end of the flanking nucleotides of the PBS sequence, or wherein the PBS sequence has a sequence comprising the a PBS sequence of Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A that corresponds to the RT template sequence, the gRNA spacer sequence, or both.
        19. The template RNA according to any of embodiments 8-18, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold of Table 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
        20. The template RNA according to any of embodiments 8-18, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold of Table 12 that corresponds to the RT template sequence, the gRNA spacer sequence, or both, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
        21. A gene modifying system for modifying DNA, comprising:
        
            (a) a first RNA comprising, from 5′ to 3, (i) a guide RNA sequence that is complementary to a first portion of the human PAH gene, wherein the guide RNA sequence has a sequence comprising the core nucleotides of a spacer sequence of Table 1A, Table 1B, Table 1C, or Table 1D, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the guide RNA sequence, or wherein the guide RNA sequence has a sequence of a spacer chosen from Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A; and (ii) a sequence (e.g., a scaffold region) that binds a gene modifying polypeptide (e.g., binds the Cas domain of the gene modifying polypeptide), and
            (b) a second RNA comprising (iii) a heterologous object sequence comprising a nucleotide substitution to introduce a mutation into a second portion of the human PAH gene (wherein optionally the heterologous object sequence comprises, from 5′ to 3′, a post-edit homology region, a mutation region, and a pre-edit homology region), (iv) a primer region comprising at least 5, 6, 7, or 8 bases of 100% identity to a third portion of the human PAH gene, and (v) an RRS (RNA binding protein recognition sequence) that binds a gene modifying protein.
        
        
        22. The gene modifying system of embodiment 21, wherein the heterologous object sequence comprises the core nucleotides of an RT template sequence from Table 3A, Table 3B, Table 3C, or Table 3D, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the RT template sequence, or wherein the heterologous object sequence comprises a sequence of an RT template sequence from Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A, or a sequence having 1, 2, or 3 substitutions thereto.
        23. The gene modifying system of embodiment 21, wherein the heterologous object sequence comprises the core nucleotides of the RT template sequence of Table 3A, Table 3B, Table 3C, or Table 3D that corresponds to the gRNA spacer sequence, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the RT template sequence, or wherein the heterologous object sequence comprises a sequence of an RT template sequence from Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A that corresponds to the gRNA spacer sequence, or a sequence having 1, 2, or 3 substitutions thereto.
        24. The gene modifying system of any one of embodiments 21-23, wherein the PBS sequence has a sequence comprising the core nucleotides of the PBS sequence from the same row of Table 3A, Table 3B, Table 3C, or Table 3D as the RT template sequence, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 5′ end of the flanking nucleotides of the PBS sequence, or wherein the PBS sequence has a sequence of a PBS sequence from Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, E5A, E6, or E6A, or a sequence having 1, 2, or 3 substitutions thereto.
        25. The gene modifying system of one of embodiments 21-23, wherein the PBS sequence has a sequence comprising the core nucleotides of a PBS sequence of Table 3A, Table 3B, Table 3C, or Table 3D that corresponds to the RT template sequence, or a sequence having 1, 2, or 3 substitutions thereto, the gRNA spacer sequence, or both, and optionally comprises one or more consecutive nucleotides starting with the 5′ end of the flanking nucleotides of the PBS sequence, or wherein the PBS sequence has a sequence of a PBS sequence from Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A that corresponds to the RT template sequence, or a sequence having 1, 2, or 3 substitutions thereto.
        26. The gene modifying system of any one of embodiments 21-25, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold of Table 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
        27. The gene modifying system of any one of embodiments 21-25, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold of Table 12 that corresponds to the RT template sequence, the gRNA spacer sequence, or both, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
        28. A gene modifying system for modifying DNA, comprising:
        
            (a) a first RNA comprising, from 5′ to 3, (i) a guide RNA sequence that is complementary to a first portion of the human PAH gene, and (ii) a sequence (e.g., a scaffold region) that binds a gene modifying polypeptide (e.g., binds the Cas domain of the gene modifying polypeptide), and
            (b) a second RNA comprising (iii) a heterologous object sequence comprising a nucleotide substitution to introduce a mutation into a second portion of the human PAH gene, wherein the heterologous object sequence comprises the core nucleotides of an RT template sequence of Table 3A, Table 3B, Table 3C, or Table 3D, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the RT template sequence, or wherein the heterologous object sequence comprises an RT sequence from Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, E5A, E6, or E6A, or a sequence having 1, 2, or 3 substitutions thereto, and (iv) a primer region comprising at least 5, 6, 7, or 8 bases of 100% homology to a third portion of the human PAH gene, and (v) an RRS (RNA binding protein recognition sequence) that binds a gene modifying protein.
        
        
        29. The gene modifying system of embodiment 28, wherein the gRNA spacer comprises the core nucleotides of a gRNA spacer sequence of Table 1A, Table 1B, Table 1C, or Table 1D, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the gRNA spacer sequence, or wherein the gRNA spacer comprises a gRNA spacer sequence from Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A, or a sequence having 1, 2, or 3 substitutions thereto.
        30. The gene modifying system of embodiment 28, wherein the heterologous object sequence comprises the core nucleotides of the gRNA spacer sequence of Table 1A, Table 1B, Table 1C, or Table 1D that corresponds to the RT template sequence, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the gRNA spacer sequence, or wherein the heterologous object sequence comprises a gRNA spacer sequence from Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A that corresponds to the RT template sequence, or a sequence having 1, 2, or 3 substitutions thereto.
        31. The gene modifying system of any one of embodiments 28-30, wherein the PBS sequence has a sequence comprising the core nucleotides of the PBS sequence from the same row of Table 3A, Table 3B, Table 3C, or Table 3D as the RT template sequence, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 5′ end of the flanking nucleotides of the PBS sequence, or wherein the PBS sequence has a sequence comprising a PBS sequence from Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A, or a sequence having 1, 2, or 3 substitutions thereto.
        32. The gene modifying system of any one of embodiments 28-30, wherein the PBS sequence has a sequence comprising the core nucleotides of a PBS sequence of Table 3A, Table 3B, Table 3C, or Table 3D that corresponds to the RT template sequence, the gRNA spacer sequence, or both, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 5′ end of the flanking nucleotides of the PBS sequence, or wherein the PBS sequence has a sequence comprising a PBS sequence from Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, E5A, E6, or E6A that corresponds to the RT template sequence, the gRNA spacer sequence, or both, or a sequence having 1, 2, or 3 substitutions thereto.
        33. The gene modifying system of any one of embodiments 28-32, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold of Table 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
        34. The gene modifying system of any one of embodiments 28-32, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold of Table 12 that corresponds to the RT template sequence, the gRNA spacer sequence, or both, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
        35. A gRNA comprising (i) a gRNA spacer sequence that is complementary to a first portion of the human PAH gene, wherein the gRNA spacer has a sequence comprising the core nucleotides of a gRNA spacer sequence of Table 1A, Table 1B, Table 1C, or Table 1D, Table 2A, Table 2B, Table 2C, or Table 2D, or Table 4A, Table 4B, Table 4C, or Table 4D, or a sequence having 1, 2, or 3 substitutions thereto and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the gRNA spacer sequence, or wherein the gRNA spacer has a sequence comprising a gRNA spacer sequence from Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A or a sequence having 1, 2, or 3 substitutions thereto; and (ii) a gRNA scaffold.
        36. The gRNA of embodiment 35, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold of Table 12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
        37. The gRNA of embodiment 35, wherein the gRNA scaffold comprises a sequence of a gRNA scaffold of Table 12 that corresponds to the gRNA spacer sequence, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
        38. A template RNA comprising: (iii) a heterologous object sequence comprising a mutation region to introduce a mutation into a second portion of the human PAH gene, wherein the heterologous object sequence comprises the core nucleotides of an RT template sequence of Table 3A, Table 3B, Table 3C, or Table 3D, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the RT template sequence, or wherein the heterologous object sequence comprises an RT template sequence from Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A or a sequence having 1, 2, or 3 substitutions thereto, and (iv) a PBS sequence comprising at least 5, 6, 7, or 8 bases of 100% homology to a third portion of the human PAH gene.
        39. The template RNA according to embodiment 38, wherein the PBS sequence has a sequence comprising the core nucleotides of the PBS sequence from the same row of Table 3A, Table 3B, Table 3C, or Table 3D as the RT template sequence, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 5′ end of the flanking nucleotides of the PBS sequence.
        40. The template RNA according to embodiment 38, wherein the PBS sequence has a sequence comprising the core nucleotides of a PBS sequence of Table 3A, Table 3B, Table 3C, or Table 3D that corresponds to the RT template sequence, or a sequence having 1, 2, or 3 substitutions thereto, and optionally comprises one or more consecutive nucleotides starting with the 5′ end of the flanking nucleotides of the PBS sequence, or wherein the PBS sequence comprises a PBS sequence from Tables 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A that corresponds to the RT template sequence, or a sequence having 1, 2, or 3 substitutions thereto.
        41. The template RNA according to any one of embodiments 1-20 or 38-40, the gene modifying system of any one of embodiments 21-35, or the gRNA of any one of embodiments 35-37, wherein the mutation introduced by the system is a W408R, Q261R, Q243R, and/or IVS10-11A>G mutation (e.g., to correct a pathogenic R408W, R261Q, R243Q, and/or IVS10-11G>A mutation) of the PAH gene.
        42. The template RNA according to any one of embodiments 1-20 or 38-41 or the gene modifying system of any one of embodiments 35-37 or 41, wherein the pre-edit sequence comprises between about 1 nucleotide to about 35 nucleotides (e.g., comprises about 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, or 30-35 nucleotides) in length.
        43. The template RNA according to any one of embodiments 1-20 or 38-42 or the gene modifying system of any one of embodiments 35-37, 41, or 42, wherein the mutation region comprises a single nucleotide.
        44. The template RNA according to any one of embodiments 1-20 or 38-42 or the gene modifying system of any one of embodiments 35-37, 41, or 42, wherein the mutation region is at least two nucleotides in length.
        45. The template RNA according to any one of embodiments 1-20, 38-42, or 44 or the gene modifying system of any one of embodiments 35-37, 41, 42, or 44, wherein the mutation region is up to 32 (e.g., up to 5, 10, 15, 20, 25, 30, or 32) nucleotides in length and comprises one, two, or three sequence differences relative to a second portion of the human PAH gene.
        46. The template RNA according to any one of embodiments 1-20, 38-42, 44, or 45 or the gene modifying system of any one of embodiments 35-37, 41, 42, 44, or 45, wherein the mutation region comprises two sequences differences relative to a second portion of the human PAH gene.
        47. The template RNA according to any one of embodiments 1-20, 38-42, or 44-46 or the gene modifying system of any one of embodiments 35-37, 41, 42, or 44-46, wherein the mutation region comprises a first region (e.g., a first nucleotide) designed to correct a pathogenic mutation in the PAH gene and a second region (e.g., a second nucleotide) designed to inactivate a PAM sequence (e.g., a “PAM-kill” mutation as described herein).
        48. The template RNA according to any one of embodiments 1-20 or 38-46 or the gene modifying system of any one of embodiments 35-37 or 41-46, wherein the mutation region comprises less than 80%, 70%, 60%, 50%, 40%, or 30% identity to corresponding portion of the human PAH gene.
        49. The template RNA of any one of the preceding embodiments, wherein the template RNA comprises one or more silent mutations (e.g., silent substitutions), e.g., as exemplified in Tables 7A-7C, 8A-8D, E6, or E6A.
        50. The template RNA of any of the preceding embodiments, wherein the mutation region comprises a first region designed to correct a pathogenic mutation in the PAH gene and a second region designed to introduce a silent substitution.
        51. The template RNA of any one of the preceding embodiments, which comprises one or more chemically modified nucleotides.
        52. A gene modifying system comprising:
        
            a template RNA of any of embodiments 1-20, 38-46, or a system of any of embodiments 35-37 or 41-46, and
            a gene modifying polypeptide, or a nucleic acid (e.g., RNA) encoding the gene modifying polypeptide.
        
        
        53. The gene modifying system of embodiment 52, wherein the gene modifying polypeptide comprises:
        
            a reverse transcriptase (RT) domain (e.g., an RT domain from a retrovirus, or a polypeptide domain having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acids sequence identity thereto); and
            a Cas domain that binds to the target DNA molecule and is heterologous to the RT domain (e.g., a Cas9 domain); and
            optionally, a linker disposed between the RT domain and the Cas domain.
        
        
        54. The gene modifying system of embodiment 53, wherein the RT domain comprises:
        
            (a) an RT domain of Table 6; or
            (b) an RT domain from a murine leukemia virus (MMLV), a porcine endogenous retrovirus (PERV); Avian reticuloendotheliosis virus (AVIRE), a feline leukemia virus (FLV), simian foamy virus (SFV) (e.g., SFV3L), bovine leukemia virus (BLV), Mason-Pfizer monkey virus (MPMV), human foamy virus (HFV), or bovine foamy/syncytial virus (BFV/BSV).
        
        
        55. The gene modifying system of embodiment 53 or 54, wherein the Cas domain comprises a Cas domain of Table 7 or Table 8.
        56. The gene modifying system of any one of embodiments 53-55, wherein the Cas domain:
        
            (a) is a Cas9 domain;
            (b) is a SpCas9 domain, a BlatCas9 domain, a Nme2Cas9 domain, a PnpCas9 domain, a SauCas9 domain, a SauCas9-KKH domain, a SauriCas9 domain, a SauriCas9-KKH domain, a ScaCas9-Sc++ domain, a SpyCas9 domain, a SpyCas9-NG domain, a SpyCas9-SpRY domain, or a St1Cas9 domain; and/or
            (c) is a Cas9 domain comprising an N670A mutation, an N611A mutation, an N605A mutation, an N580A mutation, an N588A mutation, an N872A mutation, an N863 mutation, an N622A mutation, or an H840A mutation.
        
        
        57. The gene modifying system of embodiment 56, wherein the Cas9 domain binds a PAM sequence listed in Table 7 or Table 12.
        58. The gene modifying system of embodiment 57, wherein a second portion of the human PAH gene overlaps with a PAM recognized by the Cas domain, e.g., wherein the second portion of the human PAH gene is within the PAM or wherein the PAM is within the second portion of the human PAH gene).
        59. The gene modifying system any one of embodiments 52-58, wherein the gRNA spacer is a gRNA spacer according to Table 1A, Table 1B, Table 1C, or Table 1D, and the Cas domain comprises a Cas domain listed in the same row of Table 1A, Table 1B, Table 1C, or Table 1D, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
        60. The gene modifying system of any one of embodiments 52-58, wherein the template RNA comprises a sequence of a template RNA sequence of Table 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
        61. The gene modifying system of any one of embodiments 52-60, wherein:
        
            (a) the template RNA comprises a sequence of a template RNA sequence of Tables 3A-3D, 5A-5F, 8A-8D, E3, E3A, BB, E5, ESA, E6, or E6A;
            (b) the Cas domain comprises a Cas domain of Table 7 or Table 8;
            (c) the linker comprises a linker sequence of Table 10 (e.g., of any of SEQ ID NOs: 5217, 5106, 5190, and 5218); and
            (d) the gene modifying polypeptide comprises one or two NLS sequences from Table 11 (e.g., of any of SEQ ID NOs: 5245, 5290, 5323, 5330, 5349, 5350, 5351, and 4001).
        
        
        62. The gene modifying system of any of embodiments 52-61, which produces a first nick in a first strand of the human PAH gene.
        63. The gene modifying system of embodiment 62, which further comprises a second strand-targeting gRNA that directs a second nick to the second strand of the human PAH gene.
        64. The gene modifying system of embodiment 63, wherein the second strand-targeting gRNA comprises:
        
            (i) a sequence comprising the core nucleotides of a left gRNA spacer sequence or a right gRNA spacer sequence from Table 2A, Table 2B, Table 2C, or Table 2D, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the left gRNA spacer sequence or right gRNA spacer sequence; or
            (ii) a second-strand-targeting gRNA comprising a spacer sequence of Table 6A, or a spacer sequence having 1, 2, or 3 substitutions thereto.
        
        
        65. The gene modifying system of embodiment 63, wherein the second strand-targeting gRNA comprises a sequence comprising the core nucleotides of a left gRNA spacer sequence or a right gRNA spacer sequence from Table 2A, Table 2B, Table 2C, or Table 2D that corresponds to the gRNA spacer sequence of (i), and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the left gRNA spacer sequence or right gRNA spacer sequence.
        66. The gene modifying system of embodiment 63, wherein the second strand-targeting gRNA comprises:
        
            (i) a sequence comprising the core nucleotides of a second nick gRNA sequence from Table 4A, Table 4B, Table 4C, Table 4D, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the second nick gRNA sequence; or
            (ii) a second-strand-targeting gRNA comprising a spacer sequence from Table 6A or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
        
        
        67. The gene modifying system of embodiment 63, wherein the second strand-targeting gRNA comprises a sequence comprising the core nucleotides of the second nick gRNA sequence from Table 4A, Table 4B, Table 4C, or Table 4D that corresponds to the gRNA spacer sequence of (i), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and optionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the second nick gRNA sequence.
        68. The gene modifying system of any one of embodiments 52-67, wherein the second strand-targeting gRNA has a “PAM-in orientation” with the template RNA of the gene modifying system, e.g., as exemplified in Tables 2A-2D, 4A-4D, or 6A.
        69. The gene modifying system of any one of embodiments 52-68, the second strand-targeting gRNA targets a sequence overlapping the target mutation of the template RNA.
        70. The gene modifying system of embodiment 69, wherein second strand-targeting gRNA comprises:
        
            (i) a sequence (e.g., a spacer sequence) complementary to the PAH mutation;
            (ii) a sequence (e.g., a spacer sequence) complementary to the wild-type sequence at the target locus;
            (iii) a sequence (e.g., a spacer sequence) complementary to a SNP proximal to the target locus, e.g., a SNP contained in the genomic DNA of a subject (e.g., a patient);
            (iv) a sequence (e.g., spacer sequence) complementary to or comprising one or more silent substitutions proximal to the target locus.
        
        
        71. The template RNA, gene modifying system, or gRNA, of any one of the preceding embodiments, wherein the gRNA spacer comprises about 1, 2, 3, or more flanking nucleotides of the gRNA spacer.
        72. The template RNA or gene modifying system of any one of the preceding embodiments, wherein the heterologous object sequence comprises about 2, 3, 4, 5, 10, 20, 30, 40, or more flanking nucleotides of the RT template sequence.
        73. The template RNA or gene modifying system, of any one of the preceding embodiments, wherein the heterologous object sequence comprises between about 8-30, 9-25, 10-20, 11-16, or 12-15 (e.g., about 11-16) nucleotides.
        74. The template RNA or gene modifying system, of any one of the preceding embodiments, wherein the mutation region comprises 1, 2, or 3 nucleotide positions of sequence differences relative to the corresponding portion of the human PAH gene.
        75. The template RNA or gene modifying system of any one of the preceding embodiments, wherein the mutation region comprises at least 2 nucleotide positions of sequence difference relative to the corresponding portion of the human PAH gene.
        76. The template RNA or gene modifying system, of any one of the preceding embodiments, wherein the post-edit homology region and/or pre-edit homology region comprises 100% identity to the PAH gene.
        77. The template RNA or gene modifying system of any one of the preceding embodiments, wherein the PBS sequence additionally comprises about 1, 2, 3, 4, 5, 6, 7, or more flanking nucleotides.
        78. The template RNA or gene modifying system of any one of the preceding embodiments, wherein the PBS sequence comprises about 5-20, 8-16, 8-14, 8-13, 9-13, 9-12, or 10-12 (e.g., about 9-12) nucleotides.
        79. The template RNA or gene modifying system of any one of the preceding embodiments, wherein the PBS sequence binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nick site in the PAH gene.
        80. The gene modifying system of any one of the preceding embodiments, wherein the domains of the gene modifying polypeptide are joined by a peptide linker.
        81. The gene modifying system of embodiment 80, wherein the linker comprises a sequence of a linker of Table 10 (e.g., of any of SEQ ID NOs: 5217, 5106, 5190, and 5218).
        82. The gene modifying system of any one of the preceding embodiments, wherein the gene modifying polypeptide further comprise one or more nuclear localization sequences (NLS).
        83. The gene modifying system of embodiment 82, wherein the gene modifying polypeptide comprises a first NLS and a second NLS.
        84. The gene modifying system of embodiment 82 or 83, wherein the NLS comprises a sequence of a NLS of Table 11 (e.g., of any of SEQ ID NOs: 5245, 5290, 5323, 5330, 5349, 5350, 5351, and 4001).
        85. A template RNA comprising a sequence of a template RNA of Table 4A-4D, 5A-5F, 8A-8D, E3, E3A, BB, E5, E5A, E6, or E6A, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
        86. A template RNA comprising a sequence of a template RNA of Tables 4A-4D, 5A-5F, 8A-8D, E3, E3A, BB, E5, E5A, E6, or E6A.
        87. A gene modifying system comprising:
        
            (i) a template RNA comprising a sequence of a template RNA of Table 4A, Table 4B, Table 4C, or Table 4D, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and
            (ii) a second-nick gRNA sequence from the same row of Table 4A, Table 4B, Table 4C, or Table 4D as (i), a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
        
        
        88. A gene modifying system comprising:
        
            (i) a template RNA comprising a sequence of a template RNA of Table 4A, Table 4B, Table 4C, or Table 4D; and
            (ii) a second-nick gRNA sequence from the same row of Table 4A, Table 4B, Table 4C, or Table 4D as (i).
        
        
        89. A DNA encoding the template RNA of any one of embodiments 1-20, 38-48, 71-79, 85, or 86, or the gRNA of any one of embodiments 34-37.
        90. A pharmaceutical composition, comprising the system of any one of embodiments 49-84, 87, or 88, or one or more nucleic acids encoding the same, and a pharmaceutically acceptable excipient or carrier.
        91. The pharmaceutical composition of embodiment 90, wherein the pharmaceutically acceptable excipient or carrier is selected from the group consisting of a plasmid vector, a viral vector, a vesicle, and a lipid nanoparticle.
        92. The pharmaceutical composition of embodiment 91, wherein the viral vector is an adeno-associated virus.
        93. A host cell (e.g., a mammalian cell, e.g., a human cell) comprising the template RNA or gene modifying system of any one of the preceding embodiments.
        94. A method of making the template RNA of any one of embodiments 1-20, 38-48, 71-79, 85, or 86, the method comprising synthesizing the template RNA by in vitro transcription (e.g., solid state synthesis) or by introducing a DNA encoding the template RNA into a host cell under conditions that allow for production of the template RNA.
        95. A method for modifying a target site in the human PAH gene in a cell, the method comprising contacting the cell with the gene modifying system of any one of embodiments 49-84, 87, or 88, or DNA encoding the same, thereby modifying the target site in the human PAH gene in a cell.
        96. A method for modifying a target site in the human PAH gene in a cell, the method comprising contacting the cell with: (i) the template RNA of any one of embodiments 49-84, 87, or 88, or DNA encoding the same; and (ii) a gene modifying polypeptide or a nucleic acid encoding a gene modifying polypeptide, thereby modifying the target site in the human PAH gene in a cell.
        97. A method for treating a subject having a disease or condition associated with a mutation in the human PAH gene, the method comprising administering to the subject the gene modifying system of any one of embodiments 49-84, 87, or 88, or DNA encoding the same, thereby treating the subject having a disease or condition associated with a mutation in the human PAH gene.
        98. A method for treating a subject having a disease or condition associated with a mutation in the human PAH gene, the method comprising administering to the subject the template RNA of any one of embodiments 49-84, 87, or 88, or DNA encoding the same; and (ii) a gene modifying polypeptide or a nucleic acid encoding a gene modifying polypeptide, thereby treating the subject having a disease or condition associated with a mutation in the human PAH gene.
        99. The method of embodiment 97 or 98, wherein the disease or condition is phenylketonuria (PKU).
        100. The method of any one of embodiments 97-99, wherein the subject has a R408W, R261Q, R243Q, and/or IVS10-11G>A mutation.
        101. A method for treating a subject having PKU the method comprising administering to the subject the gene modifying system of any one of embodiments 49-84, 87, or 88, or DNA encoding the same, thereby treating the subject having PKU.
        102. A method for treating a subject having PKU the method comprising administering to the subject (i) the template RNA of any one of embodiments 49-84, 87, or 88, or DNA encoding the same, and (ii) a gene modifying polypeptide or a nucleic acid encoding a gene modifying polypeptide, thereby treating the subject having PKU.
        103. The gene modifying system or method of any one of the preceding embodiments, wherein introduction of the system into a target cell results in a correction of a pathogenic mutation in the PAH gene.
        104. The gene modifying system or method of any one of the preceding embodiments, wherein the pathogenic mutation is a W408R, Q261R, Q243R, and/or IVS10-11A>G mutation, and wherein the correction comprises an amino acid substitution of R408W, R261Q, or R243Q, or a nucleic acid substitution of IVS10-11G>A.
        105. The gene modifying system or method of any of the preceding embodiments, wherein correction of the mutation occurs in at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, or more) of target nucleic acids.
        106. The gene modifying system or method of any of the preceding embodiments, wherein correction of the mutation occurs in at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, or more) of target cells.
        107. The gene modifying system or method of any of the preceding embodiments, wherein the gene modifying system comprises a second strand-targeting gRNA, and wherein correction of the mutation in a population of target cells is increased relative to a population of target cells treated with a gene modifying system comprising a template RNA without a second strand-targeting gRNA.
        108. The gene modifying system or method of any of the preceding embodiments, wherein the template RNA comprises one or more silent substitutions (e.g., as exemplified in Tables 7A, X4, and X4A), and wherein correction of the mutation in a population of target cells is increased relative to a population of target cells treated with a gene modifying system comprising a template RNA that does not comprise one or more silent substitutions.
        109. The method of any of the preceding embodiments, wherein the cell is a mammalian cell, such as a human cell.
        110. The method of any one of the preceding embodiments, wherein the subject is a human.
        111. The method of any of the preceding embodiments, wherein the contacting occurs ex vivo, e.g., wherein the cell's or subject's DNA is modified ex vivo.
        112. The method of any of the preceding embodiments, wherein the contacting occurs in vivo, e.g., wherein the cell's or subject's DNA is modified in vivo.
        113. The method of any of the preceding embodiments, wherein contacting the cell or the subject with the system comprises contacting the cell or a cell within the subject with a nucleic acid (e.g., DNA or RNA) encoding the gene modifying polypeptide under conditions that allow for production of the gene modifying polypeptide.
    
    


Additional Enumerated Embodiments


    
    
        A1. A template RNA comprising, from 5′ to 3′:
        
            (i) a gRNA spacer that is complementary to a first portion of the human PAH gene, wherein the gRNA spacer has a nucleotide sequence comprising ACCTCAATCCTTTGGGTGTA (SEQ ID NO: 16355), or a nucleotide sequence having 1, 2, or 3 substitution thereto;
            (ii) a gRNA scaffold that binds a Cas domain of a gene modifying polypeptide,
            (iii) a heterologous object sequence comprising a mutation region to correct a mutation in a second portion of the human PAH gene, and
            (iv) a primer binding site (PBS) sequence comprising at least 5 bases with 100% identity to a third portion of the human PAH gene.
        
        
        A2. The template RNA of embodiment A1, wherein the gRNA spacer has a nucleotide sequence comprising ACCTCAATCCTTTGGGTGTA (SEQ ID NO: 16355).
        A3. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of at least 30 nucleotides from the 3′ end of a sequence according to tcactcaagcctgtggtifiggtcttaggaactttgctgccacaatacctCggcccttctcagttcgctacgacccatac (SEQ ID NO: 24984), or a sequence having 1, 2, 3, or 4 substitutions thereto.
        A4. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of tcactcaagcctgtggtifiggtcttaggaactttgctgccacaatacctCggcccttctcagttcgctacgacccatac (SEQ ID NO: 24984), or comprises at least 40, 50, 60, or 70 nucleotides from the 3′ end of said sequence, or a sequence having 1, 2, 3, or 4 substitutions thereto.
        A5. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of at least 30 nucleotides from the 3′ end of a sequence according to tcactcaagcctgtggtifiggtcttaggaactttgctgccacaatacctCggcccttctcagttcgctacgacccatac (SEQ ID NO: 24984).
        A6. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of tcactcaagcctgtggtifiggtcttaggaactttgctgccacaatacctCggcccttctcagttcgctacgacccatac (SEQ ID NO: 24984), or comprises at least 40, 50, 60, or 70 nucleotides from the 3′ end of said sequence.
        A7. The template RNA of any of the preceding embodiments, wherein the PBS comprises a sequence of at least 5, 6, 7, or 8 nucleotides from the 5′ end of a sequence according to acccaaagg, or a sequence having 1 substitution thereto.
        A8. The template RNA of any of the preceding embodiments, wherein the PBS comprises a sequence of acccaaagg
        A9. A template RNA comprising, from 5′ to 3′:
        
            (i) a gRNA spacer that is complementary to a first portion of the human PAH gene, wherein the gRNA spacer has a nucleotide sequence comprising CCTCAATCCTTTGGGTGTAT (SEQ ID NO: 16332), or a nucleotide sequence having 1, 2, or 3 substitution thereto;
            (ii) a gRNA scaffold that binds a Cas domain of a gene modifying polypeptide,
            (iii) a heterologous object sequence comprising a mutation region to correct a mutation in a second portion of the human PAH gene, and
            (iv) a primer binding site (PBS) sequence comprising at least 5 bases with 100% identity to a third portion of the human PAH gene.
        
        
        A10. The template RNA of embodiment A9, wherein the gRNA spacer has a nucleotide sequence comprising CCTCAATCCTTTGGGTGTAT (SEQ ID NO: 16332).
        A11. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of at least 50 nucleotides from the 3′ end of a sequence according to tcactcaagcctgtggtifiggtcttaggaactttgctgccacaatacctCggcccttctcagttcgctacgacccata (SEQ ID NO: 24975), or a sequence having 1, 2, 3, or 4 substitutions thereto.
        A12. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of tcactcaagcctgtggtifiggtcttaggaactttgctgccacaatacctCggcccttctcagttcgctacgacccata (SEQ ID NO: 24975), or comprises at least 60 or 70 nucleotides from the 3′ end of said sequence, or a sequence having 1, 2, 3, or 4 substitutions thereto.
        A13. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of at least 50 nucleotides from the 3′ end of a sequence according to tcactcaagcctgtggtifiggtcttaggaactttgctgccacaatacctCggcccttctcagttcgctacgacccata (SEQ ID NO: 24975).
        A14. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of tcactcaagcctgtggtifiggtcttaggaactttgctgccacaatacctCggcccttctcagttcgctacgacccata (SEQ ID NO: 24975), or comprises at least 60 or 70 nucleotides from the 3′ end of said sequence.
        A15. The template RNA of any of the preceding embodiments, wherein the PBS comprises a sequence of at least 5, 6, 7, or 8 nucleotides from the 5′ end of a sequence according to cacccaaag, or a sequence having 1 substitution thereto.
        A16. The template RNA of any of the preceding embodiments, wherein the PBS comprises a sequence of cacccaaag.
        A17. A template RNA comprising, from 5′ to 3′:
        
            (i) a gRNA spacer that is complementary to a first portion of the human PAH gene, wherein the gRNA spacer has a nucleotide sequence comprising TGGGTCGTAGCGAACTGAGA (SEQ ID NO: 16102), or a nucleotide sequence having 1, 2, or 3 substitution thereto;
            (ii) a gRNA scaffold that binds a Cas domain of a gene modifying polypeptide,
            (iii) a heterologous object sequence comprising a mutation region to correct a mutation in a second portion of the human PAH gene, and
            (iv) a primer binding site (PBS) sequence comprising at least 5 bases with 100% identity to a third portion of the human PAH gene.
        
        
        A18. The template RNA of embodiment A17, wherein the gRNA spacer has a nucleotide sequence comprising TGGGTCGTAGCGAACTGAGA (SEQ ID NO: 16102).
        A19. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of at least 10 nucleotides from the 3′ end of a sequence according to tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttct (SEQ ID NO: 24863), or a sequence having 1, 2, 3, or 4 substitutions thereto.
        A20. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttct (SEQ ID NO: 24863), or comprises at least 20, 30, 40, of 50 nucleotides from the 3′ end of said sequence, or a sequence having 1, 2, 3, or 4 substitutions thereto.
        A21. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of at least 10 nucleotides from the 3′ end of a sequence according to tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttct (SEQ ID NO: 24863).
        A22. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttct (SEQ ID NO: 24863), or comprises at least 20, 30, 40, of 50 nucleotides from the 3′ end of said sequence.
        A23. The template RNA of any of the preceding embodiments, wherein the PBS comprises a sequence of at least 5, 6, 7, or 8 nucleotides from the 5′ end of a sequence according to cagttcgct, or a sequence having 1 substitution thereto.
        A24. The template RNA of any of the preceding embodiments, wherein the PBS comprises a sequence of cagttcgct.
        A25. A template RNA comprising, from 5′ to 3′:
        
            (i) a gRNA spacer that is complementary to a first portion of the human PAH gene, wherein the gRNA spacer has a nucleotide sequence comprising GGGTCGTAGCGAACTGAGAA (SEQ ID NO: 16084), or a nucleotide sequence having 1, 2, or 3 substitution thereto;
            (ii) a gRNA scaffold that binds a Cas domain of a gene modifying polypeptide,
            (iii) a heterologous object sequence comprising a mutation region to correct a mutation in a second portion of the human PAH gene, and
            (iv) a primer binding site (PBS) sequence comprising at least 5 bases with 100% identity to a third portion of the human PAH gene.
        
        
        A26. The template RNA of embodiment A25, wherein the gRNA spacer has a nucleotide sequence comprising GGGTCGTAGCGAACTGAGAA (SEQ ID NO: 16084).
        A27. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of at least 9 nucleotides from the 3′ end of a sequence according to tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttc (SEQ ID NO: 24856), or a sequence having 1, 2, 3, or 4 substitutions thereto.
        A28. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttc (SEQ ID NO: 24856), or comprises at least 10, 20, 30, 40, or 50 nucleotides from the 3′ end of said sequence, or a sequence having 1, 2, 3, or 4 substitutions thereto.
        A29. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of at least 9 nucleotides from the 3′ end of a sequence according to tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttc (SEQ ID NO: 24856).
        A30. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCggcccttc (SEQ ID NO: 24856), or comprises at least 10, 20, 30, 40, or 50 nucleotides from the 3′ end of said sequence.
        A31. The template RNA of any of the preceding embodiments, wherein the PBS comprises a sequence of at least 5, 6, 7, or 8 nucleotides from the 5′ end of a sequence according to tcagttcgc, or a sequence having 1 substitution thereto.
        A32. The template RNA of any of the preceding embodiments, wherein the PBS comprises a sequence of tcagttcgc
        A33. A template RNA comprising, from 5′ to 3′:
        
            (i) a gRNA spacer that is complementary to a first portion of the human PAH gene, wherein the gRNA spacer has a nucleotide sequence comprising TAGCGAACTGAGAAGGGCCA (SEQ ID NO: 16011), or a nucleotide sequence having 1, 2, or 3 substitution thereto;
            (ii) a gRNA scaffold that binds a Cas domain of a gene modifying polypeptide,
            (iii) a heterologous object sequence comprising a mutation region to correct a mutation in a second portion of the human PAH gene, and
            (iv) a primer binding site (PBS) sequence comprising at least 5 bases with 100% identity to a third portion of the human PAH gene.
        
        
        A34. The template RNA of embodiment A33, wherein the gRNA spacer has a nucleotide sequence comprising TAGCGAACTGAGAAGGGCCA (SEQ ID NO: 16011).
        A35. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of at least 3 nucleotides from the 3′ end of a sequence according to tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCgg (SEQ ID NO: 24817), or a sequence having 1, 2, 3, or 4 substitutions thereto.
        A36. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCgg (SEQ ID NO: 24817), or comprises at least 5, 10, 20, 30, 40, or 50 nucleotides from the 3′ end of said sequence, or a sequence having 1, 2, 3, or 4 substitutions thereto.
        A37. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of at least 3 nucleotides from the 3′ end of a sequence according to tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCgg (SEQ ID NO: 24817).
        A38. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of tcactcaagcctgtggttttggtcttaggaactttgctgccacaatacctCgg (SEQ ID NO: 24817), or comprises at least 5, 10, 20, 30, 40, or 50 nucleotides from the 3′ end of said sequence.
        A39. The template RNA of any of the preceding embodiments, wherein the PBS comprises a sequence of at least 5, 6, 7, or 8 nucleotides from the 5′ end of a sequence according to cccttctca, or a sequence having 1 substitution thereto.
        A40. The template RNA of any of the preceding embodiments, wherein the PBS comprises a sequence of cccttctca.
        A41. A template RNA comprising, from 5′ to 3′:
        
            (i) a gRNA spacer that is complementary to a first portion of the human PAH gene, wherein the gRNA spacer has a nucleotide sequence comprising ACTTTGCTGCCACAATACCT (SEQ ID NO: 16032), or a nucleotide sequence having 1, 2, or 3 substitution thereto;
            (ii) a gRNA scaffold that binds a Cas domain of a gene modifying polypeptide,
            (iii) a heterologous object sequence comprising a mutation region to correct a mutation in a second portion of the human PAH gene, and
            (iv) a primer binding site (PBS) sequence comprising at least 5 bases with 100% identity to a third portion of the human PAH gene.
        
        
        A42. The template RNA of embodiment A41, wherein the gRNA spacer has a nucleotide sequence comprising ACTTTGCTGCCACAATACCT (SEQ ID NO: 16032).
        A43. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of at least 4 nucleotides from the 3′ end of a sequence according to caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggccGagg (SEQ ID NO: 24825), or a sequence having 1, 2, 3, or 4 substitutions thereto.
        A44. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggccGagg (SEQ ID NO: 24825), or comprises at least 5, 10, 20, 30, 40, or 50 nucleotides from the 3′ end of said sequence, or a sequence having 1, 2, 3, or 4 substitutions thereto.
        A45. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of at least 4 nucleotides from the 3′ end of a sequence according to caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggccGagg (SEQ ID NO: 24825).
        A46. The template RNA of any of the preceding embodiments, wherein the heterologous object sequence comprises a sequence of caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggccGagg (SEQ ID NO: 24825), or comprises at least 5, 10, 20, 30, 40, or 50 nucleotides from the 3′ end of said sequence.
        A47. The template RNA of any of the preceding embodiments, wherein the PBS comprises a sequence of at least 5, 6, 7, 8, 9, 10, or 15 nucleotides from the 5′ end of a sequence according to tattgtggcagcaaagt (SEQ ID NO: 37633), or a sequence having 1 substitution thereto.
        A48. The template RNA of any of the preceding embodiments, wherein the PBS comprises a sequence of tattgtggcagcaaagt (SEQ ID NO: 37633).
        A49. The template RNA of any of the preceding embodiments, wherein the gRNA scaffold has a sequence according to GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAA AGTGGCACCGAGTCGGTGC (SEQ ID NO: 37627), or a sequence having at least 90% identity thereto.
        A50. The template RNA of any of the preceding embodiments, wherein the gRNA scaffold has a sequence according to
    
    









(SEQ ID NO: 37627)


GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAA


CTTGAAAAAGTGGCACCGAGTCGGTGC.






    
    
        A51. The template RNA of any of the preceding embodiments, wherein the mutation region comprises a single nucleotide.
        A52. The template RNA of any of embodiments A1-51, wherein the mutation region is at least two nucleotides in length.
        A53. The template RNA of any of the preceding embodiments, wherein the mutation region is up to 20 nucleotides in length and comprises one, two, or three sequence differences relative to the second portion of the human PAH gene.
        A54. The template RNA of any of embodiments A1-53, wherein the mutation region comprises a first region designed to correct a pathogenic mutation in the PAH gene and a second region designed to inactivate a PAM sequence.
        A55. The template RNA of any of embodiments A1-54, wherein the mutation region comprises a first region designed to correct a pathogenic mutation in the PAH gene and a second region designed to introduce a silent substitution.
        A56. The template RNA of any of the preceding embodiments, which is configured to edit a pathogenic R408W mutation in the human PAH gene.
        A57. The template RNA of embodiment A56, which is configured to convert an R408W mutation to arginine.
        A58. The template RNA of any of the preceding embodiments, which comprises one or more chemically modified nucleotides.
        A59. A gene modifying system comprising:
        
            a template RNA of any of the preceding embodiments, and
            a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide.
        
        
        A60. The gene modifying system of embodiment A59, wherein the gene modifying polypeptide comprises an RT domain having a sequence according to SEQ ID NO: 8,003, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
        A61. The gene modifying system of embodiment A59, wherein the gene modifying polypeptide comprises an RT domain having a sequence according to SEQ ID NO: 8,020, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
        A62. The gene modifying system of embodiment 5A9, wherein the gene modifying polypeptide comprises an RT domain having a sequence according to SEQ ID NO: 8,074, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
        A63. The gene modifying system of embodiment A59, wherein the gene modifying polypeptide comprises an RT domain having a sequence according to SEQ ID NO: 8,113, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
        A64. The gene modifying system of embodiment A59, wherein the gene modifying polypeptide comprises DNA binding domain having a sequence of a Cas9 nickase comprising an N863A mutation, e.g., a sequence according to SEQ ID NO: 11,096, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.
        A65. The gene modifying system of embodiment A59, which produces a first nick in a first strand of the human PAH gene.
        A66. The gene modifying system of embodiment A65, which further comprises a second strand-targeting gRNA that directs a second nick to the second strand of the human PAH gene.
        A67. The gene modifying system of embodiment A66, wherein the first nick and the second nick are 80-120 nucleotides apart.
        A68. The gene modifying system of embodiment A66, wherein the template RNA and the second strand-targeting gRNA are configured to produce an outward nick orientation.
        A69. The gene modifying system of embodiment A66, wherein the second strand-targeting gRNA comprises a spacer sequence that is complementary to a human PAH gene having a disease mutation or a wild-type sequence.
        A70. A method for modifying a target site in the human PAH gene in a cell, the method comprising contacting the cell with the gene modifying system of embodiment 59, thereby modifying the target site in the human PAH gene in a cell.
        A71. The method of embodiment A70, wherein correction of the mutation occurs in at least 30% of target nucleic acids.
        A72. A method for treating a subject having a disease or condition associated with a mutation in the human PAH gene, wherein the disease or condition is phenylketonuria (PKU) or hyperphenylalaninemia (e.g., mild or severe hyperphenylalaninemia), the method comprising administering to the subject the gene modifying system of embodiment 59, thereby treating the subject having a disease or condition associated with a mutation in the human PAH gene.
    
    





BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts a gene modifying system as described herein. The left hand diagram shows the gene modifying polypeptide, which comprises a Cas nickase domain (e.g., spCas9 N863A) and a reverse transcriptase domain (RT domain) which are linked by a linker. The right hand diagram shows the template RNA which comprises, from 5′ to 3′, a gRNA spacer, a gRNA scaffold, a heterologous object sequence, and a primer binding site sequence (PBS sequence). The heterologous object sequence can comprise a mutation region that comprises one or more sequence differences relative to the target site. The heterologous object sequence can also comprise a pre-edit homology region and a post-edit homology region, which flank the mutation region. Without wishing to be bound by theory, it is thought that the gRNA spacer of the template RNA binds to the second strand of a target site in the genome, and the gRNA scaffold of the template RNA binds to the gene modifying polypeptide, e.g., localizing the gene modifying polypeptide to the target site in the genome. It is thought that the Cas domain of the gene modifying polypeptide nicks the target site (e.g., the first strand of the target site), e.g., allowing the PBS sequence to bind to a sequence adjacent to the site to be altered on the first strand of the target site. It is thought that the RT domain of the gene modifying polypeptide uses the first strand of the target site that is bound to the complementary sequence comprising the PBS sequence of the template RNA as a primer and the heterologous object sequence of the template RNA as a template to, e.g., polymerize a sequence complementary to the heterologous object sequence. Without wishing to be bound by theory, it is thought that reverse transcription can then proceed through the pre-edit homology region, then through the mutation region, and then through the post-edit homology region, thereby producing a DNA strand comprising a mutation specified by the heterologous object sequence.
FIG. 2 is a graph showing the percent rewriting achieved using the RNAV209-013 or RNAV214-040 gene modifying polypeptides with the indicated template RNAs.
FIG. 3 is a graph showing the amount of Fah mRNA relative to wild type when template RNAs are used with the RNAV209-013 or RNAV214-040 gene modifying polypeptides.
FIG. 4 is a graph showing the percentage of Cas9-positive hepatocytes 6 hours following dosing with LNPs containing various gene modifying polypeptides and template RNAs.
FIG. 5 is a graph showing the rewrite levels in liver samples 6 days following dosing with LNPs containing various gene modifying polypeptides and template RNAs.
FIG. 6 is a graph showing wild type Fah mRNA restoration compared to littermate heterozygous mice in liver samples following dosing with LNPs containing various gene modifying polypeptides and template RNAs.
FIG. 7 is a graph showing Fah protein distribution in liver samples following dosing with LNPs containing various gene modifying polypeptides and template RNAs.
FIG. 8 is a series of western blots showing Cas9-RT Expression 6 hours after infusion of Cas9-RT mRNA+TTR guide LNP. Each lane represents an individual animal where 20 μg of tissue homogenate was added per lane. Positive control was from an in vitro cell experiment where Cas9-RT was expressed (described previously). GAPDH was used as a loading control for each sample. n=4 per group, vehicle or treated.
FIG. 9 is a graph showing gene editing of TTR locus after treatment with Cas9-RT mRNA+TTR guide LNP. Level of indels detected at the TTR locus measured by TIDE analysis of Sanger sequencing of the TTR locus where the protospacer targets.
FIG. 10 is a graph showing that TTR Serum levels decrease after treatment with Cas9-RT mRNA+TTR guide LNP. Measurement of circulating TTR levels 5 days after mice were treated with LNPs encapsulating Cas9-RT+TTR guide RNA.
FIG. 11 is a graph showing Cas9-RT Expression after infusion of Cas9-RT mRNA+TTR guide LNP. Relative expression quantified by ProteinSimple Jess capillary electrophoresis Western blot. Numbers in the symbols are animal number in group. Vehicle n=2, Cas9-RT+TTR guide n=3.
FIG. 12 is a graph showing gene editing of TTR locus after infusion of Cas9-RT mRNA+TTR guide LNP. Level of indels detected at the TTR locus were measured by amplicon sequencing of the TTR locus where the protospacer targets. Each animal had 8 different biopsies taken across the liver where amplicon sequencing measured the percentage of reads showing an indel.
FIG. 13 is a graph showing percent rewriting in primary mouse hepatocytes nucleofected with various gene modifying systems.
FIG. 14 is a graph showing percent editing in primary mouse hepatocytes nucleofected with various gene modifying systems containing second-nick gRNAs.
FIG. 15 is a heat map showing rewriting efficiency of various gene modifying systems with or without second-nick gRNAs.
FIG. 16 is a graph showing the percent of mouse hepatocytes expressing Cas9 six hours post-dosing with various gene modifying systems.
FIG. 17 is a pair of western blots showing expression of Cas9 in mouse liver samples six hours post-dosing with various gene modifying systems.
FIG. 18 is a graph showing the level of phenylalanine (Phe) present in plasma samples 7 days post-dosing with various gene modifying systems.
FIGS. 19A-19B are graphs showing percent rewriting (FIG. 19A) and percent indel (FIG. 19B) in mouse liver 7 days post-dosing with various gene modifying systems.
FIGS. 20A-20C are graphs showing percent rewriting in liver samples (FIG. 20A), levels of Phe in plasma (FIG. 20B), and percent indels in mouse liver (FIG. 20C) 7 days post-dosing with various gene modifying systems.
FIGS. 21A-21B are a pair of graphs showing percent rewriting and percent indel in liver samples (FIG. 21A) and levels of Phe in plasma (FIG. 21B) 7 days post-dosing with various gene modifying systems with or without second-nick gRNAs.
FIG. 22 is a graph showing the level of phenylalanine (Phe) in the plasma versus percent rewriting in samples obtained from mice treated with various gene modifying systems.
FIG. 23 is a graph showing percent rewriting in HEK293T cells containing the M fascicularis PAH gene for four different mutation types using template RNAs containing four different spacer sequences.
FIGS. 24A-24C are graphs showing percent rewriting (FIG. 24A) and percent indels (FIG. 24B) in mouse liver cells, or concentration of Phe in plasma (FIG. 24C) days post-dosing with LNPs comprising various gene modifying systems.
FIGS. 25A-25C are heat maps showing percent rewriting for each combination of template RNA and second strand-targeting RNA in primary human hepatocytes (FIG. 25A) and primary mouse hepatocytes (FIG. 25C) following transfection with (FIGS. 25A and 25B) or LNP delivery of (FIG. 25C) various gene modifying systems.
FIGS. 26A-26B are graphs showing percent rewriting (FIG. 26A) and percent indels (FIG. 26B) in 7- and 28-day liver samples following LNP delivery of gene modifying systems to mice.
FIG. 27 is a graph showing the concentration of Phe in 7- and 28-day plasma samples following LNP delivery of gene modifying systems to mice.
FIG. 28 is a graph showing the concentration of Phe in 7- and 28-day brain samples following LNP delivery of gene modifying systems to mice.
FIG. 29 is a graph showing the concentration of Phe in the brain versus concentration of Phe in the plasma from samples used to generate FIGS. 27 and 28.
FIGS. 30A-3011 are heat maps showing percent rewriting for each combination of template RNA and second strand-targeting RNA following mRNA delivery of gene modifying systems to primary cyno hepatocytes.
FIGS. 31A-31B are a graph stratified by silent substitution (FIG. 31A) showing percent total rewriting following mRNA delivery of various gene modifying systems utilizing the hPKU3 template RNAs comprising various silent substitutions into human iPSC-derived hepatoblasts and a chart (FIG. 31B) showing the particular silent substitutions utilized in FIG. 31A. FIG. 31B discloses SEQ ID NOS 37642-37649, respectively, in order of appearance.
FIGS. 32A-32B are a graph stratified by silent substitution (FIG. 32A) showing percent total rewriting following mRNA delivery of various gene modifying systems utilizing the hPKU4 template RNAs comprising various silent substitutions into human iPSC-derived hepatoblasts and a chart (FIG. 32B) showing the particular silent substitutions utilized in FIG. 32A. FIG. 32B discloses SEQ ID NOS 37650-37657, respectively, in order of appearance.
FIGS. 33A-33B are a graph (33A) and a chart (33B) showing are a graph stratified by silent substitution (FIG. 33A) showing percent total rewriting following mRNA delivery of various gene modifying systems utilizing the hPKU5 template RNAs comprising various silent substitutions into human iPSC-derived hepatoblasts and a chart (FIG. 33B) showing the particular silent substitutions utilized in FIG. 33A. FIG. 33B discloses SEQ ID NOS 37658-37665, respectively, in order of appearance.
FIGS. 34A-34B are a graph stratified by silent substitution (FIG. 34A) showing percent total rewriting following mRNA delivery of various gene modifying systems utilizing the hPKU6 template RNAs comprising various silent substitutions into human iPSC-derived hepatoblasts and a chart (FIG. 34B) showing the particular silent substitutions utilized in FIG. 34A.
FIG. 35 is a graph showing serum levels of Phe in mice following treatment with LNPs comprising various gene modifying systems.
FIGS. 36A-36B are graphs showing percent rewriting (FIG. 36A) and percent indels (FIG. 36B) in mouse liver following treatment with LNPs comprising various gene modifying systems.



DETAILED DESCRIPTION
Definitions
The term “expression cassette,” as used herein, refers to a nucleic acid construct comprising nucleic acid elements sufficient for the expression of the nucleic acid molecule of the instant invention.
A “gRNA spacer”, as used herein, refers to a portion of a nucleic acid that has complementarity to a target nucleic acid and can, together with a gRNA scaffold, target a Cas protein to the target nucleic acid.
A “gRNA scaffold”, as used herein, refers to a portion of a nucleic acid that can bind a Cas protein and can, together with a gRNA spacer, target the Cas protein to the target nucleic acid. In some embodiments, the gRNA scaffold comprises a crRNA sequence, tetraloop, and tracrRNA sequence.
A “gene modifying polypeptide”, as used herein, refers to a polypeptide comprising a retroviral reverse transcriptase, or a polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a retroviral reverse transcriptase, which is capable of integrating a nucleic acid sequence (e.g., a sequence provided on a template nucleic acid) into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell). In some embodiments, the gene modifying polypeptide is capable of integrating the sequence substantially without relying on host machinery. In some embodiments, the gene modifying polypeptide integrates a sequence into a random position in a genome, and in some embodiments, the gene modifying polypeptide integrates a sequence into a specific target site. In some embodiments, a gene modifying polypeptide includes one or more domains that, collectively, facilitate 1) binding the template nucleic acid, 2) binding the target DNA molecule, and 3) facilitate integration of the at least a portion of the template nucleic acid into the target DNA. Gene modifying polypeptides include both naturally occurring polypeptides as well as engineered variants of the foregoing, e.g., having one or more amino acid substitutions to the naturally occurring sequence. Gene modifying polypeptides also include heterologous constructs, e.g., where one or more of the domains recited above are heterologous to each other, whether through a heterologous fusion (or other conjugate) of otherwise wild-type domains, as well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous sub-domain or other substituted domain. Exemplary gene modifying polypeptides, and systems comprising them and methods of using them, that can be used in the methods provided herein are described, e.g., in PCT/US2021/020948, which is incorporated herein by reference with respect to gene modifying polypeptides that comprise a retroviral reverse transcriptase domain. In some embodiments, a gene modifying polypeptide integrates a sequence into a gene. In some embodiments, a gene modifying polypeptide integrates a sequence into a sequence outside of a gene. A “gene modifying system,” as used herein, refers to a system comprising a gene modifying polypeptide and a template nucleic acid.
The term “domain” as used herein refers to a structure of a biomolecule that contributes to a specified function of the biomolecule. A domain may comprise a contiguous region (e.g., a contiguous sequence) or distinct, non-contiguous regions (e.g., non-contiguous sequences) of a biomolecule. Examples of protein domains include, but are not limited to, an endonuclease domain, a DNA binding domain, a reverse transcription domain; an example of a domain of a nucleic acid is a regulatory domain, such as a transcription factor binding domain. In some embodiments, a domain (e.g., a Cas domain) can comprise two or more smaller domains (e.g., a DNA binding domain and an endonuclease domain).
As used herein, the term “exogenous”, when used with reference to a biomolecule (such as a nucleic acid sequence or polypeptide) means that the biomolecule was introduced into a host genome, cell or organism by the hand of man. For example, a nucleic acid that is as added into an existing genome, cell, tissue or subject using recombinant DNA techniques or other methods is exogenous to the existing nucleic acid sequence, cell, tissue or subject.
As used herein, “first strand” and “second strand”, as used to describe the individual DNA strands of target DNA, distinguish the two DNA strands based upon which strand the reverse transcriptase domain initiates polymerization, e.g., based upon where target primed synthesis initiates. The first strand refers to the strand of the target DNA upon which the reverse transcriptase domain initiates polymerization, e.g., where target primed synthesis initiates. The second strand refers to the other strand of the target DNA. First and second strand designations do not describe the target site DNA strands in other respects; for example, in some embodiments the first and second strands are nicked by a polypeptide described herein, but the designations ‘first’ and ‘second’ strand have no bearing on the order in which such nicks occur.
The term “heterologous,” as used herein to describe a first element in reference to a second element means that the first element and second element do not exist in nature disposed as described. For example, a heterologous polypeptide, nucleic acid molecule, construct or sequence refers to (a) a polypeptide, nucleic acid molecule or portion of a polypeptide or nucleic acid molecule sequence that is not native to a cell in which it is expressed, (b) a polypeptide or nucleic acid molecule or portion of a polypeptide or nucleic acid molecule that has been altered or mutated relative to its native state, or (c) a polypeptide or nucleic acid molecule with an altered expression as compared to the native expression levels under similar conditions. For example, a heterologous regulatory sequence (e.g., promoter, enhancer) may be used to regulate expression of a gene or a nucleic acid molecule in a way that is different than the gene or a nucleic acid molecule is normally expressed in nature. In another example, a heterologous domain of a polypeptide or nucleic acid sequence (e.g., a DNA binding domain of a polypeptide or nucleic acid encoding a DNA binding domain of a polypeptide) may be disposed relative to other domains or may be a different sequence or from a different source, relative to other domains or portions of a polypeptide or its encoding nucleic acid. In certain embodiments, a heterologous nucleic acid molecule may exist in a native host cell genome, but may have an altered expression level or have a different sequence or both. In other embodiments, heterologous nucleic acid molecules may not be endogenous to a host cell or host genome but instead may have been introduced into a host cell by transformation (e.g., transfection, electroporation), wherein the added molecule may integrate into the host genome or can exist as extra-chromosomal genetic material either transiently (e.g., mRNA) or semi-stably for more than one generation (e.g., episomal viral vector, plasmid or other self-replicating vector).
As used herein, “insertion” of a sequence into a target site refers to the net addition of DNA sequence at the target site, e.g., where there are new nucleotides in the heterologous object sequence with no cognate positions in the unedited target site. In some embodiments, a nucleotide alignment of the PBS sequence and heterologous object sequence to the target nucleic acid sequence would result in an alignment gap in the target nucleic acid sequence.
As used herein, a “deletion” generated by a heterologous object sequence in a target site refers to the net deletion of DNA sequence at the target site, e.g., where there are nucleotides in the unedited target site with no cognate positions in the heterologous object sequence. In some embodiments, a nucleotide alignment of the PBS sequence and heterologous object sequence to the target nucleic acid sequence would result in an alignment gap in the molecule comprising the PBS sequence and heterologous object sequence.
The term “inverted terminal repeats” or “ITRs” as used herein refers to AAV viral cis-elements named so because of their symmetry. These elements promote efficient multiplication of an AAV genome. It is hypothesized that the minimal elements for ITR function are a Rep-binding site (RBS; 5′-GCGCGCTCGCTCGCTC-3′ for AAV2; SEQ ID NO: 4601) and a terminal resolution site (TRS; 5′-AGTTGG-3′ for AAV2) plus a variable palindromic sequence allowing for hairpin formation. According to the present invention, an ITR comprises at least these three elements (RBS, TRS, and sequences allowing the formation of an hairpin). In addition, in the present invention, the term “ITR” refers to ITRs of known natural AAV serotypes (e.g. ITR of a serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 AAV), to chimeric ITRs formed by the fusion of ITR elements derived from different serotypes, and to functional variants thereof. “Functional variant” refers to a sequence presenting a sequence identity of at least 80%, 85%, 90%, preferably of at least 95% with a known ITR and allowing multiplication of the sequence that includes said ITR in the presence of Rep proteins.
The term “mutation region,” as used herein, refers to a region in a template RNA having one or more sequence difference relative to the corresponding sequence in a target nucleic acid. The sequence difference may comprise, for example, a substitution, insertion, frameshift, or deletion.
The term “mutated” when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence are inserted, deleted, or changed compared to a reference (e.g., native) nucleic acid sequence. A single alteration may be made at a locus (a point mutation), or multiple nucleotides may be inserted, deleted, or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the art.
“Nucleic acid molecule” refers to both RNA and DNA molecules including, without limitation, complementary DNA (“cDNA”), genomic DNA (“gDNA”), and messenger RNA (“mRNA”), and also includes synthetic nucleic acid molecules, such as those that are chemically synthesized or recombinantly produced, such as RNA templates, as described herein. The nucleic acid molecule can be double-stranded or single-stranded, circular, or linear. If single-stranded, the nucleic acid molecule can be the sense strand or the antisense strand. Unless otherwise indicated, and as an example for all sequences described herein under the general format “SEQ ID NO:,” or “nucleic acid comprising SEQ ID NO:1” refers to a nucleic acid, at least a portion which has either (i) the sequence of SEQ ID NO:1, or (ii) a sequence complimentary to SEQ ID NO:1. The choice between the two is dictated by the context in which SEQ ID NO:1 is used. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target. Nucleic acid sequences of the present disclosure may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with an analog, inter-nucleotide modifications such as uncharged linkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (for example, phosphorothioates, phosphorodithioates, etc.), pendant moieties, (for example, polypeptides), intercalators (for example, acridine, psoralen, etc.), chelators, alkylators, and modified linkages (for example, alpha anomeric nucleic acids, etc.). Also included are chemically modified bases (see, for example, Table 13), backbones (see, for example, Table 14), and modified caps (see, for example, Table 15). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of a molecule, e.g., peptide nucleic acids (PNAs). Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as modifications found in “locked” nucleic acids (LNAs). In various embodiments, the nucleic acids are in operative association with additional genetic elements, such as tissue-specific expression-control sequence(s) (e.g., tissue-specific promoters and tissue-specific microRNA recognition sequences), as well as additional elements, such as inverted repeats (e.g., inverted terminal repeats, such as elements from or derived from viruses, e.g., AAV ITRs) and tandem repeats, inverted repeats/direct repeats, homology regions (segments with various degrees of homology to a target DNA), untranslated regions (UTRs) (5′, 3′, or both 5′ and 3′ UTRs), and various combinations of the foregoing. The nucleic acid elements of the systems provided by the invention can be provided in a variety of topologies, including single-stranded, double-stranded, circular, linear, linear with open ends, linear with closed ends, and particular versions of these, such as doggybone DNA (dbDNA), closed-ended DNA (ceDNA).
As used herein, a “gene expression unit” is a nucleic acid sequence comprising at least one regulatory nucleic acid sequence operably linked to at least one effector sequence. A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be contiguous or non-contiguous. Where necessary to join two protein-coding regions, operably linked sequences may be in the same reading frame.
The terms “host genome” or “host cell”, as used herein, refer to a cell and/or its genome into which protein and/or genetic material has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell and/or genome, but to the progeny of such a cell and/or the genome of the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. A host genome or host cell may be an isolated cell or cell line grown in culture, or genomic material isolated from such a cell or cell line, or may be a host cell or host genome which composing living tissue or an organism. In some instances, a host cell may be an animal cell or a plant cell, e.g., as described herein. In certain instances, a host cell may be a mammalian cell, a human cell, avian cell, reptilian cell, bovine cell, horse cell, pig cell, goat cell, sheep cell, chicken cell, or turkey cell. In certain instances, a host cell may be a corn cell, soy cell, wheat cell, or rice cell.
As used herein, “operative association” describes a functional relationship between two nucleic acid sequences, such as a 1) promoter and 2) a heterologous object sequence, and means, in such example, the promoter and heterologous object sequence (e.g., a gene of interest) are oriented such that, under suitable conditions, the promoter drives expression of the heterologous object sequence. For instance, a template nucleic acid carrying a promoter and a heterologous object sequence may be single-stranded, e.g., either the (+) or (−) orientation. An “operative association” between the promoter and the heterologous object sequence in this template means that, regardless of whether the template nucleic acid will be transcribed in a particular state, when it is in the suitable state (e.g., is in the (+) orientation, in the presence of required catalytic factors, and NTPs, etc.), it is accurately transcribed. Operative association applies analogously to other pairs of nucleic acids, including other tissue-specific expression control sequences (such as enhancers, repressors and microRNA recognition sequences), IR/DR, ITRs, UTRs, or homology regions and heterologous object sequences or sequences encoding a retroviral RT domain.
The term “primer binding site sequence” or “PBS sequence,” as used herein, refers to a portion of a template RNA capable of binding to a region comprised in a target nucleic acid sequence. In some instances, a PBS sequence is a nucleic acid sequence comprising at least 3, 4, 5, 6, 7, or 8 bases with 100% identity to the region comprised in the target nucleic acid sequence. In some embodiments the primer region comprises at least 5, 6, 7, 8 bases with 100% identity to the region comprised in the target nucleic acid sequence. Without wishing to be bound by theory, in some embodiments when a template RNA comprises a PBS sequence and a heterologous object sequence, the PBS sequence binds to a region comprised in a target nucleic acid sequence, allowing a reverse transcriptase domain to use that region as a primer for reverse transcription, and to use the heterologous object sequence as a template for reverse transcription.
As used herein, a “stem-loop sequence” refers to a nucleic acid sequence (e.g., RNA sequence) with sufficient self-complementarity to form a stem-loop, e.g., having a stem comprising at least two (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) base pairs, and a loop with at least three (e.g., four) base pairs. The stem may comprise mismatches or bulges.
As used herein, a “tissue-specific expression-control sequence” means nucleic acid elements that increase or decrease the level of a transcript comprising the heterologous object sequence in a target tissue in a tissue-specific manner, e.g., preferentially in on-target tissue(s), relative to off-target tissue(s). In some embodiments, a tissue-specific expression-control sequence preferentially drives or represses transcription, activity, or the half-life of a transcript comprising the heterologous object sequence in the target tissue in a tissue-specific manner, e.g., preferentially in an on-target tissue(s), relative to an off-target tissue(s). Exemplary tissue-specific expression-control sequences include tissue-specific promoters, repressors, enhancers, or combinations thereof, as well as tissue-specific microRNA recognition sequences. Tissue specificity refers to on-target (tissue(s) where expression or activity of the template nucleic acid is desired or tolerable) and off-target (tissue(s) where expression or activity of the template nucleic acid is not desired or is not tolerable). For example, a tissue-specific promoter drives expression preferentially in on-target tissues, relative to off-target tissues. In contrast, a microRNA that binds the tissue-specific microRNA recognition sequences is preferentially expressed in off-target tissues, relative to on-target tissues, thereby reducing expression of a template nucleic acid in off-target tissues. Accordingly, a promoter and a microRNA recognition sequence that are specific for the same tissue, such as the target tissue, have contrasting functions (promote and repress, respectively, with concordant expression levels, i.e., high levels of the microRNA in off-target tissues and low levels in on-target tissues, while promoters drive high expression in on-target tissues and low expression in off-target tissues) with regard to the transcription, activity, or half-life of an associated sequence in that tissue.
TABLE OF CONTENTS
1) Introduction
2) Gene modifying systems


    
    
        a) Polypeptide components of gene modifying systems
        
            i) Writing domain
            ii) Endonuclease domains and DNA binding domains
            
                (1) Gene modifying polypeptides comprising Cas domains
                (2) TAL Effectors and Zinc Finger Nucleases
            
            
            iii) Linkers
            iv) Localization sequences for gene modifying systems
            v) Evolved Variants of Gene Modifying Polypeptides and Systems
            vi) Inteins
            vii) Additional domains
        
        
        b) Template nucleic acids
        
            i) gRNA spacer and gRNA scaffold
            ii) Heterologous object sequence
            iii) PBS sequence
            iv) Exemplary Template Sequences
        
        
        c) gRNAs with inducible activity
        d) Circular RNAs and Ribozymes in Gene Modifying Systems
        e) Target Nucleic Acid Site
        f) Second strand nicking

3) Production of Compositions and Systems

4) Therapeutic Applications

5) Administration and Delivery

        a) Tissue Specific Activity/Administration
        
            i) Promoters
            ii) microRNAs
        
        
        b) Viral vectors and components thereof
        c) AAV Administration
        d) Lipid Nanoparticles

6) Kits, Articles of Manufacture, and Pharmaceutical Compositions

7) Chemistry, Manufacturing, and Controls (CMC)

    
    


INTRODUCTION
This disclosure relates to methods for treating phenylketonuria (PKU) and compositions for targeting, editing, modifying or manipulating a DNA sequence (e.g., inserting a heterologous object sequence into a target site of a mammalian genome) at one or more locations in a DNA sequence in a cell, tissue or subject, e.g., in vivo or in vitro. The heterologous object DNA sequence may include, e.g., a substitution.
More specifically, the disclosure provides methods for treating PKU using reverse transcriptase-based systems for altering a genomic DNA sequence of interest, e.g., by inserting, deleting, or substituting one or more nucleotides into/from the sequence of interest.
The disclosure provides, in part, methods for treating PKU using a gene modifying system comprising a gene modifying polypeptide component and a template nucleic acid (e.g., template RNA) component. In some embodiments, a gene modifying system can be used to introduce an alteration into a target site in a genome. In some embodiments, the gene modifying polypeptide component comprises a writing domain (e.g., a reverse transcriptase domain), a DNA-binding domain, and an endonuclease domain (e.g., nickase domain). In some embodiments, the template nucleic acid (e.g., template RNA) comprises a sequence (e.g., a gRNA spacer) that binds a target site in the genome (e.g., that binds to a second strand of the target site), a sequence (e.g., a gRNA scaffold) that binds the gene modifying polypeptide component, a heterologous object sequence, and a PBS sequence. Without wishing to be bound by theory, it is thought that the template nucleic acid (e.g., template RNA) binds to the second strand of a target site in the genome, and binds to the gene modifying polypeptide component (e.g., localizing the polypeptide component to the target site in the genome). It is thought that the endonuclease (e.g., nickase) of the gene modifying polypeptide component cuts the target site (e.g., the first strand of the target site), e.g., allowing the PBS sequence to bind to a sequence adjacent to the site to be altered on the first strand of the target site. It is thought that the writing domain (e.g., reverse transcriptase domain) of the polypeptide component uses the first strand of the target site that is bound to the complementary sequence comprising the PBS sequence of the template nucleic acid as a primer and the heterologous object sequence of the template nucleic acid as a template to, e.g., polymerize a sequence complementary to the heterologous object sequence. Without wishing to be bound by theory, it is thought that selection of an appropriate heterologous object sequence can result in substitution, deletion, and/or insertion of one or more nucleotides at the target site.
Gene Modifying Systems
In some embodiments, a gene modifying system described herein comprises: (A) a gene modifying polypeptide or a nucleic acid encoding the gene modifying polypeptide, wherein the gene modifying polypeptide comprises (i) a reverse transcriptase domain, and either (x) an endonuclease domain that contains DNA binding functionality or (y) an endonuclease domain and separate DNA binding domain; and (B) a template RNA. A gene modifying polypeptide, in some embodiments, acts as a substantially autonomous protein machine capable of integrating a template nucleic acid sequence into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell), substantially without relying on host machinery. For example, the gene modifying protein may comprise a DNA-binding domain, a reverse transcriptase domain, and an endonuclease domain. In some embodiments, the DNA-binding function may involve an RNA component that directs the protein to a DNA sequence, e.g., a gRNA spacer. In other embodiments, the gene modifying polypeptide may comprise a reverse transcriptase domain and an endonuclease domain. The RNA template element of a gene modifying system is typically heterologous to the gene modifying polypeptide element and provides an object sequence to be inserted (reverse transcribed) into the host genome. In some embodiments, the gene modifying polypeptide is capable of target primed reverse transcription. In some embodiments, the gene modifying polypeptide is capable of second-strand synthesis.
In some embodiments the gene modifying system is combined with a second polypeptide. In some embodiments, the second polypeptide may comprise an endonuclease domain. In some embodiments, the second polypeptide may comprise a polymerase domain, e.g., a reverse transcriptase domain. In some embodiments, the second polypeptide may comprise a DNA-dependent DNA polymerase domain. In some embodiments, the second polypeptide aids in completion of the genome edit, e.g., by contributing to second-strand synthesis or DNA repair resolution.
A functional gene modifying polypeptide can be made up of unrelated DNA binding, reverse transcription, and endonuclease domains. This modular structure allows combining of functional domains, e.g., dCas9 (DNA binding), MMLV reverse transcriptase (reverse transcription), FokI (endonuclease). In some embodiments, multiple functional domains may arise from a single protein, e.g., Cas9 or Cas9 nickase (DNA binding, endonuclease).
In some embodiments, a gene modifying polypeptide includes one or more domains that, collectively, facilitate 1) binding the template nucleic acid, 2) binding the target DNA molecule, and 3) facilitate integration of the at least a portion of the template nucleic acid into the target DNA. In some embodiments, the gene modifying polypeptide is an engineered polypeptide that comprises one or more amino acid substitutions to a corresponding naturally occurring sequence. In some embodiments, the gene modifying polypeptide comprises two or more domains that are heterologous relative to each other, e.g., through a heterologous fusion (or other conjugate) of otherwise wild-type domains, or well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous sub-domain or other substituted domain. For instance, in some embodiments, one or more of: the RT domain is heterologous to the DBD; the DBD is heterologous to the endonuclease domain; or the RT domain is heterologous to the endonuclease domain.
In some embodiments, a template RNA molecule for use in the system comprises, from 5′ to 3′ (1) a gRNA spacer; (2) a gRNA scaffold; (3) heterologous object sequence (4) a primer binding site (PBS) sequence. In some embodiments:

    
    
        (1) Is a gRNA spacer of ˜18-22 nt, e.g., is 20 nt
        (2) Is a gRNA scaffold comprising one or more hairpin loops, e.g., 1, 2, of 3 loops for associating the template with a Cas domain, e.g., a nickase Cas9 domain. In some embodiments, the gRNA scaffold comprises the sequence, from 5′ to 3′, GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGGACCGAGTCGGTCC (SEQ ID NO: 5008).
        (3) In some embodiments, the heterologous object sequence is, e.g., 7-74, e.g., 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, or 70-80 nt or, 80-90 nt in length. In some embodiments, the first (most 5′) base of the sequence is not C.
        (4) In some embodiments, the PBS sequence that binds the target priming sequence after nicking occurs is e.g., 3-20 nt, e.g., 7-15 nt, e.g., 12-14 nt. In some embodiments, the PBS sequence has 40-60% GC content.
    
    


In some embodiments, a second gRNA associated with the system may help drive complete integration. In some embodiments, the second gRNA may target a location that is 0-200 nt away from the first-strand nick, e.g., 0-50, 50-100, 100-200 nt away from the first-strand nick. In some embodiments, the second gRNA can only bind its target sequence after the edit is made, e.g., the gRNA binds a sequence present in the heterologous object sequence, but not in the initial target sequence.
In some embodiments, a gene modifying system described herein is used to make an edit in HEK293, K562, U2OS, or HeLa cells. In some embodiment, a gene modifying system is used to make an edit in primary cells, e.g., primary cortical neurons from E18.5 mice.
In some embodiments, a gene modifying polypeptide as described herein comprises a reverse transcriptase or RT domain (e.g., as described herein) that comprises a MoMLV RT sequence or variant thereof. In embodiments, the MoMLV RT sequence comprises one or more mutations selected from D200N, L603W, T330P, T306K, W313F, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, L435G, N454K, H594Q, D653N, R110S, and K103L. In embodiments, the MoMLV RT sequence comprises a combination of mutations, such as D200N, L603W, and T330P, optionally further including T306K and/or W313F.
In some embodiments, an endonuclease domain (e.g., as described herein) nCas9, e.g., comprising an N863A mutation (e.g., in spCas9) or a H840A mutation.
In some embodiments, the heterologous object sequence (e.g., of a system as described herein) is about 1-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, or more, nucleotides in length.
In some embodiments, the RT and endonuclease domains are joined by a flexible linker, e.g., comprising the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 5006).
In some embodiments, the endonuclease domain is N-terminal relative to the RT domain. In some embodiments, the endonuclease domain is C-terminal relative to the RT domain.
In some embodiments, the system incorporates a heterologous object sequence into a target site by TPRT, e.g., as described herein.
In some embodiments, a gene modifying polypeptide comprises a DNA binding domain. In some embodiments, a gene modifying polypeptide comprises an RNA binding domain. In some embodiments, the RNA binding domain comprises an RNA binding domain of B-box protein, MS2 coat protein, dCas, or an element of a sequence of a table herein. In some embodiments, the RNA binding domain is capable of binding to a template RNA with greater affinity than a reference RNA binding domain.
In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally no more than 500, 400, 300, 200, or 100 nucleotides). In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally no more than 500, 400, 300, 200, or 100 nucleotides). In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases). In some embodiments, a gene modifying system is capable of producing a deletion of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases). In some embodiments, a gene modifying system is capable of producing a substitution into the target site of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more nucleotides. In some embodiments, a gene modifying system is capable of producing a substitution in the target site of 1-2, 2-3, 3-4, 4-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 nucleotides.
In some embodiments, the substitution is a transition mutation. In some embodiments, the substitution is a transversion mutation. In some embodiments, the substitution converts an adenine to a thymine, an adenine to a guanine, an adenine to a cytosine, a guanine to a thymine, a guanine to a cytosine, a guanine to an adenine, a thymine to a cytosine, a thymine to an adenine, a thymine to a guanine, a cytosine to an adenine, a cytosine to a guanine, or a cytosine to a thymine.
In some embodiments, an insertion, deletion, substitution, or combination thereof, increases or decreases expression (e.g. transcription or translation) of a gene. In some embodiments, an insertion, deletion, substitution, or combination thereof, increases or decreases expression (e.g. transcription or translation) of a gene by altering, adding, or deleting sequences in a promoter or enhancer, e.g. sequences that bind transcription factors. In some embodiments, an insertion, deletion, substitution, or combination thereof alters translation of a gene (e.g. alters an amino acid sequence), inserts or deletes a start or stop codon, alters or fixes the translation frame of a gene. In some embodiments, an insertion, deletion, substitution, or combination thereof alters splicing of a gene, e.g. by inserting, deleting, or altering a splice acceptor or donor site. In some embodiments, an insertion, deletion, substitution, or combination thereof alters transcript or protein half-life. In some embodiments, an insertion, deletion, substitution, or combination thereof alters protein localization in the cell (e.g. from the cytoplasm to a mitochondria, from the cytoplasm into the extracellular space (e.g. adds a secretion tag)). In some embodiments, an insertion, deletion, substitution, or combination thereof alters (e.g. improves) protein folding (e.g. to prevent accumulation of misfolded proteins). In some embodiments, an insertion, deletion, substitution, or combination thereof, alters, increases, decreases the activity of a gene, e.g. a protein encoded by the gene.
Exemplary gene modifying polypeptides, and systems comprising them and methods of using them are described, e.g., in PCT/US2021/020948, which is incorporated herein by reference with respect to retroviral RT domains, including the amino acid and nucleic acid sequences therein.
Exemplary gene modifying polypeptides and retroviral RT domain sequences are also described, e.g., in International Application No. PCT/US21/20948 filed Mar. 4, 2021, e.g., at Table 30, Table 31, and Table 44 therein; the entire application is incorporated by reference herein with respect to retroviral RTs, e.g., in said sequences and tables. Accordingly, a gene modifying polypeptide described herein may comprise an amino acid sequence according to any of the Tables mentioned in this paragraph, or a domain thereof (e.g., a retroviral RT domain), or a functional fragment or variant of any of the foregoing, or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, or 99% identity thereto.
In some embodiments, a polypeptide for use in any of the systems described herein can be a molecular reconstruction or ancestral reconstruction based upon the aligned polypeptide sequence of multiple homologous proteins. In some embodiments, a reverse transcriptase domain for use in any of the systems described herein can be a molecular reconstruction or an ancestral reconstruction, or can be modified at particular residues, based upon alignments of reverse transcriptase domains from the same or different sources. A skilled artisan can, based on the Accession numbers provided herein, align polypeptides or nucleic acid sequences, e.g., by using routine sequence analysis tools as Basic Local Alignment Search Tool (BLAST) or CD-Search for conserved domain analysis. Molecular reconstructions can be created based upon sequence consensus, e.g. using approaches described in Ivics et al., Cell 1997, 501-510; Wagstaff et al., Molecular Biology and Evolution 2013, 88-99.
Polypeptide Components of Gene Modifying Systems
In some embodiments, the gene modifying polypeptide possesses the functions of DNA target site binding, template nucleic acid (e.g., RNA) binding, DNA target site cleavage, and template nucleic acid (e.g., RNA) writing, e.g., reverse transcription. In some embodiments, each functions is contained within a distinct domain. In some embodiments, a function may be attributed to two or more domains (e.g., two or more domains, together, exhibit the functionality). In some embodiments, two or more domains may have the same or similar function (e.g., two or more domains each independently have DNA-binding functionality, e.g., for two different DNA sequences). In other embodiments, one or more domains may be capable of enabling one or more functions, e.g., a Cas9 domain enabling both DNA binding and target site cleavage. In some embodiments, the domains are all located within a single polypeptide. In some embodiments, a first domain is in one polypeptide and a second domain is in a second polypeptide. For example, in some embodiments, the sequences may be split between a first polypeptide and a second polypeptide, e.g., wherein the first polypeptide comprises a reverse transcriptase (RT) domain and wherein the second polypeptide comprises a DNA-binding domain and an endonuclease domain, e.g., a nickase domain. As a further example, in some embodiments, the first polypeptide and the second polypeptide each comprise a DNA binding domain (e.g., a first DNA binding domain and a second DNA binding domain). In some embodiments, the first and second polypeptide may be brought together post-translationally via a split-intein to form a single gene modifying polypeptide.
In some aspects, a gene modifying polypeptide described herein comprises (e.g., a system described herein comprises a gene modifying polypeptide that comprises): 1) a Cas domain (e.g., a Cas nickase domain, e.g., a Cas9 nickase domain); 2) a reverse transcriptase (RT) domain of Table D, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, wherein the RT domain is C-terminal of the Cas domain; and a linker disposed between the RT domain and the Cas domain, wherein the linker has a sequence from the same row of Table D as the RT domain, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.
In some embodiments, the RT domain has a sequence with 100% identity to the RT domain of Table D and the linker has a sequence with 100% identity to the linker sequence from the same row of Table D as the RT domain. In some embodiments, the Cas domain comprises a sequence of Table 8, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto. In some embodiments, the gene modifying polypeptide comprises an amino acid sequence according to any of SEQ ID NOs: 1-3332 in the sequence listing, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.
In some embodiments, the gene modifying polypeptide comprises a GG amino acid sequence between the Cas domain and the linker, an AG amino acid sequence between the RT domain and the second NLS, and/or a GG amino acid sequence between the linker and the RT domain. In some embodiments, the gene modifying polypeptide comprises a sequence of SEQ ID NO: 4000 which comprises the first NLS and the Cas domain, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto. In some embodiments, the gene modifying polypeptide comprises a sequence of SEQ ID NO: 4001 which comprises the second NLS, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.








Exemplary N-terminal NLS-Cas9 domain



(SEQ ID NO: 4000)



MPAAKRVKLDGGDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLF



 


DSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP


 


IFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDV


 


DKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS


 


LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVN


 


TEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFY


 


KFIKPILEKMDGTEELLVKLNREDLLRKIRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNR


 


EKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN


 


LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLK


 


EDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDR


 


EMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNF


 


MQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPE


 


NIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLINGRD


 


MYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQ


 


LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRIITKHVAQILDSRMNTKYDENDKL


 


IREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY


 


KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG


 


RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAY


 


SVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE


 


LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKILFVEQHKHYLDEI


 


IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDR


 


KRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGG 


 


Exemplary C-terminal sequence comprising an NLS


(SEQ ID NO: 4001)



AGKRTADGSEFEKRTADGSEFESPKKKAKVE







Writing Domain (RT Domain)

In certain aspects of the present invention, the writing domain of the gene modifying system possesses reverse transcriptase activity and is also referred to as a reverse transcriptase domain (a RT domain). In some embodiments, the RT domain comprises an RT catalytic portion and RNA-binding region (e.g., a region that binds the template RNA).
In some embodiments, a nucleic acid encoding the reverse transcriptase is altered from its natural sequence to have altered codon usage, e.g. improved for human cells. In some embodiments the reverse transcriptase domain is a heterologous reverse transcriptase from a retrovirus. In some embodiments, the RT domain comprising a gene modifying polypeptide has been mutated from its original amino acid sequence, e.g., has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 substitutions. In some embodiments, the RT domain is derived from the RT of a retrovirus, e.g., HIV-1 RT, Moloney Murine Leukemia Virus (MMLV) RT, avian myeloblastosis virus (AMV) RT, or Rous Sarcoma Virus (RSV) RT.
In some embodiments, the retroviral reverse transcriptase (RT) domain exhibits enhanced stringency of target-primed reverse transcription (TPRT) initiation, e.g., relative to an endogenous RT domain. In some embodiments, the RT domain initiates TPRT when the 3 nt in the target site immediately upstream of the first strand nick, e.g., the genomic DNA priming the RNA template, have at least 66% or 100% complementarity to the 3 nt of homology in the RNA template. In some embodiments, the RT domain initiates TPRT when there are less than 5 nt mismatched (e.g., less than 1, 2, 3, 4, or 5 nt mismatched) between the template RNA homology and the target DNA priming reverse transcription. In some embodiments, the RT domain is modified such that the stringency for mismatches in priming the TPRT reaction is increased, e.g., wherein the RT domain does not tolerate any mismatches or tolerates fewer mismatches in the priming region relative to a wild-type (e.g., unmodified) RT domain. In some embodiments, the RT domain comprises a HIV-1 RT domain. In embodiments, the HIV-1 RT domain initiates lower levels of synthesis even with three nucleotide mismatches relative to an alternative RT domain (e.g., as described by Jamburuthugoda and Eickbush J Mol Biol 407(5):661-672 (2011); incorporated herein by reference in its entirety). In some embodiments, the RT domain forms a dimer (e.g., a heterodimer or homodimer). In some embodiments, the RT domain is monomeric. In some embodiments, an RT domain, naturally functions as a monomer or as a dimer (e.g., heterodimer or homodimer). In some embodiments, an RT domain naturally functions as a monomer, e.g., is derived from a virus wherein it functions as a monomer. In embodiments, the RT domain is selected from an RT domain from murine leukemia virus (MLV; sometimes referred to as MoMLV) (e.g., P03355), porcine endogenous retrovirus (PERV) (e.g., UniProt Q4VFZ2), mouse mammary tumor virus (MMTV) (e.g., UniProt P03365), Avian reticuloendotheliosis virus (AVIRE) (e.g., UniProtKB accession: P03360); Feline leukemia virus (FLV or FeLV) (e.g., e.g., UniProtKB accession: P10273); Mason-Pfizer monkey virus (MPMV) (e.g., UniProt P07572), bovine leukemia virus (BLV) (e.g., UniProt P03361), human T-cell leukemia virus-1 (HTLV-1) (e.g., UniProt P03362), human foamy virus (HFV) (e.g., UniProt P14350), simian foamy virus (SFV) (e.g., SFV3L) (e.g., UniProt P23074 or P27401), or bovine foamy/syncytial virus (BFV/BSV) (e.g., UniProt O41894), or a functional fragment or variant thereof (e.g., an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity thereto). In some embodiments, an RT domain is dimeric in its natural functioning. In some embodiments, the RT domain is derived from a virus wherein it functions as a dimer. In embodiments, the RT domain is selected from an RT domain from avian sarcoma/leukemia virus (ASLV) (e.g., UniProt A0A142BKH1), Rous sarcoma virus (RSV) (e.g., UniProt P03354), avian myeloblastosis virus (AMV) (e.g., UniProt Q83133), human immunodeficiency virus type I (HIV-1) (e.g., UniProt P03369), human immunodeficiency virus type II (HIV-2) (e.g., UniProt P15833), simian immunodeficiency virus (SIV) (e.g., UniProt P05896), bovine immunodeficiency virus (BIV) (e.g., UniProt P19560), equine infectious anemia virus (EIAV) (e.g., UniProt P03371), or feline immunodeficiency virus (FIV) (e.g., UniProt P16088) (Herschhorn and Hizi Cell Mol Life Sci 67(16):2717-2747 (2010)), or a functional fragment or variant thereof (e.g., an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity thereto). Naturally heterodimeric RT domains may, in some embodiments, also be functional as homodimers. In some embodiments, dimeric RT domains are expressed as fusion proteins, e.g., as homodimeric fusion proteins or heterodimeric fusion proteins. In some embodiments, the RT function of the system is fulfilled by multiple RT domains (e.g., as described herein). In further embodiments, the multiple RT domains are fused or separate, e.g., may be on the same polypeptide or on different polypeptides.
In some embodiments, a gene modifying system described herein comprises an integrase domain, e.g., wherein the integrase domain may be part of the RT domain. In some embodiments, an RT domain (e.g., as described herein) comprises an integrase domain. In some embodiments, an RT domain (e.g., as described herein) lacks an integrase domain, or comprises an integrase domain that has been inactivated by mutation or deleted. In some embodiment, a gene modifying system described herein comprises an RNase H domain, e.g., wherein the RNase H domain may be part of the RT domain. In some embodiments, the RNase H domain is not part of the RT domain and is covalently linked via a flexible linker. In some embodiments, an RT domain (e.g., as described herein) comprises an RNase H domain, e.g., an endogenous RNAse H domain or a heterologous RNase H domain. In some embodiments, an RT domain (e.g., as described herein) lacks an RNase H domain. In some embodiments, an RT domain (e.g., as described herein) comprises an RNase H domain that has been added, deleted, mutated, or swapped for a heterologous RNase H domain. In some embodiments, the polypeptide comprises an inactivated endogenous RNase H domain. In some embodiments, an endogenous RNase H domain from one of the other domains of the polypeptide is genetically removed such that it is not included in the polypeptide, e.g., the endogenous RNase H domain is partially or completely truncated from the comprising domain. In some embodiments, mutation of an RNase H domain yields a polypeptide exhibiting lower RNase activity, e.g., as determined by the methods described in Kotewicz et al. Nucleic Acids Res 16(1):265-277 (1988) (incorporated herein by reference in its entirety), e.g., lower by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to an otherwise similar domain without the mutation. In some embodiments, RNase H activity is abolished.
In some embodiments, an RT domain is mutated to increase fidelity compared to an otherwise similar domain without the mutation. For instance, in some embodiments, a YADD (SEQ ID NO: 37635) or YMDD motif (SEQ ID NO: 37636) in an RT domain (e.g., in a reverse transcriptase) is replaced with YVDD (SEQ ID NO: 37637). In embodiments, replacement of the YADD (SEQ ID NO: 37635) or YMDD (SEQ ID NO: 37636) or YVDD (SEQ ID NO: 37637) results in higher fidelity in retroviral reverse transcriptase activity (e.g., as described in Jamburuthugoda and Eickbush J Mol Biol 2011; incorporated herein by reference in its entirety).
In some embodiments, a gene modifying polypeptide described herein comprises an RT domain having an amino acid sequence according to Table 6, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto. In some embodiments, a nucleic acid described herein encodes an RT domain having an amino acid sequence according to Table 6, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.







TABLE 6







Exemplary reverse transcriptase domains from retroviruses









RT
SEQ ID



Name
NO:
RT amino acid sequence





AVIRE_
8,001
TAPLEEEYRLFLEAPIQNVTLLEQWKREIPKVWAEINPPGLASTQAPIHVQLLSTALPVRVRQYPITLEAKRSLRETIRKFRAAGILRPVHSPWNTPLLPV


P03360

RKSGTSEYRMVQDLREVNKRVETIHPTVPNPYTLLSLLPPDRIWYSVLDLKDAFFCIPLAPESQLIFAFEWADAEEGESGQLTWTRLPQGFKNSPTLFD




EALNRDLQGFRLDHPSVSLLQYVDDLLIAADTQAACLSATRDLLMTLAELGYRVSGKKAQLCQEEVTYLGFKIHKGSRSLSNSRTQAILQIPVPKTKRQV




REFLGTIGYCRLWIPGFAELAQPLYAATRGGNDPLVWGEKEEEAFQSLKLALTQPPALALPSLDKPFQLFVEETSGAAKGVLTQALGPWKRPVAYLSK




RLDPVAAGWPRCLRAIAAAALLTREASKLTFGQDIEITSSHNLESLLRSPPDKWLTNARITQYQVLLLDPPRVRFKQTAALNPATLLPETDDTLPIHHCLD




TLDSLTSTRPDLTDQPLAQAEATLFTDGSSYIRDGKRYAGAAVVTLDSVIWAEPLPIGTSAQKAELIALTKALEWSKDKSVNIYTDSRYAFATLHVHGMIY




RERGLLTAGGKAIKNAPEILALLTAVWLPKRVAVMHCKGHQKDDAPTSTGNRRADEVAREVAIRPLSTQATIS


 


AVIRE_
8,002
TAPLEEEYRLFLEAPIQNVTLLEQWKREIPKVWAEINPPGLASTQAPIHVQLLSTALPVRVRQYPITLEAKRSLRETIRKFRAAGILRPVHSPWNTPLLPV


P03360_

RKSGTSEYRMVQDLREVNKRVETIHPTVPNPYTLLSLLPPDRIWYSVLDLKDAFFCIPLAPESQLIFAFEWADAEEGESGQLTWTRLPQGFKNSPTLFN


3mut

EALNRDLQGFRLDHPSVSLLQYVDDLLIAADTQAACLSATRDLLMTLAELGYRVSGKKAQLCQEEVTYLGFKIHKGSRSLSNSRTQAILQIPVPKTKRQV




REFLGTIGYCRLWIPGFAELAQPLYAATRPGNDPLVWGEKEEEAFQSLKLALTQPPALALPSLDKPFQLFVEETSGAAKGVLTQALGPWKRPVAYLSK




RLDPVAAGWPRCLRAIAAAALLTREASKLTFGQDIEITSSHNLESLLRSPPDKWLTNARITQYQVLLLDPPRVRFKQTAALNPATLLPETDDTLPIHHCLD




TLDSLTSTRPDLTDQPLAQAEATLFTDGSSYIRDGKRYAGAAVVTLDSVIWAEPLPIGTSAQKAELIALTKALEWSKDKSVNIYTDSRYAFATLHVHGMIY




RERGWLTAGGKAIKNAPEILALLTAVWLPKRVAVMHCKGHQKDDAPTSTGNRRADEVAREVAIRPLSTQATIS


 


AVIRE_
8,003
TAPLEEEYRLFLEAPIQNVTLLEQWKREIPKVWAEINPPGLASTQAPIHVQLLSTALPVRVRQYPITLEAKRSLRETIRKFRAAGILRPVHSPWNTPLLPV


P03360_

RKSGTSEYRMVQDLREVNKRVETIHPTVPNPYTLLSLLPPDRIWYSVLDLKDAFFCIPLAPESQLIFAFEWADAEEGESGQLTWTRLPQGFKNSPTLFN


3mutA

EALNRDLQGFRLDHPSVSLLQYVDDLLIAADTQAACLSATRDLLMTLAELGYRVSGKKAQLCQEEVTYLGFKIHKGSRSLSNSRTQAILQIPVPKTKRQV




REFLGKIGYCRLFIPGFAELAQPLYAATRPGNDPLVWGEKEEEAFQSLKLALTQPPALALPSLDKPFQLFVEETSGAAKGVLTQALGPWKRPVAYLSKR




LDPVAAGWPRCLRAIAAAALLTREASKLTFGQDIEITSSHNLESLLRSPPDKWLTNARITQYQVLLLDPPRVRFKQTAALNPATLLPETDDTLPIHHCLDT




LDSLTSTRPDLTDQPLAQAEATLFTDGSSYIRDGKRYAGAAVVTLDSVIWAEPLPIGTSAQKAELIALTKALEWSKDKSVNIYTDSRYAFATLHVHGMIY




RERGWLTAGGKAIKNAPEILALLTAVWLPKRVAVMHCKGHQKDDAPTSTGNRRADEVAREVAIRPLSTQATIS


 


BAEVM_
8,004
TVSLQDEHRLFDIPVTTSLPDVWLQDFPQAWAETGGLGRAKCQAPIIIDLKPTAVPVSIKQYPMSLEAHMGIRQHIIKFLELGVLRPCRSPWNTPLLPVK


P10272

KPGTQDYRPVQDLREINKRTVDIHPTVPNPYNLLSTLKPDYSWYTVLDLKDAFFCLPLAPQSQELFAFEWKDPERGISGQLTWTRLPQGFKNSPTLFD




EALHRDLTDFRTQHPEVTLLQYVDDLLLAAPTKKACTQGTRHLLQELGEKGYRASAKKAQICQTKVTYLGYILSEGKRWLTPGRIETVARIPPPRNPRE




VREFLGTAGFCRLWIPGFAELAAPLYALTKESTPFTWQTEHQLAFEALKKALLSAPALGLPDTSKPFTLFLDERQGIAKGVLTQKLGPWKRPVAYLSKK




LDPVAAGWPPCLRIMAATAMLVKDSAKLTLGQPLTVITPHTLEAIVRQPPDRWITNARLTHYQALLLDTDRVQFGPPVTLNPATLLPVPENQPSPHDCR




QVLAETHGTREDLKDQELPDADHTWYTDGSSYLDSGTRRAGAAVVDGHNTIWAQSLPPGTSAQKAELIALTKALELSKGKKANIYTDSRYAFATAHTH




GSIYERRGLLTSEGKEIKNKAEIIALLKALFLPQEVAIIHCPGHQKGQDPVAVGNRQADRVARQAAMAEVLTLATEPDNTSHIT


 


BAEVM_
8,005
TVSLQDEHRLFDIPVTTSLPDVWLQDFPQAWAETGGLGRAKCQAPIIIDLKPTAVPVSIKQYPMSLEAHMGIRQHIIKFLELGVLRPCRSPWNTPLLPVK


P10272_

KPGTQDYRPVQDLREINKRTVDIHPTVPNPYNLLSTLKPDYSWYTVLDLKDAFFCLPLAPQSQELFAFEWKDPERGISGQLTWTRLPQGFKNSPTLFN


3mut

EALHRDLTDFRTQHPEVTLLQYVDDLLLAAPTKKACTQGTRHLLQELGEKGYRASAKKAQICQTKVTYLGYILSEGKRWLTPGRIETVARIPPPRNPRE




VREFLGTAGFCRLWIPGFAELAAPLYALTKPSTPFTWQTEHQLAFEALKKALLSAPALGLPDTSKPFTLFLDERQGIAKGVLTQKLGPWKRPVAYLSKK




LDPVAAGWPPCLRIMAATAMLVKDSAKLTLGQPLTVITPHTLEAIVRQPPDRWITNARLTHYQALLLDTDRVQFGPPVTLNPATLLPVPENQPSPHDCR




QVLAETHGTREDLKDQELPDADHTWYTDGSSYLDSGTRRAGAAVVDGHNTIWAQSLPPGTSAQKAELIALTKALELSKGKKANIYTDSRYAFATAHTH




GSIYERRGWLTSEGKEIKNKAEIIALLKALFLPQEVAIIHCPGHQKGQDPVAVGNRQADRVARQAAMAEVLTLATEPDNTSHIT


 


BAEVM_
8,006
TVSLQDEHRLFDIPVTTSLPDVWLQDFPQAWAETGGLGRAKCQAPIIIDLKPTAVPVSIKQYPMSLEAHMGIRQHIIKFLELGVLRPCRSPWNTPLLPVK


P10272_

KPGTQDYRPVQDLREINKRTVDIHPTVPNPYNLLSTLKPDYSWYTVLDLKDAFFCLPLAPQSQELFAFEWKDPERGISGQLTWTRLPQGFKNSPTLFN


3mutA

EALHRDLTDFRTQHPEVTLLQYVDDLLLAAPTKKACTQGTRHLLQELGEKGYRASAKKAQICQTKVTYLGYILSEGKRWLTPGRIETVARIPPPRNPRE




VREFLGKAGFCRLFIPGFAELAAPLYALTKPSTPFTWQTEHQLAFEALKKALLSAPALGLPDTSKPFTLFLDERQGIAKGVLTQKLGPWKRPVAYLSKKL




DPVAAGWPPCLRIMAATAMLVKDSAKLTLGQPLTVITPHTLEAIVRQPPDRWITNARLTHYQALLLDTDRVQFGPPVTLNPATLLPVPENQPSPHDCRQ




VLAETHGTREDLKDQELPDADHTWYTDGSSYLDSGTRRAGAAVVDGHNTIWAQSLPPGTSAQKAELIALTKALELSKGKKANIYTDSRYAFATAHTHG




SIYERRGWLTSEGKEIKNKAEIIALLKALFLPQEVAIIHCPGHQKGQDPVAVGNRQADRVARQAAMAEVLTLATEPDNTSHIT


 


BLVAU_
8,007
GVLDAPPSHIGLEHLPPPPEVPQFPLNLERLQALQDLVHRSLEAGYISPWDGPGNNPVFPVRKPNGAWRFVHDLRVTNALTKPIPALSPGPPDLTAIPT


P25059

HLPHIICLDLKDAFFQIPVEDRFRSYFAFTLPTPGGLQPHRRFAWRVLPQGFINSPALFERALQEPLRQVSAAFSQSLLVSYMDDILYVSPTEEQRLQCY




QTMAAHLRDLGFQVASEKTRQTPSPVPFLGQMVHERMVTYQSLPTLQISSPISLHQLQTVLGDLQWVSRGTPTTRRPLQLLYSSLKGIDDPRAIIHLSP




EQQQGIAELRQALSHNARSRYNEQEPLLAYVHLTRAGSTLVLFQKGAQFPLAYFQTPLTDNQASPWGLLLLLGCQYLQAQALSSYAKTILKYYHNLPK




TSLDNWIQSSEDPRVQELLQLWPQISSQGIQPPGPWKTLVTRAEVFLTPQFSPEPIPAALCLFSDGAARRGAYCLWKDHLLDFQAVPAPESAQKGELA




GLLAGLAAAPPEPLNIWVDSKYLYSLLRTLVLGAWLQPDPVPSYALLYKSLLRHPAIFVGHVRSHSSASHPIASLNNYVDQL


 


BLVAU_
8,008
GVLDAPPSHIGLEHLPPPPEVPQFPLNLERLQALQDLVHRSLEAGYISPWDGPGNNPVFPVRKPNGAWRFVHDLRVTNALTKPIPALSPGPPDLTAIPT


P25059_

HLPHIICLDLKDAFFQIPVEDRFRSYFAFTLPTPGGLQPHRRFAWRVLPQGFINSPALFQRALQEPLRQVSAAFSQSLLVSYMDDILYVSPTEEQRLQCY


2mut

QTMAAHLRDLGFQVASEKTRQTPSPVPFLGQMVHERMVTYQSLPTLQISSPISLHQLQTVLGDLQWVSRGTPTTRRPLQLLYSSLKPIDDPRAIIHLSP




EQQQGIAELRQALSHNARSRYNEQEPLLAYVHLTRAGSTLVLFQKGAQFPLAYFQTPLTDNQASPWGLLLLLGCQYLQAQALSSYAKTILKYYHNLPK




TSLDNWIQSSEDPRVQELLQLWPQISSQGIQPPGPWKTLVTRAEVFLTPQFSPEPIPAALCLFSDGAARRGAYCLWKDHLLDFQAVPAPESAQKGELA




GLLAGLAAAPPEPLNIWVDSKYLYSLLRTLVLGAWLQPDPVPSYALLYKSLLRHPAIFVGHVRSHSSASHPIASLNNYVDQL


 


BLVJ_
8,009
GVLDTPPSHIGLEHLPPPPEVPQFPLNLERLQALQDLVHRSLEAGYISPWDGPGNNPVFPVRKPNGAWRFVHDLRATNALTKPIPALSPGPPDLTAIPT


P03361

HPPHIICLDLKDAFFQIPVEDRFRFYLSFTLPSPGGLQPHRRFAWRVLPQGFINSPALFERALQEPLRQVSAAFSQSLLVSYMDDILYASPTEEQRSQCY




QALAARLRDLGFQVASEKTSQTPSPVPFLGQMVHEQIVTYQSLPTLQISSPISLHQLQAVLGDLQWVSRGTPTTRRPLQLLYSSLKRHHDPRAIIQLSPE




QLQGIAELRQALSHNARSRYNEQEPLLAYVHLTRAGSTLVLFQKGAQFPLAYFQTPLTDNQASPWGLLLLLGCQYLQTQALSSYAKPILKYYHNLPKTS




LDNWIQSSEDPRVQELLQLWPQISSQGIQPPGPWKTLITRAEVFLTPQFSPDPIPAALCLFSDGATGRGAYCLWKDHLLDFQAVPAPESAQKGELAGL




LAGLAAAPPEPVNIWVDSKYLYSLLRTLVLGAWLQPDPVPSYALLYKSLLRHPAIVVGHVRSHSSASHPIASLNNYVDQL


 


BLVJ_
8,010
GVLDTPPSHIGLEHLPPPPEVPQFPLNLERLQALQDLVHRSLEAGYISPWDGPGNNPVFPVRKPNGAWRFVHDLRATNALTKPIPALSPGPPDLTAIPT


P03361_

HPPHIICLDLKDAFFQIPVEDRFRFYLSFTLPSPGGLQPHRRFAWRVLPQGFINSPALFNRALQEPLRQVSAAFSQSLLVSYMDDILYASPTEEQRSQCY


2mut

QALAARLRDLGFQVASEKTSQTPSPVPFLGQMVHEQIVTYQSLPTLQISSPISLHQLQAVLGDLQWVSRGTPTTRRPLQLLYSSLKRHHDPRAIIQLSPE




QLQGIAELRQALSHNARSRYNEQEPLLAYVHLTRAGSTLVLFQKGAQFPLAYFQTPLTDNQASPWGLLLLLGCQYLQTQALSSYAKPILKYYHNLPKTS




LDNWIQSSEDPRVQELLQLWPQISSQGIQPPGPWKTLITRAEVFLTPQFSPDPIPAALCLFSDGATGRGAYCLWKDHLLDFQAVPAPESAQKGELAGL




LAGLAAAPPEPVNIWVDSKYLYSLLRTWVLGAWLQPDPVPSYALLYKSLLRHPAIVVGHVRSHSSASHPIASLNNYVDQL


 


BLVJ_
8,011
GVLDTPPSHIGLEHLPPPPEVPQFPLNLERLQALQDLVHRSLEAGYISPWDGPGNNPVFPVRKPNGAWRFVHDLRATNALTKPIPALSPGPPDLTAPP


P03361_

THPPHIICLDLKDAFFQIPVEDRFRFYLSFTLPSPGGLQPHRRFAWRVLPQGFINSPALFQRALQEPLRQVSAAFSQSLLVSYMDDILYASPTEEQRSQC


2mutB

YQALAARLRDLGFQVASEKTSQTPSPVPFLGQMVHEQIVTYQSLPTLQISSPISLHQLQAVLGDLQWVSRGTPTTRRPLQLLYSSLKRHHDPRAIIQLSP




EQLQGIAELRQALSHNARSRYNEQEPLLAYVHLTRAGSTLVLFQKGAQFPLAYFQTPLTDNQASPWGLLLLLGCQYLQTQALSSYAKPILKYYHNLPKT




SLDNWIQSSEDPRVQELLQLWPQISSQGIQPPGPWKTLITRAEVFLTPQFSPDPIPAALCLFSDGATGRGAYCLWKDHLLDFQAVPAPESAQKGELAG




LLAGLAAAPPEPVNIWVDSKYLYSLLRTWVLGAWLQPDPVPSYALLYKSLLRHPAIVVGHVRSHSSASHPIASLNNYVDQL


 


FFV_
8,012
MDLLKPLTVERKGVKIKGYWNSQADITCVPKDLLQGEEPVRQQNVTTIHGTQEGDVYYVNLKIDGRRINTEVIGTTLDYAIITPGDVPWILKKPLELTIKLD


O93209

LEEQQGTLLNNSILSKKGKEELKQLFEKYSALWQSWENQVGHRRIRPHKIATGTVKPTPQKQYHINPKAKPDIQIVINDLLKQGVLIQKESTMNTPVYPV




PKPNGRWRMVLDYRAVNKVTPLIAVQNQHSYGILGSLFKGRYKTTIDLSNGFWAHPIVPEDYWITAFTWQGKQYCWTVLPQGFLNSPGLFTGDVVDL




LQGIPNVEVYVDDVYISHDSEKEHLEYLDILFNRLKEAGYIISLKKSNIANSIVDFLGFQITNEGRGLTDTFKEKLENITAPTTLKQLQSILGLLNFARNFIPD




FTELIAPLYALIPKSTKNYVPWQIEHSTTLETLITKLNGAEYLQGRKGDKTLIMKVNASYTTGYIRYYNEGEKKPISYVSIVFSKTELKFTELEKLLTTVHKG




LLKALDLSMGQNIHVYSPIVSMQNIQKTPQTAKKALASRWLSWLSYLEDPRIRFFYDPQMPALKDLPAVDTGKDNKKHPSNFQHIFYTDGSAITSPTKE




GHLNAGMGIVYFINKDGNLQKQQEWSISLGNHTAQFAEIAAFEFALKKCLPLGGNILVVTDSNYVAKAYNEELDVWASNGFVNNRKKPLKHISKWKSV




ADLKRLRPDVVVTHEPGHQKLDSSPHAYGNNLADQLATQASFKVH


 


FFV_
8,013
MDLLKPLTVERKGVKIKGYWNSQADITCVPKDLLQGEEPVRQQNVTTIHGTQEGDVYYVNLKIDGRRINTEVIGTTLDYAIITPGDVPWILKKPLELTIKLD


O93209_

LEEQQGTLLNNSILSKKGKEELKQLFEKYSALWQSWENQVGHRRIRPHKIATGTVKPTPQKQYHINPKAKPDIQIVINDLLKQGVLIQKESTMNTPVYPV


2mut

PKPNGRWRMVLDYRAVNKVTPLIAVQNQHSYGILGSLFKGRYKTTIDLSNGFWAHPIVPEDYWITAFTWQGKQYCWTVLPQGFLNSPGLFNGDVVDL




LQGIPNVEVYVDDVYISHDSEKEHLEYLDILFNRLKEAGYIISLKKSNIANSIVDFLGFQITNEGRGLTDTFKEKLENITAPTTLKQLQSILGLLNFARNFIPD




FTELIAPLYALIPKSPKNYVPWQIEHSTTLETLITKLNGAEYLQGRKGDKTLIMKVNASYTTGYIRYYNEGEKKPISYVSIVFSKTELKFTELEKLLTTVHKG




LLKALDLSMGQNIHVYSPIVSMQNIQKTPQTAKKALASRWLSWLSYLEDPRIRFFYDPQMPALKDLPAVDTGKDNKKHPSNFQHIFYTDGSAITSPTKE




GHLNAGMGIVYFINKDGNLQKQQEWSISLGNHTAQFAEIAAFEFALKKCLPLGGNILVVTDSNYVAKAYNEELDVWASNGFVNNRKKPLKHISKWKSV




ADLKRLRPDVVVTHEPGHQKLDSSPHAYGNNLADQLATQASFKVH


 


FFV_
8,014
MDLLKPLTVERKGVKIKGYWNSQADITCVPKDLLQGEEPVRQQNVTTIHGTQEGDVYYVNLKIDGRRINTEVIGTTLDYAIITPGDVPWILKKPLELTIKLD


O93209_

LEEQQGTLLNNSILSKKGKEELKQLFEKYSALWQSWENQVGHRRIRPHKIATGTVKPTPQKQYHINPKAKPDIQIVINDLLKQGVLIQKESTMNTPVYPV


2mutA

PKPNGRWRMVLDYRAVNKVTPLIAVQNQHSYGILGSLFKGRYKTTIDLSNGFWAHPIVPEDYWITAFTWQGKQYCWTVLPQGFLNSPGLFNGDVVDL




LQGIPNVEVYVDDVYISHDSEKEHLEYLDILFNRLKEAGYIISLKKSNIANSIVDFLGFQITNEGRGLTDTFKEKLENITAPTTLKQLQSILGKLNFARNFIPD




FTELIAPLYALIPKSPKNYVPWQIEHSTTLETLITKLNGAEYLQGRKGDKTLIMKVNASYTTGYIRYYNEGEKKPISYVSIVFSKTELKFTELEKLLTTVHKG




LLKALDLSMGQNIHVYSPIVSMQNIQKTPQTAKKALASRWLSWLSYLEDPRIRFFYDPQMPALKDLPAVDTGKDNKKHPSNFQHIFYTDGSAITSPTKE




GHLNAGMGIVYFINKDGNLQKQQEWSISLGNHTAQFAEIAAFEFALKKCLPLGGNILVVTDSNYVAKAYNEELDVWASNGFVNNRKKPLKHISKWKSV




ADLKRLRPDVVVTHEPGHQKLDSSPHAYGNNLADQLATQASFKVH


 


FFV_
8,015
VPWILKKPLELTIKLDLEEQQGTLLNNSILSKKGKEELKQLFEKYSALWQSWENQVGHRRIRPHKIATGTVKPTPQKQYHINPKAKPDIQIVINDLLKQGV


O93209-

LIQKESTMNTPVYPVPKPNGRWRMVLDYRAVNKVTPLIAVQNQHSYGILGSLFKGRYKTTIDLSNGFWAHPIVPEDYWITAFTWQGKQYCWTVLPQGF


Pro

LNSPGLFTGDVVDLLQGIPNVEVYVDDVYISHDSEKEHLEYLDILFNRLKEAGYIISLKKSNIANSIVDFLGFQITNEGRGLTDTFKEKLENITAPTTLKQLQ




SILGLLNFARNFIPDFTELIAPLYALIPKSTKNYVPWQIEHSTTLETLITKLNGAEYLQGRKGDKTLIMKVNASYTTGYIRYYNEGEKKPISYVSIVFSKTELK




FTELEKLLTTVHKGLLKALDLSMGQNIHVYSPIVSMQNIQKTPQTAKKALASRWLSWLSYLEDPRIRFFYDPQMPALKDLPAVDTGKDNKKHPSNFQHI




FYTDGSAITSPTKEGHLNAGMGIVYFINKDGNLQKQQEWSISLGNHTAQFAEIAAFEFALKKCLPLGGNILVVTDSNYVAKAYNEELDVWASNGFVNNR




KKPLKHISKWKSVADLKRLRPDVVVTHEPGHQKLDSSPHAYGNNLADQLATQASFKVH


 


FFV_
8,016
VPWILKKPLELTIKLDLEEQQGTLLNNSILSKKGKEELKQLFEKYSALWQSWENQVGHRRIRPHKIATGTVKPTPQKQYHINPKAKPDIQIVINDLLKQGV


O93209-

LIQKESTMNTPVYPVPKPNGRWRMVLDYRAVNKVTPLIAVQNQHSYGILGSLFKGRYKTTIDLSNGFWAHPIVPEDYWITAFTWQGKQYCWTVLPQGF


Pro_2mut

LNSPGLFNGDVVDLLQGIPNVEVYVDDVYISHDSEKEHLEYLDILFNRLKEAGYIISLKKSNIANSIVDFLGFQITNEGRGLTDTFKEKLENITAPTTLKQLQ




SILGLLNFARNFIPDFTELIAPLYALIPKSPKNYVPWQIEHSTTLETLITKLNGAEYLQGRKGDKTLIMKVNASYTTGYIRYYNEGEKKPISYVSIVFSKTELK




FTELEKLLTTVHKGLLKALDLSMGQNIHVYSPIVSMQNIQKTPQTAKKALASRWLSWLSYLEDPRIRFFYDPQMPALKDLPAVDTGKDNKKHPSNFQHI




FYTDGSAITSPTKEGHLNAGMGIVYFINKDGNLQKQQEWSISLGNHTAQFAEIAAFEFALKKCLPLGGNILVVTDSNYVAKAYNEELDVWASNGFVNNR




KKPLKHISKWKSVADLKRLRPDVVVTHEPGHQKLDSSPHAYGNNLADQLATQASFKVH


 


FFV_
8,017
VPWILKKPLELTIKLDLEEQQGTLLNNSILSKKGKEELKQLFEKYSALWQSWENQVGHRRIRPHKIATGTVKPTPQKQYHINPKAKPDIQIVINDLLKQGV


O93209-

LIQKESTMNTPVYPVPKPNGRWRMVLDYRAVNKVTPLIAVQNQHSYGILGSLFKGRYKTTIDLSNGFWAHPIVPEDYWITAFTWQGKQYCWTVLPQGF


Pro_2mutA

LNSPGLFNGDVVDLLQGIPNVEVYVDDVYISHDSEKEHLEYLDILFNRLKEAGYIISLKKSNIANSIVDFLGFQITNEGRGLTDTFKEKLENITAPTTLKQLQ




SILGKLNFARNFIPDFTELIAPLYALIPKSPKNYVPWQIEHSTTLETLITKLNGAEYLQGRKGDKTLIMKVNASYTTGYIRYYNEGEKKPISYVSIVFSKTELK




FTELEKLLTTVHKGLLKALDLSMGQNIHVYSPIVSMQNIQKTPQTAKKALASRWLSWLSYLEDPRIRFFYDPQMPALKDLPAVDTGKDNKKHPSNFQHI




FYTDGSAITSPTKEGHLNAGMGIVYFINKDGNLQKQQEWSISLGNHTAQFAEIAAFEFALKKCLPLGGNILVVTDSNYVAKAYNEELDVWASNGFVNNR




KKPLKHISKWKSVADLKRLRPDVVVTHEPGHQKLDSSPHAYGNNLADQLATQASFKVH


 


FLV_
8,018
TLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGTAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLP


P10273

VKKPGTEDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTL




FDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNSR




QVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFEDIKKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSK




KLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGGNHHDC




LQILAETHGTRPDLTDQPLPDADLTWYTDGSSFIRNGEREAGAAVTTESEVIWAAPLPPGTSAQRAELIALTQALKMAEGKKLTVYTDSRYAFATTHVH




GEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETHSSLTVLP


 


FLV_
8,019
TLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGTAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLP


P10273_

VKKPGTEDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTL


3mut

FNEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNSR




QVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFEDIKKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSK




KLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGGNHHDC




LQILAETHGTRPDLTDQPLPDADLTWYTDGSSFIRNGEREAGAAVTTESEVIWAAPLPPGTSAQRAELIALTQALKMAEGKKLTVYTDSRYAFATTHVH




GEIYRRRGWLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETHSSLTVLP


 


FLV_
8,020
TLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGTAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLP


P10273_

VKKPGTEDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTL


3mutA

FNEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNSR




QVREFLGKAGYCRLFIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFEDIKKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKK




LDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGGNHHDCL




QILAETHGTRPDLTDQPLPDADLTWYTDGSSFIRNGEREAGAAVTTESEVIWAAPLPPGTSAQRAELIALTQALKMAEGKKLTVYTDSRYAFATTHVHG




EIYRRRGWLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETHSSLTVLP


 


FOAMV_
8,021
MNPLQLLQPLPAEIKGTKLLAHWNSGATITCIPESFLEDEQPIKKTLIKTIHGEKQQNVYYVTFKVKGRKVEAEVIASPYEYILLSPTDVPWLTQQPLQLTIL


P14350

VPLQEYQEKILSKTALPEDQKQQLKTLFVKYDNLWQHWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQGVLTPQNSTMNTPV




YPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKYKTTLDLANGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFLNSPALFTADV




VDLLKEIPNVQVYVDDIYLSHDDPKEHVQQLEKVFQILLQAGYVVSLKKSEIGQKTVEFLGFNITKEGRGLTDTFKTKLLNITPPKDLKQLQSILGLLNFAR




NFIPNFAELVQPLYNLIASAKGKYIEWSEENTKQLNMVIEALNTASNLEERLPEQRLVIKVNTSPSAGYVRYYNETGKKPIMYLNYVFSKAELKFSMLEKL




LTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSQSPVKHPSQYEGVFYTDGSAI




KSPDPTKSNNAGMGIVHATYKPEYQVLNQWSIPLGNHTAQMAEIAAVEFACKKALKIPGPVLVITDSFYVAESANKELPYWKSNGFVNNKKKPLKHISK




WKSIAECLSMKPDITIQHEKGISLQIPVFILKGNALADKLATQGSYVVN


 


FOAMV_
8,022
MNPLQLLQPLPAEIKGTKLLAHWNSGATITCIPESFLEDEQPIKKTLIKTIHGEKQQNVYYVTFKVKGRKVEAEVIASPYEYILLSPTDVPWLTQQPLQLTIL


P14350_

VPLQEYQEKILSKTALPEDQKQQLKTLFVKYDNLWQHWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQGVLTPQNSTMNTPV


2mut

YPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKYKTTLDLANGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFLNSPALFNADV




VDLLKEIPNVQVYVDDIYLSHDDPKEHVQQLEKVFQILLQAGYVVSLKKSEIGQKTVEFLGFNITKEGRGLTDTFKTKLLNITPPKDLKQLQSILGLLNFAR




NFIPNFAELVQPLYNLIAPAKGKYIEWSEENTKQLNMVIEALNTASNLEERLPEQRLVIKVNTSPSAGYVRYYNETGKKPIMYLNYVFSKAELKFSMLEKL




LTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSQSPVKHPSQYEGVFYTDGSAI




KSPDPTKSNNAGMGIVHATYKPEYQVLNQWSIPLGNHTAQMAEIAAVEFACKKALKIPGPVLVITDSFYVAESANKELPYWKSNGFVNNKKKPLKHISK




WKSIAECLSMKPDITIQHEKGISLQIPVFILKGNALADKLATQGSYVVN


 


FOAMV_
8,023
MNPLQLLQPLPAEIKGTKLLAHWNSGATITCIPESFLEDEQPIKKTLIKTIHGEKQQNVYYVTFKVKGRKVEAEVIASPYEYILLSPTDVPWLTQQPLQLTIL


P14350_

VPLQEYQEKILSKTALPEDQKQQLKTLFVKYDNLWQHWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQGVLTPQNSTMNTPV


2mutA

YPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKYKTTLDLANGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFLNSPALFNADV




VDLLKEIPNVQVYVDDIYLSHDDPKEHVQQLEKVFQILLQAGYVVSLKKSEIGQKTVEFLGFNITKEGRGLTDTFKTKLLNITPPKDLKQLQSILGKLNFAR




NFIPNFAELVQPLYNLIAPAKGKYIEWSEENTKQLNMVIEALNTASNLEERLPEQRLVIKVNTSPSAGYVRYYNETGKKPIMYLNYVFSKAELKFSMLEKL




LTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSQSPVKHPSQYEGVFYTDGSAI




KSPDPTKSNNAGMGIVHATYKPEYQVLNQWSIPLGNHTAQMAEIAAVEFACKKALKIPGPVLVITDSFYVAESANKELPYWKSNGFVNNKKKPLKHISK




WKSIAECLSMKPDITIQHEKGISLQIPVFILKGNALADKLATQGSYVVN


 


FOAMV_
8,024
VPWLTQQPLQLTILVPLQEYQEKILSKTALPEDQKQQLKTLFVKYDNLWQHWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQG


P14350-

VLTPQNSTMNTPVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKYKTTLDLANGFWAHPITPESYWLTAFTWQGKQYCWTRLPQ


Pro

GFLNSPALFTADVVDLLKEIPNVQVYVDDIYLSHDDPKEHVQQLEKVFQILLQAGYVVSLKKSEIGQKTVEFLGFNITKEGRGLTDTFKTKLLNITPPKDLK




QLQSILGLLNFARNFIPNFAELVQPLYNLIASAKGKYIEWSEENTKQLNMVIEALNTASNLEERLPEQRLVIKVNTSPSAGYVRYYNETGKKPIMYLNYVF




SKAELKFSMLEKLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSQSPVKHPS




QYEGVFYTDGSAIKSPDPTKSNNAGMGIVHATYKPEYQVLNQWSIPLGNHTAQMAEIAAVEFACKKALKIPGPVLVITDSFYVAESANKELPYWKSNGF




VNNKKKPLKHISKWKSIAECLSMKPDITIQHEKGISLQIPVFILKGNALADKLATQGSYVVN


 


FOAMV_
8,025
VPWLTQQPLQLTILVPLQEYQEKILSKTALPEDQKQQLKTLFVKYDNLWQHWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQG


P14350-

VLTPQNSTMNTPVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKYKTTLDLANGFWAHPITPESYWLTAFTWQGKQYCWTRLPQ


Pro_2mut

GFLNSPALFNADVVDLLKEIPNVQVYVDDIYLSHDDPKEHVQQLEKVFQILLQAGYVVSLKKSEIGQKTVEFLGFNITKEGRGLTDTFKTKLLNITPPKDL




KQLQSILGLLNFARNFIPNFAELVQPLYNLIAPAKGKYIEWSEENTKQLNMVIEALNTASNLEERLPEQRLVIKVNTSPSAGYVRYYNETGKKPIMYLNYV




FSKAELKFSMLEKLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSQSPVKHP




SQYEGVFYTDGSAIKSPDPTKSNNAGMGIVHATYKPEYQVLNQWSIPLGNHTAQMAEIAAVEFACKKALKIPGPVLVITDSFYVAESANKELPYWKSNG




FVNNKKKPLKHISKWKSIAECLSMKPDITIQHEKGISLQIPVFILKGNALADKLATQGSYVVN


 


FOAMV_
8,026
VPWLTQQPLQLTILVPLQEYQEKILSKTALPEDQKQQLKTLFVKYDNLWQHWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQG


P14350-

VLTPQNSTMNTPVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKYKTTLDLANGFWAHPITPESYWLTAFTWQGKQYCWTRLPQ


Pro_2mutA

GFLNSPALFNADVVDLLKEIPNVQVYVDDIYLSHDDPKEHVQQLEKVFQILLQAGYVVSLKKSEIGQKTVEFLGFNITKEGRGLTDTFKTKLLNITPPKDL




KQLQSILGKLNFARNFIPNFAELVQPLYNLIAPAKGKYIEWSEENTKQLNMVIEALNTASNLEERLPEQRLVIKVNTSPSAGYVRYYNETGKKPIMYLNYV




FSKAELKFSMLEKLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSQSPVKHP




SQYEGVFYTDGSAIKSPDPTKSNNAGMGIVHATYKPEYQVLNQWSIPLGNHTAQMAEIAAVEFACKKALKIPGPVLVITDSFYVAESANKELPYWKSNG




FVNNKKKPLKHISKWKSIAECLSMKPDITIQHEKGISLQIPVFILKGNALADKLATQGSYVVN


 


GALV_
8,027
VLNLEEEYRLHEKPVPSSIDPSWLQLFPTVWAERAGMGLANQVPPVVVELRSGASPVAVRQYPMSKEAREGIRPHIQKFLDLGVLVPCRSPWNTPLL


P21414

PVKKPGTNDYRPVQDLREINKRVQDIHPTVPNPYNLLSSLPPSYTWYSVLDLKDAFFCLRLHPNSQPLFAFEWKDPEKGNTGQLTWTRLPQGFKNSP




TLFDEALHRDLAPFRALNPQVVLLQYVDDLLVAAPTYEDCKKGTQKLLQELSKLGYRVSAKKAQLCQREVTYLGYLLKEGKRWLTPARKATVMKIPVP




TTPRQVREFLGTAGFCRLWIPGFASLAAPLYPLTKESIPFIWTEEHQQAFDHIKKALLSAPALALPDLTKPFTLYIDERAGVARGVLTQTLGPWRRPVAY




LSKKLDPVASGWPTCLKAVAAVALLLKDADKLTLGQNVTVIASHSLESIVRQPPDRWMTNARMTHYQSLLLNERVSFAPPAVLNPATLLPVESEATPVH




RCSEILAEETGTRRDLEDQPLPGVPTWYTDGSSFITEGKRRAGAPIVDGKRTVWASSLPEGTSAQKAELVALTQALRLAEGKNINIYTDSRYAFATAHIH




GAIYKQRGLLTSAGKDIKNKEEILALLEAIHLPRRVAIIHCPGHQRGSNPVATGNRRADEAAKQAALSTRVLAGTTKP


 


GALV_
8,028
VLNLEEEYRLHEKPVPSSIDPSWLQLFPTVWAERAGMGLANQVPPVVVELRSGASPVAVRQYPMSKEAREGIRPHIQKFLDLGVLVPCRSPWNTPLL


P21414_

PVKKPGTNDYRPVQDLREINKRVQDIHPTVPNPYNLLSSLPPSYTWYSVLDLKDAFFCLRLHPNSQPLFAFEWKDPEKGNTGQLTWTRLPQGFKNSP


3mut

TLFNEALHRDLAPFRALNPQVVLLQYVDDLLVAAPTYEDCKKGTQKLLQELSKLGYRVSAKKAQLCQREVTYLGYLLKEGKRWLTPARKATVMKIPVP




TTPRQVREFLGTAGFCRLWIPGFASLAAPLYPLTKPSIPFIWTEEHQQAFDHIKKALLSAPALALPDLTKPFTLYIDERAGVARGVLTQTLGPWRRPVAY




LSKKLDPVASGWPTCLKAVAAVALLLKDADKLTLGQNVTVIASHSLESIVRQPPDRWMTNARMTHYQSLLLNERVSFAPPAVLNPATLLPVESEATPVH




RCSEILAEETGTRRDLEDQPLPGVPTWYTDGSSFITEGKRRAGAPIVDGKRTVWASSLPEGTSAQKAELVALTQALRLAEGKNINIYTDSRYAFATAHIH




GAIYKQRGWLTSAGKDIKNKEEILALLEAIHLPRRVAIIHCPGHQRGSNPVATGNRRADEAAKQAALSTRVLAGTTKP


 


GALV_
8,029
VLNLEEEYRLHEKPVPSSIDPSWLQLFPTVWAERAGMGLANQVPPVVVELRSGASPVAVRQYPMSKEAREGIRPHIQKFLDLGVLVPCRSPWNTPLL


P21414_

PVKKPGTNDYRPVQDLREINKRVQDIHPTVPNPYNLLSSLPPSYTWYSVLDLKDAFFCLRLHPNSQPLFAFEWKDPEKGNTGQLTWTRLPQGFKNSP


3mutA

TLFNEALHRDLAPFRALNPQVVLLQYVDDLLVAAPTYEDCKKGTQKLLQELSKLGYRVSAKKAQLCQREVTYLGYLLKEGKRWLTPARKATVMKIPVP




TTPRQVREFLGKAGFCRLFIPGFASLAAPLYPLTKPSIPFIWTEEHQQAFDHIKKALLSAPALALPDLTKPFTLYIDERAGVARGVLTQTLGPWRRPVAYL




SKKLDPVASGWPTCLKAVAAVALLLKDADKLTLGQNVTVIASHSLESIVRQPPDRWMTNARMTHYQSLLLNERVSFAPPAVLNPATLLPVESEATPVH




RCSEILAEETGTRRDLEDQPLPGVPTWYTDGSSFITEGKRRAGAPIVDGKRTVWASSLPEGTSAQKAELVALTQALRLAEGKNINIYTDSRYAFATAHIH




GAIYKQRGWLTSAGKDIKNKEEILALLEAIHLPRRVAIIHCPGHQRGSNPVATGNRRADEAAKQAALSTRVLAGTTKP


 


HTL1A_
8,030
AVLGLEHLPRPPQISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNPVFPVKKANGTWRFIHDLRATNSLTIDLSSSSPGPPDLSSLPTTLAHLQTI


P03362

DLRDAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYAWKVLPQGFKNSPTLFEMQLAHILQPIRQAFPQCTILQYMDDILLASPSHEDLLLLSEATMASLI




SHGLPVSENKTQQTPGTIKFLGQIISPNHLTYDAVPTVPIRSRWALPELQALLGEIQWVSKGTPTLRQPLHSLYCALQRHTDPRDQIYLNPSQVQSLVQL




RQALSQNCRSRLVQTLPLLGAIMLTLTGTTTVVFQSKEQWPLVWLHAPLPHTSQCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISTQTFNQFIQTS




DHPSVPILLHHSHRFKNLGAQTGELWNTFLKTAAPLAPVKALMPVFTLSPVIINTAPCLFSDGSTSRAAYILWDKQILSQRSFPLPPPHKSAQRAELLGLL




HGLSSARSWRCLNIFLDSKYLYHYLRTLALGTFQGRSSQAPFQALLPRLLSRKVVYLHHVRSHTNLPDPISRLNALTDALLITPVLQL


 


HTL1A_
8,031
AVLGLEHLPRPPQISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNPVFPVKKANGTWRFIHDLRATNSLTIDLSSSSPGPPDLSSLPTTLAHLQTI


P03362_

DLRDAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYAWKVLPQGFKNSPTLFQMQLAHILQPIRQAFPQCTILQYMDDILLASPSHEDLLLLSEATMASLI


2mut

SHGLPVSENKTQQTPGTIKFLGQIISPNHLTYDAVPTVPIRSRWALPELQALLGEIQWVSKGTPTLRQPLHSLYCALQPHTDPRDQIYLNPSQVQSLVQL




RQALSQNCRSRLVQTLPLLGAIMLTLTGTTTVVFQSKEQWPLVWLHAPLPHTSQCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISTQTFNQFIQTS




DHPSVPILLHHSHRFKNLGAQTGELWNTFLKTAAPLAPVKALMPVFTLSPVIINTAPCLFSDGSTSRAAYILWDKQILSQRSFPLPPPHKSAQRAELLGLL




HGLSSARSWRCLNIFLDSKYLYHYLRTLALGTFQGRSSQAPFQALLPRLLSRKVVYLHHVRSHTNLPDPISRLNALTDALLITPVLQL


 


HTL1A_
8,032
AVLGLEHLPRPPQISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNPVFPVKKANGTWRFIHDLRATNSLTIDLSSSSPGPPDLSSPPTTLAHLQTI


P03362_

DLRDAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYAWKVLPQGFKNSPTLFQMQLAHILQPIRQAFPQCTILQYMDDILLASPSHEDLLLLSEATMASLI


2mutB

SHGLPVSENKTQQTPGTIKFLGQIISPNHLTYDAVPTVPIRSRWALPELQALLGEIQWVSKGTPTLRQPLHSLYCALQPHTDPRDQIYLNPSQVQSLVQL




RQALSQNCRSRLVQTLPLLGAIMLTLTGTTTVVFQSKEQWPLVWLHAPLPHTSQCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISTQTFNQFIQTS




DHPSVPILLHHSHRFKNLGAQTGELWNTFLKTAAPLAPVKALMPVFTLSPVIINTAPCLFSDGSTSRAAYILWDKQILSQRSFPLPPPHKSAQRAELLGLL




HGLSSARSWRCLNIFLDSKYLYHYLRTLALGTFQGRSSQAPFQALLPRLLSRKVVYLHHVRSHTNLPDPISRLNALTDALLITPVLQL


 


HTL1C_
8,033
AVLGLEHLPRPPEISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNPVFPVKKANGTWRFIHDLRATNSLTIDLSSSSPGPPDLSSLPTTLAHLQTI


P14078

DLKDAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYAWRVLPQGFKNSPTLFEMQLAHILQPIRQAFPQCTILQYMDDILLASPSHADLQLLSEATMASLI




SHGLPVSENKTQQTPGTIKFLGQIISPNHLTYDAVPKVPIRSRWALPELQALLGEIQWVSKGTPTLRQPLHSLYCALQRHTDPRDQIYLNPSQVQSLVQL




RQALSQNCRSRLVQTLPLLGAIMLTLTGTTTVVFQSKQQWPLVWLHAPLPHTSQCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISTQTFNQFIQTS




DHPSVPILLHHSHRFKNLGAQTGELWNTFLKTTAPLAPVKALMPVFTLSPVIINTAPCLFSDGSTSQAAYILWDKHILSQRSFPLPPPHKSAQRAELLGLL




HGLSSARSWRCLNIFLDSKYLYHYLRTLALGTFQGRSSQAPFQALLPRLLSRKVVYLHHVRSHTNLPDPISRLNALTDALLITPVLQL


 


HTL1C_
8,034
AVLGLEHLPRPPEISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNPVFPVKKANGTWRFIHDLRATNSLTIDLSSSSPGPPDLSSLPTTLAHLQTI


P14078_

DLKDAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYAWRVLPQGFKNSPTLFQMQLAHILQPIRQAFPQCTILQYMDDILLASPSHADLQLLSEATMASLI


2mut

SHGLPVSENKTQQTPGTIKFLGQIISPNHLTYDAVPKVPIRSRWALPELQALLGEIQWVSKGTPTLRQPLHSLYCALQPHTDPRDQIYLNPSQVQSLVQL




RQALSQNCRSRLVQTLPLLGAIMLTLTGTTTVVFQSKQQWPLVWLHAPLPHTSQCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISTQTFNQFIQTS




DHPSVPILLHHSHRFKNLGAQTGELWNTFLKTTAPLAPVKALMPVFTLSPVIINTAPCLFSDGSTSQAAYILWDKHILSQRSFPLPPPHKSAQRAELLGLL




HGLSSARSWRCLNIFLDSKYLYHYLRTLALGTFQGRSSQAPFQALLPRLLSRKVVYLHHVRSHTNLPDPISRLNALTDALLITPVLQL


 


HTL1L_
8,035
GLEHLPRPPEISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNPVFPVKKANGTWRFIHDLRATNSLTVDLSSSSPGPPDLSSLPTTLAHLQTIDLK


P0C211

DAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYAWKVLPQGFKNSPTLFEMQLASILQPIRQAFPQCVILQYMDDILLASPSPEDLQQLSEATMASLISH




GLPVSQDKTQQTPGTIKFLGQIISPNHITYDAVPTVPIRSRWALPELQALLGEIQWVSKGTPTLRQPLHSLYCALQGHTDPRDQIYLNPSQVQSLMQLQ




QALSQNCRSRLAQTLPLLGAIMLTLTGTTTVVFQSKQQWPLVWLHAPLPHTSQCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISIQTFNQFIQTSD




HPSVPILLHHSHRFKNLGAQTGELWNTFLKTAAPLAPVKALTPVFTLSPIIINTAPCLFSDGSTSQAAYILWDKHILSQRSFPLPPPHKSAQQAELLGLLH




GLSSARSWHCLNIFLDSKYLYHYLRTLALGTFQGKSSQAPFQALLPRLLAHKVIYLHHVRSHTNLPDPISKLNALTDALLITPIL


 


HTL1L_
8,036
GLEHLPRPPEISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNPVFPVKKANGTWRFIHDLRATNSLTVDLSSSSPGPPDLSSLPTTLAHLQTIDLK


P0C211_

DAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYAWKVLPQGFKNSPTLFQMQLASILQPIRQAFPQCVILQYMDDILLASPSPEDLQQLSEATMASLISH


2mut

GLPVSQDKTQQTPGTIKFLGQIISPNHITYDAVPTVPIRSRWALPELQALLGEIQWVSKGTPTLRQPLHSLYCALQGHTDPRDQIYLNPSQVQSLMQLQ




QALSQNCRSRLAQTLPLLGAIMLTLTGTTTVVFQSKQQWPLVWLHAPLPHTSQCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISIQTFNQFIQTSD




HPSVPILLHHSHRFKNLGAQTGELWNTFLKTAAPLAPVKALTPVFTLSPIIINTAPCLFSDGSTSQAAYILWDKHILSQRSFPLPPPHKSAQQAELLGLLH




GLSSARSWHCLNIFLDSKYLYHYLRTLAWGTFQGKSSQAPFQALLPRLLAHKVIYLHHVRSHTNLPDPISKLNALTDALLITPIL


 


HTL1L_
8,037
GLEHLPRPPEISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNPVFPVKKANGTWRFIHDLRATNSLTVDLSSSSPGPPDLSSPPTTLAHLQTIDLK


P0C211_

DAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYAWKVLPQGFKNSPTLFQMQLASILQPIRQAFPQCVILQYMDDILLASPSPEDLQQLSEATMASLISH


2mutB

GLPVSQDKTQQTPGTIKFLGQIISPNHITYDAVPTVPIRSRWALPELQALLGEIQWVSKGTPTLRQPLHSLYCALQGHTDPRDQIYLNPSQVQSLMQLQ




QALSQNCRSRLAQTLPLLGAIMLTLTGTTTVVFQSKQQWPLVWLHAPLPHTSQCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISIQTFNQFIQTSD




HPSVPILLHHSHRFKNLGAQTGELWNTFLKTAAPLAPVKALTPVFTLSPIIINTAPCLFSDGSTSQAAYILWDKHILSQRSFPLPPPHKSAQQAELLGLLH




GLSSARSWHCLNIFLDSKYLYHYLRTLAWGTFQGKSSQAPFQALLPRLLAHKVIYLHHVRSHTNLPDPISKLNALTDALLITPIL


 


HTL32_
8,038
GLEHLPPPPEVSQFPLNPERLQALTDLVSRALEAKHIEPYQGPGNNPIFPVKKPNGKWRFIHDLRATNSVTRDLASPSPGPPDLTSLPQGLPHLRTIDLT


Q0R5R2

DAFFQIPLPTIFQPYFAFTLPQPNNYGPGTRYSWRVLPQGFKNSPTLFEQQLSHILTPVRKTFPNSLIIQYMDDILLASPAPGELAALTDKVTNALTKEGL




PLSPEKTQATPGPIHFLGQVISQDCITYETLPSINVKSTWSLAELQSMLGELQWVSKGTPVLRSSLHQLYLALRGHRDPRDTIKLTSIQVQALRTIQKALT




LNCRSRLVNQLPILALIMLRPTGTTAVLFQTKQKWPLVWLHTPHPATSLRPWGQLLANAVIILDKYSLQHYGQVCKSFHHNISNQALTYYLHTSDQSSV




AILLQHSHRFHNLGAQPSGPWRSLLQMPQIFQNIDVLRPPFTISPVVINHAPCLFSDGSASKAAFIIWDRQVIHQQVLSLPSTCSAQAGELFGLLAGLQK




SQPWVALNIFLDSKFLIGHLRRMALGAFPGPSTQCELHTQLLPLLQGKTVYVHHVRSHTLLQDPISRLNEATDALMLAPLLPL


 


HTL32_
8,039
GLEHLPPPPEVSQFPLNPERLQALTDLVSRALEAKHIEPYQGPGNNPIFPVKKPNGKWRFIHDLRATNSVTRDLASPSPGPPDLTSLPQGLPHLRTIDLT


Q0R5R2_

DAFFQIPLPTIFQPYFAFTLPQPNNYGPGTRYSWRVLPQGFKNSPTLFQQQLSHILTPVRKTFPNSLIIQYMDDILLASPAPGELAALTDKVTNALTKEGL


2mut

PLSPEKTQATPGPIHFLGQVISQDCITYETLPSINVKSTWSLAELQSMLGELQWVSKGTPVLRSSLHQLYLALRGHRDPRDTIKLTSIQVQALRTIQKALT




LNCRSRLVNQLPILALIMLRPTGTTAVLFQTKQKWPLVWLHTPHPATSLRPWGQLLANAVIILDKYSLQHYGQVCKSFHHNISNQALTYYLHTSDQSSV




AILLQHSHRFHNLGAQPSGPWRSLLQMPQIFQNIDVLRPPFTISPVVINHAPCLFSDGSASKAAFIIWDRQVIHQQVLSLPSTCSAQAGELFGLLAGLQK




SQPWVALNIFLDSKFLIGHLRRMAWGAFPGPSTQCELHTQLLPLLQGKTVYVHHVRSHTLLQDPISRLNEATDALMLAPLLPL


 


HTL32_
8,040
GLEHLPPPPEVSQFPLNPERLQALTDLVSRALEAKHIEPYQGPGNNPIFPVKKPNGKWRFIHDLRATNSVTRDLASPSPGPPDLTSPPQGLPHLRTIDL


Q0R5R2_

TDAFFQIPLPTIFQPYFAFTLPQPNNYGPGTRYSWRVLPQGFKNSPTLFQQQLSHILTPVRKTFPNSLIIQYMDDILLASPAPGELAALTDKVTNALTKEG


2mutB

LPLSPEKTQATPGPIHFLGQVISQDCITYETLPSINVKSTWSLAELQSMLGELQWVSKGTPVLRSSLHQLYLALRGHRDPRDTIKLTSIQVQALRTIQKAL




TLNCRSRLVNQLPILALIMLRPTGTTAVLFQTKQKWPLVWLHTPHPATSLRPWGQLLANAVIILDKYSLQHYGQVCKSFHHNISNQALTYYLHTSDQSS




VAILLQHSHRFHNLGAQPSGPWRSLLQMPQIFQNIDVLRPPFTISPVVINHAPCLFSDGSASKAAFIIWDRQVIHQQVLSLPSTCSAQAGELFGLLAGLQ




KSQPWVALNIFLDSKFLIGHLRRMAWGAFPGPSTQCELHTQLLPLLQGKTVYVHHVRSHTLLQDPISRLNEATDALMLAPLLPL


 


HTL3P_
8,041
GLEHLPPPPEVSQFPLNPERLQALTDLVSRALEAKHIEPYQGPGNNPIFPVKKPNGKWRFIHDLRATNSLTRDLASPSPGPPDLTSLPQDLPHLRTIDLT


Q4U0X6

DAFFQIPLPAVFQPYFAFTLPQPNNHGPGTRYSWRVLPQGFKNSPTLFEQQLSHILAPVRKAFPNSLIIQYMDDILLASPALRELTALTDKVTNALTKEGL




PMSLEKTQATPGSIHFLGQVISPDCITYETLPSIHVKSIWSLAELQSMLGELQWVSKGTPVLRSSLHQLYLALRGHRDPRDTIELTSTQVQALKTIQKALA




LNCRSRLVSQLPILALIILRPTGTTAVLFQTKQKWPLVWLHTPHPATSLRPWGQLLANAIITLDKYSLQHYGQICKSFHHNISNQALTYYLHTSDQSSVAIL




LQHSHRFHNLGAQPSGPWRSLLQVPQIFQNIDVLRPPFIISPVVIDHAPCLFSDGATSKAAFILWDKQVIHQQVLPLPSTCSAQAGELFGLLAGLQKSKP




WPALNIFLDSKFLIGHLRRMALGAFLGPSTQCDLHARLFPLLQGKTVYVHHVRSHTLLQDPISRLNEATDALMLAPLLPL


 


HTL3P_
8,042
GLEHLPPPPEVSQFPLNPERLQALTDLVSRALEAKHIEPYQGPGNNPIFPVKKPNGKWRFIHDLRATNSLTRDLASPSPGPPDLTSLPQDLPHLRTIDLT


Q4U0X6_

DAFFQIPLPAVFQPYFAFTLPQPNNHGPGTRYSWRVLPQGFKNSPTLFQQQLSHILAPVRKAFPNSLIIQYMDDILLASPALRELTALTDKVTNALTKEG


2mut

LPMSLEKTQATPGSIHFLGQVISPDCITYETLPSIHVKSIWSLAELQSMLGELQWVSKGTPVLRSSLHQLYLALRGHRDPRDTIELTSTQVQALKTIQKAL




ALNCRSRLVSQLPILALIILRPTGTTAVLFQTKQKWPLVWLHTPHPATSLRPWGQLLANAIITLDKYSLQHYGQICKSFHHNISNQALTYYLHTSDQSSVAI




LLQHSHRFHNLGAQPSGPWRSLLQVPQIFQNIDVLRPPFIISPVVIDHAPCLFSDGATSKAAFILWDKQVIHQQVLPLPSTCSAQAGELFGLLAGLQKSK




PWPALNIFLDSKFLIGHLRRMAWGAFLGPSTQCDLHARLFPLLQGKTVYVHHVRSHTLLQDPISRLNEATDALMLAPLLPL


 


HTL3P_
8,043
GLEHLPPPPEVSQFPLNPERLQALTDLVSRALEAKHIEPYQGPGNNPIFPVKKPNGKWRFIHDLRATNSLTRDLASPSPGPPDLTSPPQDLPHLRTIDLT


Q4U0X6_

DAFFQIPLPAVFQPYFAFTLPQPNNHGPGTRYSWRVLPQGFKNSPTLFQQQLSHILAPVRKAFPNSLIIQYMDDILLASPALRELTALTDKVTNALTKEG


2mutB

LPMSLEKTQATPGSIHFLGQVISPDCITYETLPSIHVKSIWSLAELQSMLGELQWVSKGTPVLRSSLHQLYLALRGHRDPRDTIELTSTQVQALKTIQKAL




ALNCRSRLVSQLPILALIILRPTGTTAVLFQTKQKWPLVWLHTPHPATSLRPWGQLLANAIITLDKYSLQHYGQICKSFHHNISNQALTYYLHTSDQSSVAI




LLQHSHRFHNLGAQPSGPWRSLLQVPQIFQNIDVLRPPFIISPVVIDHAPCLFSDGATSKAAFILWDKQVIHQQVLPLPSTCSAQAGELFGLLAGLQKSK




PWPALNIFLDSKFLIGHLRRMAWGAFLGPSTQCDLHARLFPLLQGKTVYVHHVRSHTLLQDPISRLNEATDALMLAPLLPL


 


HTLV2_
8,044
HLPPPPQVDQFPLNLPERLQALNDLVSKALEAGHIEPYSGPGNNPVFPVKKPNGKWRFIHDLRATNAITTTLTSPSPGPPDLTSLPTALPHLQTIDLTDA


P03363_

FFQIPLPKQYQPYFAFTIPQPCNYGPGTRYAWTVLPQGFKNSPTLFQQQLAAVLNPMRKMFPTSTIVQYMDDILLASPTNEELQQLSQLTLQALTTHGL


2mut

PISQEKTQQTPGQIRFLGQVISPNHITYESTPTIPIKSQWTLTELQVILGEIQWVSKGTPILRKHLQSLYSALHPYRDPRACITLTPQQLHALHAIQQALQH




NCRGRLNPALPLLGLISLSTSGTTSVIFQPKQNWPLAWLHTPHPPTSLCPWGHLLACTILTLDKYTLQHYGQLCQSFHHNMSKQALCDFLRNSPHPSV




GILIHHMGRFHNLGSQPSGPWKTLLHLPTLLQEPRLLRPIFTLSPVVLDTAPCLFSDGSPQKAAYVLWDQTILQQDITPLPSHETHSAQKGELLALICGLR




AAKPWPSLNIFLDSKYLIKYLHSLAIGAFLGTSAHQTLQAALPPLLQGKTIYLHHVRSHTNLPDPISTFNEYTDSLILAPLVPL


 


JSRV_
8,045
PLGTSDSPVTHADPIDWKSEEPVWVDQWPLTQEKLSAAQQLVQEQLRLGHIEPSTSAWNSPIFVIKKKSGKWRLLQDLRKVNETMMHMGALQPGLPT


P31623

PSAIPDKSYIIVIDLKDCFYTIPLAPQDCKRFAFSLPSVNFKEPMQRYQWRVLPQGMTNSPTLCQKFVATAIAPVRQRFPQLYLVHYMDDILLAHTDEHLL




YQAFSILKQHLSLNGLVIADEKIQTHFPYNYLGFSLYPRVYNTQLVKLQTDHLKTLNDFQKLLGDINWIRPYLKLPTYTLQPLFDILKGDSDPASPRTLSLE




GRTALQSIEEAIRQQQITYCDYQRSWGLYILPTPRAPTGVLYQDKPLRWIYLSATPTKHLLPYYELVAKIIAKGRHEAIQYFGMEPPFICVPYALEQQDWL




FQFSDNWSIAFANYPGQITHHYPSDKLLQFASSHAFIFPKIVRRQPIPEATLIFTDGSSNGTAALIINHQTYYAQTSFSSAQVVELFAVHQALLTVPTSFNL




FTDSSYVVGALQMIETVPIIGTTSPEVLNLFTLIQQVLHCRQHPCFFGHIRAHSTLPGALVQGNHTADVLTKQVFFQS


 


JSRV_
8,046
PLGTSDSPVTHADPIDWKSEEPVWVDQWPLTQEKLSAAQQLVQEQLRLGHIEPSTSAWNSPIFVIKKKSGKWRLLQDLRKVNETMMHMGALQPGLPT


P31623_

PSPIPDKSYIIVIDLKDCFYTIPLAPQDCKRFAFSLPSVNFKEPMQRYQWRVLPQGMTNSPTLCQKFVATAIAPVRQRFPQLYLVHYMDDILLAHTDEHLL


2mutB

YQAFSILKQHLSLNGLVIADEKIQTHFPYNYLGFSLYPRVYNTQLVKLQTDHLKTLNDFQKLLGDINWIRPYLKLPTYTLQPLFDILKGDSDPASPRTLSLE




GRTALQSIEEAIRQQQITYCDYQRSWGLYILPTPRAPTGVLYQDKPLRWIYLSATPTKHLLPYYELVAKIIAKGRHEAIQYFGMEPPFICVPYALEQQDWL




FQFSDNWSIAFANYPGQITHHYPSDKLLQFASSHAFIFPKIVRRQPIPEATLIFTDGSSNGTAALIINHQTYYAQTSFSSAQVVELFAVHQALLTVPTSFNL




FTDSSYVVGALQMIETVPIIGTTSPEVLNLFTLIQQVLHCRQHPCFFGHIRAHSTLPGALVQGNHTADVLTKQVFFQS


 


KORV_
8,047
TLGDQGSRGSDPLPEPRVTLTVEGIPTEFLVNTGAEHSVLTKPMGKMGSKRTVVAGATGSKVYPWTTKRLLKIGQKQVTHSFLVIPECPAPLLGRDLLT


Q9TTC1

KLKAQIQFSTEGPQVTWEDRPAMCLVLNLEEEYRLHEKPVPPSIDPSWLQLFPMVWAEKAGMGLANQVPPVVVELKSDASPVAVRQYPMSKEAREGI




RPHIQRFLDLGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLSSLPPSHTWYSVLDLKDAFFCLKLHPNSQPLFAFEW




RDPEKGNTGQLTWTRLPQGFKNSPTLFDEALHRDLASFRALNPQVVMLQYVDDLLVAAPTYRDCKEGTRRLLQELSKLGYRVSAKKAQLCREEVTYL




GYLLKGGKRWLTPARKATVMKIPTPTTPRQVREFLGTAGFCRLWIPGFASLAAPLYPLTREKVPFTWTEAHQEAFGRIKEALLSAPALALPDLTKPFAL




YVDEKEGVARGVLTQTLGPWRRPVAYLSKKLDPVASGWPTCLKAIAAVALLLKDADKLTLGQNVLVIAPHNLESIVRQPPDRWMTNARMTHYQSLLLN




ERVSFAPPAILNPATLLPVESDDTPIHICSEILAEETGTRPDLRDQPLPGVPAWYTDGSSFIMDGRRQAGAAIVDNKRTVWASNLPEGTSAQKAELIALT




QALRLAEGKSINIYTDSRYAFATAHVHGAIYKQRGLLTSAGKDIKNKEEILALLEAIHLPKRVAIIHCPGHQRGTDPVATGNRKADEAAKQAAQSTRILTET




TKN


 


KORV_
8,048
TLGDQGSRGSDPLPEPRVTLTVEGIPTEFLVNTGAEHSVLTKPMGKMGSKRTVVAGATGSKVYPWTTKRLLKIGQKQVTHSFLVIPECPAPLLGRDLLT


Q9TTC1_

KLKAQIQFSTEGPQVTWEDRPAMCLVLNLEEEYRLHEKPVPPSIDPSWLQLFPMVWAEKAGMGLANQVPPVVVELKSDASPVAVRQYPMSKEAREGI


3mut

RPHIQRFLDLGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLSSLPPSHTWYSVLDLKDAFFCLKLHPNSQPLFAFEW




RDPEKGNTGQLTWTRLPQGFKNSPTLFNEALHRDLASFRALNPQVVMLQYVDDLLVAAPTYRDCKEGTRRLLQELSKLGYRVSAKKAQLCREEVTYL




GYLLKGGKRWLTPARKATVMKIPTPTTPRQVREFLGTAGFCRLWIPGFASLAAPLYPLTRPKVPFTWTEAHQEAFGRIKEALLSAPALALPDLTKPFAL




YVDEKEGVARGVLTQTLGPWRRPVAYLSKKLDPVASGWPTCLKAIAAVALLLKDADKLTLGQNVLVIAPHNLESIVRQPPDRWMTNARMTHYQSLLLN




ERVSFAPPAILNPATLLPVESDDTPIHICSEILAEETGTRPDLRDQPLPGVPAWYTDGSSFIMDGRRQAGAAIVDNKRTVWASNLPEGTSAQKAELIALT




QALRLAEGKSINIYTDSRYAFATAHVHGAIYKQRGWLTSAGKDIKNKEEILALLEAIHLPKRVAIIHCPGHQRGTDPVATGNRKADEAAKQAAQSTRILTE




TTKN


 


KORV_
8,049
TLGDQGSRGSDPLPEPRVTLTVEGIPTEFLVNTGAEHSVLTKPMGKMGSKRTVVAGATGSKVYPWTTKRLLKIGQKQVTHSFLVIPECPAPLLGRDLLT


Q9TTC1_

KLKAQIQFSTEGPQVTWEDRPAMCLVLNLEEEYRLHEKPVPPSIDPSWLQLFPMVWAEKAGMGLANQVPPVVVELKSDASPVAVRQYPMSKEAREGI


3mutA

RPHIQRFLDLGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLSSLPPSHTWYSVLDLKDAFFCLKLHPNSQPLFAFEW




RDPEKGNTGQLTWTRLPQGFKNSPTLFNEALHRDLASFRALNPQVVMLQYVDDLLVAAPTYRDCKEGTRRLLQELSKLGYRVSAKKAQLCREEVTYL




GYLLKGGKRWLTPARKATVMKIPTPTTPRQVREFLGKAGFCRLFIPGFASLAAPLYPLTRPKVPFTWTEAHQEAFGRIKEALLSAPALALPDLTKPFALY




VDEKEGVARGVLTQTLGPWRRPVAYLSKKLDPVASGWPTCLKAIAAVALLLKDADKLTLGQNVLVIAPHNLESIVRQPPDRWMTNARMTHYQSLLLNE




RVSFAPPAILNPATLLPVESDDTPIHICSEILAEETGTRPDLRDQPLPGVPAWYTDGSSFIMDGRRQAGAAIVDNKRTVWASNLPEGTSAQKAELIALTQ




ALRLAEGKSINIYTDSRYAFATAHVHGAIYKQRGWLTSAGKDIKNKEEILALLEAIHLPKRVAIIHCPGHQRGTDPVATGNRKADEAAKQAAQSTRILTET




TKN


 


KORV_
8,050
LLGRDLLTKLKAQIQFSTEGPQVTWEDRPAMCLVLNLEEEYRLHEKPVPPSIDPSWLQLFPMVWAEKAGMGLANQVPPVVVELKSDASPVAVRQYPM


Q9TTC1-

SKEAREGIRPHIQRFLDLGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLSSLPPSHTWYSVLDLKDAFFCLKLHPNSQ


Pro

PLFAFEWRDPEKGNTGQLTWTRLPQGFKNSPTLFDEALHRDLASFRALNPQWVMLQYVDDLLVAAPTYRDCKEGTRRLLQELSKLGYRVSAKKAQLC




REEVTYLGYLLKGGKRWLTPARKATVMKIPTPTTPRQVREFLGTAGFCRLWIPGFASLAAPLYPLTREKVPFTWTEAHQEAFGRIKEALLSAPALALPD




LTKPFALYVDEKEGVARGVLTQTLGPWRRPVAYLSKKLDPVASGWPTCLKAIAAVALLLKDADKLTLGQNVLVIAPHNLESIVRQPPDRWMTNARMTH




YQSLLLNERVSFAPPAILNPATLLPVESDDTPIHICSEILAEETGTRPDLRDQPLPGVPAWYTDGSSFIMDGRRQAGAAIVDNKRTVWASNLPEGTSAQ




KAELIALTQALRLAEGKSINIYTDSRYAFATAHVHGAIYKQRGLLTSAGKDIKNKEEILALLEAIHLPKRVAIIHCPGHQRGTDPVATGNRKADEAAKQAAQ




STRILTETTKN


 


KORV_
8,051
LLGRDLLTKLKAQIQFSTEGPQVTWEDRPAMCLVLNLEEEYRLHEKPVPPSIDPSWLQLFPMVWAEKAGMGLANQVPPVVVELKSDASPVAVRQYPM


Q9TTC1-

SKEAREGIRPHIQRFLDLGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLSSLPPSHTWYSVLDLKDAFFCLKLHPNSQ


Pro_3mut

PLFAFEWRDPEKGNTGQLTWTRLPQGFKNSPTLFNEALHRDLASFRALNPQVVMLQYVDDLLVAAPTYRDCKEGTRRLLQELSKLGYRVSAKKAQLC




REEVTYLGYLLKGGKRWLTPARKATVMKIPTPTTPRQVREFLGTAGFCRLWIPGFASLAAPLYPLTRPKVPFTWTEAHQEAFGRIKEALLSAPALALPD




LTKPFALYVDEKEGVARGVLTQTLGPWRRPVAYLSKKLDPVASGWPTCLKAIAAVALLLKDADKLTLGQNVLVIAPHNLESIVRQPPDRWMTNARMTH




YQSLLLNERVSFAPPAILNPATLLPVESDDTPIHICSEILAEETGTRPDLRDQPLPGVPAWYTDGSSFIMDGRRQAGAAIVDNKRTVWASNLPEGTSAQ




KAELIALTQALRLAEGKSINIYTDSRYAFATAHVHGAIYKQRGWLTSAGKDIKNKEEILALLEAIHLPKRVAIIHCPGHQRGTDPVATGNRKADEAAKQAA




QSTRILTETTKN


 


KORV_
8,052
LLGRDLLTKLKAQIQFSTEGPQVTWEDRPAMCLVLNLEEEYRLHEKPVPPSIDPSWLQLFPMVWAEKAGMGLANQVPPVVVELKSDASPVAVRQYPM


Q9TTC1-

SKEAREGIRPHIQRFLDLGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLSSLPPSHTWYSVLDLKDAFFCLKLHPNSQ


Pro_3mutA

PLFAFEWRDPEKGNTGQLTWTRLPQGFKNSPTLFNEALHRDLASFRALNPQVVMLQYVDDLLVAAPTYRDCKEGTRRLLQELSKLGYRVSAKKAQLC




REEVTYLGYLLKGGKRWLTPARKATVMKIPTPTTPRQVREFLGKAGFCRLFIPGFASLAAPLYPLTRPKVPFTWTEAHQEAFGRIKEALLSAPALALPDL




TKPFALYVDEKEGVARGVLTQTLGPWRRPVAYLSKKLDPVASGWPTCLKAIAAVALLLKDADKLTLGQNVLVIAPHNLESIVRQPPDRWMTNARMTHY




QSLLLNERVSFAPPAILNPATLLPVESDDTPIHICSEILAEETGTRPDLRDQPLPGVPAWYTDGSSFIMDGRRQAGAAIVDNKRTVWASNLPEGTSAQK




AELIALTQALRLAEGKSINIYTDSRYAFATAHVHGAIYKQRGWLTSAGKDIKNKEEILALLEAIHLPKRVAIIHCPGHQRGTDPVATGNRKADEAAKQAAQ




STRILTETTKN


 


MLVAV_
8,053
TLNLEDEYRLYETSAEPEVSPGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEAKLGIKPHIQRLLDQGILVPCQSPWNTPLL


P03356

PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHRWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSP




TLFDEALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLLTLGNLGYRASAKKAQLCQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPK




TPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA




YLSKKLDPVAAGWPPCLRMVAAIAVLRKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEEG




APHDCLEILAETHGTRPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAF




ATAHIHGEIYRRRGLLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNRLADQAAREAAIKTPPDTSTLL


 


MLVAV_
8,054
TLNLEDEYRLYETSAEPEVSPGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEAKLGIKPHIQRLLDQGILVPCQSPWNTPLL


P03356_

PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHRWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSP


3mut

TLFNEALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLLTLGNLGYRASAKKAQLCQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPK




TPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPV




AYLSKKLDPVAAGWPPCLRMVAAIAVLRKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQAMLLDTDRVQFGPWVALNPATLLPLPEE




GAPHDCLEILAETHGTRPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYA




FATAHIHGEIYRRRGWLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNRLADQAAREAAIKTPPDTSTLL


 


MLVAV_
8,055
TLNLEDEYRLYETSAEPEVSPGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEAKLGIKPHIQRLLDQGILVPCQSPWNTPLL


P03356_

PVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHRWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSP


3mutA

TLFNEALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLLTLGNLGYRASAKKAQLCQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPK




TPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA




YLSKKLDPVAAGWPPCLRMVAAIAVLRKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQAMLLDTDRVQFGPWVALNPATLLPLPEEG




APHDCLEILAETHGTRPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAF




ATAHIHGEIYRRRGWLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNRLADQAAREAAIKTPPDTSTLL


 


MLVBM_
8,056
TLGIEDEYRLHETSTEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIQQYPMSHEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


Q7SVK7

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSPT




LFDEALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLQTLGDLGYRASAKKAQICQKQVKYLGYLLREGQRWLTEARKETVMGQPVPKT




PRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFSWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY




LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQAMLLDTDRVQFGPWVALNPATLLPLPEEGAP




HDCLEILAETHGTRPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWAGALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFAT




AHIHGEIYRRRGLLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNRLADQAAREAAIKTPPDTSTLL


 


MLVBM_
8,057
TLGIEDEYRLHETSTEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIQQYPMSHEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


Q7SVK7

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSPT




LFDEALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLQTLGDLGYRASAKKAQICQKQVKYLGYLLREGQRWLTEARKETVMGQPVPKT




PRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFSWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY




LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQAMLLDTDRVQFGPWVALNPATLLPLPEEGAP




HDCLEILAETHGTRPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWAGALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFAT




AHIHGEIYRRRGLLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNRLADQAAREAAIKTPPDTSTLL


 


MLVBM_
8,058
TLGIEDEYRLHETSTEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIQQYPMSHEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


Q7SVK7_

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSPT


3mut

LFNEALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLQTLGDLGYRASAKKAQICQKQVKYLGYLLREGQRWLTEARKETVMGQPVPKT




PRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFSWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA




YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEEGA




PHDCLEILAETHGTRPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWAGALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFA




TAHIHGEIYRRRGWLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNRLADQAAREAAIKTPPDTSTLL


 


MLVBM_
8,059
TLGIEDEYRLHETSTEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIQQYPMSHEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


Q7SVK7_

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSPT


3mut

LFNEALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLQTLGDLGYRASAKKAQICQKQVKYLGYLLREGQRWLTEARKETVMGQPVPKT




PRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFSWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA




YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEEGA




PHDCLEILAETHGTRPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWAGALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFA




TAHIHGEIYRRRGWLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNRLADQAAREAAIKTPPDTSTLL


 


MLVBM_
8,060
LGIEDEYRLHETSTEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIQQYPMSHEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPV


Q7SVK7_

KKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSPTL


3mutA_

FNEALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLQTLGDLGYRASAKKAQICQKQVKYLGYLLREGQRWLTEARKETVMGQPVPKTP


WS

RQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFSWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL




SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEEGAP




HDCLEILAETHGTRPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWAGALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFAT




AHIHGEIYRRRGWLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNRLADQAAREAAIKTPPDTSTLLI


 


MLVBM_
8,061
LGIEDEYRLHETSTEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIQQYPMSHEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPV


Q7SVK7_

KKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSPTL


3mutA_

FNEALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLQTLGDLGYRASAKKAQICQKQVKYLGYLLREGQRWLTEARKETVMGQPVPKTP


WS

RQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFSWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL




SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEEGAP




HDCLEILAETHGTRPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWAGALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFAT




AHIHGEIYRRRGWLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNRLADQAAREAAIKTPPDTSTLLI


 


MLVCB_
8,062
TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P08361

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT




LFDEALHRDLAGFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGDLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPIPKT




PRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAFQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY




LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQ




HDCLDILAEAHGTRSDLMDQPLPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFAT




AHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNSAEARGNRMADQAAREVATRETPETSTLL


 


MLVCB_
8,063
TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P08361_

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT


3mut

LFNEALHRDLAGFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGDLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPIPKT




PRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAFQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA




YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPWVALNPATLLPLPEEGL




QHDCLDILAEAHGTRSDLMDQPLPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAF




ATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNSAEARGNRMADQAAREVATRETPETSTLL


 


MLVCB_
8,064
TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P08361_

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT


3mutA

LFNEALHRDLAGFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGDLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPIPKT




PRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAFQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY




LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQ




HDCLDILAEAHGTRSDLMDQPLPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFAT




AHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNSAEARGNRMADQAAREVATRETPETSTLL


 


MLVF5_
8,065
TLNIEDEYRLHETSKGPDVPLGSTWLSDFPQAWAETGGMGLAFRQAPLIISLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P26810

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQSLFAFEWKDPEMGISGQLTWTRLPQGFKNSPT




LFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGDLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT




PRQLREFLGTAGLCRLWIPGFAEMAAPLYPLTKTGTLFKWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY




LSKKLDPVAAGWPPCLRMVAAIAVLTKDVGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPIVALNPATLLPLPEEGLQ




HDCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSFLQEGQRRAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAAGKKLNVYTDSRYAFAT




AHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNHAEARGNRMADQAAREVATRETPETSTLL


 


MLVF5_
8,066
TLNIEDEYRLHETSKGPDVPLGSTWLSDFPQAWAETGGMGLAFRQAPLIISLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P26810_

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQSLFAFEWKDPEMGISGQLTWTRLPQGFKNSPT


3mut

LFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGDLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT




PRQLREFLGTAGLCRLWIPGFAEMAAPLYPLTKPGTLFKWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY




LSKKLDPVAAGWPPCLRMVAAIAVLTKDVGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPIVALNPATLLPLPEEGLQ




HDCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSFLQEGQRRAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAAGKKLNVYTDSRYAFAT




AHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNHAEARGNRMADQAAREVATRETPETSTLL


 


MLVF5_
8,067
TLNIEDEYRLHETSKGPDVPLGSTWLSDFPQAWAETGGMGLAFRQAPLIISLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P26810_

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQSLFAFEWKDPEMGISGQLTWTRLPQGFKNSPT


3mutA

LFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGDLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT




PRQLREFLGKAGLCRLFIPGFAEMAAPLYPLTKPGTLFKWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY




LSKKLDPVAAGWPPCLRMVAAIAVLTKDVGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPIVALNPATLLPLPEEGLQ




HDCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSFLQEGQRRAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAAGKKLNVYTDSRYAFAT




AHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNHAEARGNRMADQAAREVATRETPETSTLL


 


MLVFF_
8,068
TLNIEDEYRLHETSKGPDVPLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P26809_

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQSLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT


3mut

LFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGDLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT




PRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFEWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA




YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPIVALNPATLLPLPEEGLQ




HDCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVVWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFA




TAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNRAEARGNRMADQAAREVATRETPETSTLL


 


MLVFF_
8,069
TLNIEDEYRLHETSKGPDVPLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P26809_

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQSLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT


3mutA

LFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGDLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT




PRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFEWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY




LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPIVALNPATLLPLPEEGLQ




HDCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVVWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFA




TAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNRAEARGNRMADQAAREVATRETPETSTLL


 


MLVMS_
8,070
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P03355

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT




LFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT




PRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA




YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGL




QHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFA




TAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL


 


MLVMS_
8,137
TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


reference

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT




LFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT




PRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY




LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQ




HNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFAT




AHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP


 


MLVMS_
8,071
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P03355

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT




LFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT




PRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA




YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPWVALNPATLLPLPEEGL




QHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFA




TAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL


 


MLVMS_
8,072
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P03355_

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT


3mut

LFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT




PRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA




YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPWVALNPATLLPLPEEGL




QHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFA




TAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL


 


MLVMS_
8,073
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P03355_

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT


3mut

LFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT




PRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA




YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPWVALNPATLLPLPEEGL




QHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFA




TAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL


 


MLVMS_
8,074
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P03355_

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT


3mutA_

LFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT


WS

PRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY




LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQ




HNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFAT




AHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL


 


MLVMS_
8,075
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P03355_

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT


3mutA_

LFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT


WS

PRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY




LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQ




HNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFAT




AHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL


 


MLVMS_
8,076
TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P03355_

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT


PLV919

LFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT




PRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY




LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPWVALNPATLLPLPEEGLQ




HNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFAT




AHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEF




E


 


MLVMS_
8,077
TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P03355_

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT


PLV919

LFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT




PRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY




LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQ




HNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFAT




AHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEF




E


 


MLVRD_
8,078
TLNIEDEYRLHEISTEPDVSPGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEAKLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P11227

VKKPGTNDYRPVQGLREVNKRVEDIHPTVPNPYNLLSGLPTSHRWYTVLDLKDAFFCLRLHPTSQPLFASEWRDPGMGISGQLTWTRLPQGFKNSPT




LFDEALHRGLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLKTLGNLGYRASAKKAQICQKQVKYLGYLLREGQRWLTEARKETVMGQPTPKT




PRQLREFLGTAGFCRLWIPRFAEMAAPLYPLTKTGTLFNWGPDQQKAYHEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY




LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQAMLLDTDRVQFGPWVALNPATLLPLPEEGAP




HDCLEILAETHGTEPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFATA




HIHGEIYKRRGLLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNRLADQAAREAAIKTPPDTSTLL


 


MLVRD_
8,079
TLNIEDEYRLHEISTEPDVSPGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEAKLGIKPHIQRLLDQGILVPCQSPWNTPLLP


P11227_

VKKPGTNDYRPVQGLREVNKRVEDIHPTVPNPYNLLSGLPTSHRWYTVLDLKDAFFCLRLHPTSQPLFASEWRDPGMGISGQLTWTRLPQGFKNSPT


3mut

LFNEALHRGLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLKTLGNLGYRASAKKAQICQKQVKYLGYLLREGQRWLTEARKETVMGQPTPKT




PRQLREFLGTAGFCRLWIPRFAEMAAPLYPLTKPGTLFNWGPDQQKAYHEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY




LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEEGAP




HDCLEILAETHGTEPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFATA




HIHGEIYKRRGWLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNRLADQAAREAAIKTPPDTSTLL


 


MMTVB_
8,080
WVQEISDSRPMLHIYLNGRRFLGLLNTGADKTCIAGRDWPANWPIHQTESSLQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMK


P03365

DIKVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAV




NATMHDMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDS




YIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLF




EILNGDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSK




DPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQA




EIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT


 


MMTVB_
8,081
WVQEISDSRPMLHIYLNGRRFLGLLNTGADKTCIAGRDWPANWPIHQTESSLQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMK


P03365

DIKVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAV




NATMHDMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDS




YIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLF




EILNGDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSK




DPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQA




EIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT


 


MMTVB_
8,082
WVQEISDSRPMLHIYLNGRRFLGLLNTGADKTCIAGRDWPANWPIHQTESSLQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMK


P03365_

DIKVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAV


2mut

NATMHDMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDS




YIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLF




EILNPDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSK




DPDYIWVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQA




EIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT


 


MMTVB_
8,083
VQEISDSRPMLHIYLNGRRFLGLLDTGADKTCIAGRDWPANWPIHQTESSLQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMKDI


P03365_

KVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAVNA


2mut_

TMHDMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDSYIV


WS

HYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLFEIL




NPDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTPTACLWQDGWVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSKDP




DYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQAEIV




AVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILTA


 


MMTVB_
8,084
VQEISDSRPMLHIYLNGRRFLGLLDTGADKTCIAGRDWPANWPIHQTESSLQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMKDI


P03365_

KVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAVNA


2mut_

TMHDMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDSYIV


WS

HYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLFEIL




NPDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSKDP




DYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQAEIV




AVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILTA


 


MMTVB_
8,085
WVQEISDSRPMLHIYLNGRRFLGLLNTGADKTCIAGRDWPANWPIHQTESSLQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMK


P03365_

DIKVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAV


2mutB

NATMHDMGALQPGLPSPVAPPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDS




YIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLF




EILNPDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSK




DPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQA




EIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT


 


MMTVB_
8,086
WVQEISDSRPMLHIYLNGRRFLGLLNTGADKTCIAGRDWPANWPIHQTESSLQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMK


P03365_

DIKVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAV


2mutB

NATMHDMGALQPGLPSPVAPPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDS




YIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLF




EILNPDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSK




DPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQA




EIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT


 


MMTVB_
8,087
VQEISDSRPMLHIYLNGRRFLGLLDTGADKTCIAGRDWPANWPIHQTESSLQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMKDI


P03365_

KVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAVNA


2mutB_

TMHDMGALQPGLPSPPAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDSYIV


WS

HYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLFEIL




NPDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSKDP




DYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQAEIV




AVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILTA


 


MMTVB_
8,088
VQEISDSRPMLHIYLNGRRFLGLLDTGADKTCIAGRDWPANWPIHQTESSLQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMKDI


P03365_

KVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAVNA


2mutB_

TMHDMGALQPGLPSPPAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDSYIV


WS

HYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLFEIL




NPDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSKDP




DYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQAEIV




AVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILTA


 


MMTVB_
8,089
VQEISDSRPMLHIYLNGRRFLGLLDTGADKTCIAGRDWPANWPIHQTESSLQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMKDI


P03365_

KVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAVNA


WS

TMHDMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDSYIV




HYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLFEIL




NGDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSKDP




DYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQAEIV




AVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILTA


 


MMTVB_
8,090
VQEISDSRPMLHIYLNGRRFLGLLDTGADKTCIAGRDWPANWPIHQTESSLQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMKDI


P03365_

KVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAVNA


WS

TMHDMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDSYIV




HYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLFEIL




NGDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTPTACLWQDGWVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSKDP




DYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQAEIV




AVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILTA


 


MMTVB_
8,091
GRDIMKDIKVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLL


P03365-

QDLRAVNATMHDMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVR


Pro

DKYQDSYIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTT




GELKPLFEILNGDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHR




SKELFSKDPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQ




NTAQQAEIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT


 


MMTVB_
8,092
GRDIMKDIKVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLL


P03365-

QDLRAVNATMHDMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVR


Pro

DKYQDSYIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTT




GELKPLFEILNGDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHR




SKELFSKDPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQ




NTAQQAEIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT


 


MMTVB_
8,093
GRDIMKDIKVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLL


P03365-

QDLRAVNATMHDMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVR


Pro_2mut

DKYQDSYIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTT




GELKPLFEILNPDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHR




SKELFSKDPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQ




NTAQQAEIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT


 


MMTVB_
8,094
GRDIMKDIKVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLL


P03365-

QDLRAVNATMHDMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVR


Pro_2mut

DKYQDSYIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTT




GELKPLFEILNPDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHR




SKELFSKDPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQ




NTAQQAEIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT


 


MMTVB_
8,095
GRDIMKDIKVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLL


P03365-

QDLRAVNATMHDMGALQPGLPSPVAPPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVR


Pro_2mutB

DKYQDSYIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTT




GELKPLFEILNPDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHR




SKELFSKDPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQ




NTAQQAEIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT


 


MMTVB_
8,096
GRDIMKDIKVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLL


P03365-

QDLRAVNATMHDMGALQPGLPSPVAPPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVR


Pro_2mutB

DKYQDSYIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTT




GELKPLFEILNPDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHR




SKELFSKDPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQ




NTAQQAEIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT


 


MPMV_
8,097
LTAAIDILAPQQCAEPITWKSDEPVWVDQWPLTNDKLAAAQQLVQEQLEAGHITESSSPWNTPIFVIKKKSGKWRLLQDLRAVNATMVLMGALQPGLP


P07572

SPVAIPQGYLKIIIDLKDCFFSIPLHPSDQKRFAFSLPSTNFKEPMQRFQWKVLPQGMANSPTLCQKYVATAIHKVRHAWKQMYIIHYMDDILIAGKDGQ




QVLQCFDQLKQELTAAGLHIAPEKVQLQDPYTYLGFELNGPKITNQKAVIRKDKLQTLNDFQKLLGDINWLRPYLKLTTGDLKPLFDTLKGDSDPNSHR




SLSKEALASLEKVETAIAEQFVTHINYSLPLIFLIFNTALTPTGLFWQDNPIMWIHLPASPKKVLLPYYDAIADLIILGRDHSKKYFGIEPSTIIQPYSKSQIDW




LMQNTEMWPIACASFVGILDNHYPPNKLIQFCKLHTFVFPQIISKTPLNNALLVFTDGSSTGMAAYTLTDTTIKFQTNLNSAQLVELQALIAVLSAFPNQPL




NIYTDSAYLAHSIPLLETVAQIKHISETAKLFLQCQQLIYNRSIPFYIGHVRAHSGLPGPIAQGNQRADLATKIVASNINT


 


MPMV_
8,098
LTAAIDILAPQQCAEPITWKSDEPVWVDQWPLTNDKLAAAQQLVQEQLEAGHITESSSPWNTPIFVIKKKSGKWRLLQDLRAVNATMVLMGALQPGLP


P07572_

SPVAPPQGYLKIIIDLKDCFFSIPLHPSDQKRFAFSLPSTNFKEPMQRFQWKVLPQGMANSPTLCQKYVATAIHKVRHAWKQMYIIHYMDDILIAGKDGQ


2mutB

QVLQCFDQLKQELTAAGLHIAPEKVQLQDPYTYLGFELNGPKITNQKAVIRKDKLQTLNDFQKLLGDINWLRPYLKLTTGDLKPLFDTLKPDSDPNSHRS




LSKEALASLEKVETAIAEQFVTHINYSLPLIFLIFNTALTPTGLFWQDNPIMWIHLPASPKKVLLPYYDAIADLIILGRDHSKKYFGIEPSTIIQPYSKSQIDWL




MQNTEMWPIACASFVGILDNHYPPNKLIQFCKLHTFVFPQIISKTPLNNALLVFTDGSSTGMAAYTLTDTTIKFQTNLNSAQLVELQALIAVLSAFPNQPL




NIYTDSAYLAHSIPLLETVAQIKHISETAKLFLQCQQLIYNRSIPFYIGHVRAHSGLPGPIAQGNQRADLATKIVASNINT


 


PERV_
8,099
TLQLDDEYRLYSPLVKPDQNIQFWLEQFPQAWAETAGMGLAKQVPPQVIQLKASATPVSVRQYPLSKEAQEGIRPHVQRLIQQGILVPVQSPWNTPLL


Q4VFZ2

PVRKPGTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLCALPPQRSWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPGTGRTGQLTWTRLPQGFKNS




PTIFDEALHRDLANFRIQHPQVTLLQYVDDLLLAGATKQDCLEGTKALLLELSDLGYRASAKKAQICRREVTYLGYSLRDGQRWLTEARKKTVQIPAPT




TAKQVREFLGTAGFCRLWIPGFATLAAPLYPLTKEKGEFSWAPEHQKAFDAIKKALLSAPALALPDVTKPFTLYVDERKGVARGVLTQTLGPWRRPVA




YLSKKLDPVASGWPVCLKAIAAVAILVKDADKLTLGQNITVIAPHALENIVRQPPDRWMTNARMTHYQSLLLTERVTFAPPAALNPATLLPEETDEPVTH




DCHQLLIEETGVRKDLTDIPLTGEVLTWFTDGSSYVVEGKRMAGAAVVDGTRTIWASSLPEGTSAQKAELMALTQALRLAEGKSINIYTDSRYAFATAH




VHGAIYKQRGLLTSAGREIKNKEEILSLLEALHLPKRLAIIHCPGHQKAKDPISRGNQMADRVAKQAAQGVNLL


 


PERV_
8,100
TLQLDDEYRLYSPLVKPDQNIQFWLEQFPQAWAETAGMGLAKQVPPQVIQLKASATPVSVRQYPLSKEAQEGIRPHVQRLIQQGILVPVQSPWNTPLL


Q4VFZ2

PVRKPGTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLCALPPQRSWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPGTGRTGQLTWTRLPQGFKNS




PTIFDEALHRDLANFRIQHPQVTLLQYVDDLLLAGATKQDCLEGTKALLLELSDLGYRASAKKAQICRREVTYLGYSLRDGQRWLTEARKKTVQIPAPT




TAKQVREFLGTAGFCRLWIPGFATLAAPLYPLTKEKGEFSWAPEHQKAFDAIKKALLSAPALALPDVTKPFTLYVDERKGVARGVLTQTLGPWRRPVA




YLSKKLDPVASGWPVCLKAIAAVAILVKDADKLTLGQNITVIAPHALENIVRQPPDRWMTNARMTHYQSLLLTERVTFAPPAALNPATLLPEETDEPVTH




DCHQLLIEETGVRKDLTDIPLTGEVLTWFTDGSSYVVEGKRMAGAAVVDGTRTIWASSLPEGTSAQKAELMALTQALRLAEGKSINIYTDSRYAFATAH




VHGAIYKQRGLLTSAGREIKNKEEILSLLEALHLPKRLAIIHCPGHQKAKDPISRGNQMADRVAKQAAQGVNLL


 


PERV_
8,101
TLQLDDEYRLYSPLVKPDQNIQFWLEQFPQAWAETAGMGLAKQVPPQVIQLKASATPVSVRQYPLSKEAQEGIRPHVQRLIQQGILVPVQSPWNTPLL


Q4VFZ2_

PVRKPGTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLCALPPQRSWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPGTGRTGQLTWTRLPQGFKNS


3mut

PTIFNEALHRDLANFRIQHPQVTLLQYVDDLLLAGATKQDCLEGTKALLLELSDLGYRASAKKAQICRREVTYLGYSLRDGQRWLTEARKKTVQIPAPT




TAKQVREFLGTAGFCRLWIPGFATLAAPLYPLTKPKGEFSWAPEHQKAFDAIKKALLSAPALALPDVTKPFTLYVDERKGVARGVLTQTLGPWRRPVA




YLSKKLDPVASGWPVCLKAIAAVAILVKDADKLTLGQNITVIAPHALENIVRQPPDRWMTNARMTHYQSLLLTERVTFAPPAALNPATLLPEETDEPVTH




DCHQLLIEETGVRKDLTDIPLTGEVLTWFTDGSSYVVEGKRMAGAAVVDGTRTIWASSLPEGTSAQKAELMALTQALRLAEGKSINIYTDSRYAFATAH




VHGAIYKQRGWLTSAGREIKNKEEILSLLEALHLPKRLAIIHCPGHQKAKDPISRGNQMADRVAKQAAQGVNLL


 


PERV_
8,102
TLQLDDEYRLYSPLVKPDQNIQFWLEQFPQAWAETAGMGLAKQVPPQVIQLKASATPVSVRQYPLSKEAQEGIRPHVQRLIQQGILVPVQSPWNTPLL


Q4VFZ2_

PVRKPGTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLCALPPQRSWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPGTGRTGQLTWTRLPQGFKNS


3mut

PTIFNEALHRDLANFRIQHPQVTLLQYVDDLLLAGATKQDCLEGTKALLLELSDLGYRASAKKAQICRREVTYLGYSLRDGQRWLTEARKKTVVQIPAPT




TAKQVREFLGTAGFCRLWIPGFATLAAPLYPLTKPKGEFSWAPEHQKAFDAIKKALLSAPALALPDVTKPFTLYVDERKGVARGVLTQTLGPWRRPVA




YLSKKLDPVASGWPVCLKAIAAVAILVKDADKLTLGQNITVIAPHALENIVRQPPDRWMTNARMTHYQSLLLTERVTFAPPAALNPATLLPEETDEPVTH




DCHQLLIEETGVRKDLTDIPLTGEVLTWFTDGSSYVVEGKRMAGAAVVDGTRTIWASSLPEGTSAQKAELMALTQALRLAEGKSINIYTDSRYAFATAH




VHGAIYKQRGWLTSAGREIKNKEEILSLLEALHLPKRLAIIHCPGHQKAKDPISRGNQMADRVAKQAAQGVNLL


 


PERV_
8,103
LDDEYRLYSPLVKPDQNIQFWLEQFPQAWAETAGMGLAKQVPPQVIQLKASATPVSVRQYPLSKEAQEGIRPHVQRLIQQGILVPVQSPWNTPLLPVR


Q4VFZ2_

KPGTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLCALPPQRSWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPGTGRTGQLTWTRLPQGFKNSPTIF


3mutA_

NEALHRDLANFRIQHPQVTLLQYVDDLLLAGATKQDCLEGTKALLLELSDLGYRASAKKAQICRREVTYLGYSLRDGQRWLTEARKKTVVQIPAPTTAK


WS

QVREFLGKAGFCRLFIPGFATLAAPLYPLTKPKGEFSWAPEHQKAFDAIKKALLSAPALALPDVTKPFTLYVDERKGVARGVLTQTLGPWRRPVAYLSK




KLDPVASGWPVCLKAIAAVAILVKDADKLTLGQNITVIAPHALENIVRQPPDRWMTNARMTHYQSLLLTERVTFAPPAALNPATLLPEETDEPVTHDCHQ




LLIEETGVRKDLTDIPLTGEVLTWFTDGSSYVVEGKRMAGAAVVDGTRTIWASSLPEGTSAQKAELMALTQALRLAEGKSINIYTDSRYAFATAHVHGAI




YKQRGWLTSAGREIKNKEEILSLLEALHLPKRLAIIHCPGHQKAKDPISRGNQMADRVAKQAAQGVNLLP


 


PERV_
8,104
LDDEYRLYSPLVKPDQNIQFWLEQFPQAWAETAGMGLAKQVPPQVIQLKASATPVSVRQYPLSKEAQEGIRPHVQRLIQQGILVPVQSPWNTPLLPVR


Q4VFZ2_

KPGTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLCALPPQRSWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPGTGRTGQLTWTRLPQGFKNSPTIF


3mutA_

NEALHRDLANFRIQHPQVTLLQYVDDLLLAGATKQDCLEGTKALLLELSDLGYRASAKKAQICRREVTYLGYSLRDGQRWLTEARKKTVVQIPAPTTAK


WS

QVREFLGKAGFCRLFIPGFATLAAPLYPLTKPKGEFSWAPEHQKAFDAIKKALLSAPALALPDVTKPFTLYVDERKGVARGVLTQTLGPWRRPVAYLSK




KLDPVASGWPVCLKAIAAVAILVKDADKLTLGQNITVIAPHALENIVRQPPDRWMTNARMTHYQSLLLTERVTFAPPAALNPATLLPEETDEPVTHDCHQ




LLIEETGVRKDLTDIPLTGEVLTWFTDGSSYVVEGKRMAGAAVVDGTRTIWASSLPEGTSAQKAELMALTQALRLAEGKSINIYTDSRYAFATAHVHGAI




YKQRGWLTSAGREIKNKEEILSLLEALHLPKRLAIIHCPGHQKAKDPISRGNQMADRVAKQAAQGVNLLP


 


SFV1_
8,105
MDPLQLLQPLEAEIKGTKLKAHWNSGATITCVPEAFLEDERPIQTMLIKTIHGEKQQDVYYLTFKVQGRKVEAEVLASPYDYILLNPSDVPWLMKKPLQL


P23074

TVLVPLHEYQERLLQQTALPKEQKELLQKLFLKYDALWQHWENQVGHRRIKPHNIATGTLAPRPQKQYPINPKAKPSIQIVIDDLLKQGVLIQQNSTMNT




PVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIYRGKYKTTLDLTNGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFLNSPALFTAD




WVDLLKEIPNVQAYVDDIYISHDDPQEHLEQLEKIFSILLNAGYVVSLKKSEIAQREVEFLGFNITKEGRGLTDTFKQKLLNITPPKDLKQLQSILGLLNFAR




NFIPNYSELVKPLYTIVANANGKFISWTEDNSNQLQHIISVLNQADNLEERNPETRLIIKVNSSPSAGYIRYYNEGSKRPIMYVNYIFSKAEAKFTQTEKLL




TTMHKGLIKAMDLAMGQEILVYSPIVSMTKIQRTPLPERKALPVRWITWMTYLEDPRIQFHYDKSLPELQQIPNVTEDVIAKTKHPSEFAMVFYTDGSAIK




HPDVNKSHSAGMGIAQVQFIPEYKIVHQWSIPLGDHTAQLAEIAAVEFACKKALKISGPVLIVTDSFYVAESANKELPYWKSNGFLNNKKKPLRHVSKW




KSIAECLQLKPDIIIMHEKGHQQPMTTLHTEGNNLADKLATQGSYVVH


 


SFV1_
8,106
MDPLQLLQPLEAEIKGTKLKAHWNSGATITCVPEAFLEDERPIQTMLIKTIHGEKQQDVYYLTFKVQGRKVEAEVLASPYDYILLNPSDVPWLMKKPLQL


P23074_

TVLVPLHEYQERLLQQTALPKEQKELLQKLFLKYDALWQHWENQVGHRRIKPHNIATGTLAPRPQKQYPINPKAKPSIQIVIDDLLKQGVLIQQNSTMNT


2mut

PVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIYRGKYKTTLDLTNGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFLNSPALFNAD




WVDLLKEIPNVQAYVDDIYISHDDPQEHLEQLEKIFSILLNAGYVVSLKKSEIAQREVEFLGFNITKEGRGLTDTFKQKLLNITPPKDLKQLQSILGLLNFAR




NFIPNYSELVKPLYTIVAPANGKFISWTEDNSNQLQHIISVLNQADNLEERNPETRLIIKVNSSPSAGYIRYYNEGSKRPIMYVNYIFSKAEAKFTQTEKLLT




TMHKGLIKAMDLAMGQEILVYSPIVSMTKIQRTPLPERKALPVRWITWMTYLEDPRIQFHYDKSLPELQQIPNVTEDVIAKTKHPSEFAMVFYTDGSAIKH




PDVNKSHSAGMGIAQVQFIPEYKIVHQWSIPLGDHTAQLAEIAAVEFACKKALKISGPVLIVTDSFYVAESANKELPYWKSNGFLNNKKKPLRHVSKWK




SIAECLQLKPDIIIMHEKGHQQPMTTLHTEGNNLADKLATQGSYVVH


 


SFV1_
8,107
MDPLQLLQPLEAEIKGTKLKAHWNSGATITCVPEAFLEDERPIQTMLIKTIHGEKQQDVYYLTFKVQGRKVEAEVLASPYDYILLNPSDVPWLMKKPLQL


P23074_

TVLVPLHEYQERLLQQTALPKEQKELLQKLFLKYDALWQHWENQVGHRRIKPHNIATGTLAPRPQKQYPINPKAKPSIQIVIDDLLKQGVLIQQNSTMNT


2mutA

PVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIYRGKYKTTLDLTNGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFLNSPALFNAD




WDLLKEIPNVQAYVDDIYISHDDPQEHLEQLEKIFSILLNAGYVVSLKKSEIAQREVEFLGFNITKEGRGLTDTFKQKLLNITPPKDLKQLQSILGKLNFAR




NFIPNYSELVKPLYTIVAPANGKFISWTEDNSNQLQHIISVLNQADNLEERNPETRLIIKVNSSPSAGYIRYYNEGSKRPIMYVNYIFSKAEAKFTQTEKLLT




TMHKGLIKAMDLAMGQEILVYSPIVSMTKIQRTPLPERKALPVRWITWMTYLEDPRIQFHYDKSLPELQQIPNVTEDVIAKTKHPSEFAMVFYTDGSAIKH




PDVNKSHSAGMGIAQVQFIPEYKIVHQWSIPLGDHTAQLAEIAAVEFACKKALKISGPVLIVTDSFYVAESANKELPYWKSNGFLNNKKKPLRHVSKWK




SIAECLQLKPDIIIMHEKGHQQPMTTLHTEGNNLADKLATQGSYVVH


 


SFV1_
8,108
VPWLMKKPLQLTVLVPLHEYQERLLQQTALPKEQKELLQKLFLKYDALWQHWENQVGHRRIKPHNIATGTLAPRPQKQYPINPKAKPSIQIVIDDLLKQ


P23074-

GVLIQQNSTMNTPVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIYRGKYKTTLDLTNGFWAHPITPESYWLTAFTWQGKQYCWTRLPQ


Pro

GFLNSPALFTADVVDLLKEIPNVQAYVDDIYISHDDPQEHLEQLEKIFSILLNAGYVVSLKKSEIAQREVEFLGFNITKEGRGLTDTFKQKLLNITPPKDLKQ




LQSILGLLNFARNFIPNYSELVKPLYTIVANANGKFISWTEDNSNQLQHIISVLNQADNLEERNPETRLIIKVNSSPSAGYIRYYNEGSKRPIMYVNYIFSKA




EAKFTQTEKLLTTMHKGLIKAMDLAMGQEILVYSPIVSMTKIQRTPLPERKALPVRWITWMTYLEDPRIQFHYDKSLPELQQIPNVTEDVIAKTKHPSEFA




MVFYTDGSAIKHPDVNKSHSAGMGIAQVQFIPEYKIVHQWSIPLGDHTAQLAEIAAVEFACKKALKISGPVLIVTDSFYVAESANKELPYWKSNGFLNNK




KKPLRHVSKWKSIAECLQLKPDIIIMHEKGHQQPMTTLHTEGNNLADKLATQGSYVVH


 


SFV1_
8,109
VPWLMKKPLQLTVLVPLHEYQERLLQQTALPKEQKELLQKLFLKYDALWQHWENQVGHRRIKPHNIATGTLAPRPQKQYPINPKAKPSIQIVIDDLLKQ


P23074-

GVLIQQNSTMNTPVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIYRGKYKTTLDLTNGFWAHPITPESYWLTAFTWQGKQYCWTRLPQ


Pro_2mut

GFLNSPALFNADVVDLLKEIPNVQAYVDDIYISHDDPQEHLEQLEKIFSILLNAGYVVSLKKSEIAQREVEFLGFNITKEGRGLTDTFKQKLLNITPPKDLK




QLQSILGLLNFARNFIPNYSELVKPLYTIVAPANGKFISWTEDNSNQLQHIISVLNQADNLEERNPETRLIIKVNSSPSAGYIRYYNEGSKRPIMYVNYIFSK




AEAKFTQTEKLLTTMHKGLIKAMDLAMGQEILVYSPIVSMTKIQRTPLPERKALPVRWITWMTYLEDPRIQFHYDKSLPELQQIPNVTEDVIAKTKHPSEF




AMVFYTDGSAIKHPDVNKSHSAGMGIAQVQFIPEYKIVHQWSIPLGDHTAQLAEIAAVEFACKKALKISGPVLIVTDSFYVAESANKELPYWKSNGFLNN




KKKPLRHVSKWKSIAECLQLKPDIIIMHEKGHQQPMTTLHTEGNNLADKLATQGSYVVH


 


SFV1_
8,110
VPWLMKKPLQLTVLVPLHEYQERLLQQTALPKEQKELLQKLFLKYDALWQHWENQVGHRRIKPHNIATGTLAPRPQKQYPINPKAKPSIQIVIDDLLKQ


P23074-

GVLIQQNSTMNTPVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIYRGKYKTTLDLTNGFWAHPITPESYWLTAFTWQGKQYCWTRLPQ


Pro_2mutA

GFLNSPALFNADVVDLLKEIPNVQAYVDDIYISHDDPQEHLEQLEKIFSILLNAGYVVSLKKSEIAQREVEFLGFNITKEGRGLTDTFKQKLLNITPPKDLK




QLQSILGKLNFARNFIPNYSELVKPLYTIVAPANGKFISWTEDNSNQLQHIISVLNQADNLEERNPETRLIIKVNSSPSAGYIRYYNEGSKRPIMYVNYIFSK




AEAKFTQTEKLLTTMHKGLIKAMDLAMGQEILVYSPIVSMTKIQRTPLPERKALPVRWITWMTYLEDPRIQFHYDKSLPELQQIPNVTEDVIAKTKHPSEF




AMVFYTDGSAIKHPDVNKSHSAGMGIAQVQFIPEYKIVHQWSIPLGDHTAQLAEIAAVEFACKKALKISGPVLIVTDSFYVAESANKELPYWKSNGFLNN




KKKPLRHVSKWKSIAECLQLKPDIIIMHEKGHQQPMTTLHTEGNNLADKLATQGSYVVH


 


SFV3L_
8,111
MDPLQLLQPLEAEIKGTKLKAHWNSGATITCVPQAFLEEEVPIKNIWIKTIHGEKEQPVYYLTFKIQGRKVEAEVISSPYDYILVSPSDIPWLMKKPLQLTT


P27401

LVPLQEYEERLLKQTMLTGSYKEKLQSLFLKYDALWQHWENQVGHRRIKPHHIATGTVNPRPQKQYPINPKAKASIQTVINDLLKQGVLIQQNSIMNTP




VYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIFRGKYKTTLDLSNGFWAHSITPESYWLTAFTWLGQQYCWTRLPQGFLNSPALFTADV




VDLLKEVPNVQVYVDDIYISHDDPREHLEQLEKVFSLLLNAGYVVSLKKSEIAQHEVEFLGFNITKEGRGLTETFKQKLLNITPPRDLKQLQSILGLLNFAR




NFIPNFSELVKPLYNIIATANGKYITWTTDNSQQLQNIISMLNSAENLEERNPEVRLIMKVNTSPSAGYIRFYNEFAKRPIMYLNYVYTKAEVKFTNTEKLL




TTIHKGLIKALDLGMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMSYLEDPRIQFHYDKTLPELQQVPTVTDDIIAKIKHPSEFSMVFYTDGSAIKHP




NVNKSHNAGMGIAQVQFKPEFTVINTWSIPLGDHTAQLAEVAAVEFACKKALKIDGPVLIVTDSFYVAESVNKELPYWQSNGFFNNKKKPLKHVSKWK




SIADCIQLKPDIIIIHEKGHQPTASTFHTEGNNLADKLATQGSYVVN


 


SFV3L_
8,112
MDPLQLLQPLEAEIKGTKLKAHWNSGATITCVPQAFLEEEVPIKNIWIKTIHGEKEQPVYYLTFKIQGRKVEAEVISSPYDYILVSPSDIPWLMKKPLQLTT


P27401_

LVPLQEYEERLLKQTMLTGSYKEKLQSLFLKYDALWQHWENQVGHRRIKPHHIATGTVNPRPQKQYPINPKAKASIQTVINDLLKQGVLIQQNSIMNTP


2mut

VYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIFRGKYKTTLDLSNGFWAHSITPESYWLTAFTWLGQQYCWTRLPQGFLNSPALFNADV




VDLLKEVPNVQVYVDDIYISHDDPREHLEQLEKVFSLLLNAGYVVSLKKSEIAQHEVEFLGFNITKEGRGLTETFKQKLLNITPPRDLKQLQSILGLLNFAR




NFIPNFSELVKPLYNIIATAPGKYITWTTDNSQQLQNIISMLNSAENLEERNPEVRLIMKVNTSPSAGYIRFYNEFAKRPIMYLNYVYTKAEVKFTNTEKLL




TTIHKGLIKALDLGMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMSYLEDPRIQFHYDKTLPELQQVPTVTDDIIAKIKHPSEFSMVFYTDGSAIKHP




NVNKSHNAGMGIAQVQFKPEFTVINTWSIPLGDHTAQLAEVAAVEFACKKALKIDGPVLIVTDSFYVAESVNKELPYWQSNGFFNNKKKPLKHVSKWK




SIADCIQLKPDIIIIHEKGHQPTASTFHTEGNNLADKLATQGSYVVN


 


SFV3L_
8,113
MDPLQLLQPLEAEIKGTKLKAHWNSGATITCVPQAFLEEEVPIKNIWIKTIHGEKEQPVYYLTFKIQGRKVEAEVISSPYDYILVSPSDIPWLMKKPLQLTT


P27401_

LVPLQEYEERLLKQTMLTGSYKEKLQSLFLKYDALWQHWENQVGHRRIKPHHIATGTVNPRPQKQYPINPKAKASIQTVINDLLKQGVLIQQNSIMNTP


2mutA

VYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIFRGKYKTTLDLSNGFWAHSITPESYWLTAFTWLGQQYCWTRLPQGFLNSPALFNADV




VDLLKEVPNVQVYVDDIYISHDDPREHLEQLEKVFSLLLNAGYVVSLKKSEIAQHEVEFLGFNITKEGRGLTETFKQKLLNITPPRDLKQLQSILGKLNFA




RNFIPNFSELVKPLYNIIATAPGKYITWTTDNSQQLQNIISMLNSAENLEERNPEVRLIMKVNTSPSAGYIRFYNEFAKRPIMYLNYVYTKAEVKFTNTEKL




LTTIHKGLIKALDLGMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMSYLEDPRIQFHYDKTLPELQQVPTVTDDIIAKIKHPSEFSMVFYTDGSAIKH




PNVNKSHNAGMGIAQVQFKPEFTVINTWSIPLGDHTAQLAEVAAVEFACKKALKIDGPVLIVTDSFYVAESVNKELPYWQSNGFFNNKKKPLKHVSKW




KSIADCIQLKPDIIIIHEKGHQPTASTFHTEGNNLADKLATQGSYVVN


 


SFV3L_
8,114
IPWLMKKPLQLTTLVPLQEYEERLLKQTMLTGSYKEKLQSLFLKYDALWQHWENQVGHRRIKPHHIATGTVNPRPQKQYPINPKAKASIQTVINDLLKQ


P27401-

GVLIQQNSIMNTPVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIFRGKYKTTLDLSNGFWAHSITPESYWLTAFTWLGQQYCWTRLPQ


Pro

GFLNSPALFTADVVDLLKEVPNVQVYVDDIYISHDDPREHLEQLEKVFSLLLNAGYVVSLKKSEIAQHEVEFLGFNITKEGRGLTETFKQKLLNITPPRDL




KQLQSILGLLNFARNFIPNFSELVKPLYNIIATANGKYITWTTDNSQQLQNIISMLNSAENLEERNPEVRLIMKVNTSPSAGYIRFYNEFAKRPIMYLNYVY




TKAEVKFTNTEKLLTTIHKGLIKALDLGMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMSYLEDPRIQFHYDKTLPELQQVPTVTDDIIAKIKHPSEF




SMVFYTDGSAIKHPNVNKSHNAGMGIAQVQFKPEFTVINTWSIPLGDHTAQLAEVAAVEFACKKALKIDGPVLIVTDSFYVAESVNKELPYWQSNGFFN




NKKKPLKHVSKWKSIADCIQLKPDIIIIHEKGHQPTASTFHTEGNNLADKLATQGSYVVN


 


SFV3L_
8,115
IPWLMKKPLQLTTLVPLQEYEERLLKQTMLTGSYKEKLQSLFLKYDALWQHWENQVGHRRIKPHHIATGTVNPRPQKQYPINPKAKASIQTVINDLLKQ


P27401-

GVLIQQNSIMNTPVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIFRGKYKTTLDLSNGFWAHSITPESYWLTAFTWLGQQYCWTRLPQ


Pro_2mut

GFLNSPALFNADVVDLLKEVPNVQVYVDDIYISHDDPREHLEQLEKVFSLLLNAGYVVSLKKSEIAQHEVEFLGFNITKEGRGLTETFKQKLLNITPPRDL




KQLQSILGLLNFARNFIPNFSELVKPLYNIIATAPGKYITWTTDNSQQLQNIISMLNSAENLEERNPEVRLIMKVNTSPSAGYIRFYNEFAKRPIMYLNYVY




TKAEVKFTNTEKLLTTIHKGLIKALDLGMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMSYLEDPRIQFHYDKTLPELQQVPTVTDDIIAKIKHPSEF




SMVFYTDGSAIKHPNVNKSHNAGMGIAQVQFKPEFTVINTWSIPLGDHTAQLAEVAAVEFACKKALKIDGPVLIVTDSFYVAESVNKELPYWQSNGFFN




NKKKPLKHVSKWKSIADCIQLKPDIIIIHEKGHQPTASTFHTEGNNLADKLATQGSYVVN


 


SFV3L_
8,116
IPWLMKKPLQLTTLVPLQEYEERLLKQTMLTGSYKEKLQSLFLKYDALWQHWENQVGHRRIKPHHIATGTVNPRPQKQYPINPKAKASIQTVINDLLKQ


P27401-

GVLIQQNSIMNTPVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIFRGKYKTTLDLSNGFWAHSITPESYWLTAFTWLGQQYCWTRLPQ


Pro_2mutA

GFLNSPALFNADVVDLLKEVPNVQVYVDDIYISHDDPREHLEQLEKVFSLLLNAGYVVSLKKSEIAQHEVEFLGFNITKEGRGLTETFKQKLLNITPPRDL




KQLQSILGKLNFARNFIPNFSELVKPLYNIIATAPGKYITWTTDNSQQLQNIISMLNSAENLEERNPEVRLIMKVNTSPSAGYIRFYNEFAKRPIMYLNYVY




TKAEVKFTNTEKLLTTIHKGLIKALDLGMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMSYLEDPRIQFHYDKTLPELQQVPTVTDDIIAKIKHPSEF




SMVFYTDGSAIKHPNVNKSHNAGMGIAQVQFKPEFTVINTWSIPLGDHTAQLAEVAAVEFACKKALKIDGPVLIVTDSFYVAESVNKELPYWQSNGFFN




NKKKPLKHVSKWKSIADCIQLKPDIIIIHEKGHQPTASTFHTEGNNLADKLATQGSYVVN


 


SFVCP_
8,117
MNPLQLLQPLPAEVKGTKLLAHWNSGATITCIPESFLEDEQPIKQTLIKTIHGEKQQNVYYLTFKVKGRKVEAEVIASPYEYILLSPTDVPWLTQQPLQLTI


Q87040

LVPLQEYQDRILNKTALPEEQKQQLKALFTKYDNLWQHWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQGVLTPQNSTMNTP




VYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKYKTTLDLANGFWAHPITPDSYWLTAFTWQGKQYCWTRLPQGFLNSPALFTAD




AVDLLKEVPNVQVYVDDIYLSHDNPHEHIQQLEKVFQILLQAGYVVSLKKSEIGQRTVEFLGFNITKEGRGLTDTFKTKLLNVTPPKDLKQLQSILGLLNF




ARNFIPNFAELVQTLYNLIASSKGKYIEWTEDNTKQLNKVIEALNTASNLEERLPDQRLVIKVNTSPSAGYVRYYNESGKKPIMYLNYVFSKAELKFSMLE




KLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSIPPLKHPSQYEGVFCTDGSA




IKSPDPTKSNNAGMGIVHAIYNPEYKILNQWSIPLGHHTAQMAEIAAVEFACKKALKVPGPVLVITDSFYVAESANKELPYWKSNGFVNNKKEPLKHISK




WKSIAECLSIKPDITIQHEKGHQPINTSIHTEGNALADKLATQGSYVVN


 


SFVCP_
8,118
MNPLQLLQPLPAEVKGTKLLAHWNSGATITCIPESFLEDEQPIKQTLIKTIHGEKQQNVYYLTFKVKGRKVEAEVIASPYEYILLSPTDVPWLTQQPLQLTI


Q87040_

LVPLQEYQDRINKTALPEEQKQQLKALFTKYDNLWQHWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQGVLTPQNSTMNTP


2mut

VYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKYKTTLDLANGFWAHPITPDSYWLTAFTWQGKQYCWTRLPQGFLNSPALFNAD




AVDLLKEVPNVQVYVDDIYLSHDNPHEHIQQLEKVFQILLQAGYVVSLKKSEIGQRTVEFLGFNITKEGRGLTDTFKTKLLNVTPPKDLKQLQSILGLLNF




ARNFIPNFAELVQTLYNLIASSPGKYIEWTEDNTKQLNKVIEALNTASNLEERLPDQRLVIKVNTSPSAGYVRYYNESGKKPIMYLNYVFSKAELKFSMLE




KLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSIPPLKHPSQYEGVFCTDGSA




IKSPDPTKSNNAGMGIVHAIYNPEYKILNQWSIPLGHHTAQMAEIAAVEFACKKALKVPGPVLVITDSFYVAESANKELPYWKSNGFVNNKKEPLKHISK




WKSIAECLSIKPDITIQHEKGHQPINTSIHTEGNALADKLATQGSYVVN


 


SFVCP_
8,119
MNPLQLLQPLPAEVKGTKLLAHWNSGATITCIPESFLEDEQPIKQTLIKTIHGEKQQNVYYLTFKVKGRKVEAEVIASPYEYILLSPTDVPWLTQQPLQLTI


Q87040_

LVPLQEYQDRILNKTALPEEQKQQLKALFTKYDNLWQHWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQGVLTPQNSTMNTP


2mutA

VYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKYKTTLDLANGFWAHPITPDSYWLTAFTWQGKQYCWTRLPQGFLNSPALFNAD




AVDLLKEVPNVQVYVDDIYLSHDNPHEHIQQLEKVFQILLQAGYVVSLKKSEIGQRTVEFLGFNITKEGRGLTDTFKTKLLNVTPPKDLKQLQSILGKLNF




ARNFIPNFAELVQTLYNLIASSPGKYIEWTEDNTKQLNKVIEALNTASNLEERLPDQRLVIKVNTSPSAGYVRYYNESGKKPIMYLNYVFSKAELKFSMLE




KLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSIPPLKHPSQYEGVFCTDGSA




IKSPDPTKSNNAGMGIVHAIYNPEYKILNQWSIPLGHHTAQMAEIAAVEFACKKALKVPGPVLVITDSFYVAESANKELPYWKSNGFVNNKKEPLKHISK




WKSIAECLSIKPDITIQHEKGHQPINTSIHTEGNALADKLATQGSYVVN


 


SFVCP_
8,120
VPWLTQQPLQLTILVPLQEYQDRILNKTALPEEQKQQLKALFTKYDNLWQHWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQG


Q87040-

VLTPQNSTMNTPVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKYKTTLDLANGFWAHPITPDSYWLTAFTWQGKQYCWTRLPQ


Pro

GFLNSPALFTADAVDLLKEVPNVQVYVDDIYLSHDNPHEHIQQLEKVFQILLQAGYVVSLKKSEIGQRTVEFLGFNITKEGRGLTDTFKTKLLNVTPPKDL




KQLQSILGLLNFARNFIPNFAELVQTLYNLIASSKGKYIEWTEDNTKQLNKVIEALNTASNLEERLPDQRLVIKVNTSPSAGYVRYYNESGKKPIMYLNYV




FSKAELKFSMLEKLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSIPPLKHPS




QYEGVFCTDGSAIKSPDPTKSNNAGMGIVHAIYNPEYKILNQWSIPLGHHTAQMAEIAAVEFACKKALKVPGPVLVITDSFYVAESANKELPYWKSNGF




VNNKKEPLKHISKWKSIAECLSIKPDITIQHEKGHQPINTSIHTEGNALADKLATQGSYVVN


 


SFVCP_
8,121
VPWLTQQPLQLTILVPLQEYQDRILNKTALPEEQKQQLKALFTKYDNLWQHWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQG


Q87040-

VLTPQNSTMNTPVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKYKTTLDLANGFWAHPITPDSYWLTAFTWQGKQYCWTRLPQ


Pro_2mut

GFLNSPALFNADAVDLLKEVPNVQVYVDDIYLSHDNPHEHIQQLEKVFQILLQAGYVVSLKKSEIGQRTVEFLGFNITKEGRGLTDTFKTKLLNVTPPKDL




KQLQSILGLLNFARNFIPNFAELVQTLYNLIASSPGKYIEWTEDNTKQLNKVIEALNTASNLEERLPDQRLVIKVNTSPSAGYVRYYNESGKKPIMYLNYV




FSKAELKFSMLEKLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSIPPLKHPS




QYEGVFCTDGSAIKSPDPTKSNNAGMGIVHAIYNPEYKILNQWSIPLGHHTAQMAEIAAVEFACKKALKVPGPVLVITDSFYVAESANKELPYWKSNGF




VNNKKEPLKHISKWKSIAECLSIKPDITIQHEKGHQPINTSIHTEGNALADKLATQGSYVVN


 


SFVCP_
8,122
VPWLTQQPLQLTILVPLQEYQDRILNKTALPEEQKQQLKALFTKYDNLWQHWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQG


Q87040-

VLTPQNSTMNTPVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKYKTTLDLANGFWAHPITPDSYWLTAFTWQGKQYCWTRLPQ


Pro_2mutA

GFLNSPALFNADAVDLLKEVPNVQVYVDDIYLSHDNPHEHIQQLEKVFQILLQAGYVVSLKKSEIGQRTVEFLGFNITKEGRGLTDTFKTKLLNVTPPKDL




KQLQSILGKLNFARNFIPNFAELVQTLYNLIASSPGKYIEWTEDNTKQLNKVIEALNTASNLEERLPDQRLVIKVNTSPSAGYVRYYNESGKKPIMYLNYV




FSKAELKFSMLEKLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPIRWITWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSIPPLKHPS




QYEGVFCTDGSAIKSPDPTKSNNAGMGIVHAIYNPEYKILNQWSIPLGHHTAQMAEIAAVEFACKKALKVPGPVLVITDSFYVAESANKELPYWKSNGF




VNNKKEPLKHISKWKSIAECLSIKPDITIQHEKGHQPINTSIHTEGNALADKLATQGSYVVN


 


SMRVH_
8,123
PRSRAIDIPVPHADKISWKITDPVWVDQWPLTYEKTLAAIALVQEQLAAGHIEPTNSPWNTPIFIIKKKSGSWRLLQDLRAVNKVMVPMGALQPGLPSPV


P03364

AIPLNYHKIVIDLKDCFFTIPLHPEDRPYFAFSVPQINFQSPMPRYQWKVLPQGMANSPTLCQKFVAAAIAPVRSQWPEAYILHYMDDILLACDSAEAAK




ACYAHIISCLTSYGLKIAPDKVQVSEPFSYLGFELHHQQVFTPRVCLKTDHLKTLNDFQKLLGDIQWLRPYLKLPTSALVPLNNILKGDPNPLSVRALTPE




AKQSLALINKAIQNQSVQQISYNLPLVLLLLPTPHTPTAVFWQPNGTDPTKNGSPLLWLHLPASPSKVLLTYPSLLAMLIIKGRYTGRQLFGRDPHSIIIPY




TQDQLTWLLQTSDEWAIALSSFTGDIDNHYPSDPVIQFAKLHQFIFPKITKCAPIPQATLVFTDGSSNGIAAYVIDNQPISIKSPYLSAQLVELYAILQVFTV




LAHQPFNLYTDSAYIAQSVPLLETVPFIKSSTNATPLFSKLQQLILNRQHPFFIGHLRAHLNLPGPLAEGNALADAATQIFPIISD


 


SMRVH_
8,124
PRSRAIDIPVPHADKISWKITDPVWVDQWPLTYEKTLAAIALVQEQLAAGHIEPTNSPWNTPIFIIKKKSGSWRLLQDLRAVNKVMVPMGALQPGLPSPV


P03364_

AIPLNYHKIVIDLKDCFFTIPLHPEDRPYFAFSVPQINFQSPMPRYQWKVLPQGMANSPTLCQKFVAAAIAPVRSQWPEAYILHYMDDILLACDSAEAAK


2mut

ACYAHIISCLTSYGLKIAPDKVQVSEPFSYLGFELHHQQVFTPRVCLKTDHLKTLNDFQKLLGDIQWLRPYLKLPTSALVPLNNILKPDPNPLSVRALTPE




AKQSLALINKAIQNQSVQQISYNLPLVLLLLPTPHTPTAVFWQPNGTDPTKNGSPLLWLHLPASPSKVLLTYPSLLAMLIIKGRYTGRQLFGRDPHSIIIPY




TQDQLTWLLQTSDEWAIALSSFTGDIDNHYPSDPVIQFAKLHQFIFPKITKCAPIPQATLVFTDGSSNGIAAYVIDNQPISIKSPYLSAQLVELYAILQVFTV




LAHQPFNLYTDSAYIAQSVPLLETVPFIKSSTNATPLFSKLQQLILNRQHPFFIGHLRAHLNLPGPLAEGNALADAATQIFPIISD


 


SMRVH_
8,125
PRSRAIDIPVPHADKISWKITDPVWVDQWPLTYEKTLAAIALVQEQLAAGHIEPTNSPWNTPIFIIKKKSGSWRLLQDLRAVNKVMVPMGALQPGLPSPV


P03364_

APPLNYHKIVIDLKDCFFTIPLHPEDRPYFAFSVPQINFQSPMPRYQWKVLPQGMANSPTLCQKFVAAAIAPVRSQWPEAYILHYMDDILLACDSAEAAK


2mutB

ACYAHIISCLTSYGLKIAPDKVQVSEPFSYLGFELHHQQVFTPRVCLKTDHLKTLNDFQKLLGDIQWLRPYLKLPTSALVPLNNILKPDPNPLSVRALTPE




AKQSLALINKAIQNQSVQQISYNLPLVLLLLPTPHTPTAVFWQPNGTDPTKNGSPLLWLHLPASPSKVLLTYPSLLAMLIIKGRYTGRQLFGRDPHSIIIPY




TQDQLTWLLQTSDEWAIALSSFTGDIDNHYPSDPVIQFAKLHQFIFPKITKCAPIPQATLVFTDGSSNGIAAYVIDNQPISIKSPYLSAQLVELYAILQVFTV




LAHQPFNLYTDSAYIAQSVPLLETVPFIKSSTNATPLFSKLQQLILNRQHPFFIGHLRAHLNLPGPLAEGNALADAATQIFPIISD


 


SRV2_
8,126
LATAVDILAPQRYADPITWKSDEPVWVDQWPLTQEKLAAAQQLVQEQLQAGHIIESNSPWNTPIFVIKKKSGKWRLLQDLRAVNATMVLMGALQPGLP


P51517

SPVAIPQGYFKIVIDLKDCFFTIPLQPVDQKRFAFSLPSTNFKQPMKRYQWKVLPQGMANSPTLCQKYVAAAIEPVRKSWAQMYIIHYMDDILIAGKLGE




QVLQCFAQLKQALTTTGLQIAPEKVQLQDPYTYLGFQINGPKITNQKAVIRRDKLQTLNDFQKLLGDINWLRPYLHLTTGDLKPLFDILKGDSNPNSPRS




LSEAALASLQKVETAIAEQFVTQIDYTQPLTFLIFNTTLTPTGLFWQNNPVMWVHLPASPKKVLLPYYDAIADLIILGRDNSKKYFGLEPSTIIQPYSKSQIH




WLMQNTETWPIACASYAGNIDNHYPPNKLIQFCKLHAVVFPRIISKTPLDNALLVFTDGSSTGIAAYTFEKTTVRFKTSHTSAQLVELQALIAVLSAFPHR




ALNVYTDSAYLAHSIPLLETVSHIKHISDTAKFFLQCQQLIYNRSIPFYLGHIRAHSGLPGPLSQGNHITDLATKVVATTLTT


 


SRV2_
8,127
LATAVDILAPQRYADPITWKSDEPVWVDQWPLTQEKLAAAQQLVQEQLQAGHIIESNSPWNTPIFVIKKKSGKWRLLQDLRAVNATMVLMGALQPGLP


P51517_

SPVAPPQGYFKIVIDLKDCFFTIPLQPVDQKRFAFSLPSTNFKQPMKRYQWKVLPQGMANSPTLCQKYVAAAIEPVRKSWAQMYIIHYMDDILIAGKLGE


2mutB

QVLQCFAQLKQALTTTGLQIAPEKVQLQDPYTYLGFQINGPKITNQKAVIRRDKLQTLNDFQKLLGDINWLRPYLHLTTGDLKPLFDILKGDSNPNSPRS




LSEAALASLQKVETAIAEQFVTQIDYTQPLTFLIFNTTLTPTGLFWQNNPVMWVHLPASPKKVLLPYYDAIADLIILGRDNSKKYFGLEPSTIIQPYSKSQIH




WLMQNTETWPIACASYAGNIDNHYPPNKLIQFCKLHAVVFPRIISKTPLDNALLVFTDGSSTGIAAYTFEKTTVRFKTSHTSAQLVELQALIAVLSAFPHR




ALNVYTDSAYLAHSIPLLETVSHIKHISDTAKFFLQCQQLIYNRSIPFYLGHIRAHSGLPGPLSQGNHITDLATKVVATTLTT


 


WDSV_
8,128
SCQTKNTLNIDEYLLQFPDQLWASLPTDIGRMLVPPITIKIKDNASLPSIRQYPLPKDKTEGLRPLISSLENQGILIKCHSPCNTPIFPIKKAGRDEYRMIHD


O92815

LRAINNIVAPLTAVVASPTTVLSNLAPSLHWFTVIDLSNAFFSVPIHKDSQYLFAFTFEGHQYTWTVLPQGFIHSPTLFSQALYQSLHKIKFKISSEICIYMD




DVLIASKDRDTNLKDTAVMLQHLASEGHKVSKKKLQLCQQEVVYLGQLLTPEGRKILPDRKVTVSQFQQPTTIRQIRAFLGLVGYCRHWIPEFSIHSKFL




EKQLKKDTAEPFQLDDQQVEAFNKLKHAITTAPVLWPDPAKPFQLYTSHSEHASIAVLTQKHAGRTRPIAFLSSKFDAIESGLPPCLKACASIHRSLTQA




DSFILGAPLIIYTTHAICTLLQRDRSQLVTASRFSKWEADLLRPELTFVACSAVSPAHLYMQSCENNIPPHDCVLLTHTISRPRPDLSDLPIPDPDMTLFSD




GSYTTGRGGAAVVMHRPVTDDFIIIHQQPGGASAQTAELLALAAACHLATDKTVNIYTDSRYAYGWHDFGHLWMHRGFVTSAGTPIKNHKEIEYLLKQ




IMKPKQVSVIKIEAHTKGVSMEVRGNAAADEAAKNAVFLVQR


 


WDSV_
8,129
SCQTKNTLNIDEYLLQFPDQLWASLPTDIGRMLVPPITIKIKDNASLPSIRQYPLPKDKTEGLRPLISSLENQGILIKCHSPCNTPIFPIKKAGRDEYRMIHD


O92815_

LRAINNIVAPLTAVVASPTTVLSNLAPSLHWFTVIDLSNAFFSVPIHKDSQYLFAFTFEGHQYTWTVLPQGFIHSPTLFNQALYQSLHKIKFKISSEICIYMD


2mut

DVLIASKDRDTNLKDTAVMLQHLASEGHKVSKKKLQLCQQEVVYLGQLLTPEGRKILPDRKVTVSQFQQPTTIRQIRAFLGLVGYCRHWIPEFSIHSKFL




EKQLKPDTAEPFQLDDQQVEAFNKLKHAITTAPVLVVPDPAKPFQLYTSHSEHASIAVLTQKHAGRTRPIAFLSSKFDAIESGLPPCLKACASIHRSLTQA




DSFILGAPLIIYTTHAICTLLQRDRSQLVTASRFSKWEADLLRPELTFVACSAVSPAHLYMQSCENNIPPHDCVLLTHTISRPRPDLSDLPIPDPDMTLFSD




GSYTTGRGGAAVVMHRPVTDDFIIIHQQPGGASAQTAELLALAAACHLATDKTVNIYTDSRYAYGWHDFGHLWMHRGFVTSAGTPIKNHKEIEYLLKQ




IMKPKQVSVIKIEAHTKGVSMEVRGNAAADEAAKNAVFLVQR


 


WDSV_
8,130
SCQTKNTLNIDEYLLQFPDQLWASLPTDIGRMLVPPITIKIKDNASLPSIRQYPLPKDKTEGLRPLISSLENQGILIKCHSPCNTPIFPIKKAGRDEYRMIHD


O92815_

LRAINNIVAPLTAVVASPTTVLSNLAPSLHWFTVIDLSNAFFSVPIHKDSQYLFAFTFEGHQYTWTVLPQGFIHSPTLFNQALYQSLHKIKFKISSEICIYMD


2mutA

DVLIASKDRDTNLKDTAVMLQHLASEGHKVSKKKLQLCQQEVVYLGQLLTPEGRKILPDRKVTVSQFQQPTTIRQIRAFLGKVGYCRHFIPEFSIHSKFL




EKQLKPDTAEPFQLDDQQVEAFNKLKHAITTAPVLVVPDPAKPFQLYTSHSEHASIAVLTQKHAGRTRPIAFLSSKFDAIESGLPPCLKACASIHRSLTQA




DSFILGAPLIIYTTHAICTLLQRDRSQLVTASRFSKWEADLLRPELTFVACSAVSPAHLYMQSCENNIPPHDCVLLTHTISRPRPDLSDLPIPDPDMTLFSD




GSYTTGRGGAAVVMHRPVTDDFIIIHQQPGGASAQTAELLALAAACHLATDKTVNIYTDSRYAYGWHDFGHLWMHRGFVTSAGTPIKNHKEIEYLLKQ




IMKPKQVSVIKIEAHTKGVSMEVRGNAAADEAAKNAVFLVQR


 


WMSV_
8,131
VLNLEEEYRLHEKPVPSSIDPSWLQLFPTVWAERAGMGLANQVPPVVVELRSGASPVAVRQYPMSKEAREGIRPHIQRFLDLGVLVPCQSPWNTPLL


P03359

PVKKPGTNDYRPVQDLREINKRVQDIHPTVPNPYNLLSSLPPSHTWYSVLDLKDAFFCLKLHPNSQPLFAFEWRDPEKGNTGQLTWTRLPQGFKNSP




TLFDEALHRDLAPFRALNPQVVLLQYVDDLLVAAPTYRDCKEGTQKLLQELSKLGYRVSAKKAQLCQKEVTYLGYLLKEGKRWLTPARKATVMKIPPP




TTPRQVREFLGTAGFCRLWIPGFASLAAPLYPLTKESIPFIWTEEHQKAFDRIKEALLSAPALALPDLTKPFTLYVDERAGVARGVLTQTLGPWRRPVAY




LSKKLDPVASGWPTCLKAVAAVALLLKDADKLTLGQNVTVIASHSLESIVRQPPDRWMTNARMTHYQSLLLNERVSFAPPAVLNPATLLPVESEATPVH




RCSEILAEETGTRRDLKDQPLPGVPAWYTDGSSFIAEGKRRAGAAIVDGKRTVWASSLPEGTSAQKAELVALTQALRLAEGKDINIYTDSRYAFATAHI




HGAIYKQRGLLTSAGKDIKNKEEILALLEAIHLPKRVAIIHCPGHQKGNDPVATGNRRADEAAKQAALSTRVLAETTKP


 


WMSV_
8,132
VLNLEEEYRLHEKPVPSSIDPSWLQLFPTVWAERAGMGLANQVPPVVVELRSGASPVAVRQYPMSKEAREGIRPHIQRFLDLGVLVPCQSPWNTPLL


P03359_

PVKKPGTNDYRPVQDLREINKRVQDIHPTVPNPYNLLSSLPPSHTWYSVLDLKDAFFCLKLHPNSQPLFAFEWRDPEKGNTGQLTWTRLPQGFKNSP


3mut

TLFNEALHRDLAPFRALNPQVVLLQYVDDLLVAAPTYRDCKEGTQKLLQELSKLGYRVSAKKAQLCQKEVTYLGYLLKEGKRWLTPARKATVMKIPPP




TTPRQVREFLGTAGFCRLWIPGFASLAAPLYPLTKPSIPFIWTEEHQKAFDRIKEALLSAPALALPDLTKPFTLYVDERAGVARGVLTQTLGPWRRPVAY




LSKKLDPVASGWPTCLKAVAAVALLLKDADKLTLGQNVTVIASHSLESIVRQPPDRWMTNARMTHYQSLLLNERVSFAPPAVLNPATLLPVESEATPVH




RCSEILAEETGTRRDLKDQPLPGVPAWYTDGSSFIAEGKRRAGAAIVDGKRTVWASSLPEGTSAQKAELVALTQALRLAEGKDINIYTDSRYAFATAHI




HGAIYKQRGWLTSAGKDIKNKEEILALLEAIHLPKRVAIIHCPGHQKGNDPVATGNRRADEAAKQAALSTRVLAETTKP


 


WMSV_
8,133
VLNLEEEYRLHEKPVPSSIDPSWLQLFPTVWAERAGMGLANQVPPVVVELRSGASPVAVRQYPMSKEAREGIRPHIQRFLDLGVLVPCQSPWNTPLL


P03359_

PVKKPGTNDYRPVQDLREINKRVQDIHPTVPNPYNLLSSLPPSHTWYSVLDLKDAFFCLKLHPNSQPLFAFEWRDPEKGNTGQLTWTRLPQGFKNSP


3mutA

TLFNEALHRDLAPFRALNPQVVLLQYVDDLLVAAPTYRDCKEGTQKLLQELSKLGYRVSAKKAQLCQKEVTYLGYLLKEGKRWLTPARKATVMKIPPP




TTPRQVREFLGKAGFCRLFIPGFASLAAPLYPLTKPSIPFIWTEEHQKAFDRIKEALLSAPALALPDLTKPFTLYVDERAGVARGVLTQTLGPWRRPVAY




LSKKLDPVASGWPTCLKAVAAVALLLKDADKLTLGQNVTVIASHSLESIVRQPPDRWMTNARMTHYQSLLLNERVSFAPPAVLNPATLLPVESEATPVH




RCSEILAEETGTRRDLKDQPLPGVPAWYTDGSSFIAEGKRRAGAAIVDGKRTVWASSLPEGTSAQKAELVALTQALRLAEGKDINIYTDSRYAFATAHI




HGAIYKQRGWLTSAGKDIKNKEEILALLEAIHLPKRVAIIHCPGHQKGNDPVATGNRRADEAAKQAALSTRVLAETTKP


 


XMRV6_
8,134
TLNIEDEYRLHETSKEPDVPLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


A1Z651

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT




LFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSEQDCQRGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPK




TPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA




YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQAMLLDTDRVQFGPWVALNPATLLPLPEKEA




PHDCLEILAETHGTRPDLTDQPIPDADYTWYTDGSSFLQEGQRRAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFAT




AHVHGEIYRRRGLLTSEGREIKNKNEILALLKALFLPKRLSIIHCPGHQKGNSAEARGNRMADQAAREAAMKAVLETSTLL


 


XMRV6_
8,135
TLNIEDEYRLHETSKEPDVPLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


A1Z651_

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT


3mut

LFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSEQDCQRGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPK




TPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPV




AYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQAMLLDTDRVQFGPWVALNPATLLPLPEKE




APHDCLEILAETHGTRPDLTDQPIPDADYTWYTDGSSFLQEGQRRAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAF




ATAHVHGEIYRRRGWLTSEGREIKNKNEILALLKALFLPKRLSIIHCPGHQKGNSAEARGNRMADQAAREAAMKAVLETSTLL


 


XMRV6_
8,136
TLNIEDEYRLHETSKEPDVPLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


A1Z651_

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPT


3mutA

LFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSEQDCQRGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPK




TPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVA




YLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEKEA




PHDCLEILAETHGTRPDLTDQPIPDADYTWYTDGSSFLQEGQRRAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFAT




AHVHGEIYRRRGWLTSEGREIKNKNEILALLKALFLPKRLSIIHCPGHQKGNSAEARGNRMADQAAREAAMKAVLETSTLL









In some embodiments, reverse transcriptase domains are modified, for example by site-specific mutation. In some embodiments, reverse transcriptase domains are engineered to have improved properties, e.g. SuperScript IV (SSIV) reverse transcriptase derived from the MMLV RT. In some embodiments, the reverse transcriptase domain may be engineered to have lower error rates, e.g., as described in WO2001068895, incorporated herein by reference. In some embodiments, the reverse transcriptase domain may be engineered to be more thermostable. In some embodiments, the reverse transcriptase domain may be engineered to be more processive. In some embodiments, the reverse transcriptase domain may be engineered to have tolerance to inhibitors. In some embodiments, the reverse transcriptase domain may be engineered to be faster. In some embodiments, the reverse transcriptase domain may be engineered to better tolerate modified nucleotides in the RNA template. In some embodiments, the reverse transcriptase domain may be engineered to insert modified DNA nucleotides. In some embodiments, the reverse transcriptase domain is engineered to bind a template RNA. In some embodiments, one or more mutations are chosen from D200N, L603W, T330P, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, W313F, L435G, N454K, H594Q, L671P, E69K, H8Y, T306K, or D653N in the RT domain of murine leukemia virus reverse transcriptase or a corresponding mutation at a corresponding position of another RT domain.
In some embodiments, a gene modifying polypeptide comprises the RT domain from a retroviral reverse transcriptase, e.g., a wild-type M-MLV RT, e.g., comprising the following sequence:







M-MLV (WT):


(SEQ ID NO: 5002)


TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLI


 


IPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL


 


LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT


 


VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP


 


TLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ


 


TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTP


 


KTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKA


 


YQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPV


 


AYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVE


 


ALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG


 


LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAA


 


VTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAF


 


ATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPG


 


HQKGHSAEARGNRMADQAARKAAITETPDTSTLLI






In some embodiments, a gene modifying polypeptide comprises the RT domain from a retroviral reverse transcriptase, e.g., an M-MLV RT, e.g., comprising the following sequence:







(SEQ ID NO: 5003)


TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLI


 


IPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPL


 


LPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYT


 


VLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSP


 


TLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ


 


TLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTP


 


KTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKA


 


YQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPV


 


AYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVE


 


ALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG


 


LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAA


 


VTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAF


 


ATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPG


 


HQKGHSAEARGNRMADQAARKAAITETPDTSTLL






In some embodiments, a gene modifying polypeptide comprises the RT domain from a retroviral reverse transcriptase comprising the sequence of amino acids 659-1329 of NP_057933. In embodiments, the gene modifying polypeptide further comprises one additional amino acid at the N-terminus of the sequence of amino acids 659-1329 of NP_057933, e.g., as shown below:









(SEQ ID NO: 5004)



TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMG


 



LAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQR


 



LLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNK


 



RVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRL


 



HPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFD


 



EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGT


 



RALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL


 



TEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEM


 



AAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLP


 



DLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD


 



PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAV


 



EALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNP


 



ATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHT


 



WYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR


 



AELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRR


 



RGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQK


 



GHSAEARGNRMADQAARKAA






Core RT (bold), annotated per above

RNAseH (underlined), annotated per above

In embodiments, the gene modifying polypeptide further comprises one additional amino acid at the C-terminus of the sequence of amino acids 659-1329 of NP_057933. In embodiments, the gene modifying polypeptide comprises an RNaseH1 domain (e.g., amino acids 1178-1318 of NP_057933).
In some embodiments, a retroviral reverse transcriptase domain, e.g., M-MLV RT, may comprise one or more mutations from a wild-type sequence that may improve features of the RT, e.g., thermostability, processivity, and/or template binding. In some embodiments, an M-MLV RT domain comprises, relative to the M-MLV (WT) sequence above, one or more mutations, e.g., selected from D200N, L603W, T330P, T306K, W313F, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, L435G, N454K, H594Q, D653N, R110S, K103L, e.g., a combination of mutations, such as D200N, L603W, and T330P, optionally further including T306K and W313F. In some embodiments, an M-MLV RT used herein comprises the mutations D200N, L603W, T330P, T306K and W313F. In embodiments, the mutant M-MLV RT comprises the following amino acid sequence:









M-MLV (PE2):



(SEQ ID NO: 5005)



TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMG



 



LAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQR



 



LLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNK



 



RVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRL



 



HPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFN



 



EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGT



 



RALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL



 



TEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEM



 



AAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLP



 



DLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD



 



PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHA



 



VEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVA



 



LNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDA



 



DHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTS



 



AQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEI



 



YRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPG



 



HQKGHSAEARGNRMADQAARKAAITETPDTSTLLI






In some embodiments, a writing domain (e.g., RT domain) comprises an RNA-binding domain, e.g., that specifically binds to an RNA sequence. In some embodiments, a template RNA comprises an RNA sequence that is specifically bound by the RNA-binding domain of the writing domain.
In some embodiments, the reverse transcription domain only recognizes and reverse transcribes a specific template, e.g., a template RNA of the system. In some embodiments, the template comprises a sequence or structure that enables recognition and reverse transcription by a reverse transcription domain. In some embodiments, the template comprises a sequence or structure that enables association with an RNA-binding domain of a polypeptide component of a genome engineering system described herein. In some embodiments, the genome engineering system reverse preferably transcribes a template comprising an association sequence over a template lacking an association sequence.
The writing domain may also comprise DNA-dependent DNA polymerase activity, e.g., comprise enzymatic activity capable of writing DNA into the genome from a template DNA sequence. In some embodiments, DNA-dependent DNA polymerization is employed to complete second-strand synthesis of a target site edit. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a DNA polymerase domain in the polypeptide. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a reverse transcriptase domain that is also capable of DNA-dependent DNA polymerization, e.g., second-strand synthesis. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a second polypeptide of the system. In some embodiments, the DNA-dependent DNA polymerase activity is provided by an endogenous host cell polymerase that is optionally recruited to the target site by a component of the genome engineering system.
In some embodiments, the reverse transcriptase domain has a lower probability of premature termination rate (Poff) in vitro relative to a reference reverse transcriptase domain. In some embodiments, the reference reverse transcriptase domain is a viral reverse transcriptase domain, e.g., the RT domain from M-MLV.
In some embodiments, the reverse transcriptase domain has a lower probability of premature termination rate (Poff) in vitro of less than about 5×10−3/nt, 5×10−4/nt, or 5×10−6/nt, e.g., as measured on a 1094 nt RNA. In embodiments, the in vitro premature termination rate is determined as described in Bibillo and Eickbush (2002) J Biol Chem 277(38):34836-34845 (incorporated by reference herein its entirety).
In some embodiments, the reverse transcriptase domain is able to complete at least about 30% or 50% of integrations in cells. The percent of complete integrations can be measured by dividing the number of substantially full-length integration events (e.g., genomic sites that comprise at least 98% of the expected integrated sequence) by the number of total (including substantially full-length and partial) integration events in a population of cells. In embodiments, the integrations in cells is determined (e.g., across the integration site) using long-read amplicon sequencing, e.g., as described in Karst et al. (2020) bioRxiv doi.org/10.1101/645903 (incorporated by reference herein in its entirety).
In embodiments, quantifying integrations in cells comprises counting the fraction of integrations that contain at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the DNA sequence corresponding to the template RNA (e.g., a template RNA having a length of at least 0.05, 0.1, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 3, 4, or 5 kb, e.g., a length between 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 1.0-1.2, 1.2-1.4, 1.4-1.6, 1.6-1.8, 1.8-2.0, 2-3, 3-4, or 4-5 kb).
In some embodiments, the reverse transcriptase domain is capable of polymerizing dNTPs in vitro. In embodiments, the reverse transcriptase domain is capable of polymerizing dNTPs in vitro at a rate between 0.1-50 nt/sec (e.g., between 0.1-1, 1-10, or 10-50 nt/sec). In embodiments, polymerization of dNTPs by the reverse transcriptase domain is measured by a single-molecule assay, e.g., as described in Schwartz and Quake (2009) PNAS 106(48):20294-20299 (incorporated by reference in its entirety).
In some embodiments, the reverse transcriptase domain has an in vitro error rate (e.g., misincorporation of nucleotides) of between 1×10−3-1×10−4 or 1×10−4-1×10−5 substitutions/nt, e.g., as described in Yasukawa et al. (2017) Biochem Biophys Res Commun 492(2):147-153 (incorporated herein by reference in its entirety). In some embodiments, the reverse transcriptase domain has an error rate (e.g., misincorporation of nucleotides) in cells (e.g., HEK293T cells) of between 1×10−3-1×10−4 or 1×10−4-1×10−5 substitutions/nt, e.g., by long-read amplicon sequencing, e.g., as described in Karst et al. (2020) bioRxiv doi.org/10.1101/645903 (incorporated by reference herein in its entirety).
In some embodiments, the reverse transcriptase domain is capable of performing reverse transcription of a target RNA in vitro. In some embodiments, the reverse transcriptase requires a primer of at least 3 nucleotides to initiate reverse transcription of a template. In some embodiments, reverse transcription of the target RNA is determined by detection of cDNA from the target RNA (e.g., when provided with a ssDNA primer, e.g., which anneals to the target with at least 3, 4, 5, 6, 7, 8, 9, or 10 nt at the 3′ end), e.g., as described in Bibillo and Eickbush (2002) J Biol Chem 277(38):34836-34845 (incorporated herein by reference in its entirety).
In some embodiments, the reverse transcriptase domain performs reverse transcription at least 5 or 10 times more efficiently (e.g., by cDNA production), e.g., when converting its RNA template to cDNA, for example, as compared to an RNA template lacking the protein binding motif (e.g., a 3′ UTR). In embodiments, efficiency of reverse transcription is measured as described in Yasukawa et al. (2017) Biochem Biophys Res Commun 492(2):147-153 (incorporated by reference herein in its entirety).
In some embodiments, the reverse transcriptase domain specifically binds a specific RNA template with higher frequency (e.g., about 5 or 10-fold higher frequency) than any endogenous cellular RNA, e.g., when expressed in cells (e.g., HEK293T cells). In embodiments, frequency of specific binding between the reverse transcriptase domain and the template RNA are measured by CLIP-seq, e.g., as described in Lin and Miles (2019) Nucleic Acids Res 47(11):5490-5501 (incorporated herein by reference in its entirety).
In some embodiments, an RT domain (e.g., as listed in Table 6) comprises one or more mutations as listed in Table 2 below. In some embodiment, an RT domain as listed in Table 6 comprises one, two, three, four, five, or six of the mutations listed in the corresponding row of Table 2 below.







TABLE 2







Exemplary RT domain mutations (relative to corresponding wild-


type sequences as listed in the corresponding row of Table 6)








RT Domain Name
Mutation(s)
















AVIRE_P03360








AVIRE_P03360_3mut
D200N
G330P
L605W


AVIRE_P03360_3mutA
D200N
G330P
L605W
T306K
W313F


BAEVM_P10272


BAEVM_P10272_3mut
D198N
E328P
L602W


BAEVM_P10272_3mutA
D198N
E328P
L602W
T304K
W311F


BLVAU_P25059


BLVAU_P25059_2mut
E159Q
G286P


BLVJ_P03361


BLVJ_P03361_2mut
E159Q
L524W


BLVJ_P03361_2mutB
E159Q
L524W
197P


FFV_O93209
D21N


FFV_O93209_2mut
D21N
T293N
T419P


FFV_O93209_2mutA
D21N
T293N
T419P
L393K


FFV_O93209-Pro


FFV_O93209-Pro_2mut
T207N
T333P


FFV_O93209-Pro_2mutA
T207N
T333P
L307K


FLV_P10273


FLV_P10273_3mut
D199N
L602W


FLV_P10273_3mutA
D199N
L602W
T305K
W312F


FOAMV_P14350
D24N


FOAMV_P14350_2mut
D24N
T296N
S420P


FOAMV_P14350_2mutA
D24N
T296N
S420P
L396K


FOAMV_P14350-Pro


FOAMV_P14350-Pro_2mut
T207N
S331P


FOAMV_P14350-Pro_2mutA
T207N
S331P
L307K


GALV_P21414


GALV_P21414_3mut
D198N
E328P
L600W


GALV_P21414_3mutA
D198N
E328P
L600W
T304K
W311F


HTL1A_P03362


HTL1A_P03362_2mut
E152Q
R279P


HTL1A_P03362_2mutB
E152Q
R279P
L90P


HTL1C_P14078


HTL1C_P14078_2mut
E152Q
R279P


HTL1L_P0C211


HTL1L_P0C211_2mut
E149Q
L527W


HTL1L_P0C211_2mutB
E149Q
L527W
L87P


HTL32_Q0R5R2


HTL32_Q0R5R2_2mut
E149Q
L526W


HTL32_Q0R5R2_2mutB
E149Q
L526W
L87P


HTL3P_Q4U0X6


HTL3P_Q4U0X6_2mut
E149Q
L526W


HTL3P_Q4U0X6_2mutB
E149Q
L526W
L87P


HTLV2_P03363_2mut
E147Q
G274P


JSRV_P31623


JSRV_P31623_2mutB
A100P


KORV_Q9TTC1
D32N


KORV_Q9TTC1_3mut
D32N
D322N
E452P
L724W


KORV_Q9TTC1_3mutA
D32N
D322N
E452P
L724W
T428K
W435F


KORV_Q9TTC1-Pro


KORV_Q9TTC1-Pro_3mut
D231N
E361P
L633W


KORV_Q9TTC1-Pro_3mutA
D231N
E361P
L633W
T337K
W344F


MLVAV_P03356


MLVAV_P03356_3mut
D200N
T330P
L603W


MLVAV_P03356_3mutA
D200N
T330P
L603W
T306K
W313F


MLVBM_Q7SVK7


MLVBM_Q7SVK7


MLVBM_Q7SVK7_3mut
D200N
T330P
L603W


MLVBM_Q7SVK7_3mut
D200N
T330P
L603W


MLVBM_Q7SVK7_3mutA_WS
D199N
T329P
L602W
T305K
W312F


MLVBM_Q7SVK7_3mutA_WS
D199N
T329P
L602W
T305K
W312F


MLVCB_P08361


MLVCB_P08361_3mut
D200N
T330P
L603W


MLVCB_P08361_3mutA
D200N
T330P
L603W
T306K
W313F


MLVF5_P26810


MLVF5_P26810_3mut
D200N
T330P
L603W


MLVF5_P26810_3mutA
D200N
T330P
L603W
T306K
W313F


MLVFF_P26809_3mut
D200N
T330P
L603W


MLVFF_P26809_3mutA
D200N
T330P
L603W
T306K
W313F


MLVMS_P03355


MLVMS_P03355


MLVMS_P03355_3mut
D200N
T330P
L603W


MLVMS_P03355_3mut
D200N
T330P
L603W


MLVMS_P03355_3mutA_WS
D200N
T330P
L603W
T306K
W313F


MLVMS_P03355_3mutA_WS
D200N
T330P
L603W
T306K
W313F


MLVMS_P03355_PLV919
D200N
T330P
L603W
T306K
W313F
H8Y


MLVMS_P03355_PLV919
D200N
T330P
L603W
T306K
W313F
H8Y


MLVRD_P11227


MLVRD_P11227_3mut
D200N
T330P
L603W


MMTVB_P03365
D26N


MMTVB_P03365
D26N


MMTVB_P03365_2mut
D26N
G401P


MMTVB_P03365_2mut_WS
G400P


MMTVB_P03365_2mut_WS
G400P


MMTVB_P03365_2mutB
D26N
G401P
V215P


MMTVB_P03365_2mutB
D26N
G401P
V215P


MMTVB_P03365_2mutB_WS
G400P
V212P


MMTVB_P03365_2mutB_WS
G400P
V212P


MMTVB_P03365_WS


MMTVB_P03365_WS


MMTVB_P03365-Pro


MMTVB_P03365-Pro


MMTVB_P03365-Pro_2mut
G309P


MMTVB_P03365-Pro_2mut
G309P


MMTVB_P03365-Pro_2mutB
G309P
V123P


MMTVB_P03365-Pro_2mutB
G309P
V123P


MPMV_P07572


MPMV_P07572_2mutB
G289P
I103P


PERV_Q4VFZ2


PERV_Q4VFZ2


PERV_Q4VFZ2_3mut
D199N
E329P
L602W


PERV_Q4VFZ2_3mut
D199N
E329P
L602W


PERV_Q4VFZ2_3mutA_WS
D196N
E326P
L599W
T302K
W309F


PERV_Q4VFZ2_3mutA_WS
D196N
E326P
L599W
T302K
W309F


SFV1_P23074
D24N


SFV1_P23074_2mut
D24N
T296N
N420P


SFV1_P23074_2mutA
D24N
T296N
N420P
L396K


SFV1_P23074-Pro


SFV1_P23074-Pro_2mut
T207N
N331P


SFV1_P23074-Pro_2mutA
T207N
N331P
L307K


SFV3L_P27401
D24N


SFV3L_P27401_2mut
D24N
T296N
N422P


SFV3L_P27401_2mutA
D24N
T296N
N422P
L396K


SFV3L_P27401-Pro


SFV3L_P27401-Pro_2mut
T307N
N333P


SFV3L_P27401-Pro_2mutA
T307N
N333P
L307K


SFVCP_Q87040
D24N


SFVCP_Q87040_2mut
D24N
T296N
K422P


SFVCP_Q87040_2mutA
D24N
T296N
K422P
L396K


SFVCP_Q87040-Pro


SFVCP_Q87040-Pro_2mut
T207N
K333P


SFVCP_Q87040-Pro_2mutA
T207N
K333P
L307K


SMRVH_P03364


SMRVH_P03364_2mut
G288P


SMRVH_P03364_2mutB
G288P
I102P


SRV2_P51517


SRV2_P51517_2mutB
I103P


WDSV_092815


WDSV_092815_2mut
S183N
K312P


WDSV_092815_2mutA
S183N
K312P
L288K
W295F


WMSV_P03359


WMSV_P03359_3mut
D198N
E328P
L600W


WMSV_P03359_3mutA
D198N
E328P
L600W
T304K
W311F


XMRV6_A1Z651


XMRV6_A1Z651_3mut
D200N
T330P
L603W


XMRV6_A1Z651_3mutA
D200N
T330P
L603W
T306K
W313F









Template Nucleic Acid Binding Domain

The gene modifying polypeptide typically contains regions capable of associating with the template nucleic acid (e.g., template RNA). In some embodiments, the template nucleic acid binding domain is an RNA binding domain. In some embodiments, the RNA binding domain is a modular domain that can associate with RNA molecules containing specific signatures, e.g., structural motifs. In other embodiments, the template nucleic acid binding domain (e.g., RNA binding domain) is contained within the reverse transcription domain, e.g., the reverse transcriptase-derived component has a known signature for RNA preference.
In other embodiments, the template nucleic acid binding domain (e.g., RNA binding domain) is contained within the target DNA binding domain. For example, in some embodiments, the DNA binding domain is a CRISPR-associated protein that recognizes the structure of a template nucleic acid (e.g., template RNA) comprising a gRNA. In some embodiments, a gene modifying polypeptide comprises a DNA-binding domain comprising a CRISPR-associated protein that associates with a gRNA scaffold that allows the DNA-binding domain to bind a target genomic DNA sequence. In some embodiments, the gRNA scaffold and gRNA spacer is comprised within the template nucleic acid (e.g., template RNA), thus the DNA-binding domain is also the template nucleic acid binding domain. In some embodiments, the polypeptide possesses RNA binding function in multiple domains, e.g., can bind a gRNA structure in a CRISPR-associated DNA binding domain and an additional sequence or structure in a reverse transcriptase domain.
In some embodiments, the RNA binding domain is capable of binding to a template RNA with greater affinity than a reference RNA binding domain. In some embodiments, the reference RNA binding domain is an RNA binding domain from Cas9 of S. pyogenes. In some embodiments, the RNA binding domain is capable of binding to a template RNA with an affinity between 100 pM-10 nM (e.g., between 100 pM-1 nM or 1 nM-10 nM). In some embodiments, the affinity of a RNA binding domain for its template RNA is measured in vitro, e.g., by thermophoresis, e.g., as described in Asmari et al. Methods 146:107-119 (2018) (incorporated by reference herein in its entirety). In some embodiments, the affinity of a RNA binding domain for its template RNA is measured in cells (e.g., by FRET or CLIP-Seq).
In some embodiments, the RNA binding domain is associated with the template RNA in vitro at a frequency at least about 5-fold or 10-fold higher than with a scrambled RNA. In some embodiments, the frequency of association between the RNA binding domain and the template RNA or scrambled RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019) Nucleic Acids Res 47(11):5490-5501 (incorporated by reference herein in its entirety). In some embodiments, the RNA binding domain is associated with the template RNA in cells (e.g., in HEK293T cells) at a frequency at least about 5-fold or 10-fold higher than with a scrambled RNA. In some embodiments, the frequency of association between the RNA binding domain and the template RNA or scrambled RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019), supra.
Endonuclease Domains and DNA Binding Domains
In some embodiments, a gene modifying polypeptide possesses the function of DNA target site cleavage via an endonuclease domain. In some embodiments, a gene modifying polypeptide comprises a DNA binding domain, e.g., for binding to a target nucleic acid. In some embodiments, a domain (e.g., a Cas domain) of the gene modifying polypeptide comprises two or more smaller domains, e.g., a DNA binding domain and an endonuclease domain. It is understood that when a DNA binding domain (e.g., a Cas domain) is said to bind to a target nucleic acid sequence, in some embodiments, the binding is mediated by a gRNA.
In some embodiments, a domain has two functions. For example, in some embodiments, the endonuclease domain is also a DNA-binding domain. In some embodiments, the endonuclease domain is also a template nucleic acid (e.g., template RNA) binding domain. For example, in some embodiments, a polypeptide comprises a CRISPR-associated endonuclease domain that binds a template RNA comprising a gRNA, binds a target DNA sequence (e.g., with complementarity to a portion of the gRNA), and cuts the target DNA sequence. In some embodiments, an endonuclease domain or endonuclease/DNA-binding domain from a heterologous source can be used or can be modified (e.g., by insertion, deletion, or substitution of one or more residues) in a gene modifying system described herein.
In some embodiments, a nucleic acid encoding the endonuclease domain or endonuclease/DNA binding domain is altered from its natural sequence to have altered codon usage, e.g. improved for human cells. In some embodiments, the endonuclease element is a heterologous endonuclease element, such as a Cas endonuclease (e.g., Cas9), a type-II restriction endonuclease (e.g., Fok1), a meganuclease (e.g., I-SceI), or other endonuclease domain.
In certain aspects, the DNA-binding domain of a gene modifying polypeptide described herein is selected, designed, or constructed for binding to a desired host DNA target sequence. In certain embodiments, the DNA-binding domain of the polypeptide is a heterologous DNA-binding element. In some embodiments the heterologous DNA binding element is a zinc-finger element or a TAL effector element, e.g., a zinc-finger or TAL polypeptide or functional fragment thereof. In some embodiments the heterologous DNA binding element is a sequence-guided DNA binding element, such as Cas9, Cpf1, or other CRISPR-related protein that has been altered to have no endonuclease activity. In some embodiments the heterologous DNA binding element retains endonuclease activity. In some embodiments, the heterologous DNA binding element retains partial endonuclease activity to cleave ssDNA, e.g., possesses nickase activity. In specific embodiments, the heterologous DNA-binding domain can be any one or more of Cas9, TAL domain, ZF domain, Myb domain, combinations thereof, or multiples thereof.
In some embodiments, DNA-binding domains are modified, for example by site-specific mutation, increasing or decreasing DNA-binding elements (for example, number and/or specificity of zinc fingers), etc., to alter DNA-binding specificity and affinity. In some embodiments a nucleic acid sequence encoding the DNA binding domain is altered from its natural sequence to have altered codon usage, e.g. improved for human cells. In embodiments, the DNA binding domain comprises one or more modifications relative to a wild-type DNA binding domain, e.g., a modification via directed evolution, e.g., phage-assisted continuous evolution (PACE).
In some embodiments, the DNA binding domain comprises a meganuclease domain (e.g., as described herein, e.g., in the endonuclease domain section), or a functional fragment thereof. In some embodiments, the meganuclease domain possesses endonuclease activity, e.g., double-strand cleavage and/or nickase activity. In other embodiments, the meganuclease domain has reduced activity, e.g., lacks endonuclease activity, e.g., the meganuclease is catalytically inactive. In some embodiments, a catalytically inactive meganuclease is used as a DNA binding domain, e.g., as described in Fonfara et al. Nucleic Acids Res 40(2):847-860 (2012), incorporated herein by reference in its entirety.
In some embodiments, a gene modifying polypeptide comprises a modification to a DNA-binding domain, e.g., relative to the wild-type polypeptide. In some embodiments, the DNA-binding domain comprises an addition, deletion, replacement, or modification to the amino acid sequence of the original DNA-binding domain. In some embodiments, the DNA-binding domain is modified to include a heterologous functional domain that binds specifically to a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, the functional domain replaces at least a portion (e.g., the entirety of) the prior DNA-binding domain of the polypeptide. In some embodiments, the functional domain comprises a zinc finger (e.g., a zinc finger that specifically binds to the target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, the functional domain comprises a Cas domain (e.g., a Cas domain that specifically binds to the target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, the Cas domain comprises a Cas9 or a mutant or variant thereof (e.g., as described herein). In embodiments, the Cas domain is associated with a guide RNA (gRNA), e.g., as described herein. In embodiments, the Cas domain is directed to a target nucleic acid (e.g., DNA) sequence of interest by the gRNA. In embodiments, the Cas domain is encoded in the same nucleic acid (e.g., RNA) molecule as the gRNA. In embodiments, the Cas domain is encoded in a different nucleic acid (e.g., RNA) molecule from the gRNA.
In some embodiments, the DNA binding domain is capable of binding to a target sequence (e.g., a dsDNA target sequence) with greater affinity than a reference DNA binding domain. In some embodiments, the reference DNA binding domain is a DNA binding domain from Cas9 of S. pyogenes. In some embodiments, the DNA binding domain is capable of binding to a target sequence (e.g., a dsDNA target sequence) with an affinity between 100 pM-10 nM (e.g., between 100 pM-1 nM or 1 nM-10 nM).
In some embodiments, the affinity of a DNA binding domain for its target sequence (e.g., dsDNA target sequence) is measured in vitro, e.g., by thermophoresis, e.g., as described in Asmari et al. Methods 146:107-119 (2018) (incorporated by reference herein in its entirety).
In embodiments, the DNA binding domain is capable of binding to its target sequence (e.g., dsDNA target sequence), e.g, with an affinity between 100 pM-10 nM (e.g., between 100 pM-1 nM or 1 nM-10 nM) in the presence of a molar excess of scrambled sequence competitor dsDNA, e.g., of about 100-fold molar excess.
In some embodiments, the DNA binding domain is found associated with its target sequence (e.g., dsDNA target sequence) more frequently than any other sequence in the genome of a target cell, e.g., human target cell, e.g., as measured by ChIP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010) Curr. Protoc Mol Biol Chapter 21 (incorporated herein by reference in its entirety). In some embodiments, the DNA binding domain is found associated with its target sequence (e.g., dsDNA target sequence) at least about 5-fold or 10-fold, more frequently than any other sequence in the genome of a target cell, e.g., as measured by ChIP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010), supra.
In some embodiments, the endonuclease domain has nickase activity and cleaves one strand of a target DNA. In some embodiments, nickase activity reduces the formation of double-stranded breaks at the target site. In some embodiments, the endonuclease domain creates a staggered nick structure in the first and second strands of a target DNA. In some embodiments, a staggered nick structure generates free 3′ overhangs at the target site. In some embodiments, free 3′ overhangs at the target site improve editing efficiency, e.g., by enhancing access and annealing of a 3′ homology region of a template nucleic acid. In some embodiments, a staggered nick structure reduces the formation of double-stranded breaks at the target site.
In some embodiments, the endonuclease domain cleaves both strands of a target DNA, e.g., results in blunt-end cleavage of a target with no ssDNA overhangs on either side of the cut-site. The amino acid sequence of an endonuclease domain of a gene modifying system described herein may be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the amino acid sequence of an endonuclease domain described herein, e.g., an endonuclease domain from Table 8.
In certain embodiments, the heterologous endonuclease is Fok1 or a functional fragment thereof. In certain embodiments, the heterologous endonuclease is a Holliday junction resolvase or homolog thereof, such as the Holliday junction resolving enzyme from Sulfolobus solfataricus—Ssol Hje (Govindaraju et al., Nucleic Acids Research 44:7, 2016). In certain embodiments, the heterologous endonuclease is the endonuclease of the large fragment of a spliceosomal protein, such as Prp8 (Mahbub et al., Mobile DNA 8:16, 2017). In certain embodiments, the heterologous endonuclease is derived from a CRISPR-associated protein, e.g., Cas9. In certain embodiments, the heterologous endonuclease is engineered to have only ssDNA cleavage activity, e.g., only nickase activity, e.g., be a Cas9 nickase, e.g., SpCas9 with D10A, H840A, or N863A mutations. Table 8 provides exemplary Cas proteins and mutations associated with nickase activity. In still other embodiments, homologous endonuclease domains are modified, for example by site-specific mutation, to alter DNA endonuclease activity. In still other embodiments, endonuclease domains are modified to reduce DNA-sequence specificity, e.g., by truncation to remove domains that confer DNA-sequence specificity or mutation to inactivate regions conferring DNA-sequence specificity.
In some embodiments, the endonuclease domain has nickase activity and does not form double-stranded breaks. In some embodiments, the endonuclease domain forms single-stranded breaks at a higher frequency than double-stranded breaks, e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% of the breaks are single-stranded breaks, or less than 10%, 5%, 4%, 3%, 2%, or 1% of the breaks are double-stranded breaks. In some embodiments, the endonuclease forms substantially no double-stranded breaks. In some embodiments, the endonuclease does not form detectable levels of double-stranded breaks.
In some embodiments, the endonuclease domain has nickase activity that nicks the target site DNA of the first strand; e.g., in some embodiments, the endonuclease domain cuts the genomic DNA of the target site near to the site of alteration on the strand that will be extended by the writing domain. In some embodiments, the endonuclease domain has nickase activity that nicks the target site DNA of the first strand and does not nick the target site DNA of the second strand. For example, when a polypeptide comprises a CRISPR-associated endonuclease domain having nickase activity, in some embodiments, said CRISPR-associated endonuclease domain nicks the target site DNA strand containing the PAM site (e.g., and does not nick the target site DNA strand that does not contain the PAM site). As a further example, when a polypeptide comprises a CRISPR-associated endonuclease domain having nickase activity, in some embodiments, said CRISPR-associated endonuclease domain nicks the target site DNA strand not containing the PAM site (e.g., and does not nick the target site DNA strand that contains the PAM site).
In some other embodiments, the endonuclease domain has nickase activity that nicks the target site DNA of the first strand and the second strand. Without wishing to be bound by theory, after a writing domain (e.g., RT domain) of a polypeptide described herein polymerizes (e.g., reverse transcribes) from the heterologous object sequence of a template nucleic acid (e.g., template RNA), the cellular DNA repair machinery must repair the nick on the first DNA strand. The target site DNA now contains two different sequences for the first DNA strand: one corresponding to the original genomic DNA (e.g., having a free 5′ end) and a second corresponding to that polymerized from the heterologous object sequence (e.g., having a free 3′ end). It is thought that the two different sequences equilibrate with one another, first one hybridizing the second strand, then the other, and which sequence the cellular DNA repair apparatus incorporates into its repaired target site may be a stochastic process. Without wishing to be bound by theory, it is thought that introducing an additional nick to the second-strand may bias the cellular DNA repair machinery to adopt the heterologous object sequence-based sequence more frequently than the original genomic sequence (Anzalone et al. Nature 576:149-157 (2019)). In some embodiments, the additional nick is positioned at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 nucleotides 5′ or 3′ of the target site modification (e.g., the insertion, deletion, or substitution) or to the nick on the first strand.
Alternatively or additionally, without wishing to be bound by theory, it is thought that an additional nick to the second strand may promote second-strand synthesis. In some embodiments, where the gene modifying system has inserted or substituted a portion of the first strand, synthesis of a new sequence corresponding to the insertion/substitution in the second strand is necessary.
In some embodiments, the polypeptide comprises a single domain having endonuclease activity (e.g., a single endonuclease domain) and said domain nicks both the first strand and the second strand. For example, in such an embodiment the endonuclease domain may be a CRISPR-associated endonuclease domain, and the template nucleic acid (e.g., template RNA) comprises a gRNA spacer that directs nicking of the first strand and an additional gRNA spacer that directs nicking of the second strand. In some embodiments, the polypeptide comprises a plurality of domains having endonuclease activity, and a first endonuclease domain nicks the first strand and a second endonuclease domain nicks the second strand (optionally, the first endonuclease domain does not (e.g., cannot) nick the second strand and the second endonuclease domain does not (e.g., cannot) nick the first strand).
In some embodiments, the endonuclease domain is capable of nicking a first strand and a second strand. In some embodiments, the first and second strand nicks occur at the same position in the target site but on opposite strands. In some embodiments, the second strand nick occurs in a staggered location, e.g., upstream or downstream, from the first nick. In some embodiments, the endonuclease domain generates a target site deletion if the second strand nick is upstream of the first strand nick. In some embodiments, the endonuclease domain generates a target site duplication if the second strand nick is downstream of the first strand nick. In some embodiments, the endonuclease domain generates no duplication and/or deletion if the first and second strand nicks occur in the same position of the target site. In some embodiments, the endonuclease domain has altered activity depending on protein conformation or RNA-binding status, e.g., which promotes the nicking of the first or second strand (e.g., as described in Christensen et al. PNAS 2006; incorporated by reference herein in its entirety).
In some embodiments, the endonuclease domain comprises a meganuclease, or a functional fragment thereof. In some embodiments, the endonuclease domain comprises a homing endonuclease, or a functional fragment thereof. In some embodiments, the endonuclease domain comprises a meganuclease from the LAGLIDADG (SEQ ID NO: 37638), GIY-YIG, HNH, His-Cys Box, or PD-(D/E) XK families, or a functional fragment or variant thereof, e.g., which possess conserved amino acid motifs, e.g., as indicated in the family names. In some embodiments, the endonuclease domain comprises a meganuclease, or fragment thereof, chosen from, e.g., I-SmaMI (Uniprot F7WD42), I-Seel (Uniprot P03882), I-Anil (Uniprot P03880), I-Dmol (Uniprot P21505), I-CreI (Uniprot P05725), I-Teel (Uniprot P13299), I-OnuI (Uniprot Q4VWW5), or I-Bmol (Uniprot Q9ANR6). In some embodiments, the meganuclease is naturally monomeric, e.g., I-Seel, I-Teel, or dimeric, e.g., I-CreI, in its functional form. For example, the LAGLIDADG meganucleases (SEQ ID NO: 37638) with a single copy of the LAGLIDADG motif (SEQ ID NO: 37638) generally form homodimers, whereas members with two copies of the LAGLIDADG motif (SEQ ID NO: 37638) are generally found as monomers. In some embodiments, a meganuclease that normally forms as a dimer is expressed as a fusion, e.g., the two subunits are expressed as a single ORF and, optionally, connected by a linker, e.g., an I-CreI dimer fusion (Rodriguez-Fornes et al. Gene Therapy 2020; incorporated by reference herein in its entirety). In some embodiments, a meganuclease, or a functional fragment thereof, is altered to favor nickase activity for one strand of a double-stranded DNA molecule, e.g., I-SceI (K122I and/or K223I) (Niu et al. J Mol Biol 2008), I-Anil (K227M) (McConnell Smith et al. PNAS 2009), I-Dmol (Q42A and/or K120M) (Molina et al. J Biol Chem 2015). In some embodiments, a meganuclease or functional fragment thereof possessing this preference for single-strand cleavage is used as an endonuclease domain, e.g., with nickase activity. In some embodiments, an endonuclease domain comprises a meganuclease, or a functional fragment thereof, which naturally targets or is engineered to target a safe harbor site, e.g., an I-CreI targeting SH6 site (Rodriguez-Fomes et al., supra). In some embodiments, an endonuclease domain comprises a meganuclease, or a functional fragment thereof, with a sequence tolerant catalytic domain, e.g., I-Teel recognizing the minimal motif CNNNG (Kleinstiver et al. PNAS 2012). In some embodiments, a target sequence tolerant catalytic domain is fused to a DNA binding domain, e.g., to direct activity, e.g., by fusing I-Teel to: (i) zinc fingers to create Tev-ZFEs (Kleinstiver et al. PNAS 2012), (ii) other meganucleases to create MegaTevs (Wolfs et al. Nucleic Acids Res 2014), and/or (iii) Cas9 to create TevCas9 (Wolfs et al. PNAS 2016).
In some embodiments, the endonuclease domain comprises a restriction enzyme, e.g., a Type IIS or Type IIP restriction enzyme. In some embodiments, the endonuclease domain comprises a Type IIS restriction enzyme, e.g., FokI, or a fragment or variant thereof. In some embodiments, the endonuclease domain comprises a Type IIP restriction enzyme, e.g., PvuII, or a fragment or variant thereof. In some embodiments, a dimeric restriction enzyme is expressed as a fusion such that it functions as a single chain, e.g., a FokI dimer fusion (Minczuk et al. Nucleic Acids Res 36(12):3926-3938 (2008)).
The use of additional endonuclease domains is described, for example, in Guha and Edgell Int J Mol Sci 18(22):2565 (2017), which is incorporated herein by reference in its entirety.
In some embodiments, a gene modifying polypeptide comprises a modification to an endonuclease domain, e.g., relative to a wild-type Cas protein. In some embodiments, the endonuclease domain comprises an addition, deletion, replacement, or modification to the amino acid sequence of the wild-type Cas protein. In some embodiments, the endonuclease domain is modified to include a heterologous functional domain that binds specifically to and/or induces endonuclease cleavage of a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, the endonuclease domain comprises a zinc finger. In embodiments, the endonuclease domain comprising the Cas domain is associated with a guide RNA (gRNA), e.g., as described herein. In some embodiments, the endonuclease domain is modified to include a functional domain that does not target a specific target nucleic acid (e.g., DNA) sequence. In embodiments, the endonuclease domain comprises a Fok1 domain.
In some embodiments, the endonuclease domain is associated with the target dsDNA in vitro at a frequency at least about 5-fold or 10-fold higher than with a scrambled dsDNA. In some embodiments, the endonuclease domain is associated with the target dsDNA in vitro at a frequency at least about 5-fold or 10-fold higher than with a scrambled dsDNA, e.g., in a cell (e.g., a HEK293T cell). In some embodiments, the frequency of association between the endonuclease domain and the target DNA or scrambled DNA is measured by ChIP-seq, e.g., as described in He and Pu (2010) Curr. Protoc Mol Biol Chapter 21 (incorporated by reference herein in its entirety).
In some embodiments, the endonuclease domain can catalyze the formation of a nick at a target sequence, e.g., to an increase of at least about 5-fold or 10-fold relative to a non-target sequence (e.g., relative to any other genomic sequence in the genome of the target cell). In some embodiments, the level of nick formation is determined using NickSeq, e.g., as described in Elacqua et al. (2019) bioRxiv doi.org/10.1101/867937 (incorporated herein by reference in its entirety).
In some embodiments, the endonuclease domain is capable of nicking DNA in vitro. In embodiments, the nick results in an exposed base. In embodiments, the exposed base can be detected using a nuclease sensitivity assay, e.g., as described in Chaudhry and Weinfeld (1995) Nucleic Acids Res 23(19):3805-3809 (incorporated by reference herein in its entirety). In embodiments, the level of exposed bases (e.g., detected by the nuclease sensitivity assay) is increased by at least 10%, 50%, or more relative to a reference endonuclease domain. In some embodiments, the reference endonuclease domain is an endonuclease domain from Cas9 of S. pyogenes. 
In some embodiments, the endonuclease domain is capable of nicking DNA in a cell. In embodiments, the endonuclease domain is capable of nicking DNA in a HEK293T cell. In embodiments, an unrepaired nick that undergoes replication in the absence of Rad51 results in increased NHEJ rates at the site of the nick, which can be detected, e.g., by using a Rad51 inhibition assay, e.g., as described in Bothmer et al. (2017) Nat Commun 8:13905 (incorporated by reference herein in its entirety). In embodiments, NHEJ rates are increased above 0-5%. In embodiments, NHEJ rates are increased to 20-70% (e.g., between 30%-60% or 40-50%), e.g., upon Rad51 inhibition.
In some embodiments, the endonuclease domain releases the target after cleavage. In some embodiments, release of the target is indicated indirectly by assessing for multiple turnovers by the enzyme, e.g., as described in Yourik at al. RNA 25(1):35-44 (2019) (incorporated herein by reference in its entirety) and shown in FIG. 2. In some embodiments, the kexp of an endonuclease domain is 1×10−3-1×10−5 min−1 as measured by such methods.
In some embodiments, the endonuclease domain has a catalytic efficiency (kcat/Km) greater than about 1×108 s−1 M−1 in vitro. In embodiments, the endonuclease domain has a catalytic efficiency greater than about 1×105, 1×106, 1×107, or 1×108, s−1 M−1 in vitro. In embodiments, catalytic efficiency is determined as described in Chen et al. (2018) Science 360(6387):436-439 (incorporated herein by reference in its entirety). In some embodiments, the endonuclease domain has a catalytic efficiency (kcat/Km) greater than about 1×108 s−1 M−1 in cells. In embodiments, the endonuclease domain has a catalytic efficiency greater than about 1×105, 1×106, 1×107, or 1×108 s−1 M−1 in cells.
Gene Modifying Polypeptides Comprising Cas Domains
In some embodiments, a gene modifying polypeptide described herein comprises a Cas domain. In some embodiments, the Cas domain can direct the gene modifying polypeptide to a target site specified by a gRNA spacer, thereby modifying a target nucleic acid sequence in “cis”. In some embodiments, a gene modifying polypeptide is fused to a Cas domain. In some embodiments, a gene modifying polypeptide comprises a CRISPR/Cas domain (also referred to herein as a CRISPR-associated protein). In some embodiments, a CRISPR/Cas domain comprises a protein involved in the clustered regulatory interspaced short palindromic repeat (CRISPR) system, e.g., a Cas protein, and optionally binds a guide RNA, e.g., single guide RNA (sgRNA).
CRISPR systems are adaptive defense systems originally discovered in bacteria and archaea. CRISPR systems use RNA-guided nucleases termed CRISPR-associated or “Cas” endonucleases (e. g., Cas9 or Cpf1) to cleave foreign DNA. For example, in a typical CRISPR-Cas system, an endonuclease is directed to a target nucleotide sequence (e. g., a site in the genome that is to be sequence-edited) by sequence-specific, non-coding “guide RNAs” that target single- or double-stranded DNA sequences. Three classes (I-III) of CRISPR systems have been identified. The class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). One class II CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and a trans-activating crRNA (“tracrRNA”). The crRNA contains a “spacer” sequence, a typically about 20-nucleotide RNA sequence that corresponds to a target DNA sequence (“protospacer”). In the wild-type system, and in some engineered systems, crRNA also contains a region that binds to the tracrRNA to form a partially double-stranded structure that is cleaved by RNase III, resulting in a crRNA/tracrRNA hybrid molecule. A crRNA/tracrRNA hybrid then directs the Cas endonuclease to recognize and cleave a target DNA sequence. A target DNA sequence is generally adjacent to a “protospacer adjacent motif” (“PAM”) that is specific for a given Cas endonuclease and required for cleavage activity at a target site matching the spacer of the crRNA. CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements, e.g., as listed for exemplary Cas enzymes in Table 7; examples of PAM sequences include 5′-NGG (Streptococcus pyogenes), 5′-NNAGAA (Streptococcus thermophilus CRISPR1), 5′-NGGNG (Streptococcus thermophilus CRISPR3), and 5′-NNNGATT (Neisseria meningiditis). Some endonucleases, e.g., Cas9 endonucleases, are associated with G-rich PAM sites, e. g., 5′-NGG), and perform blunt-end cleaving of the target DNA at a location 3 nucleotides upstream from (5′ from) the PAM site. Another class II CRISPR system includes the type V endonuclease Cpf1, which is smaller than Cas9; examples include AsCpf1 (from Acidaminococcus sp.) and LbCpf1 (from Lachnospiraceae sp.). Cpf1-associated CRISPR arrays are processed into mature crRNAs without the requirement of a tracrRNA; in other words, a Cpf1 system, in some embodiments, comprises only Cpf1 nuclease and a crRNA to cleave a target DNA sequence. Cpf1 endonucleases, are typically associated with T-rich PAM sites, e. g., 5′-TTN. Cpf1 can also recognize a 5′-CTA PAM motif. Cpf1 typically cleaves a target DNA by introducing an offset or staggered double-strand break with a 4- or 5-nucleotide 5′ overhang, for example, cleaving a target DNA with a 5-nucleotide offset or staggered cut located 18 nucleotides downstream from (3′ from) from a PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complimentary strand; the 5-nucleotide overhang that results from such offset cleavage allows more precise genome editing by DNA insertion by homologous recombination than by insertion at blunt-end cleaved DNA. See, e.g., Zetsche et al. (2015) Cell, 163:759-771.
A variety of CRISPR associated (Cas) genes or proteins can be used in the technologies provided by the present disclosure and the choice of Cas protein will depend upon the particular conditions of the method. Specific examples of Cas proteins include class II systems including Cas1, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9, Cas10, Cpf1, C2C1, or C2C3. In some embodiments, a Cas protein, e.g., a Cas9 protein, may be from any of a variety of prokaryotic species. In some embodiments a particular Cas protein, e.g., a particular Cas9 protein, is selected to recognize a particular protospacer-adjacent motif (PAM) sequence. In some embodiments, a DNA-binding domain or endonuclease domain includes a sequence targeting polypeptide, such as a Cas protein, e.g., Cas9. In certain embodiments a Cas protein, e.g., a Cas9 protein, may be obtained from a bacteria or archaea or synthesized using known methods. In certain embodiments, a Cas protein may be from a gram-positive bacteria or a gram-negative bacteria. In certain embodiments, a Cas protein may be from a Streptococcus (e.g., a S. pyogenes, or a S. thermophilus), a Francisella (e.g., an F. novicida), a Staphylococcus (e.g., an S. aureus), an Acidaminococcus (e.g., an Acidaminococcus sp. BV3L6), a Neisseria (e.g., an N. meningitidis), a Cryptococcus, a Corynebacterium, a Haemophilus, a Eubacterium, a Pasteurella, a Prevotella, a Veillonella, or a Marinobacter. 
In some embodiments, a gene modifying polypeptide may comprise the amino acid sequence of SEQ ID NO: 4000 below, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In embodiments, the amino acid sequence of SEQ ID NO: 4000 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned at the N-terminal end of the gene modifying polypeptide. In embodiments, the amino acid sequence of SEQ ID NO: 4000 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids of the N-terminal end of the gene modifying polypeptide.









Exemplary N-terminal NLS-Cas9 domain



(SEQ ID NO: 4000)



MPAAKRVKLDGGDKKYSIGLDIGTNSVGWAVITDEYKVPS



 



KKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR



 



RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE



 



EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD



 



KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQ



 



LVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIA



 



QLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLS



 



KDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILR



 



VNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEK



 



YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT



 



EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQ



 



EDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMT



 



RKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK



 



VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQK



 



KAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED



 



RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTL



 



FEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRK



 



LINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFK



 



EDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVD



 



ELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIE



 



EGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVD



 



QELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARG



 



KSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER



 



GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE



 



NDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAH



 



DAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKS



 



EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIET



 



NGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGF



 



SKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLV



 



VAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK



 



GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE



 



LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHY



 



LDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQ



 



AENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDA



 



TLIHQSITGLYETRIDLSQLGGDGG






In some embodiments, a gene modifying polypeptide may comprise the amino acid sequence of SEQ ID NO: 4001 below, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In embodiments, the amino acid sequence of SEQ ID NO: 4001 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned at the C-terminal end of the gene modifying polypeptide. In embodiments, the amino acid sequence of SEQ ID NO: 4001 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids of the C-terminal end of the gene modifying polypeptide.







Exemplary C-terminal sequence comprising an NLS


(SEQ ID NO: 4001)


AGKRTADGSEFEKRTADGSEFESPKKKAKVE


 


Exemplary benchmarking sequence


(SEQ ID NO: 4002)


MPAAKRVKLDGGDKKYSIGLDIGTNSVGWAVITDEYKVPS


 


KKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR


 


RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE


 


DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKA


 


DLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLV


 


QTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQL


 


PGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD


 


TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVN


 


TEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKE


 


IFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEEL


 


LVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF


 


YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKS


 


EETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP


 


KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV


 


DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNA


 


SLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDR


 


EMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING


 


IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQ


 


KAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK


 


VMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIK


 


ELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD


 


INRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDN


 


VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLS


 


ELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKL


 


IREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL


 


NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI


 


GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET


 


GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKES


 


ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV


 


EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE


 


VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP


 


SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI


 


IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI


 


IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH


 


QSITGLYETRIDLSQLGGDGGSGGSSGGSSGSETPGTSES


 


ATPESSGGSSGGSSGGTLNIEDEYRLHETSKEPDVSLGST


 


WLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQY


 


PMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKP


 


GTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQ


 


WYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLT


 


WTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVD


 


DLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQK


 


QVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFL


 


GKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAY


 


QEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQK


 


LGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAG


 


KLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALL


 


LDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHG


 


TRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTET


 


EVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDS


 


RYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKAL


 


FLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITET


 


PDTSTLLIENSSPSGGSKRTADGSEFEAGKRTADGSEFEK


 


RTADGSEFESPKKKAKVE






In some embodiments, a gene modifying polypeptide may comprise a Cas domain as listed in Table 7 or 8, or a functional fragment thereof, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto.







TABLE 7







CRISPR/Cas Proteins, Species, and Mutations



















Mutations







Mutations to
to make





# of

alter PAM
catalytically


Name
Enzyme
Species
AAs
PAM
recognition
dead





FnCas9
Cas9
Francisella
1629
5′-NGG-3′
Wt
D11A/H969A/




novicida



N995A


 


FnCas9
Cas9
Francisella
1629
5′-YG-3′
E1369R/E1449H/
D11A/H969A/


RHA

novicida


R1556A
N995A


 


SaCas9
Cas9
Staphylococcus
1053
5′-NNGRRT-
Wt
D10A/H557A




aureus

3′




 


SaCas9
Cas9
Staphylococcus
1053
5′-NNNRRT-
E782K/N968K/
D10A/H557A


KKH

aureus

3′
R1015H



 


SpCas9
Cas9
Streptococcus
1368
5′-NGG-3′
Wt
D10A/D839A/




pyogenes



H840A/N863A


 


SpCas9
Cas9
Streptococcus
1368
5′-NGA-3′
D1135V/R1335Q/
D10A/D839A/


VQR

pyogenes


T1337R
H840A/N863A


 


AsCpf1
Cpf1
Acidaminococcus
1307
5′-TYCV-3′
S542R/K607R
E993A


RR

sp. BV3L6






 


AsCpf1
Cpf1
Acidaminococcus
1307
5′-TATV-3′
S542R/K548V/
E993A


RVR

sp. BV3L6


N552R



 


FnCpf1
Cpf1
Francisella
1300
5′-NTTN-3′
Wt
D917A/E1006A/




novicida



D1255A


 


NmCas9
Cas9
Neisseria
1082
5′-
Wt
D16A/D587A/




meningitidis

NNNGATT-

H588A/N611A






3′
















TABLE 8







Amino Acid Sequences of CRISPR/ Cas Proteins, Species, and Mutations
















SEQ






Parental

ID
Nickase
Nickase
Nickase


Variant
Host(s)
Protein Sequence
NO:
(HNH)
(HNH)
(RuvC)





Nme2Cas9
Neisseria
MAAFKPNPINYILGLDIGIASVGWAMVEIDEEENPIRLID
9,001
N611A
H588A
D16A



meningitidis
LGVRVFERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLL








RARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDR








KLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLK








GVANNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYS








HTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLM








TQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWL








TKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQA








RKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRAL








EKEGLKDKKSPLNLSSELQDEIGTAFSLFKTDEDITGRLK








DRVQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKR








YDEACAEIYGDHYGKKNTEEKIYLPPIPADEIRNPVVLRA








LSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKEIE








KRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYE








QQHGKCLYSGKEINLVRLNEKGYVEIDHALPFSRTWDDSF








NNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVE








TSRFPRSKKQRILLQKFDEDGFKECNLNDTRYVNRFLCQF








VADHILLTGKGKRRVFASNGQITNLLRGFWGLRKVRAEND








RHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDK








ETGKVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEA








DTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSG








AHKDTLRSAKRFVKHNEKISVKRVWLTEIKLADLENMVNY








KNGREIELYEALKARLEAYGGNAKQAFDPKDNPFYKKGGQ








LVKAVRVEKTQESGVLLNKKNAYTIADNGDMVRVDVFCKV








DKKGKNQYFIVPIYAWQVAENILPDIDCKGYRIDDSYTFC








FSLHKYDLIAFQKDEKSKVEFAYYINCDSSNGRFYLAWHD








KGSKEQQFRISTQNLVLIQKYQVNELGKEIRPCRLKKRPP








VR






 


PpnCas9
Pasteurella
MQNNPLNYILGLDLGIASIGWAVVEIDEESSPIRLIDVGV
9,002
N605A
H582A
D13A



pneumotropica
RTFERAEVAKTGESLALSRRLARSSRRLIKRRAERLKKAK








RLLKAEKILHSIDEKLPINVWQLRVKGLKEKLERQEWAAV








LLHLSKHRGYLSQRKNEGKSDNKELGALLSGIASNHQMLQ








SSEYRTPAEIAVKKFQVEEGHIRNQRGSYTHTFSRLDLLA








EMELLFQRQAELGNSYTSTTLLENLTALLMWQKPALAGDA








ILKMLGKCTFEPSEYKAAKNSYSAERFVWLTKLNNLRILE








NGTERALNDNERFALLEQPYEKSKLTYAQVRAMLALSDNA








IFKGVRYLGEDKKTVESKTTLIEMKFYHQIRKTLGSAELK








KEWNELKGNSDLLDEIGTAFSLYKTDDDICRYLEGKLPER








VLNALLENLNFDKFIQLSLKALHQILPLMLQGQRYDEAVS








AIYGDHYGKKSTETTRLLPTIPADEIRNPVVLRTLTQARK








VINAVVRLYGSPARIHIETAREVGKSYQDRKKLEKQQEDN








RKQRESAVKKFKEMFPHFVGEPKGKDILKMRLYELQQAKC








LYSGKSLELHRLLEKGYVEVDHALPFSRTWDDSFNNKVLV








LANENQNKGNLTPYEWLDGKNNSERWQHFVVRVQTSGFSY








AKKQRILNHKLDEKGFIERNLNDTRYVARFLCNFIADNML








LVGKGKRNVFASNGQITALLRHRWGLQKVREQNDRHHALD








AVVVACSTVAMQQKITRFVRYNEGNVFSGERIDRETGEII








PLHFPSPWAFFKENVEIRIFSENPKLELENRLPDYPQYNH








EWVQPLFVSRMPTRKMTGQGHMETVKSAKRLNEGLSVLKV








PLTQLKLSDLERMVNRDREIALYESLKARLEQFGNDPAKA








FAEPFYKKGGALVKAVRLEQTQKSGVLVRDGNGVADNASM








VRVDVFTKGGKYFLVPIYTWQVAKGILPNRAATQGKDEND








WDIMDEMATFQFSLCQNDLIKLVTKKKTIFGYFNGLNRAT








SNINIKEHDLDKSKGKLGIYLEVGVKLAISLEKYQVDELG








KNIRPCRPTKRQHVR






 


SauCas9
Staphylococcus
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEAN
9,003
N580A
H557A
D10A



aureus
VENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDH








SELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHN








VNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKK








DGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT








YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYF








PEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEK








FQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGK








PEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQS








SEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI








NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTL








VDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAR








EKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYL








IEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIP








RSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKIS








YETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKD








FINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGF








TSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKK








LDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQI








KHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTL








IVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKL








KLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKI








KYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDN








GVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQA








EFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDIT








YREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYE








VKSKKHPQIIKKG






 


SauCas9-
Staphylococcus
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEAN
9,004
N580A
H557A
D10A


KKH
aureus
VENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDH








SELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHN








VNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKK








DGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT








YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYF








PEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEK








FQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGK








PEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQS








SEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI








NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTL








VDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAR








EKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYL








IEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIP








RSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKIS








YETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKD








FINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGF








TSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKK








LDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQI








KHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTL








IVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKL








KLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKI








KYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDN








GVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQA








EFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDIT








YREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYE








VKSKKHPQIIKKG






 


SauriCas9
Staphylococcus
MQENQQKQNYILGLDIGITSVGYGLIDSKTREVIDAGVRL
9,005
N588A
H565A
D15A



auricularis
FPEADSENNSNRRSKRGARRLKRRRIHRLNRVKDLLADYQ








MIDLNNVPKSTDPYTIRVKGLREPLTKEEFAIALLHIAKR








RGLHNISVSMGDEEQDNELSTKQQLQKNAQQLQDKYVCEL








QLERLTNINKVRGEKNRFKTEDFVKEVKQLCETQRQYHNI








DDQFIQQYIDLVSTRREYFEGPGNGSPYGWDGDLLKWYEK








LMGRCTYFPEELRSVKYAYSADLFNALNDLNNLVVTRDDN








PKLEYYEKYHIIENVFKQKKNPTLKQIAKEIGVQDYDIRG








YRITKSGKPQFTSFKLYHDLKNIFEQAKYLEDVEMLDEIA








KILTIYQDEISIKKALDQLPELLTESEKSQIAQLTGYTGT








HRLSLKCIHIVIDELWESPENQMEIFTRLNLKPKKVEMSE








IDSIPTTLVDEFILSPVVKRAFIQSIKVINAVINRFGLPE








DIIIELAREKNSKDRRKFINKLQKQNEATRKKIEQLLAKY








GNTNAKYMIEKIKLHDMQEGKCLYSLEAIPLEDLLSNPTH








YEVDHIIPRSVSFDNSLNNKVLVKQSENSKKGNRTPYQYL








SSNESKISYNQFKQHILNLSKAKDRISKKKRDMLLEERDI








NKFEVQKEFINRNLVDTRYATRELSNLLKTYFSTHDYAVK








VKTINGGFTNHLRKVWDFKKHRNHGYKHHAEDALVIANAD








FLFKTHKALRRTDKILEQPGLEVNDTTVKVDTEEKYQELF








ETPKQVKNIKQFRDFKYSHRVDKKPNRQLINDTLYSTREI








DGETYVVQTLKDLYAKDNEKVKKLFTERPQKILMYQHDPK








TFEKLMTILNQYAEAKNPLAAYYEDKGEYVTKYAKKGNGP








AIHKIKYIDKKLGSYLDVSNKYPETQNKLVKLSLKSFRFD








IYKCEQGYKMVSIGYLDVLKKDNYYYIPKDKYEAEKQKKK








IKESDLFVGSFYYNDLIMYEDELFRVIGVNSDINNLVELN








MVDITYKDFCEVNNVTGEKRIKKTIGKRVVLIEKYTTDIL








GNLYKTPLPKKPQLIFKRGEL






 


SauriCas9-
Staphylococcus
MQENQQKQNYILGLDIGITSVGYGLIDSKTREVIDAGVRL
9,006
N588A
H565A
D15A


KKH
auricularis
FPEADSENNSNRRSKRGARRLKRRRIHRLNRVKDLLADYQ








MIDLNNVPKSTDPYTIRVKGLREPLTKEEFAIALLHIAKR








RGLHNISVSMGDEEQDNELSTKQQLQKNAQQLQDKYVCEL








QLERLTNINKVRGEKNRFKTEDFVKEVKQLCETQRQYHNI








DDQFIQQYIDLVSTRREYFEGPGNGSPYGWDGDLLKWYEK








LMGRCTYFPEELRSVKYAYSADLFNALNDLNNLVVTRDDN








PKLEYYEKYHIIENVFKQKKNPTLKQIAKEIGVQDYDIRG








YRITKSGKPQFTSFKLYHDLKNIFEQAKYLEDVEMLDEIA








KILTIYQDEISIKKALDQLPELLTESEKSQIAQLTGYTGT








HRLSLKCIHIVIDELWESPENQMEIFTRLNLKPKKVEMSE








IDSIPTTLVDEFILSPVVKRAFIQSIKVINAVINRFGLPE








DIIIELAREKNSKDRRKFINKLQKQNEATRKKIEQLLAKY








GNTNAKYMIEKIKLHDMQEGKCLYSLEAIPLEDLLSNPTH








YEVDHIIPRSVSFDNSLNNKVLVKQSENSKKGNRTPYQYL








SSNESKISYNQFKQHILNLSKAKDRISKKKRDMLLEERDI








NKFEVQKEFINRNLVDTRYATRELSNLLKTYFSTHDYAVK








VKTINGGFTNHLRKVWDFKKHRNHGYKHHAEDALVIANAD








FLFKTHKALRRTDKILEQPGLEVNDTTVKVDTEEKYQELF








ETPKQVKNIKQFRDFKYSHRVDKKPNRKLINDTLYSTREI








DGETYVVQTLKDLYAKDNEKVKKLFTERPQKILMYQHDPK








TFEKLMTILNQYAEAKNPLAAYYEDKGEYVTKYAKKGNGP








AIHKIKYIDKKLGSYLDVSNKYPETQNKLVKLSLKSFRFD








IYKCEQGYKMVSIGYLDVLKKDNYYYIPKDKYEAEKQKKK








IKESDLFVGSFYKNDLIMYEDELFRVIGVNSDINNLVELN








MVDITYKDFCEVNNVTGEKHIKKTIGKRVVLIEKYTTDIL








GNLYKTPLPKKPQLIFKRGEL






 


ScaCas9-
Streptococcus
MEKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTNR
9,007
N872A
H849A
D10A


Sc++
canis
KSIKKNLMGALLFDSGETAEATRLKRTARRRYTRRKNRIR








YLQEIFANEMAKLDDSFFQRLEESFLVEEDKKNERHPIFG








NLADEVAYHRNYPTIYHLRKKLADSPEKADLRLIYLALAH








IIKFRGHFLIEGKLNAENSDVAKLFYQLIQTYNQLFEESP








LDEIEVDAKGILSARLSKSKRLEKLIAVFPNEKKNGLFGN








IIALALGLTPNFKSNFDLTEDAKLQLSKDTYDDDLDELLG








QIGDQYADLFSAAKNLSDAILLSDILRSNSEVTKAPLSAS








MVKRYDEHHQDLALLKTLVRQQFPEKYAEIFKDDTKNGYA








GYVGADKKLRKRSGKLATEEEFYKFIKPILEKMDGAEELL








AKLNRDDLLRKQRTFDNGSIPHQIHLKELHAILRRQEEFY








PFLKENREKIEKILTFRIPYYVGPLARGNSRFAWLTRKSE








EAITPWNFEEVVDKGASAQSFIERMTNFDEQLPNKKVLPK








HSLLYEYFTVYNELTKVKYVTERMRKPEFLSGEQKKAIVD








LLFKTNRKVTVKQLKEDYFKKIECFDSVEIIGVEDRFNAS








LGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE








MIEERLKTYAHLFDDKVMKQLKRRHYTGWGRLSRKMINGI








RDKQSGKTILDFLKSDGFSNRNFMQLIHDDSLTFKEEIEK








AQVSGQGDSLHEQIADLAGSPAIKKGILQTVKIVDELVKV








MGHKPENIVIEMARENQTTTKGLQQSRERKKRIEEGIKEL








ESQILKENPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN








RLSDYDVDHIVPQSFIKDDSIDNKVLTRSVENRGKSDNVP








SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEA








DKAGFIKRQLVETRQITKHVARILDSRMNTKRDKNDKPIR








EVKVITLKSKLVSDFRKDFQLYKVRDINNYHHAHDAYLNA








VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK








ATAKRFFYSNIMNFFKTEVKLANGEIRKRPLIETNGETGE








VVWNKEKDFATVRKVLAMPQVNIVKKTEVQTGGFSKESIL








SKRESAKLIPRKKGWDTRKYGGFGSPTVAYSILVVAKVEK








GKAKKLKSVKVLVGITIMEKGSYEKDPIGFLEAKGYKDIK








KELIFKLPKYSLFELENGRRRMLASAKELQKANELVLPQH








LVRLLYYTQNISATTGSNNLGYIEQHREEFKEIFEKIIDF








SEKYILKNKVNSNLKSSFDEQFAVSDSILLSNSFVSLLKY








TSFGASGGFTFLDLDVKQGRLRYQTVTEVLDATLIYQSIT








GLYETRTDLSQLGGD






 


SpyCas9
Streptococcus
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
9,008
N863A
H840A
D10A



pyogenes
HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC








YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG








NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH








MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP








INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN








LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA








QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS








MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA








GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR








KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI








EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE








VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV








YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT








VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI








IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA








HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL








DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL








HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV








IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP








VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH








IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK








NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ








LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS








KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK








YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS








NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF








ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI








ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV








KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK








YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS








HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV








ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA








PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI








DLSQLGGD






 


SpyCas9-
Streptococcus
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
9,009
N863A
H840A
D10A


NG
pyogenes
HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC








YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG








NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH








MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP








INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN








LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA








QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS








MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA








GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR








KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI








EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE








VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV








YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT








VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI








IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA








HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL








DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL








HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV








IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP








VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH








IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK








NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ








LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS








KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK








YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS








NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF








ATVRKVLSMPQVNIVKKTEVQTGGFSKESIRPKRNSDKLI








ARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSV








KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK








YSLFELENGRKRMLASARFLQKGNELALPSKYVNFLYLAS








HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV








ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA








PRAFKYFDTTIDRKVYRSTKEVLDATLIHQSITGLYETRI








DLSQLGGD






 


SpyCas9-
Streptococcus
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
9,010
N863A
H840A
D10A


SpRY
pyogenes
HSIKKNLIGALLFDSGETAERTRLKRTARRRYTRRKNRIC








YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG








NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH








MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP








INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN








LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA








QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS








MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA








GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR








KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI








EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE








VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV








YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT








VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI








IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA








HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL








DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL








HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV








IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP








VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH








IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK








NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ








LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS








KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK








YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS








NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF








ATVRKVLSMPQVNIVKKTEVQTGGFSKESIRPKRNSDKLI








ARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSV








KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK








YSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLAS








HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV








ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTRLGA








PRAFKYFDTTIDPKQYRSTKEVLDATLIHQSITGLYETRI








DLSQLGGD






 


St1Cas9
Streptococcus
MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQA
9,011
N622A
H599A
D9A



thermophilus
ENNLVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFT








KISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISY








LDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQ








TYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQ








QEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGR








YRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNL








LNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLF








KYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLE








TLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGS








FSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELY








ETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNP








VVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEK








KAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHK








QLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHI








LPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDA








WSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFI








ERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTS








QLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNT








LVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLK








SKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKA








DETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQ








TFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYI








RKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQ








SVSPWRADVYFNKTTGKYEILGLKYADLQFEKGTGTYKIS








QEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQ








LFRFLSRTMPKQKHYVELKPYDKQKFEGGEALIKVLGNVA








NSGQCKKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLD








F






 


BlatCas9
Brevibacillus
MAYTMGIDVGIASCGWAIVDLERQRIIDIGVRTFEKAENP
9,012
N607A
H584A
D8A



laterosporus
KNGEALAVPRREARSSRRRLRRKKHRIERLKHMFVRNGLA








VDIQHLEQTLRSQNEIDVWQLRVDGLDRMLTQKEWLRVLI








HLAQRRGFQSNRKTDGSSEDGQVLVNVTENDRLMEEKDYR








TVAEMMVKDEKFSDHKRNKNGNYHGVVSRSSLLVEIHTLF








ETQRQHHNSLASKDFELEYVNIWSAQRPVATKDQIEKMIG








TCTFLPKEKRAPKASWHFQYFMLLQTINHIRITNVQGTRS








LNKEEIEQVVNMALTKSKVSYHDTRKILDLSEEYQFVGLD








YGKEDEKKKVESKETIIKLDDYHKLNKIFNEVELAKGETW








EADDYDTVAYALTFFKDDEDIRDYLQNKYKDSKNRLVKNL








ANKEYTNELIGKVSTLSFRKVGHLSLKALRKIIPFLEQGM








TYDKACQAAGFDFQGISKKKRSVVLPVIDQISNPVVNRAL








TQTRKVINALIKKYGSPETIHIETARELSKTFDERKNITK








DYKENRDKNEHAKKHLSELGIINPTGLDIVKYKLWCEQQG








RCMYSNQPISFERLKESGYTEVDHIIPYSRSMNDSYNNRV








LVMTRENREKGNQTPFEYMGNDTQRWYEFEQRVTTNPQIK








KEKRQNLLLKGFTNRRELEMLERNLNDTRYITKYLSHFIS








TNLEFSPSDKKKKVVNTSGRITSHLRSRWGLEKNRGQNDL








HHAMDAIVIAVTSDSFIQQVTNYYKRKERRELNGDDKFPL








PWKFFREEVIARLSPNPKEQIEALPNHFYSEDELADLQPI








FVSRMPKRSITGEAHQAQFRRVVGKTKEGKNITAKKTALV








DISYDKNGDFNMYGRETDPATYEAIKERYLEFGGNVKKAF








STDLHKPKKDGTKGPLIKSVRIMENKTLVHPVNKGKGVVY








NSSIVRTDVFQRKEKYYLLPVYVTDVTKGKLPNKVIVAKK








GYHDWIEVDDSFTFLFSLYPNDLIFIRQNPKKKISLKKRI








ESHSISDSKEVQEIHAYYKGVDSSTAAIEFIIHDGSYYAK








GVGVQNLDCFEKYQVDILGNYFKVKGEKRLELETSDSNHK








GKDVNSIKSTSR






 


cCas9-v16
Staphylococcus
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEAN
9,013
N580A
H557A
D10A



aureus
VENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDH








SELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHN








VNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKK








DGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT








YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYF








PEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEK








FQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGK








PEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQS








SEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI








NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTL








VDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAR








EKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYL








IEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIP








RSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKIS








YETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKD








FINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGF








TSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKK








LDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQI








KHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTL








IVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKL








KLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKI








KYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDN








GVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQA








EFIASFYKNDLIKINGELYRVIGVNSDKNNLIEVNMIDIT








YREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYE








VKSKKHPQIIKKG






 


cCas9-v17
Staphylococcus
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEAN
9,014
N580A

D10A



aureus
VENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDH


H557A





SELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHN








VNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKK








DGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT








YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYF








PEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEK








FQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGK








PEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQS








SEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI








NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTL








VDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAR








EKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYL








IEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIP








RSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKIS








YETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKD








FINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGF








TSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKK








LDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQI








KHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTL








IVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKL








KLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKI








KYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDN








GVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQA








EFIASFYKNDLIKINGELYRVIGVNNSTRNIVELNMIDIT








YREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYE








VKSKKHPQIIKKG






 


cCas9-v21
Staphylococcus
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEAN
9,015
N580A
H557A
D10A



aureus
VENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDH








SELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHN








VNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKK








DGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT








YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYF








PEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEK








FQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGK








PEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQS








SEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI








NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTL








VDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAR








EKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYL








IEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIP








RSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKIS








YETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKD








FINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGF








TSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKK








LDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQI








KHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTL








IVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKL








KLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKI








KYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDN








GVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQA








EFIASFYKNDLIKINGELYRVIGVNSDDRNIIELNMIDIT








YREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYE








VKSKKHPQIIKKG






 


cCas9-v42
Staphylococcus
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEAN
9,016
N580A
H557A
D10A



aureus
VENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDH








SELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHN








VNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKK








DGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT








YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYF








PEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEK








FQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGK








PEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQS








SEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI








NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTL








VDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAR








EKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYL








IEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIP








RSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKIS








YETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKD








FINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGF








TSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKK








LDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQI








KHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTL








IVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKL








KLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKI








KYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDN








GVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQA








EFIASFYKNDLIKINGELYRVIGVNNNRLNKIELNMIDIT








YREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYE








VKSKKHPQIIKKG






 


CdiCas9
Corynebacterium
MKYHVGIDVGTFSVGLAAIEVDDAGMPIKTLSLVSHIHDS
9,017
N597A
H573A
D8A



diphtheriae
GLDPDEIKSAVTRLASSGIARRTRRLYRRKRRRLQQLDKF








IQRQGWPVIELEDYSDPLYPWKVRAELAASYIADEKERGE








KLSVALRHIARHRGWRNPYAKVSSLYLPDGPSDAFKAIRE








EIKRASGQPVPETATVGQMVTLCELGTLKLRGEGGVLSAR








LQQSDYAREIQEICRMQEIGQELYRKIIDVVFAAESPKG








SASSRVGKDPLQPGKNRALKASDAFQRYRIAALIGNLRVR








VDGEKRILSVEEKNLVFDHLVNLTPKKEPEWVTIAEILGI








DRGQLIGTATMTDDGERAGARPPTHDTNRSIVNSRIAPLV








DWWKTASALEQHAMVKALSNAEVDDFDSPEGAKVQAFFAD








LDDDVHAKLDSLHLPVGRAAYSEDTLVRLTRRMLSDGVDL








YTARLQEFGIEPSWTPPTPRIGEPVGNPAVDRVLKTVSRW








LESATKTWGAPERVIIEHVREGFVTEKRAREMDGDMRRRA








ARNAKLFQEMQEKLNVQGKPSRADLWRYQSVQRQNCQCAY








CGSPITFSNSEMDHIVPRAGQGSTNTRENLVAVCHRCNQS








KGNTPFAIWAKNTSIEGVSVKEAVERTRHWVTDTGMRSTD








FKKFTKAVVERFQRATMDEEIDARSMESVAWMANELRSRV








AQHFASHGTTVRVYRGSLTAEARRASGISGKLKFFDGVGK








SRLDRRHHAIDAAVIAFTSDYVAETLAVRSNLKQSQAHRQ








EAPQWREFTGKDAEHRAAWRVWCQKMEKLSALLTEDLRDD








RVVVMSNVRLRLGNGSAHKETIGKLSKVKLSSQLSVSDID








KASSEALWCALTREPGFDPKEGLPANPERHIRVNGTHVYA








GDNIGLFPVSAGSIALRGGYAELGSSFHHARVYKITSGKK








PAFAMLRVYTIDLLPYRNQDLFSVELKPQTMSMRQAEKKL








RDALATGNAEYLGWLVVDDELVVDTSKIATDQVKAVEAEL








GTIRRWRVDGFFSPSKLRLRPLQMSKEGIKKESAPELSKI








IDRPGWLPAVNKLFSDGNVTVVRRDSLGRVRLESTAHLPV








TWKVQ






 


CjeCas9
Campylobacter
MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKT
9,018
N582A
H559A
D8A



jejuni
GESLALPRRLARSARKRLARRKARLNHLKHLIANEFKLNY








EDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDFAR








VILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQ








SVGEYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFL








KDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRALKDFS








HLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKN








TEGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYE








FKGEKGTYFIEFKKYKEFIKALGEHNLSQDDLNEIAKDIT








LIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKA








LKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNE








TYYKDEVTNPVVLRAIKEYRKVLNALLKKYGKVHKINIEL








AREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGLKIN








SKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHI








YPYSRSFDDSYMNKVLVFTKQNQEKLNQTPFEAFGNDSAK








WQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDRNLNDT








RYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVE








AKSGMLTSALRHTWGFSAKDRNNHLHHAIDAVIIAYANNS








IVKAFSDFKKEQESNSAELYAKKISELDYKNKRKFFEPFS








GFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQ








SYGGKEGVLKALELGKIRKVNGKIVKNGDMFRVDIFKHKK








TNKFYAVPIYTMDFALKVLPNKAVARSKKGEIKDWILMDE








NYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLI








VSKHDNKFETLSKNQKILFKNANEKEVIAKSIGIQNLKVF








EKYIVSALGEVTKAEFRQREDFKK






 


GeoCas9
Geobacillus
MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENP
9,019
N605A
H582A
D8A



stearo-
QTGESLALPRRLARSARRRLRRRKHRLERIRRLVIREGIL







thermophilus
TKEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLLH








LAKRRGFKSNRKSERSNKENSTMLKHIEENRAILSSYRTV








GEMIVKDPKFALHKRNKGENYTNTIARDDLEREIRLIFSK








QREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGFC








TFEPKEKRAPKATYTFQSFIAWEHINKLRLISPSGARGLT








DEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYD








RGESRKQNENIRFLELDAYHQIRKAVDKVYGKGKSSSFLP








IDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPNLANK








VYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYS








SACERAGYTFTGPKKKQKTMLLPNIPPIANPVVMRALTQA








RKVVNAIIKKYGSPVSIHIELARDLSQTFDERRKTKKEQD








ENRKKNETAIRQLMEYGLTLNPTGHDIVKFKLWSEQNGRC








AYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNKVLV








LTRENREKGNRIPAEYLGVGTERWQQFETFVLTNKQFSKK








KRDRLLRLHYDENEETEFKNRNLNDTRYISRFFANFIREH








LKFAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHH








AVDAVIVACTTPSDIAKVTAFYQRREQNKELAKKTEPHFP








QPWPHFADELRARLSKHPKESIKALNLGNYDDQKLESLQP








VFVSRMPKRSVTGAAHQETLRRYVGIDERSGKIQTVVKTK








LSEIKLDASGHFPMYGKESDPRTYEAIRQRLLEHNNDPKK








AFQEPLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTV








AYNSNIVRVDVFEKDGKYYCVPVYTMDIMKGILPNKAIEP








NKPYSEWKEMTEDYTFRFSLYPNDLIRIELPREKTVKTAA








GEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSR








TLKRFEKYQVDVLGNIYKVRGEKRVGLASSAHSKPGKTIR








PLQSTRD






 


iSpyMacCa
Streptococcus
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
9,020
N863A
H840A
D10A


s9
spp.
HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC








YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG








NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH








MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP








INASGVDAKAILSARLSKSRKLENLIAQLPGEKKNGLFGN








LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA








QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS








MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA








GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLR








KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI








EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE








VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV








YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT








VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI








IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA








HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL








DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL








HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV








IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP








VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH








IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK








NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ








LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS








KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK








YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS








NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF








ATVRKVLSMPQVNIVKKTEIQTVGQNGGLFDDNPKSPLEV








TPSKLVPLKKELNPKKYGGYQKPTTAYPVLLITDTKQLIP








ISVMNKKQFEQNPVKFLRDRGYQQVGKNDFIKLPKYTLVD








IGDGIKRLWASSKEIHKGNQLVVSKKSQILLYHAHHLDSD








LSNDYLQNHNQQFDVLFNEIISFSKKCKLGKEHIQKIENV








YSNKKNSASIEELAESFIKLLGFTQLGATSPFNFLGVKLN








QKQYKGKKDYILPCTEGTLIRQSITGLYETRVDLSKIGED








SGGSGGSKRTADGSEFES






 


NmeCas9
Neisseria
MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLID
9,021
N611A
H588A
D16A



meningitidis
LGVRVFERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLL








RTRRLLKREGVLQAANFDENGLIKSLPNTPWQLRAAALDR








KLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLK








GVAGNAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYS








HTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLM








TQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWL








TKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQA








RKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRAL








EKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLK








DRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKR








YDEACAEIYGDHYGKKNTEEKIYLPPIPADEIRNPVVLRA








LSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKEIE








KRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYE








QQHGKCLYSGKEINLGRLNEKGYVEIDHALPFSRTWDDSF








NNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVE








TSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQF








VADRMRLTGKGKKRVFASNGQITNLLRGFWGLRKVRAEND








RHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDK








ETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEA








DTLEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSG








QGHMETVKSAKRLDEGVSVLRVPLTQLKLKDLEKMVNRER








EPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQV








KAVRVEQVQKTGVWVRNHNGIADNATMVRVDVFEKGDKYY








LVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFS








LHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHK








IGKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRPP








VR






 


ScaCas9
Streptococcus
MEKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTNR
9,022
N872A
H849A
D10A



canis
KSIKKNLMGALLFDSGETAEATRLKRTARRRYTRRKNRIR








YLQEIFANEMAKLDDSFFQRLEESFLVEEDKKNERHPIFG








NLADEVAYHRNYPTIYHLRKKLADSPEKADLRLIYLALAH








IIKFRGHFLIEGKLNAENSDVAKLFYQLIQTYNQLFEESP








LDEIEVDAKGILSARLSKSKRLEKLIAVFPNEKKNGLFGN








IIALALGLTPNFKSNFDLTEDAKLQLSKDTYDDDLDELLG








QIGDQYADLFSAAKNLSDAILLSDILRSNSEVTKAPLSAS








MVKRYDEHHQDLALLKTLVRQQFPEKYAEIFKDDTKNGYA








GYVGIGIKHRKRTTKLATQEEFYKFIKPILEKMDGAEELL








AKLNRDDLLRKQRTFDNGSIPHQIHLKELHAILRRQEEFY








PFLKENREKIEKILTFRIPYYVGPLARGNSRFAWLTRKSE








EAITPWNFEEVVDKGASAQSFIERMTNFDEQLPNKKVLPK








HSLLYEYFTVYNELTKVKYVTERMRKPEFLSGEQKKAIVD








LLFKTNRKVTVKQLKEDYFKKIECFDSVEIIGVEDRFNAS








LGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE








MIEERLKTYAHLFDDKVMKQLKRRHYTGWGRLSRKMINGI








RDKQSGKTILDFLKSDGFSNRNFMQLIHDDSLTFKEEIEK








AQVSGQGDSLHEQIADLAGSPAIKKGILQTVKIVDELVKV








MGHKPENIVIEMARENQTTTKGLQQSRERKKRIEEGIKEL








ESQILKENPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN








RLSDYDVDHIVPQSFIKDDSIDNKVLTRSVENRGKSDNVP








SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEA








DKAGFIKRQLVETRQITKHVARILDSRMNTKRDKNDKPIR








EVKVITLKSKLVSDFRKDFQLYKVRDINNYHHAHDAYLNA








VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK








ATAKRFFYSNIMNFFKTEVKLANGEIRKRPLIETNGETGE








VVWNKEKDFATVRKVLAMPQVNIVKKTEVQTGGFSKESIL








SKRESAKLIPRKKGWDTRKYGGFGSPTVAYSILVVAKVEK








GKAKKLKSVKVLVGITIMEKGSYEKDPIGFLEAKGYKDIK








KELIFKLPKYSLFELENGRRRMLASATELQKANELVLPQH








LVRLLYYTQNISATTGSNNLGYIEQHREEFKEIFEKIIDF








SEKYILKNKVNSNLKSSFDEQFAVSDSILLSNSFVSLLKY








TSFGASGGFTFLDLDVKQGRLRYQTVTEVLDATLIYQSIT








GLYETRTDLSQLGGD






 


ScaCas9-
Streptococcus
MEKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTNR
9,023
N872A
H849A
D10A


HiFi-Sc++
canis
KSIKKNLMGALLFDSGETAEATRLKRTARRRYTRRKNRIR








YLQEIFANEMAKLDDSFFQRLEESFLVEEDKKNERHPIFG








NLADEVAYHRNYPTIYHLRKKLADSPEKADLRLIYLALAH








IIKFRGHFLIEGKLNAENSDVAKLFYQLIQTYNQLFEESP








LDEIEVDAKGILSARLSKSKRLEKLIAVFPNEKKNGLFGN








IIALALGLTPNFKSNFDLTEDAKLQLSKDTYDDDLDELLG








QIGDQYADLFSAAKNLSDAILLSDILRSNSEVTKAPLSAS








MVKRYDEHHQDLALLKTLVRQQFPEKYAEIFKDDTKNGYA








GYVGADKKLRKRSGKLATEEEFYKFIKPILEKMDGAEELL








AKLNRDDLLRKQRTFDNGSIPHQIHLKELHAILRRQEEFY








PFLKENREKIEKILTFRIPYYVGPLARGNSRFAWLTRKSE








EAITPWNFEEVVDKGASAQSFIERMTNFDEQLPNKKVLPK








HSLLYEYFTVYNELTKVKYVTERMRKPEFLSGEQKKAIVD








LLFKTNRKVTVKQLKEDYFKKIECFDSVEIIGVEDRFNAS








LGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE








MIEERLKTYAHLFDDKVMKQLKRRHYTGWGRLSRKMINGI








RDKQSGKTILDFLKSDGFSNANFMQLIHDDSLTFKEEIEK








AQVSGQGDSLHEQIADLAGSPAIKKGILQTVKIVDELVKV








MGHKPENIVIEMARENQTTTKGLQQSRERKKRIEEGIKEL








ESQILKENPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN








RLSDYDVDHIVPQSFIKDDSIDNKVLTRSVENRGKSDNVP








SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEA








DKAGFIKRQLVETRQITKHVARILDSRMNTKRDKNDKPIR








EVKVITLKSKLVSDFRKDFQLYKVRDINNYHHAHDAYLNA








VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK








ATAKRFFYSNIMNFFKTEVKLANGEIRKRPLIETNGETGE








VVWNKEKDFATVRKVLAMPQVNIVKKTEVQTGGFSKESIL








SKRESAKLIPRKKGWDTRKYGGFGSPTVAYSILVVAKVEK








GKAKKLKSVKVLVGITIMEKGSYEKDPIGFLEAKGYKDIK








KELIFKLPKYSLFELENGRRRMLASAKELQKANELVLPQH








LVRLLYYTQNISATTGSNNLGYIEQHREEFKEIFEKIIDF








SEKYILKNKVNSNLKSSFDEQFAVSDSILLSNSFVSLLKY








TSFGASGGFTFLDLDVKQGRLRYQTVTEVLDATLIYQSIT








GLYETRTDLSQLGGD






 


SpyCas9-
Streptococcus
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
9,024
N863A
H840A
D10A


3var-NRRH
pyogenes
HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC








YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG








NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH








MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP








INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN








LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA








QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS








MVKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA








GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR








KQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKI








EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE








VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV








YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT








VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI








IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA








HLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTIL








DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL








HEHIANLAGSPAIKKGILQTVKVVDELVKVMGGHKPENIV








IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP








VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH








IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK








NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ








LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS








KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK








YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS








NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF








ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNSDKLI








ARKKDWDPKKYGGFNSPTAAYSVLVVAKVEKGKSKKLKSV








KELLGITIMERSSFEKNPIGFLEAKGYKEVKKDLIIKLPK








YSLFELENGRKRMLASAGVLHKGNELALPSKYVNFLYLAS








HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV








ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGV








PAAFKYFDTTIDKKRYTSTKEVLDATLIHQSITGLYETRI








DLSQLGGD






 


SpyCas9-
Streptococcus
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
9,025
N863A
H840A
D10A


3var-NRTH
pyogenes
HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC








YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG








NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH








MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP








INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN








LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA








QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS








MVKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA








GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR








KQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKI








EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE








VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV








YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT








VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI








IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA








HLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTIL








DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL








HEHIANLAGSPAIKKGILQTVKVVDELVKVMGGHKPENIV








IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP








VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH








IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK








NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ








LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS








KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK








YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS








NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF








ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNSDKLI








ARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSV








KELLGITIMERSSFEKNPIGFLEAKGYKEVKKDLIIKLPK








YSLFELENGRKRMLASASVLHKGNELALPSKYVNFLYLAS








HYEKLKGSSEDNKQKQLFVEQHKHYLDEIIEQISEFSKRV








ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA








SAAFKYFDTTIGRKLYTSTKEVLDATLIHQSITGLYETRI








DLSQLGGD






 


SpyCas9-
Streptococcus
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
9,026
N863A
H840A
D10A


3var-NRCH
pyogenes
HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC








YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG








NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH








MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP








INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN








LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA








QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS








MVKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA








GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR








KQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKI








EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE








VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV








YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT








VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI








IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA








HLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTIL








DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL








HEHIANLAGSPAIKKGILQTVKVVDELVKVMGGHKPENIV








IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP








VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH








IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK








NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ








LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS








KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK








YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS








NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF








ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNSDKLI








ARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSV








KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK








YSLFELENGRKRMLASAGVLQKGNELALPSKYVNFLYLAS








HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV








ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA








PAAFKYFDTTINRKQYNTTKEVLDATLIRQSITGLYETRI








DLSQLGGD






 


SpyCas9-
Streptococcus
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
9,027
N863A
H840A
D10A


HF1
pyogenes
HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC








YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG








NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH








MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP








INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN








LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA








QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS








MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA








GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR








KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI








EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE








VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV








YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT








VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI








IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA








HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL








DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL








HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV








IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP








VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH








IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK








NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ








LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS








KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK








YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS








NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF








ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI








ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV








KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK








YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS








HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV








ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA








PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI








DLSQLGGD






 


SpyCas9-
Streptococcus
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
9,028
N863A
H840A
D10A


QQR1
pyogenes
HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC








YLQEIFSNEMAKVDDSFFHRLEESFL








VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDST








DKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI








QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLI








AQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQL








SKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL








RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEK








YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT








EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQ








EDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMT








RKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK








VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKK








AIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDR








FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLF








EDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKL








INGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE








DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDE








LVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE








GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ








ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGK








SDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG








GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEN








DKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD








AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSE








QEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN








GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS








KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV








AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG








YKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNEL








ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYL








DEIIEQISEFSKRVILADAQLDKVLSAYNKHRDKPIREQA








ENIIHLFTLTNLGAPAAFKYFDTTFKQKQYRSTKEVLDAT








LIHQSITGLYETRIDLSQLGGD






 


SpyCas9-
Streptococcus
MDKKYSIGLDIGTNSVGW
9,029
N863A
H840A
D10A


SpG
pyogenes
AVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET








AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFF








HRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL








RKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDN








SDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK








SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL








AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD








AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKAL








VRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP








ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE








LHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG








NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNF








DKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA








FLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSV








EISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED








IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTG








WGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH








DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGIL








QTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSR








ERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQN








GRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLT








RSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD








NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSR








MNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREI








NNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV








RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIR








KRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT








EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFLWPT








VAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP








IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAK








QLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLF








VEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR








DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS








TKEVLDATLIHQSITGLYETRIDLSQLGGD






 


SpyCas9-
Streptococcus
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
9,030
N863A
H840A
D10A


VQR
pyogenes
HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC








YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG








NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH








MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP








INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN








LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA








QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS








MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA








GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR








KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI








EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE








VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV








YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT








VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI








IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA








HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL








DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL








HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV








IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP








VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH








IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK








NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ








LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS








KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK








YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS








NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF








ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI








ARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSV








KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK








YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS








HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV








ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA








PAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRI








DLSQLGGD






 


SpyCas9-
Streptococcus
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
9,031
N863A
H840A
D10A


VRER
pyogenes
HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC








YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG








NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH








MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP








INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN








LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA








QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS








MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA








GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR








KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI








EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE








VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV








YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT








VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI








IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA








HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL








DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL








HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV








IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP








VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH








IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK








NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ








LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS








KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK








YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS








NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF








ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI








ARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSV








KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK








YSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLAS








HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV








ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA








PAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRI








DLSQLGGD






 


SpyCas9-
Streptococcus
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
9,032
N863A
H840A
D10A


xCas
pyogenes
HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC








YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG








NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH








MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP








INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN








LIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLA








QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS








MIKLYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA








GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR








KQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKI








EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEK








VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV








YNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVT








VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI








IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA








HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL








DFLKSDGFANRNFIQLIHDDSLTFKEDIQKAQVSGQGDSL








HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV








IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP








VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH








IVPQSF






 


SpyCas9-
Streptococcus
LKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQL
9,033
N863A
H840A
D10A


xCas-NG
pyogenes
LNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQ








ITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF








RKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES








EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFF








KTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKV








LSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDW








DPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI








TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL








ENGRKRMLASAGVLQKGNELALPSKYVNFLYLASHYEKLK








GSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADAN








LDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY








FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG








GDMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNT








DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNR








ICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPI








FGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL








AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE








NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF








GNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNL








LAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLS








ASMIKLYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNG








YAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDL








LRKQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNRE








KIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNF








EKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF








TVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRK








VTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL








KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT








YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKT








ILDFLKSDGFANRNFIQLIHDDSLTFKEDIQKAQVSGQGD








SLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPEN








IVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE








HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDV








DHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKK








MKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK








RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITL








KSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALI








KKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFF








YSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGR








DFATVRKVLSMPQVNIVKKTEVQTGGFSKESIRPKRNSDK








LIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLK








SVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKL








PKYSLFELENGRKRMLASARFLQKGNELALPSKYVNFLYL








ASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSK








RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNL








GAPRAFKYFDTTIDRKVYRSTKEVLDATLIHQSITGLYET








RIDLSQLGGD






 


St1Cas9-
Streptococcus
MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQA
9,034
N622A
H599A
D9A


CNRZ1066
thermophilus
ENNLVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFT








KISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISY








LDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQ








TYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQ








QEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGR








YRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNL








LNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLF








KYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLE








TLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGS








FSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELY








ETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNP








VVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEK








KAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHK








QLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHI








LPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDA








WSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFI








ERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTS








QLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNT








LVSYSEEQLLDIETGELISDDEYKESVFKAPYQHFVDTLK








SKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKK








DETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQ








TFEKVIEPILENYPNKQMNEKGKEVPCNPFLKYKEEHGYI








RKYSKKGNGPEIKSLKYYDSKLLGNPIDITPENSKNKVVL








QSLKPWRTDVYFNKATGKYEILGLKYADLQFEKGTGTYKI








SQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQ








QLFRFLSRTLPKQKHYVELKPYDKQKFEGGEALIKVLGNV








ANGGQCIKGLAKSNISIYKVRTDVLGNQHIIKNEGDKPKL








DF






 


St1Cas9-
Streptococcus
MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQA
9,035
N622A
H599A
D9A


LMG1831
thermophilus
ENNLVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFT








KISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISY








LDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQ








TYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQ








QEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGR








YRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNL








LNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLF








KYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLE








TLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGS








FSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELY








ETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNP








VVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEK








KAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHK








QLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHI








LPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDA








WSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFI








ERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTS








QLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNT








LVSYSEEQLLDIETGELISDDEYKESVFKAPYQHFVDTLK








SKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKK








DETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQ








TFEKVIEPILENYPNKQMNEKGKEVPCNPFLKYKEEHGYI








RKYSKKGNGPEIKSLKYYDSKLLGNPIDITPENSKNKVVL








QSLKPWRTDVYFNKNTGKYEILGLKYADLQFEKKTGTYKI








SQEKYNGIMKEEGVDSDSEFKFTLYKNDLLLVKDTETKEQ








QLFRFLSRTMPNVKYYVELKPYSKDKFEKNESLIEILGSA








DKSGRCIKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKL








DF






 


St1Cas9-
Streptococcus
MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQA
9,036
N622A
H599A
D9A


MTH17CL3
thermophilus
ENNLVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFT






96

KISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISY








LDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQ








TYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQ








QEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGR








YRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNL








LNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLF








KYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLE








TLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGS








FSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELY








ETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNP








VVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEK








KAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHK








QLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHI








LPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDA








WSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFI








ERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTS








QLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNT








LVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLK








SKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKA








DETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQ








TFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYI








RKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQ








SLKPWRTDVYFNKNTGKYEILGLKYSDMQFEKGTGKYSIS








KEQYENIKVREGVDENSEFKFTLYKNDLLLLKDSENGEQI








LLRFTSRNDTSKHYVELKPYNRQKFEGSEYLIKSLGTVAK








GGQCIKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLDF






 


St1Cas9-
Streptococcus
MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQA
9,037
N622A
H599A
D9A


TH1477
thermophilus
ENNLVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFT








KISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISY








LDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQ








TYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQ








QEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGR








YRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNL








LNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLF








KYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLE








TLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGS








FSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELY








ETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNP








VVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEK








KAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHK








QLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHI








LPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDA








WSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFI








ERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTS








QLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNT








LVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLK








SKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKA








DETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQ








TFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYI








RKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQ








SLKPWRTDVYFNKNTGKYEILGLKYSDMQFEKGTGKYSIS








KEQYENIKVREGVDENSEFKFTLYKNDLLLLKDSENGEQI








LLRFTSRNDTSKHYVELKPYNRQKFEGSEYLIKSLGTVVK








GGRCIKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLDF






 


SRGN3.1
Staphylococcus
MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEAN
9,038
N585A
H562A
D10A



spp.
VENNEGRRSKRGSRRLKRRRIHRLERVKLLLTEYDLINKE








QIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHN








VDVAADKEETASDSLSTKDQINKNAKFLESRYVCELQKER








LENEGHVRGVENRFLTKDIVREAKKIIDTQMQYYPEIDET








FKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMG








HCTYFPQELRSVKYAYSADLFNALNDLNNLIIQRDNSEKL








EYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIKGYRI








TKSGTPEFTSFKLFHDLKKVVKDHAILDDIDLLNQIAEIL








TIYQDKDSIVAELGQLEYLMSEADKQSISELTGYTGTHSL








SLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQR








IPTDMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDII








IELARENNSDDRKKFINNLQKKNEATRKRINEIIGQTGNQ








NAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEV








DHIIPRSVSFDNSYHNKVLVKQSENSKKSNLTPYQYFNSG








KSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKF








EVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKT








INGSFTDYLRKVWKFKKERNHGYKHHAEDALIIANADFLF








KENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFIIP








KQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNS








TYIVQTIKDIYAKDNTTLKKQFDKSPEKFLMYQHDPRTFE








KLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVK








SLKYIGNKLGSHLDVTHQFKSSTKKLVKLSIKNYRFDVYL








TEKGYKFVTIAYLNVFKKDNYYYIPKDKYQELKEKKKIKD








TDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYD








IKYKDYCEINNIKGEPRIKKTIGKKTESIEKFTTDVLGNL








YLHSTEKAPQLIFKRGL






 


SRGN3.3
Staphylococcus
MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEAN
9,039
N585A
H562A
D10A



spp.
VENNEGRRSKRGSRRLKRRRIHRLERVKLLLTEYDLINKE








QIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHN








VDVAADKEETASDSLSTKDQINKNAKFLESRYVCELQKER








LENEGHVRGVENRFLTKDIVREAKKIIDTQMQYYPEIDET








FKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMG








HCTYFPQELRSVKYAYSADLFNALNDLNNLIIQRDNSEKL








EYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIKGYRI








TKSGTPEFTSFKLFHDLKKVVKDHAILDDIDLLNQIAEIL








TIYQDKDSIVAELGQLEYLMSEADKQSISELTGYTGTHSL








SLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQR








IPTDMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDII








IELARENNSDDRKKFINNLQKKNEATRKRINEIIGQTGNQ








NAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEV








DHIIPRSVSFDNSYHNKVLVKQSENSKKSNLTPYQYFNSG








KSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKF








EVQKEFINRNLVDTRYATRELTSYLKAYFSANNMDVKVKT








INGSFTNHLRKVWRFDKYRNHGYKHHAEDALIIANADFLF








KENKKLQNTNKILEKPTIENNTKKVTVEKEEDYNNVFETP








KLVEDIKQYRDYKFSHRVDKKPNRQLINDTLYSTRMKDEH








DYIVQTITDIYGKDNTNLKKQFNKNPEKFLMYQNDPKTFE








KLSIIMKQYSDEKNPLAKYYEETGEYLTKYSKKNNGPIVK








KIKLLGNKVGNHLDVTNKYENSTKKLVKLSIKNYRFDVYL








TEKGYKFVTIAYLNVFKKDNYYYIPKDKYQELKEKKKIKD








TDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYD








IKYKDYCEINNIKGEPRIKKTIGKKTESIEKFTTDVLGNL








YLHSTEKAPQLIFKRGL









In some embodiments, a Cas protein requires a protospacer adjacent motif (PAM) to be present in or adjacent to a target DNA sequence for the Cas protein to bind and/or function. In some embodiments, the PAM is or comprises, from 5′ to 3′, NGG, YG, NNGRRT, NNNRRT, NGA, TYCV, TATV, NTTN, or NNNGATT, where N stands for any nucleotide, Y stands for C or T, R stands for A or G, and V stands for A or C or G. In some embodiments, a Cas protein is a protein listed in Table 7 or 8. In some embodiments, a Cas protein comprises one or more mutations altering its PAM. In some embodiments, a Cas protein comprises E1369R, E1449H, and R1556A mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises E782K, N968K, and R1015H mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises D1135V, R1335Q, and T1337R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R and K607R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R, K548V, and N552R mutations or analogous substitutions to the amino acids corresponding to said positions. Exemplary advances in the engineering of Cas enzymes to recognize altered PAM sequences are reviewed in Collias et al Nature Communications 12:555 (2021), incorporated herein by reference in its entirety.
In some embodiments, the Cas protein is catalytically active and cuts one or both strands of the target DNA site. In some embodiments, cutting the target DNA site is followed by formation of an alteration, e.g., an insertion or deletion, e.g., by the cellular repair machinery.
In some embodiments, the Cas protein is modified to deactivate or partially deactivate the nuclease, e.g., nuclease-deficient Cas9. Whereas wild-type Cas9 generates double-strand breaks (DSBs) at specific DNA sequences targeted by a gRNA, a number of CRISPR endonucleases having modified functionalities are available, for example: a “nickase” version of Cas9 that has been partially deactivated generates only a single-strand break; a catalytically inactive Cas9 (“dCas9”) does not cut target DNA. In some embodiments, dCas9 binding to a DNA sequence may interfere with transcription at that site by steric hindrance. In some embodiments, dCas9 binding to an anchor sequence may interfere with (e.g., decrease or prevent) genomic complex (e.g., ASMC) formation and/or maintenance. In some embodiments, a DNA-binding domain comprises a catalytically inactive Cas9, e.g., dCas9. Many catalytically inactive Cas9 proteins are known in the art. In some embodiments, dCas9 comprises mutations in each endonuclease domain of the Cas protein, e.g., D10A and H840A or N863A mutations. In some embodiments, a catalytically inactive or partially inactive CRISPR/Cas domain comprises a Cas protein comprising one or more mutations, e.g., one or more of the mutations listed in Table 7. In some embodiments, a Cas protein described on a given row of Table 7 comprises one, two, three, or all of the mutations listed in the same row of Table 7. In some embodiments, a Cas protein, e.g., not described in Table 7, comprises one, two, three, or all of the mutations listed in a row of Table 7 or a corresponding mutation at a corresponding site in that Cas protein.
In some embodiments, a catalytically inactive, e.g., dCas9, or partially deactivated Cas9 protein comprises a D11 mutation (e.g., D11A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H969 mutation (e.g., H969A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a N995 mutation (e.g., N995A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises mutations at one, two, or three of positions D11, H969, and N995 (e.g., D11A, H969A, and N995A mutations) or analogous substitutions to the amino acids corresponding to said positions.
In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D10 mutation (e.g., a D10A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H557 mutation (e.g., a H557A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D10 mutation (e.g., a D10A mutation) and a H557 mutation (e.g., a H557A mutation) or analogous substitutions to the amino acids corresponding to said positions.
In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D839 mutation (e.g., a D839A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H840 mutation (e.g., a H840A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a N863 mutation (e.g., a N863A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D10 mutation (e.g., D10A), a D839 mutation (e.g., D839A), a H840 mutation (e.g., H840A), and a N863 mutation (e.g., N863A) or analogous substitutions to the amino acids corresponding to said positions.
In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a E993 mutation (e.g., a E993A mutation) or an analogous substitution to the amino acid corresponding to said position.
In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D917 mutation (e.g., a D917A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a a E1006 mutation (e.g., a E1006A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D1255 mutation (e.g., a D1255A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D917 mutation (e.g., D917A), a E1006 mutation (e.g., E1006A), and a D1255 mutation (e.g., D1255A) or analogous substitutions to the amino acids corresponding to said positions.
In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D16 mutation (e.g., a D16A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D587 mutation (e.g., a D587A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a partially deactivated Cas domain has nickase activity. In some embodiments, a partially deactivated Cas9 domain is a Cas9 nickase domain. In some embodiments, the catalytically inactive Cas domain or dead Cas domain produces no detectable double strand break formation. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H588 mutation (e.g., a H588A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a N611 mutation (e.g., a N611A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D16 mutation (e.g., D16A), a D587 mutation (e.g., D587A), a H588 mutation (e.g., H588A), and a N611 mutation (e.g., N611A) or analogous substitutions to the amino acids corresponding to said positions.
In some embodiments, a DNA-binding domain or endonuclease domain may comprise a Cas molecule comprising or linked (e.g., covalently) to a gRNA (e.g., a template nucleic acid, e.g., template RNA, comprising a gRNA).
In some embodiments, an endonuclease domain or DNA binding domain comprises a Streptococcus pyogenes Cas9 (SpCas9) or a functional fragment or variant thereof. In some embodiments, the endonuclease domain or DNA binding domain comprises a modified SpCas9. In embodiments, the modified SpCas9 comprises a modification that alters protospacer-adjacent motif (PAM) specificity. In embodiments, the PAM has specificity for the nucleic acid sequence 5′-NGT-3′. In embodiments, the modified SpCas9 comprises one or more amino acid substitutions, e.g., at one or more of positions L1111, D1135, G1218, E1219, A1322, of R1335, e.g., selected from L1111R, D1135V, G1218R, E1219F, A1322R, R1335V. In embodiments, the modified SpCas9 comprises the amino acid substitution T1337R and one or more additional amino acid substitutions, e.g., selected from L1111, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T1337, T1337L, T1337Q, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereto. In embodiments, the modified SpCas9 comprises: (i) one or more amino acid substitutions selected from D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q, and T1337; and (ii) one or more amino acid substitutions selected from L1111R, G1218R, E1219F, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereto.
In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas domain, e.g., a Cas9 domain. In embodiments, the endonuclease domain or DNA binding domain comprises a nuclease-active Cas domain, a Cas nickase (nCas) domain, or a nuclease-inactive Cas (dCas) domain. In embodiments, the endonuclease domain or DNA binding domain comprises a nuclease-active Cas9 domain, a Cas9 nickase (nCas9) domain, or a nuclease-inactive Cas9 (dCas9) domain. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas9 domain of Cas9 (e.g., dCas9 and nCas9), Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, or Cas12i. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas9 (e.g., dCas9 and nCas9), Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, or Cas12i. In some embodiments, the endonuclease domain or DNA binding domain comprises an S. pyogenes or an S. thermophilus Cas9, or a functional fragment thereof. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas9 sequence, e.g., as described in Chylinski, Rhun, and Charpentier (2013) RNA Biology 10:5, 726-737; incorporated herein by reference. In some embodiments, the endonuclease domain or DNA binding domain comprises the HNH nuclease subdomain and/or the RuvC1 subdomain of a Cas, e.g., Cas9, e.g., as described herein, or a variant thereof. In some embodiments, the endonuclease domain or DNA binding domain comprises Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, or Cas12i. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas polypeptide (e.g., enzyme), or a functional fragment thereof. In embodiments, the Cas polypeptide (e.g., enzyme) is selected from Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cash, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csn1 or Csx12), Cas10, Cas10d, Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12b/C2c1, Cas12c/C2c3, SpCas9(K855A), eSpCas9(1.1), SpCas9-HF1, hyper accurate Cas9 variant (HypaCas9), homologues thereof, modified or engineered versions thereof, and/or functional fragments thereof. In embodiments, the Cas9 comprises one or more substitutions, e.g., selected from H840A, D10A, P475A, W476A, N477A, D1125A, W1126A, and D1127A. In embodiments, the Cas9 comprises one or more mutations at positions selected from: D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987, e.g., one or more substitutions selected from D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and/or D986A. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas (e.g., Cas9) sequence from Corynebacterium ulcerans, Corynebacterium diphtheria, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus iniae, Belliella baltica, Psychroflexus torquis, Streptococcus thermophilus, Listeria innocua, Campylobacter jejuni, Neisseria meningitidis, Streptococcus pyogenes, or Staphylococcus aureus, or a fragment or variant thereof.
In some embodiments, the endonuclease domain or DNA binding domain comprises a Cpf1 domain, e.g., comprising one or more substitutions, e.g., at position D917, E1006A, D1255 or any combination thereof, e.g., selected from D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, and D917A/E1006A/D1255A.
In some embodiments, the endonuclease domain or DNA binding domain comprises spCas9, spCas9-VRQR, spCas9-VRER, xCas9 (sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL.
In some embodiments, a gene modifying polypeptide has an endonuclease domain comprising a Cas9 nickase, e.g., Cas9 H840A. In embodiments, the Cas9 H840A has the following amino acid sequence:









Cas9 nickase (H840A):



(SEQ ID NO: 11,001)



DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS



 



IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL



 



QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI



 



VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI



 



KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN



 



ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI



 



ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI



 



GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI



 



KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY



 



IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ



 



RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK



 



ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV



 



DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN



 



ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK



 



QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK



 



DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL



 



FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF



 



LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE



 



HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE



 



MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE



 



NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIV



 



PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY



 



WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV



 



ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL



 



VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP



 



KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI



 



MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT



 



VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR



 



KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKE



 



LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS



 



LFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY



 



EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL



 



ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA



 



AFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL



 



SQLGGD






In some embodiments, a gene modifying polypeptide comprises a dCas9 sequence comprising a D10A and/or H840A mutation, e.g., the following sequence:







(SEQ ID NO: 5007)


SMDKKYSIGLAIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIG


 


ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFH


 


RLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKA


 


DLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEN


 


PINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLT


 


PNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDA


 


ILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKE


 


IFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL


 


RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIP


 


YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFD


 


KNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV


 


DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLK


 


IIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK


 


QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHD


 


DSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK


 


VMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEH


 


PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKD


 


DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN


 


LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKL


 


IREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIK


 


KYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE


 


ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE


 


VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV


 


EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLP


 


KYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP


 


EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRD


 


KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH


 


QSITGLYETRIDLSQLGGD






TAL Effectors and Zinc Finger Nucleases

In some embodiments, an endonuclease domain or DNA-binding domain comprises a TAL effector molecule. A TAL effector molecule, e.g., a TAL effector molecule that specifically binds a DNA sequence, typically comprises a plurality of TAL effector domains or fragments thereof, and optionally one or more additional portions of naturally occurring TAL effectors (e.g., N- and/or C-terminal of the plurality of TAL effector domains). Many TAL effectors are known to those of skill in the art and are commercially available, e.g., from Thermo Fisher Scientific.
Naturally occurring TALEs are natural effector proteins secreted by numerous species of bacterial pathogens including the plant pathogen Xanthomonas which modulates gene expression in host plants and facilitates bacterial colonization and survival. The specific binding of TAL effectors is based on a central repeat domain of tandemly arranged nearly identical repeats of typically 33 or 34 amino acids (the repeat-variable di-residues, RVD domain).
Members of the TAL effectors family differ mainly in the number and order of their repeats. The number of repeats typically ranges from 1.5 to 33.5 repeats and the C-terminal repeat is usually shorter in length (e.g., about 20 amino acids) and is generally referred to as a “half-repeat.” Each repeat of the TAL effector generally features a one-repeat-to-one-base-pair correlation with different repeat types exhibiting different base-pair specificity (one repeat recognizes one base-pair on the target gene sequence). Generally, the smaller the number of repeats, the weaker the protein-DNA interactions. A number of 6.5 repeats has been shown to be sufficient to activate transcription of a reporter gene (Scholze et al., 2010).
Repeat to repeat variations occur predominantly at amino acid positions 12 and 13, which have therefore been termed “hypervariable” and which are responsible for the specificity of the interaction with the target DNA promoter sequence, as shown in Table 9 listing exemplary repeat variable diresidues (RVD) and their correspondence to nucleic acid base targets.







TABLE 9







RVDs and Nucleic Acid Base Specificity








Target
Possible RVD Amino Acid Combinations























A
NI
NN
NI
HI
KI










G
NN
GN
SN
VN
LN
DN
QN
EN
HN
RH
NK
AN
FN


C
HD
RD
KD
ND
AD


T
NG
HG
VG
IG
EG
MG
YG
AA
EP
VA
QG
KG
RG









Accordingly, it is possible to modify the repeats of a TAL effector to target specific DNA sequences. Further studies have shown that the RVD NK can target G. Target sites of TAL effectors also tend to include a T flanking the 5′ base targeted by the first repeat, but the exact mechanism of this recognition is not known. More than 113 TAL effector sequences are known to date. Non-limiting examples of TAL effectors from Xanthomonas include, Hax2, Hax3, Hax4, AvrXa7, AvrXa10 and AvrBs3.
Accordingly, the TAL effector domain of a TAL effector molecule described herein may be derived from a TAL effector from any bacterial species (e.g., Xanthomonas species such as the African strain of Xanthomonas oryzae pv. Oryzae (Yu et al. 2011), Xanthomonas campestris pv. raphani strain 756C and Xanthomonas oryzae pv. oryzicola strain BLS256 (Bogdanove et al. 2011). In some embodiments, the TAL effector domain comprises an RVD domain as well as flanking sequence(s) (sequences on the N-terminal and/or C-terminal side of the RVD domain) also from the naturally occurring TAL effector. It may comprise more or fewer repeats than the RVD of the naturally occurring TAL effector. The TAL effector molecule can be designed to target a given DNA sequence based on the above code and others known in the art. The number of TAL effector domains (e.g., repeats (monomers or modules)) and their specific sequence can be selected based on the desired DNA target sequence. For example, TAL effector domains, e.g., repeats, may be removed or added in order to suit a specific target sequence. In an embodiment, the TAL effector molecule of the present invention comprises between 6.5 and 33.5 TAL effector domains, e.g., repeats. In an embodiment, TAL effector molecule of the present invention comprises between 8 and 33.5 TAL effector domains, e.g., repeats, e.g., between 10 and 25 TAL effector domains, e.g., repeats, e.g., between 10 and 14 TAL effector domains, e.g., repeats.
In some embodiments, the TAL effector molecule comprises TAL effector domains that correspond to a perfect match to the DNA target sequence. In some embodiments, a mismatch between a repeat and a target base-pair on the DNA target sequence is permitted as along as it allows for the function of the polypeptide comprising the TAL effector molecule. In general, TALE binding is inversely correlated with the number of mismatches. In some embodiments, the TAL effector molecule of a polypeptide of the present invention comprises no more than 7 mismatches, 6 mismatches, 5 mismatches, 4 mismatches, 3 mismatches, 2 mismatches, or 1 mismatch, and optionally no mismatch, with the target DNA sequence. Without wishing to be bound by theory, in general the smaller the number of TAL effector domains in the TAL effector molecule, the smaller the number of mismatches will be tolerated and still allow for the function of the polypeptide comprising the TAL effector molecule. The binding affinity is thought to depend on the sum of matching repeat-DNA combinations. For example, TAL effector molecules having 25 TAL effector domains or more may be able to tolerate up to 7 mismatches.
In addition to the TAL effector domains, the TAL effector molecule of the present invention may comprise additional sequences derived from a naturally occurring TAL effector. The length of the C-terminal and/or N-terminal sequence(s) included on each side of the TAL effector domain portion of the TAL effector molecule can vary and be selected by one skilled in the art, for example based on the studies of Zhang et al. (2011). Zhang et al., have characterized a number of C-terminal and N-terminal truncation mutants in Hax3 derived TAL-effector based proteins and have identified key elements, which contribute to optimal binding to the target sequence and thus activation of transcription. Generally, it was found that transcriptional activity is inversely correlated with the length of N-terminus. Regarding the C-terminus, an important element for DNA binding residues within the first 68 amino acids of the Hax 3 sequence was identified. Accordingly, in some embodiments, the first 68 amino acids on the C-terminal side of the TAL effector domains of the naturally occurring TAL effector is included in the TAL effector molecule. Accordingly, in an embodiment, a TAL effector molecule comprises 1) one or more TAL effector domains derived from a naturally occurring TAL effector; 2) at least 70, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260, 270, 280 or more amino acids from the naturally occurring TAL effector on the N-terminal side of the TAL effector domains; and/or 3) at least 68, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260 or more amino acids from the naturally occurring TAL effector on the C-terminal side of the TAL effector domains.
In some embodiments, an endonuclease domain or DNA-binding domain is or comprises a Zn finger molecule. A Zn finger molecule comprises a Zn finger protein, e.g., a naturally occurring Zn finger protein or engineered Zn finger protein, or fragment thereof. Many Zn finger proteins are known to those of skill in the art and are commercially available, e.g., from Sigma-Aldrich.
In some embodiments, a Zn finger molecule comprises a non-naturally occurring Zn finger protein that is engineered to bind to a target DNA sequence of choice. See, for example, Beerli, et al. (2002) Nature Biotechnol. 20:135-141; Pabo, et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan, et al. (2001) Nature Biotechnol. 19:656-660; Segal, et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo, et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528; 2005/0267061, all incorporated herein by reference in their entireties.
An engineered Zn finger protein may have a novel binding specificity, compared to a naturally-occurring Zn finger protein. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual Zn finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated by reference herein in their entireties.
Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as International Patent Publication Nos. WO 98/37186; WO 98/53057; WO 00/27878; and WO 01/88197 and GB 2,338,237. In addition, enhancement of binding specificity for zinc finger proteins has been described, for example, in International Patent Publication No. WO 02/077227.
In addition, as disclosed in these and other references, zinc finger domains and/or multi-fingered zinc finger proteins may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in co-owned International Patent Publication No. WO 02/077227.
Zn finger proteins and methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and described in detail in U.S. Pat. Nos. 6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; and 6,200,759; International Patent Publication Nos. WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197; WO 02/099084; WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536; and WO 03/016496.
In addition, as disclosed in these and other references, Zn finger proteins and/or multi-fingered Zn finger proteins may be linked together, e.g., as a fusion protein, using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length. The Zn finger molecules described herein may include any combination of suitable linkers between the individual zinc finger proteins and/or multi-fingered Zn finger proteins of the Zn finger molecule.
In certain embodiments, the DNA-binding domain or endonuclease domain comprises a Zn finger molecule comprising an engineered zinc finger protein that binds (in a sequence-specific manner) to a target DNA sequence. In some embodiments, the Zn finger molecule comprises one Zn finger protein or fragment thereof. In other embodiments, the Zn finger molecule comprises a plurality of Zn finger proteins (or fragments thereof), e.g., 2, 3, 4, 5, 6 or more Zn finger proteins (and optionally no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 Zn finger proteins). In some embodiments, the Zn finger molecule comprises at least three Zn finger proteins. In some embodiments, the Zn finger molecule comprises four, five or six fingers. In some embodiments, the Zn finger molecule comprises 8, 9, 10, 11 or 12 fingers. In some embodiments, a Zn finger molecule comprising three Zn finger proteins recognizes a target DNA sequence comprising 9 or 10 nucleotides. In some embodiments, a Zn finger molecule comprising four Zn finger proteins recognizes a target DNA sequence comprising 12 to 14 nucleotides. In some embodiments, a Zn finger molecule comprising six Zn finger proteins recognizes a target DNA sequence comprising 18 to 21 nucleotides.
In some embodiments, a Zn finger molecule comprises a two-handed Zn finger protein. Two handed zinc finger proteins are those proteins in which two clusters of zinc finger proteins are separated by intervening amino acids so that the two zinc finger domains bind to two discontinuous target DNA sequences. An example of a two handed type of zinc finger binding protein is SIP1, where a cluster of four zinc finger proteins is located at the amino terminus of the protein and a cluster of three Zn finger proteins is located at the carboxyl terminus (see Remade, et al. (1999) EMBO Journal 18(18):5073-5084). Each cluster of zinc fingers in these proteins is able to bind to a unique target sequence and the spacing between the two target sequences can comprise many nucleotides.
Linkers
In some embodiments, a gene modifying polypeptide may comprise a linker, e.g., a peptide linker, e.g., a linker as described in Table 10. In some embodiments, a gene modifying polypeptide comprises, in an N-terminal to C-terminal direction, a Cas domain (e.g., a Cas domain of Table 8), a linker of Table 10 (or a sequence having at least 70%, 80%, 85%, 90%, 95%, or 99% identity thereto), and an RT domain (e.g., an RT domain of Table 6). In some embodiments, a gene modifying polypeptide comprises a flexible linker between the endonuclease and the RT domain, e.g., a linker comprising the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 11,002). In some embodiments, an RT domain of a gene modifying polypeptide may be located C-terminal to the endonuclease domain. In some embodiments, an RT domain of a gene modifying polypeptide may be located N-terminal to the endonuclease domain.







TABLE 10







Exemplary linker sequences











SEQ



Amino Acid Sequence
ID NO






GGS



 



GGSGGS
5102


 



GGSGGSGGS
5103


 



GGSGGSGGSGGS
5104


 



GGSGGSGGSGGSGGS
5105


 



GGSGGSGGSGGSGGSGGS
5106


 



GGGGS
5107


 



GGGGSGGGGS
5108


 



GGGGSGGGGSGGGGS
5109


 



GGGGGGGGSGGGGSGGGGS
5110


 



GGGGGGGGSGGGGSGGGGSGGGGS
5111


 



GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS
5112


 



GGG



 



GGGG
5114


 



GGGGG
5115


 



GGGGGG
5116


 



GGGGGGG
5117


 



GGGGGGGG
5118


 



GSS



 



GSSGSS
5120


 



GSSGSSGSS
5121


 



GSSGSSGSSGSS
5122


 



GSSGSSGSSGSSGSS
5123


 



GSSGSSGSSGSSGSSGSS
5124


 



EAAAK
5125


 



EAAAKEAAAK
5126


 



EAAAKEAAAKEAAAK
5127


 



EAAAKEAAAKEAAAKEAAAK
5128


 



EAAAKEAAAKEAAAKEAAAKEAAAK
5129


 



EAAAKEAAAKEAAAKEAAAKEAAAKEAAAK
5130


 



PAP



 



PAPAP
5132


 



PAPAPAP
5133


 



PAPAPAPAP
5134


 



PAPAPAPAPAP
5135


 



PAPAPAPAPAPAP
5136


 



GGSGGG
5137


 



GGGGGS
5138


 



GGSGSS
5139


 



GSSGGS
5140


 



GGSEAAAK
5141


 



EAAAKGGS
5142


 



GGSPAP
5143


 



PAPGGS
5144


 



GGGGSS
5145


 



GSSGGG
5146


 



GGGEAAAK
5147


 



EAAAKGGG
5148


 



GGGPAP
5149


 



PAPGGG
5150


 



GSSEAAAK
5151


 



EAAAKGSS
5152


 



GSSPAP
5153


 



PAPGSS
5154


 



EAAAKPAP
5155


 



PAPEAAAK
5156


 



GGSGGGGSS
5157


 



GGSGSSGGG
5158


 



GGGGGSGSS
5159


 



GGGGSSGGS
5160


 



GSSGGSGGG
5161


 



GSSGGGGGS
5162


 



GGSGGGEAAAK
5163


 



GGSEAAAKGGG
5164


 



GGGGGSEAAAK
5165


 



GGGEAAAKGGS
5166


 



EAAAKGGSGGG
5167


 



EAAAKGGGGGS
5168


 



GGSGGGPAP
5169


 



GGSPAPGGG
5170


 



GGGGGSPAP
5171


 



GGGPAPGGS
5172


 



PAPGGSGGG
5173


 



PAPGGGGGS
5174


 



GGSGSSEAAAK
5175


 



GGSEAAAKGSS
5176


 



GSSGGSEAAAK
5177


 



GSSEAAAKGGS
5178


 



EAAAKGGSGSS
5179


 



EAAAKGSSGGS
5180


 



GGSGSSPAP
5181


 



GGSPAPGSS
5182


 



GSSGGSPAP
5183


 



GSSPAPGGS
5184


 



PAPGGSGSS
5185


 



PAPGSSGGS
5186


 



GGSEAAAKPAP
5187


 



GGSPAPEAAAK
5188


 



EAAAKGGSPAP
5189


 



EAAAKPAPGGS
5190


 



PAPGGSEAAAK
5191


 



PAPEAAAKGGS
5192


 



GGGGSSEAAAK
5193


 



GGGEAAAKGSS
5194


 



GSSGGGEAAAK
5195


 



GSSEAAAKGGG
5196


 



EAAAKGGGGSS
5197


 



EAAAKGSSGGG
5198


 



GGGGSSPAP
5199


 



GGGPAPGSS
5200


 



GSSGGGPAP
5201


 



GSSPAPGGG
5202


 



PAPGGGGSS
5203


 



PAPGSSGGG
5204


 



GGGEAAAKPAP
5205


 



GGGPAPEAAAK
5206


 



EAAAKGGGPAP
5207


 



EAAAKPAPGGG
5208


 



PAPGGGEAAAK
5209


 



PAPEAAAKGGG
5210


 



GSSEAAAKPAP
5211


 



GSSPAPEAAAK
5212


 



EAAAKGSSPAP
5213


 



EAAAKPAPGSS
5214


 



PAPGSSEAAAK
5215


 



PAPEAAAKGSS
5216


 



AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAA
5217



AKEAAAKEAAAKA



 



GGGGSEAAAKGGGGS
5218


 



EAAAKGGGGSEAAAK
5219


 



SGSETPGTSESATPES
5220


 



GSAGSAAGSGEF
5221


 



SGGSSGGSSGSETPGTSESATPESSGGSSGGSS
5222









In some embodiments, a linker of a gene modifying polypeptide comprises a motif chosen from: (SGGS)n (SEQ ID NO: 5025), (GGGS)n (SEQ ID NO: 5026), (GGGGS)n (SEQ ID NO: 5027), (G)n, (EAAAK)n (SEQ ID NO: 5028), (GGS)n, or (XP)n.
Gene Modifying Polypeptide Selection by Pooled Screening
Candidate gene modifying polypeptides may be screened to evaluate a candidate's gene editing ability. For example, an RNA gene modifying system designed for the targeted editing of a coding sequence in the human genome may be used. In certain embodiments, such a gene modifying system may be used in conjunction with a pooled screening approach.
For example, a library of gene modifying polypeptide candidates and a template guide RNA (tgRNA) may be introduced into mammalian cells to test the candidates' gene editing abilities by a pooled screening approach. In specific embodiments, a library of gene modifying polypeptide candidates is introduced into mammalian cells followed by introduction of the tgRNA into the cells.
Representative, non-limiting examples of mammalian cells that may be used in screening include HEK293T cells, U2OS cells, HeLa cells, HepG2 cells, Huh7 cells, K562 cells, or iPS cells.
A gene modifying polypeptide candidate may comprise 1) a Cas-nuclease, for example a wild-type Cas nuclease, e.g., a wild-type Cas9 nuclease, a mutant Cas nuclease, e.g., a Cas nickase, for example, a Cas9 nickase such as a Cas9 N863A nickase, or a Cas nuclease selected from Table 7 or Table 8, 2) a peptide linker, e.g., a sequence from Table D or Table 10, that may exhibit varying degrees of length, flexibility, hydrophobicity, and/or secondary structure; and 3) a reverse transcriptase (RT), e.g. an RT domain from Table D or Table 6. A gene modifying polypeptide candidate library comprises: a plurality of different gene modifying polypeptide candidates that differ from each other with respect to one, two or all three of the Cas nuclease, peptide linker or RT domain components, or a plurality of nucleic acid expression vectors that encode such gene modifying polypeptide candidates.
For screening of gene modifying polypeptide candidates, a two-component system may be used that comprises a gene modifying polypeptide component and a tgRNA component. A gene modifying component may comprise, for example, an expression vector, e.g., an expression plasmid or lentiviral vector, that encodes a gene modifying polypeptide candidate, for example, comprises a human codon-optimized nucleic acid that encodes a gene modifying polypeptide candidate, e.g., a Cas-linker-RT fusion as described above. In a particular embodiment, a lentiviral cassette is utilized that comprises: (i) a promoter for expression in mammalian cells, e.g., a CMV promoter; (ii) a gene modifying library candidate, e.g. a Cas-linker-RT fusion comprising a Cas nuclease of Table 7 or Table 8, a peptide linker of Table 10, and an RT of Table 6, for example a Cas-linker-RT fusion as in Table D; (iii) a self-cleaving polypeptide, e.g., a T2A peptide; (iv) a marker enabling selection in mammalian cells, e.g., a puromycin resistance gene; and (v) a termination signal, e.g., a poly A tail.
The tgRNA component may comprise a tgRNA or expression vector, e.g., an expression plasmid, that produces the tgRNA, for example, utilizes a U6 promoter to drive expression of the tgRNA, wherein the tgRNA is a non-coding RNA sequence that is recognized by Cas and localizes it to the genomic locus of interest, and that also templates reverse transcription of the desired edit into the genome by the RT domain.
To prepare a pool of cells expressing gene modifying polypeptide library candidates, mammalian cells, e.g., HEK293T or U2OS cells, may be transduced with pooled gene modifying polypeptide candidate expression vector preparations, e.g., lentiviral preparations, of the gene modifying candidate polypeptide library. In a particular embodiment, lentiviral plasmids are utilized, and HEK293 Lenti-X cells are seeded in 15 cm plates (˜12×106 cells) prior to lentiviral plasmid transfection. In such an embodiment, lentiviral plasmid transfection may be performed using the Lentiviral Packaging Mix (Biosettia) and transfection of the plasmid DNA for the gene modifying candidate library is performed the following day using Lipofectamine 2000 and Opti-MEM media according to the manufacturer's protocol. In such an embodiment, extracellular DNA may be removed by a full media change the next day and virus-containing media may be harvested 48 hours after. Lentiviral media may be concentrated using Lenti-X Concentrator (TaKaRa Biosciences) and 5 mL lentiviral aliquots may be made and stored at −80° C. Lentiviral titering is performed by enumerating colony forming units post-selection, e.g., post Puromycin selection.
For monitoring gene editing of a target DNA, mammalian cells, e.g., HEK293T or U2OS cells, carrying a target DNA may be utilized. In other embodiments for monitoring gene editing of a target DNA, mammalian cells, e.g., HEK293T or U2OS cells, carrying a target DNA genomic landing pad may be utilized. In particular embodiments, the target DNA genomic landing pad may comprise a gene to be edited for treatment of a disease or disorder of interest. In other particular embodiments, the target DNA is a gene sequence that expresses a protein that exhibits detectable characteristics that may be monitored to determine whether gene editing has occurred. For example, in certain embodiments, a blue fluorescence protein (BFP)- or green fluorescence protein (GFP)-expressing genomic landing pad is utilized. In certain embodiments, mammalian cells, e.g., HEK293T or U2OS cells, comprising a target DNA, e.g., a target DNA genomic landing pad, are seeded in culture plates at 500×-3000× cells per gene modifying library candidate and transduced at a 0.2-0.3 multiplicity of infection (MOI) to minimize multiple infections per cell. Puromycin (2.5 ug/mL) may be added 48 hours post infection to allow for selection of infected cells. In such an embodiment, cells may be kept under puromycin selection for at least 7 days and then scaled up for tgRNA introduction, e.g., tgRNA electroporation.
To ascertain whether gene editing occurs, mammalian cells containing a target DNA to be edited may be infected with gene modifying polypeptide library candidates then transfected with tgRNA designed for use in editing of the target DNA. Subsequently, the cells may be analyzed to determine whether editing of the target locus has occurred according to the designed outcome, or whether no editing or imperfect editing has occurred, e.g., by using cell sorting and sequence analysis.
In a particular embodiment, to ascertain whether genome editing occurs, BFP- or GFP-expressing mammalian cells, e.g., HEK293T or U2OS cells, may be infected with gene modifying library candidates and then transfected or electroporated with tgRNA plasmid or RNA, e.g., by electroporation of 250,000 cells/well with 200 ng of a tgRNA plasmid designed to convert BFP-to-GFP or GFP-to-BFP, at a cell count ensuring >250×-1000× coverage per library candidate. In such an embodiment, the genome-editing capacity of the various constructs in this assay may be assessed by sorting the cells by Fluorescence-Activated Cell Sorting (FACS) for expression of the color-converted fluorescent protein (FP) at 4-10 days post-electroporation. Cells are sorted and harvested as distinct populations of unedited cells (exhibiting original florescence protein signal), edited cells (exhibiting converted fluorescence protein signal), and imperfect edit (exhibiting no florescence protein signal) cells. A sample of unsorted cells may also be harvested as the input population to determine candidate enrichment during analysis.
To determine which gene modifying library candidates exhibit genome-editing capacity in an assay, genomic DNA (gDNA) is harvested from the sorted cell populations, and analyzed by sequencing the gene modifying library candidates in each population. Briefly, gene modifying candidates may be amplified from the genome using primers specific to the gene modifying polypeptide expression vector, e.g., the lentiviral cassette, amplified in a second round of PCR to dilute genomic DNA, and then sequenced, for example, sequenced by a next-generation sequencing platform. After quality control of sequencing reads, reads of at least about 1500 nucleotides and generally no more than about 3200 nucleotides are mapped to the gene modifying polypeptide library sequences and those containing a minimum of about an 80% match to a library sequence are considered to be successfully aligned to a given candidate for purposes of this pooled screen. In order to identify candidates capable of performing gene editing in the assay, e.g., the BFP-to-GFP or GFP-to-BFP edit, the read count of each library candidate in the edited population is compared to its read count in the initial, unsorted population.
For purposes of pooled screening, gene modifying candidates with genome-editing capacity are identified based on enrichment in the edited (converted FP) population relative to unsorted (input) cells. In some embodiments, an enrichment of at least 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or at least 100-fold over the input indicates potentially useful gene editing activity, e.g., at least 2-fold enrichment. In some embodiments, the enrichment is converted to a log-value by taking the log base 2 of the enrichment ratio. In some embodiments, a log 2 enrichment score of at least 0, 1, 2, 3, 4, 5, 5.5, 6.0, 6.2, 6.3, 6.4, 6.5, or at least 6.6 indicates potentially useful gene editing activity, e.g., a log 2 enrichment score of at least 1.0. In particular embodiments, enrichment values observed for gene modifying candidates may be compared to enrichment values observed under similar conditions utilizing a reference, e.g., Element ID No: 17380.
In some embodiments, multiple tgRNAs may be used to screen the gene modifying candidate library. In particular embodiments, a plurality of tgRNAs may be utilized to optimize template/Cas-linker-RT fusion pairs, e.g., for gene editing of particular target genes, for example, gene targets for the treatment of disease. In specific embodiments, a pooled approach to screening gene modifying candidates may be performed using a multiplicity of different tgRNAs in an arrayed format.
In some embodiments, multiple types of edits, e.g., insertions, substitutions, and/or deletions of different lengths, may be used to screen the gene modifying candidate library.
In some embodiments, multiple target sequences, e.g., different fluorescent proteins, may be used to screen the gene modifying candidate library. In some embodiments, multiple target sequences, e.g., different fluorescent proteins, may be used to screen the gene modifying candidate library. In some embodiments, multiple cell types, e.g., HEK293T or U20S, may be used to screen the gene modifying candidate library. The person of ordinary skill in the art will appreciate that a given candidate may exhibit altered editing capacity or even the gain or loss of any observable or useful activity across different conditions, including tgRNA sequence (e.g., nucleotide modifications, PBS length, RT template length), target sequence, target location, type of edit, location of mutation relative to the first-strand nick of the gene modifying polypeptide, or cell type. Thus, in some embodiments, gene modifying library candidates are screened across multiple parameters, e.g., with at least two distinct tgRNAs in at least two cell types, and gene editing activity is identified by enrichment in any single condition. In other embodiments, a candidate with more robust activity across different tgRNA and cell types is identified by enrichment in at least two conditions, e.g., in all conditions screened. For clarity, candidates found to exhibit little to no enrichment under any given condition are not assumed to be inactive across all conditions and may be screened with different parameters or reconfigured at the polypeptide level, e.g., by swapping, shuffling, or evolving domains (e.g., RT domain), linkers, or other signals (e.g., NLS).
Sequences of Exemplary Cas9-Linker-RT Fusions
In some embodiments, a gene modifying polypeptide comprises a linker sequence and an RT sequence. In some embodiments, a gene modifying polypeptide comprises a linker sequence as listed in Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises the amino acid sequence of an RT domain as listed in Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a linker sequence as listed in Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and the amino acid sequence of an RT domain as listed in Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker sequence as listed in a row of Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and (ii) the amino acid sequence of an RT domain as listed in the same row of Table D, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
Exemplary Gene Modifying Polypeptides
In some embodiments, a gene modifying polypeptide (e.g., a gene modifying polypeptide that is part of a system described herein) comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 80% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 90% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 95% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-7743. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.
In some embodiments, a gene modifying polypeptide comprises an amino acid sequence as listed in Table A1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.
In some embodiments, a gene modifying polypeptide comprises an amino acid sequence as listed in Table T1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a linker comprising a linker sequence as listed in Table T1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an RT domain comprising an RT domain sequence as listed in Table T1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Table T1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in the same row of Table T1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.







TABLE T1







Selection of exemplary gene modifying polypeptides










SEQ ID NO:





for Full

SEQ ID



Polypeptide

NO: of



Sequence
Linker Sequence
linker
RT name





1372
AEAAAKEAAAKEAAAK
15,401
AVIRE_P03360_



EAAAKALEAEAAAKEA

3mutA



AAKEAAAKEAAAKA




 


1197
AEAAAKEAAAKEAAAK
15,402
FLV_P10273_



EAAAKALEAEAAAKEA

3mutA



AAKEAAAKEAAAKA




 


2784
AEAAAKEAAAKEAAAK
15,403
MLVMS_P03355_



EAAAKALEAEAAAKEA

3mutA_WS



AAKEAAAKEAAAKA




 


 647
AEAAAKEAAAKEAAAK
15,404
SFV3L_P27401_



EAAAKALEAEAAAKEA

2mutA



AAKEAAAKEAAAKA









In some embodiments, a gene modifying polypeptide comprises an amino acid sequence as listed in Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a linker comprising a linker sequence as listed in Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an RT domain comprising an RT domain sequence as listed in Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in the same row of Table T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.







TABLE T2







Selection of exemplary gene modifying polypeptides










SEQ ID NO:





for Full

SEQ ID



Polypeptide

NO: of



Sequence
Linker Sequence
linker
RT name





2311
GGGGSGGGGSGGGGSGGGGS
15,405
MLVCB_P08361_3mutA


 


1373
GGGGGGGGSGGGGSGGGGSGGGGSGGGGS
15,406
AVIRE_P03360_3mutA


 


2644
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS
15,407
MLVMS_P03355_PLV919


 


2304
GSSGSSGSSGSSGSSGSS
15,408
MLVCB_P08361_3mutA


 


2325
EAAAKEAAAKEAAAKEAAAK
15,409
MLVCB_P08361_3mutA


 


2322
EAAAKEAAAKEAAAKEAAAKEAAAKEAAAK
15,410
MLVCB_P08361_3mutA


 


2187
PAPAPAPAPAP
15,411
MLVBM_Q7SVK7_3mut


 


2309
PAPAPAPAPAPAP
15,412
MLVCB_P08361_3mutA


 


2534
PAPAPAPAPAPAP
15,413
MLVFF_P26809_3mutA


 


2797
PAPAPAPAPAPAP
15,414
MLVMS_P03355_3mutA_WS


 


3084
PAPAPAPAPAPAP
15,415
MLVMS_P03355_3mutA_WS


 


2868
PAPAPAPAPAPAP
15,416
MLVMS_P03355_PLV919


 


 126
EAAAKGGG
15,417
PERV_Q4VFZ2_3mut


 


 306
EAAAKGGG
15,418
PERV_Q4VFZ2_3mut


 


1410
PAPGGG
15,419
AVIRE_P03360_3mutA


 


 804
GGGGSSGGS
15,420
WMSV_P03359_3mut


 


1937
GGGGGSEAAAK
15,421
BAEVM_P10272_3mutA


 


2721
GGGEAAAKGGS
15,422
MLVMS_P03355_3mut


 


3018
GGGEAAAKGGS
15,423
MLVMS_P03355_3mut


 


1018
GGGEAAAKGGS
15,424
XMRV6_A1Z651_3mutA


 


2317
GGSGGGPAP
15,425
MLVCB_P08361_3mutA


 


2649
PAPGGSGGG
15,426
MLVMS_P03355_PLV919


 


2878
PAPGGSGGG
15,427
MLVMS_P03355_PLV919


 


 912
GGSEAAAKPAP
15,428
WMSV_P03359_3mutA


 


2338
GGSPAPEAAAK
15,429
MLVCB_P08361_3mutA


 


2527
GGSPAPEAAAK
15,430
MLVFF_P26809_3mutA


 


 141
EAAAKGGSPAP
15,431
PERV_Q4VFZ2_3mut


 


 341
EAAAKGGSPAP
15,432
PERV_Q4VFZ2_3mut


 


2315
EAAAKPAPGGS
15,433
MLVCB_P08361_3mutA


 


3080
EAAAKPAPGGS
15,434
MLVMS_P03355_3mutA_WS


 


2688
GGGGSSEAAAK
15,435
MLVMS_P03355_PLV919


 


2885
GGGGSSEAAAK
15,436
MLVMS_P03355_PLV919


 


2810
GSSGGGEAAAK
15,437
MLVMS_P03355_3mutA_WS


 


3057
GSSGGGEAAAK
15,438
MLVMS_P03355_3mutA_WS


 


1861
GSSEAAAKGGG
15,439
MLVAV_P03356_3mutA


 


3056
GSSGGGPAP
15,440
MLVMS_P03355_3mutA_WS


 


1038
GSSPAPGGG
15,441
XMRV6_A1Z651_3mutA


 


2308
PAPGGGGSS
15,442
MLVCB_P08361_3mutA


 


1672
GGGEAAAKPAP
15,443
KORV_Q9TTC1-Pro_3mutA


 


2526
GGGEAAAKPAP
15,444
MLVFF_P26809_3mutA


 


1938
GGGPAPEAAAK
15,445
BAEVM_P10272_3mutA


 


2641
GSSEAAAKPAP
15,446
MLVMS_P03355_PLV919


 


2891
GSSEAAAKPAP
15,447
MLVMS_P03355_PLV919


 


1225
GSSPAPEAAAK
15,448
FLV_P10273_3mutA


 


2839
GSSPAPEAAAK
15,449
MLVMS_P03355_3mutA_WS


 


3127
GSSPAPEAAAK
15,450
MLVMS_P03355_3mutA_WS


 


2798
PAPGSSEAAAK
15,451
MLVMS_P03355_3mutA_WS


 


3091
PAPGSSEAAAK
15,452
MLVMS_P03355_3mutA_WS


 


1372
AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAA
15,453
AVIRE_P03360_3mutA



AKEAAAKEAAAKA




 


1197
AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAA
15,454
FLV_P10273_3mutA



AKEAAAKEAAAKA




 


2611
AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAA
15,455
MLVMS_P03355_PLV919



AKEAAAKEAAAKA




 


2784
AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAA
15,456
MLVMS_P03355_3mutA_WS



AKEAAAKEAAAKA




 


 480
AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAA
15,457
SFV1_P23074_2mutA



AKEAAAKEAAAKA




 


 647
AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAA
15,458
SFV3L_P27401_2mutA



AKEAAAKEAAAKA




 


1006
AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAA
15,459
XMRV6_A1Z651_3mutA



AKEAAAKEAAAKA




 


2518
SGSETPGTSESATPES
15,460
MLVFF_P26809_3mutA









Subsequences of Exemplary Gene Modifying Polypeptides

In some embodiments, the gene modifying polypeptide comprises, in N-terminal to C-terminal order, one or more (e.g., 1, 2, 3, 4, 5, or all 6) of an N-terminal methionine residue, a first nuclear localization signal (NLS), a DNA binding domain, a linker, an RT domain, and/or a second NLS. In some embodiments, a gene modifying polypeptide comprises, in N-terminal to C-terminal order, a NLS (e.g., a first NLS), a DNA binding domain, a linker, and an RT domain, wherein the linker and RT domain are the linker and RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said linker and RT domain. In some embodiments, a gene modifying polypeptide comprises, in N-terminal to C-terminal order, a DNA binding domain, a linker, an RT domain, and an NLS (e.g., a second NLS) wherein the linker and RT domain are the linker and RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said linker and RT domain. In some embodiments, a gene modifying polypeptide comprises, in N-terminal to C-terminal order, a first NLS, a DNA binding domain, a linker, an RT domain, and a second NLS, wherein the linker and RT domain are the linker and RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said linker and RT domain. In some embodiments, the gene modifying polypeptide further comprises an N-terminal methionine residue.
In some embodiments, the gene modifying polypeptide comprises, in N-terminal to C-terminal order, one or more (e.g., 1, 2, 3, 4, 5, or all 6) of an N-terminal methionine residue, a first nuclear localization signal (NLS) (e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and/or as listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto), a DNA binding domain (e.g., a Cas domain, e.g., a SpyCas9 domain, e.g., as listed in Table 8, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; or a DNA binding domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and/or as listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto), a linker (e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and/or as listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto), an RT domain (e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and/or as listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto), and a second NLS (e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and/or as listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the gene modifying polypeptide further comprises (e.g., C-terminal to the second NLS) a T2A sequence and/or a puromycin sequence (e.g., of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743 and/or as listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto). In some embodiments, a nucleic acid encoding a gene modifying polypeptide (e.g., as described herein) encodes a T2A sequence, e.g., wherein the T2A sequence is situated between a region encoding the gene modifying polypeptide and a second region, wherein the second region optionally encodes a selectable marker, e.g., puromycin.
In certain embodiments, the first NLS comprises a first NLS sequence of a gene modifying polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the first NLS comprises a first NLS sequence of a gene modifying polypeptide as listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the first NLS sequence comprises a C-myc NLS. In certain embodiments, the first NLS comprises the amino acid sequence PAAKRVKLD (SEQ ID NO: 11,095), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.
In certain embodiments, the gene modifying polypeptide further comprises a spacer sequence between the first NLS and the DNA binding domain. In certain embodiments, the spacer sequence between the first NLS and the DNA binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the spacer sequence between the first NLS and the DNA binding domain comprises the amino acid sequence GG.
In certain embodiments, the DNA binding domain comprises a DNA binding domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the DNA binding domain comprises a DNA binding domain of a gene modifying polypeptide as listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the DNA binding domain comprises a Cas domain (e.g., as listed in Table 8). In certain embodiments, the DNA binding domain comprises the amino acid sequence of a SpyCas9 polypeptide (e.g., as listed in Table 8, e.g., a Cas9 N863A polypeptide), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the DNA binding domain comprises the amino acid sequence:







(SEQ ID NO: 11,096)


DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL


 


LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL


 


EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL


 


RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI


 


NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN


 


FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL


 


LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF


 


FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK


 


QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY


 


VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN


 


LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL


 


LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII


 


KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL


 


KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS


 


LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM


 


GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV


 


ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS


 


IDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT


 


KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR


 


EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY


 


PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT


 


LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ


 


TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK


 


GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY


 


SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED


 


NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP


 


IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS


 


ITGLYETRIDLSQLGGD,






or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.

In certain embodiments, the gene modifying polypeptide further comprises a spacer sequence between the DNA binding domain and the linker. In certain embodiments, the spacer sequence between the DNA binding domain and the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the spacer sequence between the DNA binding domain and the linker comprises the amino acid sequence GG.
In certain embodiments, the linker comprises a linker sequence of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the linker comprises a linker sequence of a gene modifying polypeptide as listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the linker comprises an amino acid sequence as listed in Table D or 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.
In certain embodiments, the gene modifying polypeptide further comprises a spacer sequence between the linker and the RT domain. In certain embodiments, the spacer sequence between the linker and the RT domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the spacer sequence between the linker and the RT domain comprises the amino acid sequence GG.
In certain embodiments, the RT domain comprises a RT domain sequence of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the RT domain comprises a RT domain sequence of a gene modifying polypeptide as listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the RT domain comprises an amino acid sequence as listed in Table D or 6, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain has a length of about 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 amino acids.
In certain embodiments, the gene modifying polypeptide further comprises a spacer sequence between the RT domain and the second NLS. In certain embodiments, the spacer sequence between the RT domain and the second NLS comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the spacer sequence between the RT domain and the second NLS comprises the amino acid sequence AG.
In certain embodiments, the second NLS comprises a second NLS sequence of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743. In certain embodiments, the second NLS comprises a second NLS sequence of a gene modifying polypeptide as listed in any of Tables A1, T1, or T2. In certain embodiments, the second NLS sequence comprises a plurality of partial NLS sequences. In embodiments, the NLS sequence, e.g., the second NLS sequence, comprises a first partial NLS sequence, e.g., comprising the amino acid sequence KRTADGSEFE (SEQ ID NO: 11,097), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In embodiments, the NLS sequence, e.g., the second NLS sequence, comprises a second partial NLS sequence. In embodiments, the NLS sequence, e.g., the second NLS sequence, comprises an SV40A5 NLS, e.g., a bipartite SV40A5 NLS, e.g., comprising the amino acid sequence KRTADGSEFESPKKKAKVE (SEQ ID NO: 11,098), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the NLS sequence, e.g., the second NLS sequence, comprises the amino acid sequence KRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 11,099), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.
In certain embodiments, the gene modifying polypeptide further comprises a spacer sequence between the second NLS and the T2A sequence and/or puromycin sequence. In certain embodiments, the spacer sequence between the second NLS and the T2A sequence and/or puromycin sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the spacer sequence between the second NLS and the T2A sequence and/or puromycin sequence comprises the amino acid sequence GSG.
Linkers and RT Domains
In some embodiments, the gene modifying polypeptide comprises a linker (e.g., as described herein) and an RT domain (e.g., as described herein). In certain embodiments, the gene modifying polypeptide comprises, in N-terminal to C-terminal order, a linker (e.g., as described herein) and an RT domain (e.g., as described herein).
In certain embodiments, the linker comprises a linker sequence as listed in Table 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the linker comprises a linker sequence of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the linker comprises a linker sequence of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the linker comprises a linker sequence of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the linker comprises a linker sequence of an exemplary gene modifying polypeptide listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the RT domain comprises an RT domain sequence as listed in Table 6, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the RT domain comprises an RT domain sequence of an exemplary gene modifying polypeptide listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.
In some embodiments, a gene modifying polypeptide comprises a portion of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion.
In some embodiments, a gene modifying polypeptide comprises a linker of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said linker. In some embodiments, a gene modifying polypeptide comprises a linker of a gene modifying polypeptide of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said linker. In some embodiments, a gene modifying polypeptide comprises a linker of a gene modifying polypeptide of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said linker. In some embodiments, a gene modifying polypeptide comprises a linker of a gene modifying polypeptide as listed in any of Tables A1, T1, or T2, or a linker comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.
In some embodiments, a gene modifying polypeptide comprises an RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said RT domain. In some embodiments, a gene modifying polypeptide comprises an RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity said RT domain. In some embodiments, a gene modifying polypeptide comprises an RT domain of a gene modifying polypeptide of any one of SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity said RT domain. In some embodiments, a gene modifying polypeptide comprises an RT domain of a gene modifying polypeptide as listed in any of Tables A1, T1, or T2, or an RT domain comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.
In certain embodiments, the linker and the RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto) of a gene modifying polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-7743. In certain embodiments, the linker and the RT domain of a gene modifying polypeptide comprise amino acid sequences of a linker and RT domain having at least 80% identity to the linker and RT domains of any one of SEQ ID NOs: 1-7743. In certain embodiments, the linker and the RT domain of a gene modifying polypeptide comprise amino acid sequences of a linker and RT domain having at least 90% identity to the linker and RT domains of any one of SEQ ID NOs: 1-7743. In certain embodiments, the linker and the RT domain of a gene modifying polypeptide comprise amino acid sequences of a linker and RT domain having at least 95% identity to the linker and RT domains of any one of SEQ ID NOs: 1-7743. In certain embodiments, the linker and the RT domain of a gene modifying polypeptide comprise amino acid sequences of a linker and RT domain having at least 99% identity to the linker and RT domains of any one of SEQ ID NOs: 1-7743. In certain embodiments, the linker and the RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto) of a gene modifying polypeptide having the amino acid sequence of any one of SEQ ID NOs: 6001-7743. In certain embodiments, the linker and the RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto) of a gene modifying polypeptide having the amino acid sequence of any one of SEQ ID NOs: 4501-4541. In certain embodiments, the linker and the RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto) from a single row of any of Tables A1, T1, or T2 (e.g., from a single exemplary gene modifying polypeptide as listed in any of Tables A1, T1, or T2).
In certain embodiments, the linker and the RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto) from two different amino acid sequences selected from SEQ ID NOs: 1-7743. In certain embodiments, the linker and the RT domain of a gene modifying polypeptide comprise the amino acid sequences of a linker and RT domain (or amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto) from different rows of any of Tables A1, T1, or T2.
In certain embodiments, the gene modifying polypeptide further comprises a first NLS (e.g., a 5′ NLS), e.g., as described herein. In certain embodiments, the gene modifying polypeptide further comprises a second NLS (e.g., a 3′ NLS), e.g., as described herein. In certain embodiments, the gene modifying polypeptide further comprises an N-terminal methionine residue.
RT Families and Mutants
In certain embodiments, a gene modifying polypeptide comprises comprises the amino acid sequence of an RT domain sequence from a family selected from: AVIRE, BAEVM, FFV, FLY, FOAMY, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, MLVMS, PERV, SFV1, SFV3L, WMSV, XMRV6, BLVAU, BLVJ, HTL1A, HTL1C, HTL1L, HTL32, HTL3P, HTLV2, JSRV, MLVFS, MLVRD, MMTVB, MPMV, SFVCP, SMRVH, SRV1, SRV2, and WDSV. In certain embodiments, a gene modifying polypeptide comprises comprises the amino acid sequence of an RT domain sequence from a family selected from: AVIRE, BAEVM, FFV, FLY, FOAMY, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, MLVMS, PERV, SFV1, SFV3L, WMSV, and XMRV6.
In certain embodiments, a gene modifying polypeptide comprises comprises the amino acid sequence of an RT domain sequence from an MLVMS RT domain. In embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 1 of Table M1, or a point mutation corresponding thereto. In embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 3 of Table M1 (Gen1 MLVMS), or a point mutation corresponding thereto. In embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations at an amino acid position of the RT domain as listed in columns 1 and 2 of Table M2, or an amino acid position corresponding thereto.
In certain embodiments, a gene modifying polypeptide comprises comprises the amino acid sequence of an RT domain sequence from an AVIRE RT domain. In embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 2 of Table M1, or a point mutation corresponding thereto. In embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 4 of Table M1 (Gen2 AVIRE), or a point mutation corresponding thereto. In embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations at an amino acid position of the RT domain as listed in columns 3 and 4 of Table M2, or an amino acid position corresponding thereto. In certain embodiments, the RT domain comprises an IENSSP (SEQ ID NO: 37639) (e.g., at the C-terminus).







TABLE M1







Exemplary point mutations in MLVMS and AVIRE RT domains










RT-linker filing
Corresponding
Gen1 MLVMS
Gen2 AVIRE


(MLVMS)
AVIRE
(PLV4921)
(PLV10990)















H8Y



P51L
Q51L




S67R
T67R




E67K
E67K




E69K
E69K




T197A
T197A




D200N
D200N
D200N
D200N


H204R
N204R




E302K
E302K






T306K
T306K


F309N
Y309N




W313F
W313F
W313F
W313F


T330P
G330P
T330P
G330P


L435G
T436G




N454K
N455K




D524G
D526G




E562Q
E564Q




D583N
D585N




H594Q
H596Q




L603W
L605W
L603W
L605W


D653N
D655N




L671P
L673P













IENSSP (SEQ ID NO: 37639) at




C-term
















TABLE M2







Positions that can be mutated in exemplary 


MLVMS and AVIRE RT domains


WT residue & position













MLVMS

AVIRE




position #

position #



MLVMS aa
*
AVIRE aa
*
















H
8
Y
8



P
51
Q
51



S
67
T
67



E
69
E
69



T
197
T
197



D
200
D
200



H
204
N
204



E
302
E
302



T
306
T
306



F
309
Y
309



W
313
W
313



T
330
G
330



L
435
T
436



N
454
N
455



D
524
D
526



E
562
E
564



D
583
D
585



H
594
H
596



L
603
L
605



D
653
D
655



L
671
S
673










In certain embodiments, a gene modifying polypeptide comprises a gamma retrovirus derived RT domain. In certain embodiments, the gamma retrovirus-derived RT domain of a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from a family selected from: AVIRE, BAEVM, FFV, FLY, FOAMY, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, MLVMS, PERV, SFV1, SFV3L, WMSV, and XMRV6. In some embodiments, the gamma retrovirus-derived RT domain of a gene modifying polypeptide is not derived from PERV. In some embodiments, said RT includes one, two, three, four, five, six or more mutations shown in Table 2 and corresponding to mutations D200N, L603W, T330P, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, W313F, L435G, N454K, H594Q, L671P, E69K, or D653N in the RT domain of murine leukemia virus reverse transcriptase. In some embodiments, the gene modifying polypeptide further comprises a linker having at least 99% identity to a linker domains of any one of SEQ ID NOs: 1-7743. In some embodiments, the gene modifying polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217 or SEQ ID NO:11,041.
In embodiments, the RT domain comprises the amino acid sequence of an RT domain of an AVIRE RT (e.g., an AVIRE P03360 sequence, e.g., SEQ ID NO: 8001), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of an AVIRE RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, G330P, L605W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of an AVIRE RT further comprising one, two, or three mutations selected from the group consisting of D200N, G330P, and L605W, or a corresponding position in a homologous RT domain.
In embodiments, the RT domain comprises the amino acid sequence of an RT domain of a BAEVM RT (e.g., an BAEVM_P10272 sequence, e.g., SEQ ID NO: 8004), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of a BAEVM RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L602W, T304K, and W311F, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a BAEVM RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L602W, or a corresponding position in a homologous RT domain.
In embodiments, the RT domain comprises the amino acid sequence of an RT domain of an FFV RT (e.g., an FFV_O93209 sequence, e.g., SEQ ID NO: 8012), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of an FFV RT further comprising one, two, three, or four mutations selected from the group consisting of D21N, T293N, T419P, and L393K, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of an FFV RT further comprising one, two, or three mutations selected from the group consisting of D21N, T293N, and T419P, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of an FFV RT further comprising the mutation D21N. In some embodiments, the RT domain comprises the amino acid sequence of an FFV RT further comprising one, two, or three mutations selected from the group consisting of T207N, T333P, and L307K, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of an FFV RT further comprising one or two mutations selected from the group consisting of T207N and T333P, or a corresponding position in a homologous RT domain.
In embodiments, the RT domain comprises the amino acid sequence of an RT domain of an FLV RT (e.g., an FLV_P10273 sequence, e.g., SEQ ID NO: 8019), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of an FLV RT further comprising one, two, three, or four mutations selected from the group consisting of D199N, L602W, T305K, and W312F, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of an FLV RT further comprising one or two mutations selected from the group consisting of D199N and L602W, or a corresponding position in a homologous RT domain.
In embodiments, the RT domain comprises the amino acid sequence of an RT domain of a FOAMV RT (e.g., an FOAMV_P14350 sequence, e.g., SEQ ID NO: 8021), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of an FOAMV RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, S420P, and L396K, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of an FOAMV RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and S420P, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of an FOAMV RT further comprising the mutation D24N, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of an FOAMV RT further comprising one, two, or three mutations selected from the group consisting of T207N, S331P, and L307K, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of an FOAMV RT further comprising one or two mutations selected from the group consisting of T207N and S331P, or a corresponding position in a homologous RT domain.
In embodiments, the RT domain comprises the amino acid sequence of an RT domain of a GALV RT (e.g., an GALV_P21414 sequence, e.g., SEQ ID NO: 8027), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L600W, T304K, and W311F, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L600W, or a corresponding position in a homologous RT domain.
In embodiments, the RT domain comprises the amino acid sequence of an RT domain of a KORV RT (e.g., an KORV_Q9TTC1 sequence, e.g., SEQ ID NO: 8047), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, three, four, five, or six mutations selected from the group consisting of D32N, D322N, E452P, L274W, T428K, and W435F, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, three, or four mutations selected from the group consisting of D32N, D322N, E452P, and L274W, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a GALV RT further comprising the mutation D32N. In some embodiments, the RT domain comprises the amino acid sequence of a KORV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D231N, E361P, L633W, T337K, and W344F, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a KORV RT further comprising one, two, or three mutations selected from the group consisting of D231N, E361P, and L633W, or a corresponding position in a homologous RT domain.
In embodiments, the RT domain comprises the amino acid sequence of an RT domain of a MLVAV RT (e.g., an MLVAV_P03356 sequence, e.g., SEQ ID NO: 8053), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of a MLVAV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a MLVAV RT further comprising one, two, or three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain.
In embodiments, the RT domain comprises the amino acid sequence of an RT domain of a MLVBM RT (e.g., an MLVBM_Q7SVK7 sequence, e.g., SEQ ID NO: 8056), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of a MLVBM RT further comprising one, two, three, four, or five mutations selected from the group consisting of D199N, T329P, L602W, T305K, and W312F, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a MLVBM RT further comprising one, two, and three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain.
In embodiments, the RT domain comprises the amino acid sequence of an RT domain of a MLVCB RT (e.g., an MLVCB_P08361 sequence, e.g., SEQ ID NO: 8062), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of a MLVCB RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a MLVCB RT further comprising one, two, and three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain.
In embodiments, the RT domain comprises the amino acid sequence of an RT domain of a MLVFF RT, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of a MLVFF RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a MLVFF RT further comprising one, two, and three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain.
In embodiments, the RT domain comprises the amino acid sequence of an RT domain of a MLVMS RT (e.g., an MLVMS reference sequence, e.g., SEQ ID NO: 8137; or an MLVMS_P03355 sequence, e.g., SEQ ID NO: 8070), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of a MLVMS RT further comprising one, two, three, four, five, or six mutations selected from the group consisting of D200N, T330P, L603W, T306K, W313F, and H8Y, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a MLVMS RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a MLVMS RT further comprising one, two, or three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain.
In embodiments, the RT domain comprises the amino acid sequence of an RT domain of a PERV RT (e.g., an PERV_Q4VFZ2 sequence, e.g., SEQ ID NO: 8099), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of a PERV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D196N, E326P, L599W, T302K, and W309F, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a PERV RT further comprising one, two, or three mutations selected from the group consisting of D196N, E326P, and L599W, or a corresponding position in a homologous RT domain.
In embodiments, the RT domain comprises the amino acid sequence of an RT domain of a SFV1 RT (e.g., an SFV1_P23074 sequence, e.g., SEQ ID NO: 8105), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of a SFV1 RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, N420P, and L396K, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a SFV1 RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and N420P, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a SFV1 RT further comprising the D24N, or a corresponding position in a homologous RT domain.
In embodiments, the RT domain comprises the amino acid sequence of an RT domain of a SFV3L RT (e.g., an SFV3L_P27401 sequence, e.g., SEQ ID NO: 8111), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of a SFV3L RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, N422P, and L396K, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a SFV3L RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and N422P, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a SFV3L RT further comprising the mutation D24N, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a SFV3L RT further comprising one, two, or three mutations selected from the group consisting of T307N, N333P, and L307K, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a SFV3L RT further comprising one or two mutations selected from the group consisting of T307N and N333P, or a corresponding position in a homologous RT domain.
In embodiments, the RT domain comprises the amino acid sequence of an RT domain of a WMSV RT (e.g., an WMSV_P03359 sequence, e.g., SEQ ID NO: 8131), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of a WMSV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L600W, T304K, and W311F, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a WMSV RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L600W, or a corresponding position in a homologous RT domain.
In embodiments, the RT domain comprises the amino acid sequence of an RT domain of a XMRV6 RT (e.g., an XMRV6_A1Z651 sequence, e.g., SEQ ID NO: 8134), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of a XMRV6 RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of a XMRV6 RT further comprising one, two, or three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain.
In certain embodiments, the RT domain of a gene modifying polypeptide comprises the amino acid sequence of an RT domain of an AVIRE RT, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In embodiments, the RT domain comprises the amino acid sequence of an RT domain comprised in a sequence listed in column 1 of Table A5, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the gene modifying polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217 or SEQ ID NO:11,041.
In certain embodiments, the RT domain of a gene modifying polypeptide comprises the amino acid sequence of an RT domain of an MLVMS RT, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In embodiments, the RT domain comprises the amino acid sequence of an RT domain comprised in a sequence listed in any of columns 2-6 of Table A5, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the gene modifying polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217 or SEQ ID NO:11,041.







TABLE A5







Exemplary gene modifying polypeptides comprising 


an AVIRE RT domain or an MLVMS RT domain.








AVIRE SEQ ID NOs:
MLVMS SEQ ID NOs:















1
2704
3007
3038
2638
2930


2
2706
3007
3038
2639
2930


3
2708
3008
3039
2639
2931


4
2709
3008
3039
2640
2931


5
2709
3009
3040
2640
2932


6
2710
3010
3040
2641
2932


7
2957
3010
3041
2641
2933


9
2957
3011
3041
2642
2933


10
2958
3012
3042
2642
2934


12
2959
3012
3042
2643
2934


13
2960
3013
3043
2643
2935


14
2962
3013
3043
2644
2935


6076
6042
3014
3044
2644
2936


6143
6068
3014
3044
2645
2936


6200
6097
3015
3045
2645
2937


6254
6136
3015
3045
2646
2937


6274
6156
3016
3046
2646
2938


6315
6215
3016
3046
2647
2938


6328
6216
3017
3047
2647
2939


6337
6301
3018
3047
2648
2939


6403
6352
3018
3048
2648
2940


6420
6365
3019
3048
2649
2940


6440
6411
3019
3049
2649
2941


6513
6436
3020
3049
2650
2941


6552
6458
3020
3050
2650
2942


6613
6459
3021
3051
2651
2942


6671
6524
3021
3051
2651
2943


6822
6562
3022
3052
2652
2943


6840
6563
3023
3052
2652
2944


6884
6699
3023
3053
2653
2945


6907
6865
3024
3053
2653
2945


6970
7022
3024
3054
2654
2946


7025
7037
3025
3054
2655
2946


7052
7088
3025
3055
2655
2947


7078
7116
3026
3055
2656
2947


7243
7175
3026
3056
2656
2948


7253
7200
3027
3056
2657
2948


7318
7206
3027
3057
2657
2949


7379
7277
3028
3057
2658
2949


7486
7294
3028
3058
2658
2950


7524
7330
3029
3058
2659
2950


7668
7411
3030
3059
2659
2951


7680
7455
3030
3059
2660
2951


7720
7477
3031
3060
2660
2952


1137
7511
3031
3060
2661
2952


1138
7538
3032
3061
2661
2953


1139
7559
3032
3061
2662
2953


1140
7560
3033
3062
2662
2954


1141
7593
3033
3062
2663
2954


1142
7594
3034
3063
2663
2955


1143
7607
3034
3063
2664
2955


1144
7623
6025
3064
2664
6485


1145
7638
6041
3064
2665
6486


1146
7717
6043
3065
2665
6504


1147
7731
6098
3065
2666
6505


1148
7732
6099
3066
2666
6595


1149
2711
6180
3066
2667
6596


1150
2711
6182
3067
2667
6751


1151
2712
6237
3067
2668
6752


1152
2712
6238
3068
2668
6777


1153
2713
6311
3068
2669
6778


1154
2713
6312
3069
2669
7172


1155
2714
6578
3069
2670
7174


1156
2714
6579
3070
2670
7313


1157
2715
6663
3070
2671
7314


1158
2715
6664
3071
2671



1159
2716
6708
3071
2672



1160
2716
6709
3072
2672



1161
2717
6809
3072
2673



1162
2717
6831
3073
2673



1163
2718
6832
3073
2674



1164
2718
6864
3074
2674



1165
2719
6866
3074
2675



1166
2719
7089
3075
2675



1167
2720
7157
3075
2676



6015
2720
7159
3076
2676



6029
2721
7173
3076
2677



6045
2721
7176
3077
2677



6077
2722
7293
3077
2678



6129
2722
7295
3078
2678



6144
2723
7343
3078
2679



6164
2723
7393
3079
2680



6201
2724
7394
3079
2680



6227
2724
7425
3080
2681



6244
2725
7426
3080
2681



6250
2725
7444
3081
2682



6264
2726
7445
3081
2682



6289
2726
7476
3082
2683



6304
2727
7478
3082
2683



6316
2727
7496
3083
2684



6384
2728
7497
3083
2684



6421
2728
7537
3084
2685



6441
2729
7539
3084
2685



6492
2729
2780
3085
2686



6514
2730
2780
3085
2686



6530
2730
2781
3086
2687



6569
2731
2781
3086
2687



6584
2731
2782
3087
2688



6621
2732
2782
3087
2688



6651
2732
2783
3088
2689



6659
2733
2783
3088
2689



6683
2734
2784
3089
2690



6703
2734
2784
3089
2690



6727
2735
2785
3090
2691



6732
2735
2785
3090
2692



6745
2736
2786
3091
2692



6755
2736
2786
3091
2693



6784
2737
2787
3092
2693



6817
2737
2787
3092
2694



6823
2738
2788
3093
2694



6841
2739
2788
3093
2695



6871
2740
2789
3094
2695



6885
2740
2789
3095
2696



6898
2741
2790
3095
2696



6908
2741
2790
3096
2697



6933
2742
2791
3096
2697



6971
2742
2791
3097
2698



7009
2743
2792
3097
2698



7018
2743
2792
3098
2699



7045
2744
2793
3098
2699



7053
2744
2793
3099
2700



7068
2745
2794
3099
2700



7079
2745
2794
3100
2701



7096
2746
2795
3100
2701



7104
2746
2795
3101
2702



7122
2747
2796
3101
2702



7151
2747
2796
3102
2703



7163
2748
2797
3102
2703



7181
2748
2797
3103
2862



7244
2749
2798
3103
2862



7273
2750
2798
3104
2863



7319
2750
2799
3104
2863



7336
2751
2799
3105
2864



7380
2751
2800
3105
2864



7402
2752
2800
3106
2865



7462
2752
2801
3106
2865



7487
2753
2801
3107
2866



7525
2753
2802
3107
2866



7569
2754
2802
3108
2867



7626
2754
2803
3108
2867



7689
2755
2803
3109
2868



7707
2755
2804
3109
2868



7721
2756
2804
3110
2869



1371
2756
2805
3110
2869



1372
2757
2805
3111
2870



1373
2758
2806
3111
2870



1374
2758
2806
3112
2871



1375
2759
2807
3112
2871



1376
2759
2807
3113
2872



1377
2760
2808
3113
2872



1378
2760
2808
3114
2873



1379
2761
2809
3114
2873



1380
2761
2809
3115
2874



1381
2762
2810
3115
2874



1382
2762
2810
3116
2875



1383
2763
2811
3116
2875



1384
2763
2811
3117
2876



1385
2764
2812
3117
2876



1386
2764
2812
3118
2877



1387
2765
2813
3118
2877



1388
2765
2813
3119
2878



1389
2766
2814
3119
2878



1390
2766
2814
3120
2879



1391
2767
2815
3120
2879



1392
2767
2815
3121
2880



1393
2768
2816
3121
2880



1394
2768
2816
3122
2881



1395
2769
2817
3122
2881



1396
2769
2817
3123
2882



1397
2770
2818
3123
2882



1398
2770
2818
3124
2883



1399
2771
2819
3124
2883



1400
2771
2819
3125
2884



1401
2772
2820
3125
2884



1402
2773
2820
3126
2885



1403
2773
2821
3126
2885



1404
2774
2821
3127
2886



1405
2774
2822
3127
2886



1406
2775
2822
3128
2887



1407
2775
2823
3128
2887



1408
2776
2823
3129
2888



1409
2776
2824
3129
2888



1410
2777
2824
3130
2889



1411
2777
2825
3130
2889



1412
2778
2825
3131
2890



1413
2779
2826
3131
2890



1414
2779
2826
3132
2891



1415
2965
2827
3133
2891



1416
2965
2827
3133
2892



1417
2966
2828
3134
2893



1418
2966
2828
3134
2893



1419
2967
2829
3135
2894



1420
2968
2829
3135
2894



1421
2968
2830
3136
2895



1422
2969
2830
3136
2895



1423
2969
2831
6181
2896



1424
2970
2831
6183
2896



1425
2970
2832
6284
2897



1426
2971
2832
6285
2897



1427
2971
2833
6760
2898



1428
2972
2833
6761
2898



1429
2972
2834
7036
2899



1430
2973
2834
7038
2899



1431
2974
2835
7158
2900



1432
2974
2835
7160
2900



1433
2975
2836
2610
2901



1434
2976
2836
2610
2901



1435
2976
2837
2611
2902



1436
2977
2837
2611
2902



1437
2977
2838
2612
2903



1439
2978
2838
2612
2903



1440
2978
2839
2613
2904



1441
2979
2839
2613
2904



1442
2979
2840
2614
2905



1443
2980
2840
2614
2905



1444
2980
2841
2615
2906



1445
2981
2841
2615
2906



1446
2981
2842
2616
2907



1447
2982
2842
2616
2907



6001
2982
2843
2617
2908



6030
2983
2843
2617
2908



6078
2983
2844
2618
2909



6108
2984
2844
2618
2909



6130
2985
2845
2619
2910



6165
2985
2845
2619
2910



6265
2986
2846
2620
2911



6275
2987
2846
2620
2911



6305
2987
2847
2621
2912



6329
2988
2847
2621
2912



6370
2988
2848
2622
2913



6385
2989
2848
2622
2913



6404
2989
2849
2623
2914



6531
2990
2849
2623
2914



6585
2990
2850
2624
2915



6622
2991
2850
2624
2915



6652
2991
2851
2625
2916



6733
2992
2851
2625
2916



6756
2992
2852
2626
2917



6765
2993
2852
2626
2917



6798
2993
2853
2627
2918



6824
2994
2853
2627
2919



6972
2994
2854
2628
2919



7046
2995
2854
2628
2920



7054
2995
2855
2629
2920



7069
2996
2855
2629
2921



7080
2996
2856
2630
2921



7105
2997
2856
2630
2922



7123
2998
2857
2631
2922



7143
2998
2857
2631
2923



7152
2999
2858
2632
2923



7204
2999
2858
2632
2924



7320
3001
2859
2633
2924



7351
3001
2859
2633
2925



7381
3002
2860
2634
2925



7403
3002
2860
2634
2926



7438
3003
2861
2635
2926



7488
3003
2861
2635
2927



7500
3004
3035
2636
2927



7526
3004
3036
2636
2928



7588
3005
3036
2637
2928



7612
3005
3037
2637
2929



7627
3006
3037
2638
2929









Systems

In an aspect, the disclosure relates to a system comprising nucleic acid molecule encoding a gene modifying polypeptide (e.g., as described herein) and a template nucleic acid (e.g., a template RNA, e.g., as described herein). In certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises one or more silent mutations in the coding region (e.g., in the sequence encoding the RT domain) relative to a nucleic acid molecule as described herein. In certain embodiments, the system further comprises a gRNA (e.g., a gRNA that binds to a polypeptide that induces a nick, e.g., in the opposite strand of the target DNA bound by the gene modifying polypeptide).
In certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide encodes a polypeptide as listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.
In certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 1-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion. In certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 6001-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion. In certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding a portion of an amino acid sequence selected from SEQ ID NOs: 4501-4541, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion. In certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding a portion of a polypeptide listed in any of Tables A1, T1, or T2, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion.
In certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding the linker of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding the linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding the linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding the linker of a polypeptide as listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.
In certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding the RT domain of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding the RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding the RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises a sequence encoding the RT domain of a polypeptide as listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.
In an aspect, the disclosure relates to a system comprising a gene modifying polypeptide (e.g., as described herein) and a template nucleic acid (e.g., a template RNA, e.g., as described herein).
In certain embodiments, the gene modifying polypeptide comprises a polypeptide having an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the gene modifying polypeptide comprises a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the gene modifying polypeptide comprises a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the gene modifying polypeptide comprises a polypeptide as listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.
In certain embodiments, the gene modifying polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 1-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion. In certain embodiments, the gene modifying polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 6001-7743, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion. In certain embodiments, the gene modifying polypeptide comprises a portion of an amino acid sequence selected from SEQ ID NOs: 4501-4541, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion. In certain embodiments, the gene modifying polypeptide comprises a portion of a polypeptide listed in any of Tables A1, T1, or T2, wherein the portion comprises a linker and RT domain, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to said portion.
In certain embodiments, the gene modifying polypeptide comprises the linker of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the gene modifying polypeptide comprises a sequence encoding the linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the gene modifying polypeptide comprises a sequence encoding the linker of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the gene modifying polypeptide comprises the linker of a polypeptide as listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.
In certain embodiments, the gene modifying polypeptide comprises the RT domain of an amino acid sequence selected from SEQ ID NOs: 1-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the gene modifying polypeptide comprises a sequence encoding the RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 6001-7743, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the gene modifying polypeptide comprises a sequence encoding the RT domain of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 4501-4541, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the gene modifying polypeptide comprises the RT domain of a polypeptide as listed in any of Tables A1, T1, or T2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto.







Lengthy table referenced here




US12544458-20260210-T00001


Please refer to the end of the specification for access instructions.






Localization Sequences for Gene Modifying Systems

In certain embodiments, a gene editor system RNA further comprises an intracellular localization sequence, e.g., a nuclear localization sequence (NLS). In some embodiments, a gene modifying polypeptide comprises an NLS as comprised in SEQ ID NO: 4000 and/or SEQ ID NO: 4001, or an NLS having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
The nuclear localization sequence may be an RNA sequence that promotes the import of the RNA into the nucleus. In certain embodiments the nuclear localization signal is located on the template RNA. In certain embodiments, the gene modifying polypeptide is encoded on a first RNA, and the template RNA is a second, separate, RNA, and the nuclear localization signal is located on the template RNA and not on an RNA encoding the gene modifying polypeptide. While not wishing to be bound by theory, in some embodiments, the RNA encoding the gene modifying polypeptide is targeted primarily to the cytoplasm to promote its translation, while the template RNA is targeted primarily to the nucleus to promote insertion into the genome. In some embodiments the nuclear localization signal is at the 3′ end, 5′ end, or in an internal region of the template RNA. In some embodiments the nuclear localization signal is 3′ of the heterologous sequence (e.g., is directly 3′ of the heterologous sequence) or is 5′ of the heterologous sequence (e.g., is directly 5′ of the heterologous sequence). In some embodiments the nuclear localization signal is placed outside of the 5′ UTR or outside of the 3′ UTR of the template RNA. In some embodiments the nuclear localization signal is placed between the 5′ UTR and the 3′ UTR, wherein optionally the nuclear localization signal is not transcribed with the transgene (e.g., the nuclear localization signal is an anti-sense orientation or is downstream of a transcriptional termination signal or polyadenylation signal). In some embodiments the nuclear localization sequence is situated inside of an intron. In some embodiments a plurality of the same or different nuclear localization signals are in the RNA, e.g., in the template RNA. In some embodiments the nuclear localization signal is less than 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 bp in length. Various RNA nuclear localization sequences can be used. For example, Lubelsky and Ulitsky, Nature 555 (107-111), 2018 describe RNA sequences which drive RNA localization into the nucleus. In some embodiments, the nuclear localization signal is a SINE-derived nuclear RNA localization (SIRLOIN) signal. In some embodiments the nuclear localization signal binds a nuclear-enriched protein. In some embodiments the nuclear localization signal binds the HNRNPK protein. In some embodiments the nuclear localization signal is rich in pyrimidines, e.g., is a C/T rich, C/U rich, C rich, T rich, or U rich region. In some embodiments the nuclear localization signal is derived from a long non-coding RNA. In some embodiments the nuclear localization signal is derived from MALAT1 long non-coding RNA or is the 600 nucleotide M region of MALAT1 (described in Miyagawa et al., RNA 18, (738-751), 2012). In some embodiments the nuclear localization signal is derived from BORG long non-coding RNA or is a AGCCC motif (described in Zhang et al., Molecular and Cellular Biology 34, 2318-2329 (2014). In some embodiments the nuclear localization sequence is described in Shukla et al., The EMBO Journal e98452 (2018). In some embodiments the nuclear localization signal is derived from a retrovirus.
In some embodiments, a polypeptide described herein comprises one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, for example a nuclear localization sequence (NLS). In some embodiments, the NLS is a bipartite NLS. In some embodiments, an NLS facilitates the import of a protein comprising an NLS into the cell nucleus. In some embodiments, the NLS is fused to the N-terminus of a gene modifying polypeptide as described herein. In some embodiments, the NLS is fused to the C-terminus of the gene modifying polypeptide. In some embodiments, the NLS is fused to the N-terminus or the C-terminus of a Cas domain. In some embodiments, a linker sequence is disposed between the NLS and the neighboring domain of the gene modifying polypeptide.
In some embodiments, an NLS comprises the amino acid sequence MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 5009), PKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 5010), RKSGKIAAIWKRPRKPKKKRKV (SEQ ID NO: 5011) KRTADGSEFESPKKKRKV (SEQ ID NO: 5012), KKTELQTTNAENKTKKL (SEQ ID NO: 5013), or KRGINDRNFWRGENGRKTR (SEQ ID NO: 5014), KRPAATKKAGQAKKKK (SEQ ID NO: 5015), PAAKRVKLD (SEQ ID NO: 4644), KRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 4649), KRTADGSEFE (SEQ ID NO: 4650), KRTADGSEFESPKKKAKVE (SEQ ID NO: 11098), AGKRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 4651), or a functional fragment or variant thereof. Exemplary NLS sequences are also described in PCT/EP2000/011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, an NLS comprises an amino acid sequence as disclosed in Table 11. An NLS of this table may be utilized with one or more copies in a polypeptide in one or more locations in a polypeptide, e.g., 1, 2, 3 or more copies of an NLS in an N-terminal domain, between peptide domains, in a C-terminal domain, or in a combination of locations, in order to improve subcellular localization to the nucleus. Multiple unique sequences may be used within a single polypeptide. Sequences may be naturally monopartite or bipartite, e.g., having one or two stretches of basic amino acids, or may be used as chimeric bipartite sequences. Sequence references correspond to UniProt accession numbers, except where indicated as SeqNLS for sequences mined using a subcellular localization prediction algorithm (Lin et al BMC Bioinformat 13:157 (2012), incorporated herein by reference in its entirety).







TABLE 11







Exemplary nuclear localization


signals for use in gene modifying systems











SEQ


Sequence
Sequence References
ID No.





AHFKISGEKRPSTDPGKKAK
Q76IQ7
5223


NPKKKKKKDP




 


AHRAKKMSKTHA
P21827
5224


 


ASPEYVNLPINGNG
SeqNLS
5225


 


CTKRPRW
O88622, Q86W56, Q9QYM2, O02776
5226


 


DKAKRVSRNKSEKKRR
O15516, Q5RAK8, Q91YB2, Q91YB0,
5227



Q8QGQ6, O08785, Q9WVS9, Q6YGZ4



 


EELRLKEELLKGIYA
Q9QY16, Q9UHL0, Q2TBP1, Q9QY15
5228


 


EEQLRRRKNSRLNNTG
G5EFF5
5229


 


EVLKVIRTGKRKKKAWKR
SeqNLS
5230


MVTKVC




 


HHHHHHHHHHHHQPH
Q63934, G3V7L5, Q12837
5231


 


HKKKHPDASVNFSEFSK
P10103, Q4R844, P12682, B0CM99,
5232



A9RA84, Q6YKA4, P09429, P63159,




Q08IE6, P63158, Q9YH06, B1MTB0



 


HKRTKK
Q2R2D5
5233


 


IINGRKLKLKKSRRRSSQTS
SeqNLS
5234


NNSFTSRRS




 


KAEQERRK
Q8LH59
5235


 


KEKRKRREELFIEQKKRK
SeqNLS
5236


 


KKGKDEWFSRGKKP
P30999
5237


 


KKGPSVQKRKKT
Q6ZN17
5238


 


KKKTVINDLLHYKKEK
SeqNLS, P32354
5239


 


KKNGGKGKNKPSAKIKK
SeqNLS
5240


 


KKPKWDDFKKKKK
Q15397, Q8BKS9, Q562C7
5241


 


KKRKKD
SeqNLS, Q91Z62, Q1A730, Q969P5,
5242



Q2KHT6, Q9CPU7



 


KKRRKRRRK
SeqNLS
5243


 


KKRRRRARK
Q9UMS6, D4A702, Q91YE8
5244


 


KKSKRGR
Q9UBS0
5245


 


KKSRKRGS
B4FG96
5246


 


KKSTALSRELGKIMRRR
SeqNLS, P32354
5247


 


KKSYQDPEIIAHSRPRK
Q9U7C9
5248


 


KKTGKNRKLKSKRVKTR
Q9Z301, O54943, Q8K3T2
5249


 


KKVSIAGQSGKLWRWKR
Q6YUL8
5250


 


KKYENVVIKRSPRKRGRPR
SeqNLS
5251


K




 


KNKKRK
SeqNLS
5252


 


KPKKKR
SeqNLS
5253


 


KRAMKDDSHGNSTSPKRRK
Q0E671
5254


 


KRANSNLVAAYEKAKKK
P23508
5255


 


KRASEDTTSGSPPKKSSAGP
Q9BZZ5, Q5R644
5256


KR




 


KRFKRRWMVRKMKTKK
SeqNLS
5257


 


KRGLNSSFETSPKKVK
Q8IV63
5258


 


KRGNSSIGPNDLSKRKQRK
SeqNLS
5259


K




 


KRIHSVSLSQSQIDPSKKVK
SeqNLS
5260


RAK




 


KRKGKLKNKGSKRKK
O15381
5261


 


KRRRRRRREKRKR
Q96GM8
5262


 


KRSNDRTYSPEEEKORRA
Q91ZF2
5263


 


KRTVATNGDASGAHRAKK
SeqNLS
5264


MSK




 


KRVYNKGEDEQEHLPKGKK
SeqNLS
5265


R




 


KSGKAPRRRAVSMDNSNK
Q9WVH4, O43524
5266


 


KVNFLDMSLDDIIIYKELE
Q9P127
5267


 


KVQHRIAKKTTRRRR
Q9DXE6
5268


 


LSPSLSPL
Q9Y261, P32182, P35583
5269


 


MDSLLMNRRKFLYQFKNVR
Q9GZX7
5270


WAKGRRETYLC




 


MPQNEYIELHRKRYGYRLD
SeqNLS
5271


YHEKKRKKESREAHERSKK




AKKMIGLKAKLYHK




 


MVQLRPRASR
SeqNLS
5272


 


NNKLLAKRRKGGASPKDDP
Q965G5
5273


MDDIK




 


NYKRPMDGTYGPPAKRHEG
O14497, A2BH40
5274


E




 


PDTKRAKLDSSETTMVKKK
SeqNLS
5275


 


PEKRTKI
SeqNLS
5276


 


PGGRGKKK
Q719N1, Q9UBP0, A2VDN5
5277


 


PGKMDKGEHRQERRDRPY
Q01844, Q61545
5278


 


PKKGDKYDKTD
Q45FA5
5279


 


PKKKSRK
O35914, Q01954
5280


 


PKKNKPE
Q22663
5281


 


PKKRAKV
P04295, P89438
5282


 


PKPKKLKVE
P55263, P55262, P55264, Q64640
5283


 


PKRGRGR
Q9FYS5, Q43386
5284


 


PKRRLVDDA
P0C797
5285


 


PKRRRTY
SeqNLS
5286


 


PLFKRR
A8X6H4, Q9TXJ0
5287


 


PLRKAKR
Q86WB0, Q5R8V9
5288


 


PPAKRKCIF
Q6AZ28, O75928, Q8C5D8
5289


 


PPARRRRL
Q8NAG6
5290


 


PPKKKRKV
Q3L6L5, P03070, P14999, P03071
5291


 


PPNKRMKVKH
Q8BN78
5292


 


PPRIYPQLPSAPT
P0C799
5293


 


PQRSPFPKSSVKR
SeqNLS
5294


 


PRPRKVPR
P0C799
5295


 


PRRRVQRKR
SeqNLS, Q5R448, Q5TAQ9
5296


 


PRRVRLK
Q58DJ0, P56477, Q13568
5297


 


PSRKRPR
Q62315, Q5F363, Q92833
5298


 


PSSKKRKV
SeqNLS
5299


 


PTKKRVK
P07664
5300


 


QRPGPYDRP
SeqNLS
5301


 


RGKGGKGLGKGGAKRHRK
SeqNLS
5302


 


RKAGKGGGGHKTTKKRSA
B4FG96
5303


KDEKVP




 


RKIKLKRAK
A1L3G9
5304


 


RKIKRKRAK
B9X187
5305


 


RKKEAPGPREELRSRGR
O35126, P54258, Q5IS70, P54259
5306


 


RKKRKGK
SeqNLS, Q29243, Q62165, Q28685,
5307



O18738, Q9TSZ6, Q14118



 


RKKRRQRRR
P04326, P69697, P69698, P05907,
5308



P20879, P04613, P19553, P0C1J9,




P20893, P12506, P04612, Q73370,




P0C1K0, P05906, P35965, P04609,




P04610, P04614, P04608, P05905



 


RKKSIPLSIKNLKRKHKRKK
Q9C0C9
5309


NKITR




 


RKLVKPKNTKMKTKLRTNP
Q14190
5310


Y




 


RKRLILSDKGQLDWKK
SeqNLS, Q91Z62, Q1A730, Q2KHT6,
5311



Q9CPU7



 


RKRLKSK
Q13309
5312


 


RKRRVRDNM
Q8QPH4, Q809M7, A8C8X1, Q2VNC5,
5313



Q38SQ0, O89749, Q6DNQ9, Q809L9,




Q0A429, Q20NV3, P16509, P16505,




Q6DNQ5, P16506, Q6XT06, P26118,




Q2ICQ2, Q2RCG8, Q0A2D0, Q0A2H9,




Q9IQ46, Q809M3, Q6J847, Q6J856,




B4URE4, A4GCM7, Q0A440, P26120,




P16511,



 


RKRSPKDKKEKDLDGAGKR
Q7RTP6
5314


RKT




 


RKRTPRVDGQTGENDMNK
O94851
5315


RRRK




 


RLPVRRRRRR
P04499, P12541, P03269, P48313,
5316



P03270



 


RLRFRKPKSK
P69469
5317


 


RQQRKR
Q14980
5318


 


RRDLNSSFETSPKKVK
Q8K3G5
5319


 


RRDRAKLR
Q9SLB8
5320


 


RRGDGRRR
Q80WE1, Q5R9B4, Q06787, P35922
5321


 


RRGRKRKAEKQ
Q812D1, Q5XXA9, Q99JF8, Q8MJG1,
5322



Q66T72, O75475



 


RRKKRR
Q0VD86, Q58DS6, Q5R6G2, Q9ERI5,
5323



Q6AYK2, Q6NYC1



 


RRKRSKSEDMDSVESKRRR
Q7TT18
5324


 


RRKRSR
Q99PU7, D3ZHS6, Q92560, A2VDM8
5325


 


RRPKGKTLQKRKPK
Q6ZN17
5326


 


RRRGFERFGPDNMGRKRK
Q63014, Q9DBR0
5327


 


RRRGKNKVAAQNCRK
SeqNLS
5328


 


RRRKRR
Q5FVH8, Q6MZT1, Q08DH5, Q8BQP9
5329


 


RRRQKQKGGASRRR
SeqNLS
5330


 


RRRREGPRARRRR
P08313, P10231
5331


 


RRTIRLKLVYDKCDRSCKIQ
SeqNLS
5332


KKNRNKCQYCRFHKCLSVG




MSHNAIRFGRMPRSEKAKL




KAE




 


RRVPQRKEVSRCRKCRK
Q5RJN4, Q32L09, Q8CAK3, Q9NUL5
5333


 


RVGGRRQAVECIEDLLNEP
P03255
5334


GQPLDLSCKRPRP




 


RVVKLRIAP
P52639, Q8JMN0
5335


 


RVVRRR
P70278
5336


 


SKRKTKISRKTR
Q5RAY1, O00443
5337


 


SYVKTVPNRTRTYIKL
P21935
5338


 


TGKNEAKKRKIA
P52739, Q8K3J5, Q5RAU9
5339


 


TLSPASSPSSVSCPVIPASTD
SeqNLS
5340


ESPGSALNI




 


VSKKQRTGKKIH
P52739, Q8K3J5, Q5RAU9
5341


 


SPKKKRKVE

5342


 


KRTAD GSEFE SPKKKRKVE

5343


 


PAAKRVKLD

5344


 


PKKKRKV

5345


 


MDSLLMNRRKFLYQFKNVR

5346


WAKGRRETYLC




 


SPKKKRKVEAS

5347


 


MAPKKKRKVGIHRGVP

5348


 


KRTADGSEFEKRTADGSEFE

5349


SPKKKAKVE




 


KRTADGSEFE

5350


 


KRTADGSEFESPKKKAKVE

5351


 


AGKRTADGSEFEKRTADGS

4001


EFESPKKKAKVE









In some embodiments, the NLS is a bipartite NLS. A bipartite NLS typically comprises two basic amino acid clusters separated by a spacer sequence (which may be, e.g., about 10 amino acids in length). A monopartite NLS typically lacks a spacer. An example of a bipartite NLS is the nucleoplasmin NLS, having the sequence KR[PAATKKAGQA]KKKK (SEQ ID NO: 5015), wherein the spacer is bracketed. Another exemplary bipartite NLS has the sequence PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 5016). Exemplary NLSs are described in International Application WO2020051561, which is herein incorporated by reference in its entirety, including for its disclosures regarding nuclear localization sequences.
In certain embodiments, a gene editor system polypeptide (e.g., a gene modifying polypeptide as described herein) further comprises an intracellular localization sequence, e.g., a nuclear localization sequence and/or a nucleolar localization sequence. The nuclear localization sequence and/or nucleolar localization sequence may be amino acid sequences that promote the import of the protein into the nucleus and/or nucleolus, where it can promote integration of heterologous sequence into the genome. In certain embodiments, a gene editor system polypeptide (e.g., (e.g., a gene modifying polypeptide as described herein) further comprises a nucleolar localization sequence. In certain embodiments, the gene modifying polypeptide is encoded on a first RNA, and the template RNA is a second, separate, RNA, and the nucleolar localization signal is encoded on the RNA encoding the gene modifying polypeptide and not on the template RNA. In some embodiments, the nucleolar localization signal is located at the N-terminus, C-terminus, or in an internal region of the polypeptide. In some embodiments, a plurality of the same or different nucleolar localization signals are used. In some embodiments, the nuclear localization signal is less than 5, 10, 25, 50, 75, or 100 amino acids in length. Various polypeptide nucleolar localization signals can be used. For example, Yang et al., Journal of Biomedical Science 22, 33 (2015), describe a nuclear localization signal that also functions as a nucleolar localization signal. In some embodiments, the nucleolar localization signal may also be a nuclear localization signal. In some embodiments, the nucleolar localization signal may overlap with a nuclear localization signal. In some embodiments, the nucleolar localization signal may comprise a stretch of basic residues. In some embodiments, the nucleolar localization signal may be rich in arginine and lysine residues. In some embodiments, the nucleolar localization signal may be derived from a protein that is enriched in the nucleolus. In some embodiments, the nucleolar localization signal may be derived from a protein enriched at ribosomal RNA loci. In some embodiments, the nucleolar localization signal may be derived from a protein that binds rRNA. In some embodiments, the nucleolar localization signal may be derived from MSP58. In some embodiments, the nucleolar localization signal may be a monopartite motif. In some embodiments, the nucleolar localization signal may be a bipartite motif. In some embodiments, the nucleolar localization signal may consist of a multiple monopartite or bipartite motifs. In some embodiments, the nucleolar localization signal may consist of a mix of monopartite and bipartite motifs. In some embodiments, the nucleolar localization signal may be a dual bipartite motif. In some embodiments, the nucleolar localization motif may be a KRASSQALGTIPKRRSSSRFIKRKK (SEQ ID NO: 5017). In some embodiments, the nucleolar localization signal may be derived from nuclear factor-κB-inducing kinase. In some embodiments, the nucleolar localization signal may be an RKKRKKK motif (SEQ ID NO: 5018) (described in Birbach et al., Journal of Cell Science, 117 (3615-3624), 2004).
Evolved Variants of Gene Modifying Polypeptides and Systems
In some embodiments, the invention provides evolved variants of gene modifying polypeptides as described herein. Evolved variants can, in some embodiments, be produced by mutagenizing a reference gene modifying polypeptide, or one of the fragments or domains comprised therein. In some embodiments, one or more of the domains (e.g., the reverse transcriptase domain) is evolved. One or more of such evolved variant domains can, in some embodiments, be evolved alone or together with other domains. An evolved variant domain or domains may, in some embodiments, be combined with unevolved cognate component(s) or evolved variants of the cognate component(s), e.g., which may have been evolved in either a parallel or serial manner.
In some embodiments, the process of mutagenizing a reference gene modifying polypeptide, or fragment or domain thereof, comprises mutagenizing the reference gene modifying polypeptide or fragment or domain thereof. In embodiments, the mutagenesis comprises a continuous evolution method (e.g., PACE) or non-continuous evolution method (e.g., PANCE), e.g., as described herein. In some embodiments, the evolved gene modifying polypeptide, or a fragment or domain thereof, comprises one or more amino acid variations introduced into its amino acid sequence relative to the amino acid sequence of the reference gene modifying polypeptide, or fragment or domain thereof. In embodiments, amino acid sequence variations may include one or more mutated residues (e.g., conservative substitutions, non-conservative substitutions, or a combination thereof) within the amino acid sequence of a reference gene modifying polypeptide, e.g., as a result of a change in the nucleotide sequence encoding the gene modifying polypeptide that results in, e.g., a change in the codon at any particular position in the coding sequence, the deletion of one or more amino acids (e.g., a truncated protein), the insertion of one or more amino acids, or any combination of the foregoing. The evolved variant gene modifying polypeptide may include variants in one or more components or domains of the gene modifying polypeptide (e.g., variants introduced into a reverse transcriptase domain).
In some aspects, the disclosure provides gene modifying polypeptides, systems, kits, and methods using or comprising an evolved variant of a gene modifying polypeptide, e.g., employs an evolved variant of a gene modifying polypeptide or a gene modifying polypeptide produced or producible by PACE or PANCE. In embodiments, the unevolved reference gene modifying polypeptide is a gene modifying polypeptide as disclosed herein.
The term “phage-assisted continuous evolution (PACE),” as used herein, generally refers to continuous evolution that employs phage as viral vectors. Examples of PACE technology have been described, for example, in International PCT Application No. PCT/US 2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. Pat. No. 9,023,594, issued May 5, 2015; U.S. Pat. No. 9,771,574, issued Sep. 26, 2017; U.S. Pat. No. 9,394,537, issued Jul. 19, 2016; International PCT Application, PCT/US2015/012022, filed Jan. 20, 2015, published as WO 2015/134121 on Sep. 11, 2015; U.S. Pat. No. 10,179,911, issued Jan. 15, 2019; and International PCT Application, PCT/US2016/027795, filed Apr. 15, 2016, published as WO 2016/168631 on Oct. 20, 2016, the entire contents of each of which are incorporated herein by reference.
The term “phage-assisted non-continuous evolution (PANCE),” as used herein, generally refers to non-continuous evolution that employs phage as viral vectors. Examples of PANCE technology have been described, for example, in Suzuki T. et al, Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase, Nat Chem Biol. 13(12): 1261-1266 (2017), incorporated herein by reference in its entirety. Briefly, PANCE is a technique for rapid in vivo directed evolution using serial flask transfers of evolving selection phage (SP), which contain a gene of interest to be evolved, across fresh host cells (e.g., E. coli cells). Genes inside the host cell may be held constant while genes contained in the SP continuously evolve. Following phage growth, an aliquot of infected cells may be used to transfect a subsequent flask containing host E. coli. This process can be repeated and/or continued until the desired phenotype is evolved, e.g., for as many transfers as desired.
Methods of applying PACE and PANCE to gene modifying polypeptides may be readily appreciated by the skilled artisan by reference to, inter alia, the foregoing references. Additional exemplary methods for directing continuous evolution of genome-modifying proteins or systems, e.g., in a population of host cells, e.g., using phage particles, can be applied to generate evolved variants of gene modifying polypeptides, or fragments or subdomains thereof. Non-limiting examples of such methods are described in International PCT Application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. Pat. No. 9,023,594, issued May 5, 2015; U.S. Pat. No. 9,771,574, issued Sep. 26, 2017; U.S. Pat. No. 9,394,537, issued Jul. 19, 2016; International PCT Application, PCT/US2015/012022, filed Jan. 20, 2015, published as WO 2015/134121 on Sep. 11, 2015; U.S. Pat. No. 10,179,911, issued Jan. 15, 2019; International Application No. PCT/US2019/37216, filed Jun. 14, 2019, International Patent Publication WO 2019/023680, published Jan. 31, 2019, International PCT Application, PCT/US2016/027795, filed Apr. 15, 2016, published as WO 2016/168631 on Oct. 20, 2016, and International Patent Publication No. PCT/US2019/47996, filed Aug. 23, 2019, each of which is incorporated herein by reference in its entirety.
In some non-limiting illustrative embodiments, a method of evolution of a evolved variant gene modifying polypeptide, of a fragment or domain thereof, comprises: (a) contacting a population of host cells with a population of viral vectors comprising the gene of interest (the starting gene modifying polypeptide or fragment or domain thereof), wherein: (1) the host cell is amenable to infection by the viral vector; (2) the host cell expresses viral genes required for the generation of viral particles; (3) the expression of at least one viral gene required for the production of an infectious viral particle is dependent on a function of the gene of interest; and/or (4) the viral vector allows for expression of the protein in the host cell, and can be replicated and packaged into a viral particle by the host cell. In some embodiments, the method comprises (b) contacting the host cells with a mutagen, using host cells with mutations that elevate mutation rate (e.g., either by carrying a mutation plasmid or some genome modification—e.g., proofing-impaired DNA polymerase, SOS genes, such as UmuC, UmuD′, and/or RecA, which mutations, if plasmid-bound, may be under control of an inducible promoter), or a combination thereof. In some embodiments, the method comprises (c) incubating the population of host cells under conditions allowing for viral replication and the production of viral particles, wherein host cells are removed from the host cell population, and fresh, uninfected host cells are introduced into the population of host cells, thus replenishing the population of host cells and creating a flow of host cells. In some embodiments, the cells are incubated under conditions allowing for the gene of interest to acquire a mutation. In some embodiments, the method further comprises (d) isolating a mutated version of the viral vector, encoding an evolved gene product (e.g., an evolved variant gene modifying polypeptide, or fragment or domain thereof), from the population of host cells.
The skilled artisan will appreciate a variety of features employable within the above-described framework. For example, in some embodiments, the viral vector or the phage is a filamentous phage, for example, an M13 phage, e.g., an M13 selection phage. In certain embodiments, the gene required for the production of infectious viral particles is the M13 gene III (gIII) In embodiments, the phage may lack a functional gIII, but otherwise comprise gI, gII, gIV, gV, gVI, gVII, gVIII, gIX, and a gX. In some embodiments, the generation of infectious VSV particles involves the envelope protein VSV-G. Various embodiments can use different retroviral vectors, for example, Murine Leukemia Virus vectors, or Lentiviral vectors. In embodiments, the retroviral vectors can efficiently be packaged with VSV-G envelope protein, e.g., as a substitute for the native envelope protein of the virus.
In some embodiments, host cells are incubated according to a suitable number of viral life cycles, e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least, 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 7500, at least 10000, or more consecutive viral life cycles, which in on illustrative and non-limiting examples of M13 phage is 10-20 minutes per virus life cycle. Similarly, conditions can be modulated to adjust the time a host cell remains in a population of host cells, e.g., about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 120, about 150, or about 180 minutes. Host cell populations can be controlled in part by density of the host cells, or, in some embodiments, the host cell density in an inflow, e.g., 103 cells/ml, about 104 cells/ml, about 105 cells/ml, about 5-105 cells/ml, about 106 cells/ml, about 5-106 cells/ml, about 107 cells/ml, about 5-107 cells/ml, about 108 cells/ml, about 5-108 cells/ml, about 109 cells/ml, about 5·109 cells/ml, about 1010 cells/ml, or about 5·1010 cells/ml.
Inteins
In some embodiments, as described in more detail below, an intein-N (intN) domain may be fused to the N-terminal portion of a first domain of a gene modifying polypeptide described herein, and an intein-C (intC) domain may be fused to the C-terminal portion of a second domain of a gene modifying polypeptide described herein for the joining of the N-terminal portion to the C-terminal portion, thereby joining the first and second domains. In some embodiments, the first and second domains are each independently chosen from a DNA binding domain, an RNA binding domain, an RT domain, and an endonuclease domain.
Inteins can occur as self-splicing protein intron (e.g., peptide), e.g., which ligates flanking N-terminal and C-terminal exteins (e.g., fragments to be joined). An intein may, in some instances, comprise a fragment of a protein that is able to excise itself and join the remaining fragments (the exteins) with a peptide bond in a process known as protein splicing. Inteins are also referred to as “protein introns.” The process of an intein excising itself and joining the remaining portions of the protein is herein termed “protein splicing” or “intein-mediated protein splicing.”
In some embodiments, an intein of a precursor protein (an intein containing protein prior to intein-mediated protein splicing) comes from two genes. Such intein is referred to herein as a split intein (e.g., split intein-N and split intein-C). Accordingly, an intein-based approach may be used to join a first polypeptide sequence and a second polypeptide sequence together. For example, in cyanobacteria, DnaE, the catalytic subunit a of DNA polymerase III, is encoded by two separate genes, dnaE-n and dnaE-c. An intein-N domain, such as that encoded by the dnaE-n gene, when situated as part of a first polypeptide sequence, may join the first polypeptide sequence with a second polypeptide sequence, wherein the second polypeptide sequence comprises an intein-C domain, such as that encoded by the dnaE-c gene. Accordingly, in some embodiments, a protein can be made by providing nucleic acid encoding the first and second polypeptide sequences (e.g., wherein a first nucleic acid molecule encodes the first polypeptide sequence and a second nucleic acid molecule encodes the second polypeptide sequence), and the nucleic acid is introduced into the cell under conditions that allow for production of the first and second polypeptide sequences, and for joining of the first to the second polypeptide sequence via an intein-based mechanism.
Use of inteins for joining heterologous protein fragments is described, for example, in Wood et al., J. Biol. Chem.289(21); 14512-9 (2014) (incorporated herein by reference in its entirety). For example, when fused to separate protein fragments, the inteins IntN and IntC may recognize each other, splice themselves out, and/or simultaneously ligate the flanking N- and C-terminal exteins of the protein fragments to which they were fused, thereby reconstituting a full-length protein from the two protein fragments.
In some embodiments, a synthetic intein based on the dnaE intein, the Cfa-N (e.g., split intein-N) and Cfa-C (e.g., split intein-C) intein pair, is used. Examples of such inteins have been described, e.g., in Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5 (incorporated herein by reference in its entirety). Non-limiting examples of intein pairs that may be used in accordance with the present disclosure include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein and Cne Prp8 intein (e.g., as described in U.S. Pat. No. 8,394,604, incorporated herein by reference.
In some embodiments involving a split Cas9, an intein-N domain and an intein-C domain may be fused to the N-terminal portion of the split Cas9 and the C-terminal portion of a split Cas9, respectively, for the joining of the N-terminal portion of the split Cas9 and the C-terminal portion of the split Cas9. For example, in some embodiments, an intein-N is fused to the C-terminus of the N-terminal portion of the split Cas9, i.e., to form a structure of N—[N-terminal portion of the split Cas9]-[intein-N]˜C. In some embodiments, an intein-C is fused to the N-terminus of the C-terminal portion of the split Cas9, i.e., to form a structure of N-[intein-C]˜[C-terminal portion of the split Cas9]-C. The mechanism of intein-mediated protein splicing for joining the proteins the inteins are fused to (e.g., split Cas9) is described in Shah et al., Chem Sci. 2014; 5(1):446-461, incorporated herein by reference. Methods for designing and using inteins are known in the art and described, for example by WO2020051561, WO2014004336, WO2017132580, US20150344549, and US20180127780, each of which is incorporated herein by reference in their entirety.
In some embodiments, a split refers to a division into two or more fragments. In some embodiments, a split Cas9 protein or split Cas9 comprises a Cas9 protein that is provided as an N-terminal fragment and a C-terminal fragment encoded by two separate nucleotide sequences. The polypeptides corresponding to the N-terminal portion and the C-terminal portion of the Cas9 protein may be spliced to form a reconstituted Cas9 protein. In embodiments, the Cas9 protein is divided into two fragments within a disordered region of the protein, e.g., as described in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-949, 2014, or as described in Jiang et al. (2016) Science 351: 867-871 and PDB file: 5F9R (each of which is incorporated herein by reference in its entirety). A disordered region may be determined by one or more protein structure determination techniques known in the art, including, without limitation, X-ray crystallography, NMR spectroscopy, electron microscopy (e.g., cryoEM), and/or in silico protein modeling. In some embodiments, the protein is divided into two fragments at any C, T, A, or S, e.g., within a region of SpCas9 between amino acids A292-G364, F445-K483, or E565-T637, or at corresponding positions in any other Cas9, Cas9 variant (e.g., nCas9, dCas9), or other napDNAbp. In some embodiments, protein is divided into two fragments at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, the process of dividing the protein into two fragments is referred to as splitting the protein.
In some embodiments, a protein fragment ranges from about 2-1000 amino acids (e.g., between 2-10, 10-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 amino acids) in length. In some embodiments, a protein fragment ranges from about 5-500 amino acids (e.g., between 5-10, 10-50, 50-100, 100-200, 200-300, 300-400, or 400-500 amino acids) in length. In some embodiments, a protein fragment ranges from about 20-200 amino acids (e.g., between 20-30, 30-40, 40-50, 50-100, or 100-200 amino acids) in length.
In some embodiments, a portion or fragment of a gene modifying polypeptide is fused to an intein. The nuclease can be fused to the N-terminus or the C-terminus of the intein. In some embodiments, a portion or fragment of a fusion protein is fused to an intein and fused to an AAV capsid protein. The intein, nuclease and capsid protein can be fused together in any arrangement (e.g., nuclease-intein-capsid, intein-nuclease-capsid, capsid-intein-nuclease, etc.). In some embodiments, the N-terminus of an intein is fused to the C-terminus of a fusion protein and the C-terminus of the intein is fused to the N-terminus of an AAV capsid protein.
In some embodiments, an endonuclease domain (e.g., a nickase Cas9 domain) is fused to intein-N and a polypeptide comprising an RT domain is fused to an intein-C.
Exemplary nucleotide and amino acid sequences of intein-N domains and compatible intein-C domains are provided below:







DnaE Intein-N DNA:


(SEQ ID NO: 5029)


TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATGGCCTTCTG


 


CCAATCGGGAAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCT


 


GTCGATAACAATGGTAACATTTATACTCAGCCAGTTGCCCAGTGGCAC


 


GACCGGGGAGAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGT


 


CTCATTAGGGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAG


 


ATGCTGCCTATAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGA


 


GTTGACAACCTTCCTAAT


 


DnaE Intein-N Protein:


(SEQ ID NO: 5030)


CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWH


 


DRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMR


 


VDNLPN


 


DnaE Intein-C DNA:


(SEQ ID NO: 5031)


ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAAACGTTTAT


 


GATATTGGAGTCGAAAGAGATCACAACTTTGCTCTGAAGAACGGATTC


 


ATAGCTTCTAAT


 


DnaE Intein-C Protein:


(SEQ ID NO: 5032)


MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN


 


Cfa-N DNA:


(SEQ ID NO: 5033)


TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTG


 


CCTATTGGAAAGATTGTCGAAGAGAGAATTGAATGCACAGTATATACT


 


GTAGACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGGCAC


 


AATCGCGGCGAACAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGC


 


ATCATACGAGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAG


 


ATGTTGCCAATAGATGAGATATTCGAGCGGGGCTTGGATCTCAAACAA


 


GTGGATGGATTG CCA


 


Cfa-N Protein:


(SEQ ID NO: 5034)


CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWH


 


NRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQ


 


VDGLP


 


Cfa-C DNA:


(SEQ ID NO: 5035)


ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAG


 


AGGAAAGTAAAGATAATATCTCGAAAAAGTCTTGGTACCCAAAATGTC


 


TATGATATTGGAGTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGT


 


CTCGTAGCCAGCAAC


 


Cfa-C Protein:


(SEQ ID NO: 5036)


MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNG


 


LVASN






Additional Domains

The gene modifying polypeptide can bind a target DNA sequence and template nucleic acid (e.g., template RNA), nick the target site, and write (e.g., reverse transcribe) the template into DNA, resulting in a modification of the target site. In some embodiments, additional domains may be added to the polypeptide to enhance the efficiency of the process. In some embodiments, the gene modifying polypeptide may contain an additional DNA ligation domain to join reverse transcribed DNA to the DNA of the target site. In some embodiments, the polypeptide may comprise a heterologous RNA-binding domain. In some embodiments, the polypeptide may comprise a domain having 5′ to 3′ exonuclease activity (e.g., wherein the 5′ to 3′ exonuclease activity increases repair of the alteration of the target site, e.g., in favor of alteration over the original genomic sequence). In some embodiments, the polypeptide may comprise a domain having 3′ to 5′ exonuclease activity, e.g., proof-reading activity. In some embodiments, the writing domain, e.g., RT domain, has 3′ to 5′ exonuclease activity, e.g., proof-reading activity.
Template Nucleic Acids
The gene modifying systems described herein can modify a host target DNA site using a template nucleic acid sequence. In some embodiments, the gene modifying systems described herein transcribe an RNA sequence template into host target DNA sites by target-primed reverse transcription (TPRT). By modifying DNA sequence(s) via reverse transcription of the RNA sequence template directly into the host genome, the gene modifying system can insert an object sequence into a target genome without the need for exogenous DNA sequences to be introduced into the host cell (unlike, for example, CRISPR systems), as well as eliminate an exogenous DNA insertion step. The gene modifying system can also delete a sequence from the target genome or introduce a substitution using an object sequence. Therefore, the gene modifying system provides a platform for the use of customized RNA sequence templates containing object sequences, e.g., sequences comprising heterologous gene coding and/or function information.
In some embodiments, the template nucleic acid comprises one or more sequence (e.g., 2 sequences) that binds the gene modifying polypeptide.
In some embodiments a system or method described herein comprises a single template nucleic acid (e.g., template RNA). In some embodiments a system or method described herein comprises a plurality of template nucleic acids (e.g., template RNAs). For example, a system described herein comprises a first RNA comprising (e.g., from 5′ to 3′) a sequence that binds the gene modifying polypeptide (e.g., the DNA-binding domain and/or the endonuclease domain, e.g., a gRNA) and a sequence that binds a target site (e.g., a second strand of a site in a target genome), and a second RNA (e.g., a template RNA) comprising (e.g., from 5′ to 3′) optionally a sequence that binds the gene modifying polypeptide (e.g., that specifically binds the RT domain), a heterologous object sequence, and a PBS sequence. In some embodiments, when the system comprises a plurality of nucleic acids, each nucleic acid comprises a conjugating domain. In some embodiments, a conjugating domain enables association of nucleic acid molecules, e.g., by hybridization of complementary sequences. For example, in some embodiments a first RNA comprises a first conjugating domain and a second RNA comprises a second conjugating domain, and the first and second conjugating domains are capable of hybridizing to one another, e.g., under stringent conditions. In some embodiments, the stringent conditions for hybridization include hybridization in 4× sodium chloride/sodium citrate (SSC), at about 65 C, followed by a wash in 1×SSC, at about 65 C.
In some embodiments, the template nucleic acid comprises RNA. In some embodiments, the template nucleic acid comprises DNA (e.g., single stranded or double stranded DNA).
In some embodiments, the template nucleic acid comprises one or more (e.g., 2) homology domains that have homology to the target sequence. In some embodiments, the homology domains are about 10-20, 20-50, or 50-100 nucleotides in length.
In some embodiments, a template RNA can comprise a gRNA sequence, e.g., to direct the gene modifying polypeptide to a target site of interest. In some embodiments, a template RNA comprises (e.g., from 5′ to 3′) (i) optionally a gRNA spacer that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a gRNA scaffold that binds a polypeptide described herein (e.g., a gene modifying polypeptide or a Cas polypeptide), (iii) a heterologous object sequence comprising a mutation region (optionally the heterologous object sequence comprises, from 5′ to 3′, a first homology region, a mutation region, and a second homology region), and (iv) a primer binding site (PBS) sequence comprising a 3′ target homology domain.
The template nucleic acid (e.g., template RNA) component of a genome editing system described herein typically is able to bind the gene modifying polypeptide of the system. In some embodiments the template nucleic acid (e.g., template RNA) has a 3′ region that is capable of binding a gene modifying polypeptide. The binding region, e.g., 3′ region, may be a structured RNA region, e.g., having at least 1, 2 or 3 hairpin loops, capable of binding the gene modifying polypeptide of the system. The binding region may associate the template nucleic acid (e.g., template RNA) with any of the polypeptide modules. In some embodiments, the binding region of the template nucleic acid (e.g., template RNA) may associate with an RNA-binding domain in the polypeptide. In some embodiments, the binding region of the template nucleic acid (e.g., template RNA) may associate with the reverse transcription domain of the gene modifying polypeptide (e.g., specifically bind to the RT domain). In some embodiments, the template nucleic acid (e.g., template RNA) may associate with the DNA binding domain of the polypeptide, e.g., a gRNA associating with a Cas9-derived DNA binding domain. In some embodiments, the binding region may also provide DNA target recognition, e.g., a gRNA hybridizing to the target DNA sequence and binding the polypeptide, e.g., a Cas9 domain. In some embodiments, the template nucleic acid (e.g., template RNA) may associate with multiple components of the polypeptide, e.g., DNA binding domain and reverse transcription domain.
In some embodiments the template RNA has a poly-A tail at the 3′ end. In some embodiments the template RNA does not have a poly-A tail at the 3′ end.
In some embodiments, the template nucleic acid is a template RNA. In some embodiments, the template RNA comprises one or more modified nucleotides. For example, in some embodiments, the template RNA comprises one or more deoxyribonucleotides. In some embodiments, regions of the template RNA are replaced by DNA nucleotides, e.g., to enhance stability of the molecule. For example, the 3′ end of the template may comprise DNA nucleotides, while the rest of the template comprises RNA nucleotides that can be reverse transcribed. For instance, in some embodiments, the heterologous object sequence is primarily or wholly made up of RNA nucleotides (e.g., at least 90%, 95%, 98%, or 99% RNA nucleotides). In some embodiments, the PBS sequence is primarily or wholly made up of DNA nucleotides (e.g., at least 90%, 95%, 98%, or 99% DNA nucleotides). In other embodiments, the heterologous object sequence for writing into the genome may comprise DNA nucleotides. In some embodiments, the DNA nucleotides in the template are copied into the genome by a domain capable of DNA-dependent DNA polymerase activity. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a DNA polymerase domain in the polypeptide. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a reverse transcriptase domain that is also capable of DNA-dependent DNA polymerization, e.g., second strand synthesis. In some embodiments, the template molecule is composed of only DNA nucleotides.
In some embodiments, a system described herein comprises two nucleic acids which together comprise the sequences of a template RNA described herein. In some embodiments, the two nucleic acids are associated with each other non-covalently, e.g., directly associated with each other (e.g., via base pairing), or indirectly associated as part of a complex comprising one or more additional molecule.
A template RNA described herein may comprise, from 5′ to 3′: (1) a gRNA spacer; (2) a gRNA scaffold; (3) heterologous object sequence (4) a primer binding site (PBS) sequence. Each of these components is now described in more detail.
gRNA Spacer and gRNA Scaffold
A template RNA described herein may comprise a gRNA spacer that directs the gene modifying system to a target nucleic acid, and a gRNA scaffold that promotes association of the template RNA with the Cas domain of the gene modifying polypeptide. The systems described herein can also comprise a gRNA that is not part of a template nucleic acid. For example, a gRNA that comprises a gRNA spacer and gRNA scaffold, but not a heterologous object sequence or a PBS sequence, can be used, e.g., to induce second strand nicking, e.g., as described in the section herein entitled “Second Strand Nicking”.
In some embodiments, the gRNA is a short synthetic RNA composed of a scaffold sequence that participates in CRISPR-associated protein binding and a user-defined ˜20 nucleotide targeting sequence for a genomic target. The structure of a complete gRNA was described by Nishimasu et al. Cell 156, P935-949 (2014). The gRNA (also referred to as sgRNA for single-guide RNA) consists of crRNA- and tracrRNA-derived sequences connected by an artificial tetraloop. The crRNA sequence can be divided into guide (20 nt) and repeat (12 nt) regions, whereas the tracrRNA sequence can be divided into anti-repeat (14 nt) and three tracrRNA stem loops (Nishimasu et al. Cell 156, P935-949 (2014)). In practice, guide RNA sequences are generally designed to have a length of between 17-24 nucleotides (e.g., 19, 20, or 21 nucleotides) and be complementary to a targeted nucleic acid sequence. Custom gRNA generators and algorithms are available commercially for use in the design of effective guide RNAs. In some embodiments, the gRNA comprises two RNA components from the native CRISPR system, e.g. crRNA and tracrRNA. As is well known in the art, the gRNA may also comprise a chimeric, single guide RNA (sgRNA) containing sequence from both a tracrRNA (for binding the nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing/binding). Chemically modified sgRNAs have also been demonstrated to be effective for use with CRISPR-associated proteins; see, for example, Hendel et al. (2015) Nature Biotechnol., 985-991. In some embodiments, a gRNA spacer comprises a nucleic acid sequence that is complementary to a DNA sequence associated with a target gene.
In some embodiments, the region of the template nucleic acid, e.g., template RNA, comprising the gRNA adopts an underwound ribbon-like structure of gRNA bound to target DNA (e.g., as described in Mulepati et al. Science 19 Sep. 2014: Vol. 345, Issue 6203, pp. 1479-1484). Without wishing to be bound by theory, this non-canonical structure is thought to be facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid. Thus, in some embodiments, the region of the template nucleic acid, e.g., template RNA, comprising the gRNA may tolerate increased mismatching with the target site at some interval, e.g., every sixth base. In some embodiments, the region of the template nucleic acid, e.g., template RNA, comprising the gRNA comprising homology to the target site may possess wobble positions at a regular interval, e.g., every sixth base, that do not need to base pair with the target site.
In some embodiments, the template nucleic acid (e.g., template RNA) has at least 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 bases of at least 80%, 85%, 90%, 95%, 99%, or 100% homology to the target site, e.g., at the 5′ end, e.g., comprising a gRNA spacer sequence of length appropriate to the Cas9 domain of the gene modifying polypeptide (Table 8).
In some embodiments, a Cas9 derivative with enhanced activity may be used in the gene modification polypeptide. In some embodiments, a Cas9 derivative may comprise mutations that improve activity of the HNH endonuclease domain, e.g., SpyCas9 R221K, N394K, or mutations that improve R-loop formation, e.g., SpyCas9 L1245V, or comprise a combination of such mutations, e.g., SpyCas9 R221K/N394K, SpyCas9 N394K/L1245V, SpyCas9 R221K/L1245V, or SpyCas9 R221K/N394K/L1245V (see, e.g., Spencer and Zhang Sci Rep 7:16836 (2017), the Cas9 derivatives and comprising mutations of which are incorporated herein by reference). In some embodiments, a Cas9 derivative may comprise one or more types of mutations described herein, e.g., PAM-modifying mutations, protein stabilizing mutations, activity enhancing mutations, and/or mutations partially or fully inactivating one or two endonuclease domains relative to the parental enzyme (e.g., one or more mutations to abolish endonuclease activity towards one or both strands of a target DNA, e.g., a nickase or catalytically dead enzyme). In some embodiments, a Cas9 enzyme used in a system described herein may comprise mutations that confer nickase activity toward the enzyme (e.g., SpyCas9 N863A or H840A) in addition to mutations improving catalytic efficiency (e.g., SpyCas9 R221K, N394K, and/or L1245V). In some embodiments, a Cas9 enzyme used in a system described herein is a SpyCas9 enzyme or derivative that further comprises an N863A mutation to confer nickase activity in addition to R221K and N394K mutations to improve catalytic efficiency.
Table 12 provides parameters to define components for designing gRNA and/or Template RNAs to apply Cas variants listed in Table 8 for gene modifying. The cut site indicates the validated or predicted protospacer adjacent motif (PAM) requirements, validated or predicted location of cut site (relative to the most upstream base of the PAM site). The gRNA for a given enzyme can be assembled by concatenating the crRNA, Tetraloop, and tracrRNA sequences, and further adding a 5′ spacer of a length within Spacer (min) and Spacer (max) that matches a protospacer at a target site. Further, the predicted location of the ssDNA nick at the target is important for designing a PBS sequence of a Template RNA that can anneal to the sequence immediately 5′ of the nick in order to initiate target primed reverse transcription. In some embodiments, a gRNA scaffold described herein comprises a nucleic acid sequence comprising, in the 5′ to 3′ direction, a crRNA of Table 12, a tetraloop from the same row of Table 12, and a tracrRNA from the same row of Table 12, or a sequence having at least 70%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the gRNA or template RNA comprising the scaffold further comprises a gRNA spacer having a length within the Spacer (min) and Spacer (max) indicated in the same row of Table 12. In some embodiments, the gRNA or template RNA having a sequence according to Table 12 is comprised by a system that further comprises a gene modifying polypeptide, wherein the gene modifying polypeptide comprises a Cas domain described in the same row of Table 12.







TABLE 12







Parameters to define components for designing gRNA and/or Template RNAs


to apply Cas variants listed in Table 8 in gene modifying systems.





















Spacer
Spacer

SEQ ID
Tetra-

SEQ ID


Variant
PAM(s)
Cut
Tier
(min)
(max)
crRNA
NO:
loop
tracrRNA
NO:





Nme2Cas9
NNNNCC
-3
1
22
24
GTTGTAGC
10,051
GAAA
CGAAATGAGAACCGTTGCTACAATAAGGC
10,151








TCCCTTTCT


CGTCTGAAAAGATGTGCCGCAACGCTCTG









CATTTCG


CCCCTTAAAGCTTCTGCTTTAAGGGGCATC












GTTTA



 


PpnCas9
NNNNRTT

1
21
24
GTTGTAGC
10,052
GAAA
GCGAAATGAAAAACGTTGTTACAATAAGA
10,152








TCCCTTTTT


GATGAATTTCTCGCAAAGCTCTGCCTCTTG









CATTTCGC


AAATTTCGGTTTCAAGAGGCATC



 


SauCas9
NNGRR;
-3
1
21
23
GTTTTAGT
10,053
GAAA
CAGAATCTACTAAAACAAGGCAAAATGCC
10,153



NNGRRT




ACTCTG


GTGTTTATCTCGTCAACTTGTTGGCGAGA



 


SauCas9-
NNNRR;
-3
1
21
21
GTTTTAGT
10,054
GAAA
CAGAATCTACTAAAACAAGGCAAAATGCC
10,154


KKH
NNNRRT




ACTCTG


GTGTTTATCTCGTCAACTTGTTGGCGAGA



 


SauriCas9
NNGG
-3
1
21
21
GTTTTAGT
10,055
GAAA
CAGAATCTACTAAAACAAGGCAAAATGCC
10,155








ACTCTG


GTGTTTATCTCGTCAACTTGTTGGCGAGA



 


SauriCas9-
NNRG
-3
1
21
21
GTTTTAGT
10,056
GAAA
CAGAATCTACTAAAACAAGGCAAAATGCC
10,156


KKH





ACTCTG


GTGTTTATCTCGTCAACTTGTTGGCGAGA



 


ScaCas9-
NNG
-3
1
20
20
GTTTTAGA
10,057
GAAA
TAGCAAGTTAAAATAAGGCTAGTCCGTTA
10,157


Sc++





GCTA


TCAACTTGAAAAAGTGGCACCGAGTCGGT












GC



 


SpyCas9
NGG
-3
1
20
20
GTTTTAGA
10,058
GAAA
TAGCAAGTTAAAATAAGGCTAGTCCGTTA
10,158








GCTA


TCAACTTGAAAAAGTGGCACCGAGTCGGT












GC



 


SpyCas9_
NGG
-3
1
20
20
GTTTTAGA
10,058
GAAA
TAGCAAGTTAAAATAAGGCTAGTCCGTTA
10,193


i_v1





GCTA


TCAACTTGGACTTCGGTCCAAGTGGCACC












GAGTCGGTGC



 


SpyCas9_
NGG
-3
1
20
20
GTTTTAGA
10,058
GAAA
TAGCAAGTTAAAATAAGGCTAGTCCGTTA
10,194


i_v2





GCTA


TCAACTTGGAGCTTGCTCCAAGTGGCACC












GAGTCGGTGC



 


SpyCas9_
NGG
-3
1
20
20
GTTTTAGA
10,058
GAAA
GTTTTAGAGCTAGAAATAGCAAGTTAAAA
10,195


i_v3





GCTA


TAAGGCTAGTCCGTTATCGACTTGAAAAA












GTCGCACCGAGTCGGTGC



 


SpyCas9-
NG
-3
1
20
20
GTTTTAGA
10,059
GAAA
TAGCAAGTTAAAATAAGGCTAGTCCGTTA
10,159


NG
(NGG =




GCTA


TCAACTTGAAAAAGTGGCACCGAGTCGGT




NGA =







GC




NGT >












NGC)











 


SpyCas9-
NRN > NYN
-3
1
20
20
GTTTTAGA
10,060
GAAA
TAGCAAGTTAAAATAAGGCTAGTCCGTTA
10,160


SpRY





GCTA


TCAACTTGAAAAAGTGGCACCGAGTCGGT












GC



 


St1Cas9
NNAGAAW >
-3
1
20
20
GTCTTTGTA
10,061
GTAC
CAGAAGCTACAAAGATAAGGCTTCATGCC
10,161



NNAGGAW =




CTCTG


GAAATCAACACCCTGTCATTTTATGGCAG




NNGGAAW







GGTGTTTT



 


BlatCas9
NNNNCNAA >
-3
1
19
23
GCTATAGT
10,062
GAAA
GGTAAGTTGCTATAGTAAGGGCAACAGAC
10,162



NNNNCNDD >




TCCTTACT


CCGAGGCGTTGGGGATCGCCTAGCCCGTG




NNNNC







TTTACGGGCTCTCCCCATATTCAAAATAAT












GACAGACGAGCACCTTGGAGCATTTATCT












CCGAGGTGCT



 


cCas9-v16
NNVACT;
-3
2
21
21
GTCTTAGT
10,063
GAAA
CAGAATCTACTAAGACAAGGCAAAATGCC
10,163



NNVATGM;




ACTCTG


GTGTTTATCTCGTCAACTTGTTGGCGAGA




NNVATT;












NNVGCT;












NNVGTG;












NNVGTT











 


cCas9-v17
NNVRRN
-3
2
21
21
GTCTTAGT
10,064
GAAA
CAGAATCTACTAAGACAAGGCAAAATGCC
10,164








ACTCTG


GTGTTTATCTCGTCAACTTGTTGGCGAGA



 


cCas9-v21
NNVACT;
-3
2
21
21
GTCTTAGT
10,065
GAAA
CAGAATCTACTAAGACAAGGCAAAATGCC
10,165



NNVATGM;




ACTCTG


GTGTTTATCTCGTCAACTTGTTGGCGAGA




NNVATT;












NNVGCT;












NNVGTG;












NNVGTT











 


cCas9-v42
NNVRRN
-3
2
21
21
GTCTTAGT
10,066
GAAA
CAGAATCTACTAAGACAAGGCAAAATGCC
10,166








ACTCTG


GTGTTTATCTCGTCAACTTGTTGGCGAGA



 


CdiCas9
NNRHHHY;

2
22
22
ACTGGGGT
10,067
GAAA
CTGAACCTCAGTAAGCATTGGCTCGTTTCC
10,167



NNRAAAY




TCAG


AATGTTGATTGCTCCGCCGGTGCTCCTTAT












TTTTAAGGGCGCCGGC



 


CjeCas9
NNNNRYAC
-3
2
21
23
GTTTTAGTC
10,068
GAAA
AGGGACTAAAATAAAGAGTTTGCGGGACT
10,168








CCT


CTGCGGGGTTACAATCCCCTAAAACCGC



 


GeoCas9
NNNNCRAA

2
21
23
GTCATAGT
10,069
GAAA
TCAGGGTTACTATGATAAGGGCTTTCTGCC
10,169








TCCCCTGA


TAAGGCAGACTGACCCGCGGCGTTGGGG












ATCGCCTGTCGCCCGCTTTTGGCGGGCATT












CCCCATCCTT



 


iSpyMacCas9
NAAN
-3
2
19
21
GTTTTAGA
10,070
GAAA
TAGCAAGTTAAAATAAGGCTAGTCCGTTA
10,170








GCTA


TCAACTTGAAAAAGTGGCACCGAGTCGGT












GC



 


NmeCas9
NNNNGAYT;
-3
2
20
24
GTTGTAGC
10,071
GAAA
CGAAATGAGAACCGTTGCTACAATAAGGC
10,171



NNNNGYTT;




TCCCTTTCT


CGTCTGAAAAGATGTGCCGCAACGCTCTG




NNNNGAYA;




CATTTCG


CCCCTTAAAGCTTCTGCTTTAAGGGGCATC




NNNNGTCT







GTTTA



 


ScaCas9
NNG
-3
2
20
20
GTTTTAGA
10,072
GAAA
TAGCAAGTTAAAATAAGGCTAGTCCGTTA
10,172








GCTA


TCAACTTGAAAAAGTGGCACCGAGTCGGT












GC



 


ScaCas9-
NNG
-3
2
20
20
GTTTTAGA
10,073
GAAA
TAGCAAGTTAAAATAAGGCTAGTCCGTTA
10,173


HiFi-Sc++





GCTA


TCAACTTGAAAAAGTGGCACCGAGTCGGT












GC



 


SpyCas9-
NRRH
-3
2
20
20
GTTTAAGA
10,074
GAAA
CAGCATAGCAAGTTTAAATAAGGCTAGTC
10,174


3var-NRRH





GCTATGCT


CGTTATCAACTTGAAAAAGTGGCACCGAG









G


TCGGTGC



 


SpyCas9-
NRTH
-3
2
20
20
GTTTAAGA
10,075
GAAA
CAGCATAGCAAGTTTAAATAAGGCTAGTC
10,175


3var-NRTH





GCTATGCT


CGTTATCAACTTGAAAAAGTGGCACCGAG









G


TCGGTGC



 


SpyCas9-
NRCH
-3
2
20
20
GTTTAAGA
10,076
GAAA
CAGCATAGCAAGTTTAAATAAGGCTAGTC
10,176


3var-NRCH





GCTATGCT


CGTTATCAACTTGAAAAAGTGGCACCGAG









G


TCGGTGC



 


SpyCas9-
NGG
-3
2
20
20
GTTTTAGA
10,077
GAAA
TAGCAAGTTAAAATAAGGCTAGTCCGTTA
10,177


HF1





GCTA


TCAACTTGAAAAAGTGGCACCGAGTCGGT












GC



 


SpyCas9-
NAAG
-3
2
20
20
GTTTTAGA
10,078
GAAA
TAGCAAGTTAAAATAAGGCTAGTCCGTTA
10,178


QQR1





GCTA


TCAACTTGAAAAAGTGGCACCGAGTCGGT












GC



 


SpyCas9-
NGN
-3
2
20
20
GTTTTAGA
10,079
GAAA
TAGCAAGTTAAAATAAGGCTAGTCCGTTA
10,179


SpG





GCTA


TCAACTTGAAAAAGTGGCACCGAGTCGGT












GC



 


SpyCas9-
NGAN
-3
2
20
20
GTTTTAGA
10,080
GAAA
TAGCAAGTTAAAATAAGGCTAGTCCGTTA
10,180


VQR





GCTA


TCAACTTGAAAAAGTGGCACCGAGTCGGT












GC



 


SpyCas9-
NGCG
-3
2
20
20
GTTTTAGA
10,081
GAAA
TAGCAAGTTAAAATAAGGCTAGTCCGTTA
10,181


VRER





GCTA


TCAACTTGAAAAAGTGGCACCGAGTCGGT












GC



 


SpyCas9-
NG;GAA;
-3
2
20
20
GTTTAAGA
10,082
GAAA
CAGCATAGCAAGTTTAAATAAGGCTAGTC
10,182


xCas
GAT




GCTATGCT


CGTTATCAACTTGAAAAAGTGGCACCGAG









G


TCGGTGC



 


SpyCas9-
NG
-3
2
20
20
GTTTAAGA
10,083
GAAA
CAGCATAGCAAGTTTAAATAAGGCTAGTC
10,183


xCas-NG





GCTATGCT


CGTTATCAACTTGAAAAAGTGGCACCGAG









G


TCGGTGC



 


St1Cas9-
NNACAA
-3
2
20
20
GTCTTTGTA
10,084
GTAC
CAGAAGCTACAAAGATAAGGCTTCATGCC
10,184


CNRZ1066





CTCTG


GAAATCAACACCCTGTCATTTTATGGCAG












GGTGTTTT



 


St1Cas9-
NNGCAA
-3
2
20
20
GTCTTTGTA
10,085
GTAC
CAGAAGCTACAAAGATAAGGCTTCATGCC
10,185


LMG1831





CTCTG


GAAATCAACACCCTGTCATTTTATGGCAG












GGTGTTTT



 


St1Cas9-
NNAAAA
-3
2
20
20
GTCTTTGTA
10,086
GTAC
CAGAAGCTACAAAGATAAGGCTTCATGCC
10,186


MTH17CL396





CTCTG


GAAATCAACACCCTGTCATTTTATGGCAG












GGTGTTTT



 


St1Cas9-
NNGAAA
-3
2
20
20
GTCTTTGTA
10,087
GTAC
CAGAAGCTACAAAGATAAGGCTTCATGCC
10,187


TH1477





CTCTG


GAAATCAACACCCTGTCATTTTATGGCAG












GGTGTTTT



 


SRGN3.1
NNGG

1
21
23
GTTTTAGT
10,088
GAAA
CAGAATCTACTGAAACAAGACAATATGTC
10,188








ACTCTG


GTGTTTATCCCATCAATTTATTGGTGGGAT












TTT



 


sRGN3.3
NNGG

1
21
23
GTTTTAGT
10,089
GAAA
CAGAATCTACTGAAACAAGACAATATGTC
10,189








ACTCTG


GTGTTTATCCCATCAATTTATTGGTGGGAT












TTT









Herein, when an RNA sequence (e.g., a template RNA sequence) is said to comprise a particular sequence (e.g., a sequence of Table 12 or a portion thereof) that comprises thymine (T), it is of course understood that the RNA sequence may (and frequently does) comprise uracil (U) in place of T. For instance, the RNA sequence may comprise U at every position shown as T in the sequence in Table 12. More specifically, the present disclosure provides an RNA sequence according to every gRNA scaffold sequence of Table 12, wherein the RNA sequence has a U in place of each T in the sequence in Table 12. Additionally, it is understood that terminal Us and Ts may optionally be added or removed from tracrRNA sequences and may be modified or unmodified when provided as RNA. Without wishing to be bound by example, versions of gRNA scaffold sequences alternative to those exemplified in Table 12 may also function with the different Cas9 enzymes or derivatives thereof exemplified in Table 8, e.g., alternate gRNA scaffold sequences with nucleotide additions, substitutions, or deletions, e.g., sequences with stem-loop structures added or removed. It is contemplated herein that the gRNA scaffold sequences represent a component of gene modifying systems that can be similarly optimized for a given system, Cas-RT fusion polypeptide, indication, target mutation, template RNA, or delivery vehicle.
Heterologous Object Sequence
A template RNA described herein may comprise a heterologous object sequence that the gene modifying polypeptide can use as a template for reverse transcription, to write a desired sequence into the target nucleic acid. In some embodiments, the heterologous object sequence comprises, from 5′ to 3′, a post-edit homology region, the mutation region, and a pre-edit homology region. Without wishing to be bound by theory, an RT performing reverse transcription on the template RNA first reverse transcribes the pre-edit homology region, then the mutation region, and then the post-edit homology region, thereby creating a DNA strand comprising the desired mutation with a homology region on either side.
In some embodiments, the heterologous object sequence is at least 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 120, 140, 160, 180, 200, 500, or 1,000 nucleotides (nts) in length, or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 kilobases in length. In some embodiments, the heterologous object sequence is no more than 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 120, 140, 160, 180, 200, 500, 1,000, or 2000 nucleotides (nts) in length, or no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, or 3 kilobases in length. In some embodiments, the heterologous object sequence is 30-1000, 40-1000, 50-1000, 60-1000, 70-1000, 74-1000, 75-1000, 76-1000, 77-1000, 78-1000, 79-1000, 80-1000, 85-1000, 90-1000, 100-1000, 120-1000, 140-1000, 160-1000, 180-1000, 200-1000, 500-1000, 30-500, 40-500, 50-500, 60-500, 70-500, 74-500, 75-500, 76-500, 77-500, 78-500, 79-500, 80-500, 85-500, 90-500, 100-500, 120-500, 140-500, 160-500, 180-500, 200-500, 30-200, 40-200, 50-200, 60-200, 70-200, 74-200, 75-200, 76-200, 77-200, 78-200, 79-200, 80-200, 85-200, 90-200, 100-200, 120-200, 140-200, 160-200, 180-200, 30-100, 40-100, 50-100, 60-100, 70-100, 74-100, 75-100, 76-100, 77-100, 78-100, 79-100, 80-100, 85-100, or 90-100 nucleotides (nts) in length, or 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-15, 6-10, 6-9, 6-8, 6-7, 7-20, 7-15, 7-10, 7-9, 7-8, 8-20, 8-15, 8-10, 8-9, 9-20, 9-15, 9-10, 10-15, 10-20, or 15-20 kilobases in length. In some embodiments, the heterologous object sequence is 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, or 10-20 nt in length, e.g., 10-80, 10-50, or 10-20 nt in length, e.g., about 10-20 nt in length. In some embodiments, the heterologous object sequence is 8-30, 9-25, 10-20, 11-16, or 12-15 nucleotides in length, e.g., is 11-16 nt in length. Without wishing to be bound by theory, in some embodiments, a larger insertion size, larger region of editing (e.g., the distance between a first edit/substitution and a second edit/substitution in the target region), and/or greater number of desired edits (e.g., mismatches of the heterologous object sequence to the target genome), may result in a longer optimal heterologous object sequence.
In certain embodiments, the template nucleic acid comprises a customized RNA sequence template which can be identified, designed, engineered and constructed to contain sequences altering or specifying host genome function, for example by introducing a heterologous coding region into a genome; affecting or causing exon structure/alternative splicing, e.g., leading to exon skipping of one or more exons; causing disruption of an endogenous gene, e.g., creating a genetic knockout; causing transcriptional activation of an endogenous gene; causing epigenetic regulation of an endogenous DNA; causing up-regulation of one or more operably linked genes, e.g., leading to gene activation or overexpression; causing down-regulation of one or more operably linked genes, e.g., creating a genetic knock-down; etc. In certain embodiments, a customized RNA sequence template can be engineered to contain sequences coding for exons and/or transgenes, provide binding sites for transcription factor activators, repressors, enhancers, etc., and combinations thereof. In some embodiments, a customized template can be engineered to encode a nucleic acid or peptide tag to be expressed in an endogenous RNA transcript or endogenous protein operably linked to the target site. In other embodiments, the coding sequence can be further customized with splice donor sites, splice acceptor sites, or poly-A tails.
The template nucleic acid (e.g., template RNA) of the system typically comprises an object sequence (e.g., a heterologous object sequence) for writing a desired sequence into a target DNA. The object sequence may be coding or non-coding. The template nucleic acid (e.g., template RNA) can be designed to result in insertions, mutations, or deletions at the target DNA locus. In some embodiments, the template nucleic acid (e.g., template RNA) may be designed to cause an insertion in the target DNA. For example, the template nucleic acid (e.g., template RNA) may contain a heterologous sequence, wherein the reverse transcription will result in insertion of the heterologous sequence into the target DNA. In other embodiments, the RNA template may be designed to introduce a deletion into the target DNA. For example, the template nucleic acid (e.g., template RNA) may match the target DNA upstream and downstream of the desired deletion, wherein the reverse transcription will result in the copying of the upstream and downstream sequences from the template nucleic acid (e.g., template RNA) without the intervening sequence, e.g., causing deletion of the intervening sequence. In other embodiments, the template nucleic acid (e.g., template RNA) may be designed to introduce an edit into the target DNA. For example, the template RNA may match the target DNA sequence with the exception of one or more nucleotides, wherein the reverse transcription will result in the copying of these edits into the target DNA, e.g., resulting in mutations, e.g., transition or transversion mutations.
In some embodiments, writing of an object sequence into a target site results in the substitution of nucleotides, e.g., where the full length of the object sequence corresponds to a matching length of the target site with one or more mismatched bases. In some embodiments, a heterologous object sequence may be designed such that a combination of sequence alterations may occur, e.g., a simultaneous addition and deletion, addition and substitution, or deletion and substitution.
In some embodiments, the heterologous object sequence may contain an open reading frame or a fragment of an open reading frame. In some embodiments the heterologous object sequence has a Kozak sequence. In some embodiments the heterologous object sequence has an internal ribosome entry site. In some embodiments the heterologous object sequence has a self-cleaving peptide such as a T2A or P2A site. In some embodiments the heterologous object sequence has a start codon. In some embodiments the template RNA has a splice acceptor site. In some embodiments the template RNA has a splice donor site. Exemplary splice acceptor and splice donor sites are described in WO2016044416, incorporated herein by reference in its entirety. Exemplary splice acceptor site sequences are known to those of skill in the art. In some embodiments the template RNA has a microRNA binding site downstream of the stop codon. In some embodiments the template RNA has a polyA tail downstream of the stop codon of an open reading frame. In some embodiments the template RNA comprises one or more exons. In some embodiments the template RNA comprises one or more introns. In some embodiments the template RNA comprises a eukaryotic transcriptional terminator. In some embodiments the template RNA comprises an enhanced translation element or a translation enhancing element. In some embodiments the RNA comprises the human T-cell leukemia virus (HTLV-1) R region. In some embodiments the RNA comprises a posttranscriptional regulatory element that enhances nuclear export, such as that of Hepatitis B Virus (HPRE) or Woodchuck Hepatitis Virus (WPRE).
In some embodiments, the heterologous object sequence may contain a non-coding sequence. For example, the template nucleic acid (e.g., template RNA) may comprise a regulatory element, e.g., a promoter or enhancer sequence or miRNA binding site. In some embodiments, integration of the object sequence at a target site will result in upregulation of an endogenous gene. In some embodiments, integration of the object sequence at a target site will result in downregulation of an endogenous gene. In some embodiments the template nucleic acid (e.g., template RNA) comprises a tissue specific promoter or enhancer, each of which may be unidirectional or bidirectional. In some embodiments the promoter is an RNA polymerase I promoter, RNA polymerase II promoter, or RNA polymerase III promoter. In some embodiments the promoter comprises a TATA element. In some embodiments the promoter comprises a B recognition element. In some embodiments the promoter has one or more binding sites for transcription factors.
In some embodiments, the template nucleic acid (e.g., template RNA) comprises a site that coordinates epigenetic modification. In some embodiments, the template nucleic acid (e.g., template RNA) comprises a chromatin insulator. For example, the template nucleic acid (e.g., template RNA) comprises a CTCF site or a site targeted for DNA methylation.
In some embodiments, the template nucleic acid (e.g., template RNA) comprises a gene expression unit composed of at least one regulatory region operably linked to an effector sequence. The effector sequence may be a sequence that is transcribed into RNA (e.g., a coding sequence or a non-coding sequence such as a sequence encoding a micro RNA).
In some embodiments, the heterologous object sequence of the template nucleic acid (e.g., template RNA) is inserted into a target genome in an endogenous intron. In some embodiments, the heterologous object sequence of the template nucleic acid (e.g., template RNA) is inserted into a target genome and thereby acts as a new exon. In some embodiments, the insertion of the heterologous object sequence into the target genome results in replacement of a natural exon or the skipping of a natural exon.
The template nucleic acid (e.g., template RNA) can be designed to result in insertions, mutations, or deletions at the target DNA locus. In some embodiments, the template nucleic acid (e.g., template RNA) may be designed to cause an insertion in the target DNA. For example, the template nucleic acid (e.g., template RNA) may contain a heterologous object sequence, wherein the reverse transcription will result in insertion of the heterologous object sequence into the target DNA. In other embodiments, the RNA template may be designed to write a deletion into the target DNA. For example, the template nucleic acid (e.g., template RNA) may match the target DNA upstream and downstream of the desired deletion, wherein the reverse transcription will result in the copying of the upstream and downstream sequences from the template nucleic acid (e.g., template RNA) without the intervening sequence, e.g., causing deletion of the intervening sequence. In other embodiments, the template nucleic acid (e.g., template RNA) may be designed to write an edit into the target DNA. For example, the template RNA may match the target DNA sequence with the exception of one or more nucleotides, wherein the reverse transcription will result in the copying of these edits into the target DNA, e.g., resulting in mutations, e.g., transition or transversion mutations.
In some embodiments, the pre-edit homology domain comprises a nucleic acid sequence having 100% sequence identity with a nucleic acid sequence comprised in a target nucleic acid molecule.
In some embodiments, the post-edit homology domain comprises a nucleic acid sequence having 100% sequence identity with a nucleic acid sequence comprised in a target nucleic acid molecule.
PBS Sequence
In some embodiments, a template nucleic acid (e.g., template RNA) comprises a PBS sequence. In some embodiments, a PBS sequence is disposed 3′ of the heterologous object sequence and is complementary to a sequence adjacent to a site to be modified by a system described herein, or comprises no more than 1, 2, 3, 4, or 5 mismatches to a sequence complementary to the sequence adjacent to a site to be modified by the system/gene modifying polypeptide. In some embodiments, the PBS sequence binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nick site in the target nucleic acid molecule. In some embodiments, binding of the PBS sequence to the target nucleic acid molecule permits initiation of target-primed reverse transcription (TPRT), e.g., with the 3′ homology domain acting as a primer for TPRT. In some embodiments, the PBS sequence is 3-5, 5-10, 10-30, 10-25, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-30, 11-25, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-30, 12-25, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-30, 13-25, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-30, 14-25, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-30, 15-25, 15-20, 15-19, 15-18, 15-17, 15-16, 16-30, 16-25, 16-20, 16-19, 16-18, 16-17, 17-30, 17-25, 17-20, 17-19, 17-18, 18-30, 18-25, 18-20, 18-19, 19-30, 19-25, 19-20, 20-30, 20-25, or 25-30 nucleotides in length, e.g., 10-17, 12-16, or 12-14 nucleotides in length. In some embodiments, the PBS sequence is 5-20, 8-16, 8-14, 8-13, 9-13, 9-12, or 10-12 nucleotides in length, e.g., 9-12 nucleotides in length.
The template nucleic acid (e.g., template RNA) may have some homology to the target DNA. In some embodiments, the template nucleic acid (e.g., template RNA) PBS sequence domain may serve as an annealing region to the target DNA, such that the target DNA is positioned to prime the reverse transcription of the template nucleic acid (e.g., template RNA). In some embodiments the template nucleic acid (e.g., template RNA) has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200 or more bases of exact homology to the target DNA at the 3′ end of the RNA. In some embodiments the template nucleic acid (e.g., template RNA) has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200 or more bases of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homology to the target DNA, e.g., at the 5′ end of the template nucleic acid (e.g., template RNA).
Exemplary Template Sequences
In some embodiments of the systems and methods herein, the template RNA comprises a gRNA spacer comprising the core nucleotides of a gRNA spacer sequence of Table 1A, Table 1B, Table 1C, or Table 1D. In some embodiments, the gRNA spacer additionally comprises one or more (e.g., 2, 3, or all) consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the gRNA spacer. In some embodiments, the template RNA comprising a sequence of Table 11A, Table 1B, Table 1C, or Table 1D is comprised by a system that further comprises a gene modifying polypeptide having an RT domain listed in the same line of Table 11A, Table 1B, Table 1C, or Table 1D. RT domain amino acid sequences can be found, e.g., in Table 6 herein.







TABLE 1A







Exemplary gRNA spacer Cas pairs for correcting


the pathogenic R408W mutation


Table 1A provides a gRNA database for correcting the pathogenic


R408W mutation in PAH. List of spacers, PAMs, and Cas variants


for generating a nick at an appropriate position to enable


installation of a desired genomic edit with a gene modifying system.


The spacers in this table are designed to be used with a gene


modifying polypeptide comprising a nickase variant of the Cas


species indicated in the table. Tables 2A, 3A, and 4A detail the


other components of the system and are organized such that the


ID number shown here in Column 1 (“ID”) is meant to correspond


to the same ID number in Tables 2A, 3A, and 4A.
















SEQ






PAM

ID


Overlaps


ID
sequence
gRNA spacer
NO
Cas species
distance
mutation





   1
CCC
TTGCTGCCACAATACCTTGG
17033
SpyCas9-
  0
0






SpRY




 


   2
GG
AGCGAACTGAGAAGGGCCAA
17034
SpyCas9-NG
  1
0


 


   3
GG
AGCGAACTGAGAAGGGCCAA
17035
SpyCas9-xCas
  1
0


 


   4
GG
AGCGAACTGAGAAGGGCCAA
17036
SpyCas9-
  1
0






xCas-NG




 


   5
GGT
AGCGAACTGAGAAGGGCCAA
17037
SpyCas9-SpG
  1
0


 


   6
GGT
AGCGAACTGAGAAGGGCCAA
17038
SpyCas9-
  1
0






SpRY




 


   7
GCC
TTTGCTGCCACAATACCTTG
17039
SpyCas9-
  1
0






SpRY




 


   8
GGTA
AGCGAACTGAGAAGGGCCAA
17040
SpyCas9-
  1
0






3var-NRTH




 


   9
AGGTATT
CGTAGCGAACTGAGAAGGGCCA
17041
CdiCas9
  2
0


 


  10
AGGTATT
gtcGTAGCGAACTGAGAAGGGCCA
17042
PpnCas9
  2
0


 


  11
AGG
TAGCGAACTGAGAAGGGCCA
17043
ScaCas9
  2
0


 


  12
AGG
TAGCGAACTGAGAAGGGCCA
17044
ScaCas9-
  2
0






HiFi-Sc++




 


  13
AGG
TAGCGAACTGAGAAGGGCCA
17045
ScaCas9-Sc++
  2
0


 


  14
AGG
TAGCGAACTGAGAAGGGCCA
17046
SpyCas9
  2
0


 


  15
AGG
TAGCGAACTGAGAAGGGCCA
17047
SpyCas9-HF1
  2
0


 


  16
AGG
TAGCGAACTGAGAAGGGCCA
17048
SpyCas9-SpG
  2
0


 


  17
AGG
TAGCGAACTGAGAAGGGCCA
17049
SpyCas9-
  2
0






SpRY




 


  18
GG
CTTTGCTGCCACAATACCTT
17050
SpyCas9-NG
  2
0


 


  19
GG
CTTTGCTGCCACAATACCTT
17051
SpyCas9-xCas
  2
0


 


  20
GG
CTTTGCTGCCACAATACCTT
17052
SpyCas9-
  2
0






xCas-NG




 


  21
AG
TAGCGAACTGAGAAGGGCCA
17053
SpyCas9-NG
  2
0


 


  22
AG
TAGCGAACTGAGAAGGGCCA
17054
SpyCas9-xCas
  2
0


 


  23
AG
TAGCGAACTGAGAAGGGCCA
17055
SpyCas9-
  2
0






xCas-NG




 


  24
GGC
CTTTGCTGCCACAATACCTT
17056
SpyCas9-SpG
  2
0


 


  25
GGC
CTTTGCTGCCACAATACCTT
17057
SpyCas9-
  2
0






SpRY




 


  26
GGCCC
gaacTTTGCTGCCACAATACCTT
17058
BlatCas9
  2
0


 


  27
AGGT
TAGCGAACTGAGAAGGGCCA
17059
SpyCas9-
  2
0






3var-NRRH




 


  28
GGCC
CTTTGCTGCCACAATACCTT
17060
SpyCas9-
  2
0






3var-NRCH




 


  29
tGGCCC
agGAACTTTGCTGCCACAATACCT
17061
Nme2Cas9
  3
1


 


  30
aAGG
CGTAGCGAACTGAGAAGGGCC
17062
SauriCas9
  3
1


 


  31
aAGG
CGTAGCGAACTGAGAAGGGCC
17063
SauriCas9-
  3
1






KKH




 


  32
tGG
ACTTTGCTGCCACAATACCT
17064
ScaCas9
  3
1


 


  33
tGG
ACTTTGCTGCCACAATACCT
17065
ScaCas9-
  3
1






HiFi-Sc++




 


  34
tGG
ACTTTGCTGCCACAATACCT
17066
ScaCas9-Sc++
  3
1


 


  35
tGG
ACTTTGCTGCCACAATACCT
17067
SpyCas9
  3
1


 


  36
tGG
ACTTTGCTGCCACAATACCT
17068
SpyCas9-HF1
  3
1


 


  37
tGG
ACTTTGCTGCCACAATACCT
17069
SpyCas9-SpG
  3
1


 


  38
tGG
ACTTTGCTGCCACAATACCT
17070
SpyCas9-
  3
1






SpRY




 


  39
aAG
GTAGCGAACTGAGAAGGGCC
17071
ScaCas9
  3
1


 


  40
aAG
GTAGCGAACTGAGAAGGGCC
17072
ScaCas9-
  3
1






HiFi-Sc++




 


  41
aAG
GTAGCGAACTGAGAAGGGCC
17073
ScaCas9-Sc++
  3
1


 


  42
aAG
GTAGCGAACTGAGAAGGGCC
17074
SpyCas9-
  3
1






SpRY




 


  43
tG
ACTTTGCTGCCACAATACCT
17075
SpyCas9-NG
  3
1


 


  44
tG
ACTTTGCTGCCACAATACCT
17076
SpyCas9-xCas
  3
1


 


  45
tG
ACTTTGCTGCCACAATACCT
17077
SpyCas9-
  3
1






xCas-NG




 


  46
tGGCCCTT
ggaaCTTTGCTGCCACAATACCT
17078
BlatCas9
  3
1


 


  47
tGGCC
ggaaCTTTGCTGCCACAATACCT
17079
BlatCas9
  3
1


 


  48
tGGCCCT
GAACTTTGCTGCCACAATACCT
17080
CdiCas9
  3
1


 


  49
tGGC
ACTTTGCTGCCACAATACCT
17081
SpyCas9-
  3
1






3var-NRRH




 


  50
TtGGCC
taGGAACTTTGCTGCCACAATACC
17082
Nme2Cas9
  4
1


 


  51
CaAGG
TCGTAGCGAACTGAGAAGGGC
17083
SauCas9KKH
  4
1


 


  52
CaAGGT
TCGTAGCGAACTGAGAAGGGC
17084
SauCas9KKH
  4
1


 


  53
CaAGGT
TCGTAGCGAACTGAGAAGGGC
17085
cCas9-v17
  4
1


 


  54
CaAGGT
TCGTAGCGAACTGAGAAGGGC
17086
cCas9-v42
  4
1


 


  55
TtGG
GAACTTTGCTGCCACAATACC
17087
SauriCas9
  4
1


 


  56
TtGG
GAACTTTGCTGCCACAATACC
17088
SauriCas9-
  4
1






KKH




 


  57
CaAG
TCGTAGCGAACTGAGAAGGGC
17089
SauriCas9-
  4
1






KKH




 


  58
CaAG
CGTAGCGAACTGAGAAGGGC
17090
SpyCas9-
  4
1






QQR1




 


  59
CaAG
tcGTAGCGAACTGAGAAGGGC
17091
iSpyMacCas9
  4
1


 


  60
TtG
AACTTTGCTGCCACAATACC
17092
ScaCas9
  4
1


 


  61
TtG
AACTTTGCTGCCACAATACC
17093
ScaCas9-
  4
1






HiFi-Sc++




 


  62
TtG
AACTTTGCTGCCACAATACC
17094
ScaCas9-Sc++
  4
1


 


  63
TtG
AACTTTGCTGCCACAATACC
17095
SpyCas9-
  4
1






SpRY




 


  64
CaA
CGTAGCGAACTGAGAAGGGC
17096
SpyCas9-
  4
1






SpRY




 


  65
TtGGC
aggaACTTTGCTGCCACAATACC
17097
BlatCas9
  4
1


 


  66
CCaAG
GTCGTAGCGAACTGAGAAGGG
17098
SauCas9KKH
  5
1


 


  67
CTtGG
GGAACTTTGCTGCCACAATAC
17099
SauCas9KKH
  5
1


 


  68
CCa
TCGTAGCGAACTGAGAAGGG
17100
SpyCas9-
  5
1






SpRY




 


  69
CTt
GAACTTTGCTGCCACAATAC
17101
SpyCas9-
  5
1






SpRY




 


  70
CCaAGG
GTCGTAGCGAACTGAGAAGGG
17102
cCas9-v17
  5
1


 


  71
CCaAGG
GTCGTAGCGAACTGAGAAGGG
17103
cCas9-v42
  5
1


 


  72
GCCaA
GGTCGTAGCGAACTGAGAAGG
17104
SauCas9KKH
  6
1


 


  73
GCC
GTCGTAGCGAACTGAGAAGG
17105
SpyCas9-
  6
0






SpRY




 


  74
CCT
GGAACTTTGCTGCCACAATA
17106
SpyCas9-
  6
0






SpRY




 


  75
GCCaAG
GGTCGTAGCGAACTGAGAAGG
17107
cCas9-v17
  6
1


 


  76
GCCaAG
GGTCGTAGCGAACTGAGAAGG
17108
cCas9-v42
  6
1


 


  77
GG
GGTCGTAGCGAACTGAGAAG
17109
SpyCas9-NG
  7
0


 


  78
GG
GGTCGTAGCGAACTGAGAAG
17110
SpyCas9-xCas
  7
0


 


  79
GG
GGTCGTAGCGAACTGAGAAG
17111
SpyCas9-
  7
0






xCas-NG




 


  80
GGC
GGTCGTAGCGAACTGAGAAG
17112
SpyCas9-SpG
  7
0


 


  81
GGC
GGTCGTAGCGAACTGAGAAG
17113
SpyCas9-
  7
0






SpRY




 


  82
ACC
AGGAACTTTGCTGCCACAAT
17114
SpyCas9-
  7
0






SpRY




 


  83
GGCC
GGTCGTAGCGAACTGAGAAG
17115
SpyCas9-
  7
0






3var-NRCH




 


  84
GGG
GGGTCGTAGCGAACTGAGAA
17116
ScaCas9
  8
0


 


  85
GGG
GGGTCGTAGCGAACTGAGAA
17117
ScaCas9-
  8
0






HiFi-Sc++




 


  86
GGG
GGGTCGTAGCGAACTGAGAA
17118
ScaCas9-Sc++
  8
0


 


  87
GGG
GGGTCGTAGCGAACTGAGAA
17119
SpyCas9
  8
0


 


  88
GGG
GGGTCGTAGCGAACTGAGAA
17120
SpyCas9-HF1
  8
0


 


  89
GGG
GGGTCGTAGCGAACTGAGAA
17121
SpyCas9-SpG
  8
0


 


  90
GGG
GGGTCGTAGCGAACTGAGAA
17122
SpyCas9-
  8
0






SpRY




 


  91
GG
GGGTCGTAGCGAACTGAGAA
17123
SpyCas9-NG
  8
0


 


  92
GG
GGGTCGTAGCGAACTGAGAA
17124
SpyCas9-xCas
  8
0


 


  93
GG
GGGTCGTAGCGAACTGAGAA
17125
SpyCas9-
  8
0






xCas-NG




 


  94
TAC
TAGGAACTTTGCTGCCACAA
17126
SpyCas9-
  8
0






SpRY




 


  95
GGGCCaAG
tatgGGTCGTAGCGAACTGAGAA
17127
BlatCas9
  8
1


 


  96
GGGCC
tatgGGTCGTAGCGAACTGAGAA
17128
BlatCas9
  8
0


 


  97
GGGC
GGGTCGTAGCGAACTGAGAA
17129
SpyCas9-
  8
0






3var-NRRH




 


  98
TACC
TAGGAACTTTGCTGCCACAA
17130
SpyCas9-
  8
0






3var-NRCH




 


  99
AGGGCC
tgTATGGGTCGTAGCGAACTGAGA
17131
Nme2Cas9
  9
0


 


 100
AGGG
ATGGGTCGTAGCGAACTGAGA
17132
SauriCas9
  9
0


 


 101
AGGG
ATGGGTCGTAGCGAACTGAGA
17133
SauriCas9-
  9
0






KKH




 


 102
AGG
TGGGTCGTAGCGAACTGAGA
17134
ScaCas9
  9
0


 


 103
AGG
TGGGTCGTAGCGAACTGAGA
17135
ScaCas9-
  9
0






HiFi-Sc++




 


 104
AGG
TGGGTCGTAGCGAACTGAGA
17136
ScaCas9-Sc++
  9
0


 


 105
AGG
TGGGTCGTAGCGAACTGAGA
17137
SpyCas9
  9
0


 


 106
AGG
TGGGTCGTAGCGAACTGAGA
17138
SpyCas9-HF1
  9
0


 


 107
AGG
TGGGTCGTAGCGAACTGAGA
17139
SpyCas9-SpG
  9
0


 


 108
AGG
TGGGTCGTAGCGAACTGAGA
17140
SpyCas9-
  9
0






SpRY




 


 109
AG
TGGGTCGTAGCGAACTGAGA
17141
SpyCas9-NG
  9
0


 


 110
AG
TGGGTCGTAGCGAACTGAGA
17142
SpyCas9-xCas
  9
0


 


 111
AG
TGGGTCGTAGCGAACTGAGA
17143
SpyCas9-
  9
0






xCas-NG




 


 112
ATA
TTAGGAACTTTGCTGCCACA
17144
SpyCas9-
  9
0






SpRY




 


 113
AGGGCCaA
gtatGGGTCGTAGCGAACTGAGA
17145
BlatCas9
  9
1


 


 114
AGGGCCaA
gtatGGGTCGTAGCGAACTGAGA
17146
BlatCas9
  9
1


 


 115
ATACCTtG
gtctTAGGAACTTTGCTGCCACA
17147
BlatCas9
  9
1


 


 116
AGGGC
gtatGGGTCGTAGCGAACTGAGA
17148
BlatCas9
  9
0


 


 117
ATACC
gtctTAGGAACTTTGCTGCCACA
17149
BlatCas9
  9
0


 


 118
ATACCTt
TCTTAGGAACTTTGCTGCCACA
17150
CdiCas9
  9
1


 


 119
AATACC
tgGTCTTAGGAACTTTGCTGCCAC
17151
Nme2Cas9
 10
0


 


 120
AAGGG
tgTATGGGTCGTAGCGAACTGAG
17152
SauCas9
 10
0


 


 121
AAGGG
TATGGGTCGTAGCGAACTGAG
17153
SauCas9KKH
 10
0


 


 122
AAGG
TATGGGTCGTAGCGAACTGAG
17154
SauriCas9
 10
0


 


 123
AAGG
TATGGGTCGTAGCGAACTGAG
17155
SauriCas9-
 10
0






KKH




 


 124
AAG
ATGGGTCGTAGCGAACTGAG
17156
ScaCas9
 10
0


 


 125
AAG
ATGGGTCGTAGCGAACTGAG
17157
ScaCas9-
 10
0






HiFi-Sc++




 


 126
AAG
ATGGGTCGTAGCGAACTGAG
17158
ScaCas9-Sc++
 10
0


 


 127
AAG
ATGGGTCGTAGCGAACTGAG
17159
SpyCas9-
 10
0






SpRY




 


 128
AAT
CTTAGGAACTTTGCTGCCAC
17160
SpyCas9-
 10
0






SpRY




 


 129
AATACCTt
ggtcTTAGGAACTTTGCTGCCAC
17161
BlatCas9
 10
1


 


 130
AATAC
ggtcTTAGGAACTTTGCTGCCAC
17162
BlatCas9
 10
0


 


 131
AAGGGC
TATGGGTCGTAGCGAACTGAG
17163
cCas9-v17
 10
0


 


 132
AAGGGC
TATGGGTCGTAGCGAACTGAG
17164
cCas9-v42
 10
0


 


 133
AATA
CTTAGGAACTTTGCTGCCAC
17165
SpyCas9-
 10
0






3var-NRTH




 


 134
GAAGG
GTATGGGTCGTAGCGAACTGA
17166
SauCas9KKH
 11
0


 


 135
GAAG
GTATGGGTCGTAGCGAACTGA
17167
SauriCas9-
 11
0






KKH




 


 136
GAAG
TATGGGTCGTAGCGAACTGA
17168
SpyCas9-
 11
0






QQR1




 


 137
GAAG
gtATGGGTCGTAGCGAACTGA
17169
iSpy MacCas9
 11
0


 


 138
GAA
TATGGGTCGTAGCGAACTGA
17170
SpyCas9-
 11
0






SpRY




 


 139
GAA
TATGGGTCGTAGCGAACTGA
17171
SpyCas9-xCas
 11
0


 


 140
CAA
TCTTAGGAACTTTGCTGCCA
17172
SpyCas9-
 11
0






SpRY




 


 141
GAAGGG
GTATGGGTCGTAGCGAACTGA
17173
cCas9-v17
 11
0


 


 142
GAAGGG
GTATGGGTCGTAGCGAACTGA
17174
cCas9-v42
 11
0


 


 143
CAATACC
GGTCTTAGGAACTTTGCTGCCA
17175
CdiCas9
 11
0


 


 144
CAAT
TCTTAGGAACTTTGCTGCCA
17176
SpyCas9-
 11
0






3var-NRRH




 


 145
CAAT
gtCTTAGGAACTTTGCTGCCA
17177
iSpyMacCas9
 11
0


 


 146
AGAAG
TGTATGGGTCGTAGCGAACTG
17178
SauCas9KKH
 12
0


 


 147
AG
GTATGGGTCGTAGCGAACTG
17179
SpyCas9-NG
 12
0


 


 148
AG
GTATGGGTCGTAGCGAACTG
17180
SpyCas9-xCas
 12
0


 


 149
AG
GTATGGGTCGTAGCGAACTG
17181
SpyCas9-
 12
0






xCas-NG




 


 150
AGA
GTATGGGTCGTAGCGAACTG
17182
SpyCas9-SpG
 12
0


 


 151
AGA
GTATGGGTCGTAGCGAACTG
17183
SpyCas9-
 12
0






SpRY




 


 152
ACA
GTCTTAGGAACTTTGCTGCC
17184
SpyCas9-
 12
0






SpRY




 


 153
AGAAGG
TGTATGGGTCGTAGCGAACTG
17185
cCas9-v17
 12
0


 


 154
AGAAGG
TGTATGGGTCGTAGCGAACTG
17186
cCas9-v42
 12
0


 


 155
ACAATAC
TGGTCTTAGGAACTTTGCTGCC
17187
CdiCas9
 12
0


 


 156
AGAA
GTATGGGTCGTAGCGAACTG
17188
SpyCas9-
 12
0






3var-NRRH




 


 157
AGAA
GTATGGGTCGTAGCGAACTG
17189
SpyCas9-
 12
0






VQR




 


 158
GAGAA
ggGTGTATGGGTCGTAGCGAACT
17190
SauCas9
 13
0


 


 159
GAGAA
GTGTATGGGTCGTAGCGAACT
17191
SauCas9KKH
 13
0


 


 160
CACAA
TGGTCTTAGGAACTTTGCTGC
17192
SauCas9KKH
 13
0


 


 161
CACAAT
TGGTCTTAGGAACTTTGCTGC
17193
SauCas9KKH
 13
0


 


 162
CACAAT
TGGTCTTAGGAACTTTGCTGC
17194
cCas9-v17
 13
0


 


 163
CACAAT
TGGTCTTAGGAACTTTGCTGC
17195
cCas9-v42
 13
0


 


 164
GAG
TGTATGGGTCGTAGCGAACT
17196
ScaCas9
 13
0


 


 165
GAG
TGTATGGGTCGTAGCGAACT
17197
ScaCas9-
 13
0






HiFi-Sc++




 


 166
GAG
TGTATGGGTCGTAGCGAACT
17198
ScaCas9-Sc++
 13
0


 


 167
GAG
TGTATGGGTCGTAGCGAACT
17199
SpyCas9-
 13
0






SpRY




 


 168
CAC
GGTCTTAGGAACTTTGCTGC
17200
SpyCas9-
 13
0






SpRY




 


 169
GAGAAG
GTGTATGGGTCGTAGCGAACT
17201
cCas9-v17
 13
0


 


 170
GAGAAG
GTGTATGGGTCGTAGCGAACT
17202
cCas9-v42
 13
0


 


 171
CACAATAC
ttTGGTCTTAGGAACTTTGCTGC
17203
CjeCas9
 13
0


 


 172
GAGA
TGTATGGGTCGTAGCGAACT
17204
SpyCas9-
 13
0






3var-NRRH




 


 173
CACA
GGTCTTAGGAACTTTGCTGC
17205
SpyCas9-
 13
0






3var-NRCH




 


 174
TGAGA
GGTGTATGGGTCGTAGCGAAC
17206
SauCas9KKH
 14
0


 


 175
TGAG
GGTGTATGGGTCGTAGCGAAC
17207
SauriCas9-
 14
0






KKH




 


 176
TGAG
GTGTATGGGTCGTAGCGAAC
17208
SpyCas9-
 14
0






VQR




 


 177
TG
GTGTATGGGTCGTAGCGAAC
17209
SpyCas9-NG
 14
0


 


 178
TG
GTGTATGGGTCGTAGCGAAC
17210
SpyCas9-xCas
 14
0


 


 179
TG
GTGTATGGGTCGTAGCGAAC
17211
SpyCas9-
 14
0






xCas-NG




 


 180
TGA
GTGTATGGGTCGTAGCGAAC
17212
SpyCas9-SpG
 14
0


 


 181
TGA
GTGTATGGGTCGTAGCGAAC
17213
SpyCas9-
 14
0






SpRY




 


 182
CCA
TGGTCTTAGGAACTTTGCTG
17214
SpyCas9-
 14
0






SpRY




 


 183
TGAGAA
GGTGTATGGGTCGTAGCGAAC
17215
cCas9-v17
 14
0


 


 184
TGAGAA
GGTGTATGGGTCGTAGCGAAC
17216
cCas9-v42
 14
0


 


 185
CCACAAT
TTTGGTCTTAGGAACTTTGCTG
17217
CdiCas9
 14
0


 


 186
CCACAA
TGGTCTTAGGAACTTTGCTG
17218
St1Cas9-
 14
0






CNRZ1066




 


 187
CTGAG
ttGGGTGTATGGGTCGTAGCGAA
17219
SauCas9
 15
0


 


 188
CTGAG
GGGTGTATGGGTCGTAGCGAA
17220
SauCas9KKH
 15
0


 


 189
CTG
GGTGTATGGGTCGTAGCGAA
17221
ScaCas9
 15
0


 


 190
CTG
GGTGTATGGGTCGTAGCGAA
17222
ScaCas9-
 15
0






HiFi-Sc++




 


 191
CTG
GGTGTATGGGTCGTAGCGAA
17223
ScaCas9-Sc++
 15
0


 


 192
CTG
GGTGTATGGGTCGTAGCGAA
17224
SpyCas9-
 15
0






SpRY




 


 193
GCC
TTGGTCTTAGGAACTTTGCT
17225
SpyCas9-
 15
0






SpRY




 


 194
GCCACAAT
gtttTGGTCTTAGGAACTTTGCT
17226
BlatCas9
 15
0


 


 195
GCCAC
gtttTGGTCTTAGGAACTTTGCT
17227
BlatCas9
 15
0


 


 196
CTGAGA
GGGTGTATGGGTCGTAGCGAA
17228
cCas9-v17
 15
0


 


 197
CTGAGA
GGGTGTATGGGTCGTAGCGAA
17229
cCas9-v42
 15
0


 


 198
ACTGA
TGGGTGTATGGGTCGTAGCGA
17230
SauCas9KKH
 16
0


 


 199
TG
TTTGGTCTTAGGAACTTTGC
17231
SpyCas9-NG
 16
0


 


 200
TG
TTTGGTCTTAGGAACTTTGC
17232
SpyCas9-xCas
 16
0


 


 201
TG
TTTGGTCTTAGGAACTTTGC
17233
SpyCas9-
 16
0






xCas-NG




 


 202
TGC
TTTGGTCTTAGGAACTTTGC
17234
SpyCas9-SpG
 16
0


 


 203
TGC
TTTGGTCTTAGGAACTTTGC
17235
SpyCas9-
 16
0






SpRY




 


 204
ACT
GGGTGTATGGGTCGTAGCGA
17236
SpyCas9-
 16
0






SpRY




 


 205
TGCC
TTTGGTCTTAGGAACTTTGC
17237
SpyCas9-
 16
0






3var-NRCH




 


 206
CTG
TTTTGGTCTTAGGAACTTTG
17238
ScaCas9
 17
0


 


 207
CTG
TTTTGGTCTTAGGAACTTTG
17239
ScaCas9-
 17
0






HiFi-Sc++




 


 208
CTG
TTTTGGTCTTAGGAACTTTG
17240
ScaCas9-Sc++
 17
0


 


 209
CTG
TTTTGGTCTTAGGAACTTTG
17241
SpyCas9-
 17
0






SpRY




 


 210
AAC
TGGGTGTATGGGTCGTAGCG
17242
SpyCas9-
 17
0






SpRY




 


 211
CTGCC
tggtTTTGGTCTTAGGAACTTTG
17243
BlatCas9
 17
0


 


 212
CTGCCAC
GGTTTTGGTCTTAGGAACTTTG
17244
CdiCas9
 17
0


 


 213
AACT
TGGGTGTATGGGTCGTAGCG
17245
SpyCas9-
 17
0






3var-NRCH




 


 214
GCTGCC
tgTGGTTTTGGTCTTAGGAACTTT
17246
Nme2Cas9
 18
0


 


 215
GAA
TTGGGTGTATGGGTCGTAGC
17247
SpyCas9-
 18
0






SpRY




 


 216
GAA
TTGGGTGTATGGGTCGTAGC
17248
SpyCas9-xCas
 18
0


 


 217
GCT
GTTTTGGTCTTAGGAACTTT
17249
SpyCas9-
 18
0






SpRY




 


 218
GCTGC
gtggTTTTGGTCTTAGGAACTTT
17250
BlatCas9
 18
0


 


 219
GAAC
TTGGGTGTATGGGTCGTAGC
17251
SpyCas9-
 18
0






3var-NRRH




 


 220
GAAC
ttTGGGTGTATGGGTCGTAGC
17252
iSpyMacCas9
 18
0


 


 221
CG
TTTGGGTGTATGGGTCGTAG
17253
SpyCas9-NG
 19
0


 


 222
CG
TTTGGGTGTATGGGTCGTAG
17254
SpyCas9-xCas
 19
0


 


 223
CG
TTTGGGTGTATGGGTCGTAG
17255
SpyCas9-
 19
0






xCas-NG




 


 224
TG
GGTTTTGGTCTTAGGAACTT
17256
SpyCas9-NG
 19
0


 


 225
TG
GGTTTTGGTCTTAGGAACTT
17257
SpyCas9-xCas
 19
0


 


 226
TG
GGTTTTGGTCTTAGGAACTT
17258
SpyCas9-
 19
0






xCas-NG




 


 227
CGA
TTTGGGTGTATGGGTCGTAG
17259
SpyCas9-SpG
 19
0


 


 228
CGA
TTTGGGTGTATGGGTCGTAG
17260
SpyCas9-
 19
0






SpRY




 


 229
TGC
GGTTTTGGTCTTAGGAACTT
17261
SpyCas9-SpG
 19
0


 


 230
TGC
GGTTTTGGTCTTAGGAACTT
17262
SpyCas9-
 19
0






SpRY




 


 231
CGAACTGA
tcctTTGGGTGTATGGGTCGTAG
17263
BlatCas9
 19
0


 


 232
CGAAC
tcctTTGGGTGTATGGGTCGTAG
17264
BlatCas9
 19
0


 


 233
CGAACT
CTTTGGGTGTATGGGTCGTAG
17265
cCas9-v16
 19
0


 


 234
CGAACT
CTTTGGGTGTATGGGTCGTAG
17266
cCas9-v21
 19
0


 


 235
CGAA
TTTGGGTGTATGGGTCGTAG
17267
SpyCas9-
 19
0






3var-NRRH




 


 236
CGAA
TTTGGGTGTATGGGTCGTAG
17268
SpyCas9-
 19
0






VQR




 


 237
TGCT
GGTTTTGGTCTTAGGAACTT
17269
SpyCas9-
 19
0






3var-NRCH




 


 238
GCGAA
atCCTTTGGGTGTATGGGTCGTA
17270
SauCas9
 20
0


 


 239
GCGAA
CCTTTGGGTGTATGGGTCGTA
17271
SauCas9KKH
 20
0


 


 240
GCG
CTTTGGGTGTATGGGTCGTA
17272
ScaCas9
 20
0


 


 241
GCG
CTTTGGGTGTATGGGTCGTA
17273
ScaCas9-
 20
0






HiFi-Sc++




 


 242
GCG
CTTTGGGTGTATGGGTCGTA
17274
ScaCas9-Sc++
 20
0


 


 243
GCG
CTTTGGGTGTATGGGTCGTA
17275
SpyCas9-
 20
0






SpRY




 


 244
TTG
TGGTTTTGGTCTTAGGAACT
17276
ScaCas9
 20
0


 


 245
TTG
TGGTTTTGGTCTTAGGAACT
17277
ScaCas9-
 20
0






HiFi-Sc++




 


 246
TTG
TGGTTTTGGTCTTAGGAACT
17278
ScaCas9-Sc++
 20
0


 


 247
TTG
TGGTTTTGGTCTTAGGAACT
17279
SpyCas9-
 20
0






SpRY




 


 248
GCGAAC
CCTTTGGGTGTATGGGTCGTA
17280
cCas9-v17
 20
0


 


 249
GCGAAC
CCTTTGGGTGTATGGGTCGTA
17281
cCas9-v42
 20
0


 


 250
GCGAACT
TCCTTTGGGTGTATGGGTCGTA
17282
CdiCas9
 20
0


 


 251
AGCGA
TCCTTTGGGTGTATGGGTCGT
17283
SauCas9KKH
 21
0


 


 252
AG
CCTTTGGGTGTATGGGTCGT
17284
SpyCas9-NG
 21
0


 


 253
AG
CCTTTGGGTGTATGGGTCGT
17285
SpyCas9-xCas
 21
0


 


 254
AG
CCTTTGGGTGTATGGGTCGT
17286
SpyCas9-
 21
0






xCas-NG




 


 255
AGC
CCTTTGGGTGTATGGGTCGT
17287
SpyCas9-SpG
 21
0


 


 256
AGC
CCTTTGGGTGTATGGGTCGT
17288
SpyCas9-
 21
0






SpRY




 


 257
TTT
GTGGTTTTGGTCTTAGGAAC
17289
SpyCas9-
 21
0






SpRY




 


 258
TTTGC
cctgTGGTTTTGGTCTTAGGAAC
17290
BlatCas9
 21
0


 


 259
AGCGAA
TCCTTTGGGTGTATGGGTCGT
17291
cCas9-v17
 21
0


 


 260
AGCGAA
TCCTTTGGGTGTATGGGTCGT
17292
cCas9-v42
 21
0


 


 261
AGCG
CCTTTGGGTGTATGGGTCGT
17293
SpyCas9-
 21
0






VRER




 


 262
TAG
TCCTTTGGGTGTATGGGTCG
17294
ScaCas9
 22
0


 


 263
TAG
TCCTTTGGGTGTATGGGTCG
17295
ScaCas9-
 22
0






HiFi-Sc++




 


 264
TAG
TCCTTTGGGTGTATGGGTCG
17296
ScaCas9-Sc++
 22
0


 


 265
TAG
TCCTTTGGGTGTATGGGTCG
17297
SpyCas9-
 22
0






SpRY




 


 266
CTT
TGTGGTTTTGGTCTTAGGAA
17298
SpyCas9-
 22
0






SpRY




 


 267
TAGC
TCCTTTGGGTGTATGGGTCG
17299
SpyCas9-
 22
0






3var-NRRH




 


 268
GTAG
AATCCTTTGGGTGTATGGGTC
17300
SauriCas9-
 23
0






KKH




 


 269
GTA
ATCCTTTGGGTGTATGGGTC
17301
SpyCas9-
 23
0






SpRY




 


 270
ACT
CTGTGGTTTTGGTCTTAGGA
17302
SpyCas9-
 23
0






SpRY




 


 271
GTAGCGAA
tcaaTCCTTTGGGTGTATGGGTC
17303
BlatCas9
 23
0


 


 272
GTAGCGAA
tcaaTCCTTTGGGTGTATGGGTC
17304
BlatCas9
 23
0


 


 273
GTAGCGAA
tcAATCCTTTGGGTGTATGGGTC
17305
GeoCas9
 23
0


 


 274
GTAGC
tcaaTCCTTTGGGTGTATGGGTC
17306
BlatCas9
 23
0


 


 275
CGTAG
CAATCCTTTGGGTGTATGGGT
17307
SauCas9KKH
 24
0


 


 276
CG
AATCCTTTGGGTGTATGGGT
17308
SpyCas9-NG
 24
0


 


 277
CG
AATCCTTTGGGTGTATGGGT
17309
SpyCas9-xCas
 24
0


 


 278
CG
AATCCTTTGGGTGTATGGGT
17310
SpyCas9-
 24
0






xCas-NG




 


 279
CGT
AATCCTTTGGGTGTATGGGT
17311
SpyCas9-SpG
 24
0


 


 280
CGT
AATCCTTTGGGTGTATGGGT
17312
SpyCas9-
 24
0






SpRY




 


 281
AAC
CCTGTGGTTTTGGTCTTAGG
17313
SpyCas9-
 24
0






SpRY




 


 282
CGTA
AATCCTTTGGGTGTATGGGT
17314
SpyCas9-
 24
0






3var-NRTH




 


 283
AACT
CCTGTGGTTTTGGTCTTAGG
17315
SpyCas9-
 24
0






3var-NRCH




 


 284
TCG
CAATCCTTTGGGTGTATGGG
17316
ScaCas9
 25
0


 


 285
TCG
CAATCCTTTGGGTGTATGGG
17317
ScaCas9-
 25
0






HiFi-Sc++




 


 286
TCG
CAATCCTTTGGGTGTATGGG
17318
ScaCas9-Sc++
 25
0


 


 287
TCG
CAATCCTTTGGGTGTATGGG
17319
SpyCas9-
 25
0






SpRY




 


 288
GAA
GCCTGTGGTTTTGGTCTTAG
17320
SpyCas9-
 25
0






SpRY




 


 289
GAA
GCCTGTGGTTTTGGTCTTAG
17321
SpyCas9-xCas
 25
0


 


 290
GAACTTT
AAGCCTGTGGTTTTGGTCTTAG
17322
CdiCas9
 25
0


 


 291
GAAC
GCCTGTGGTTTTGGTCTTAG
17323
SpyCas9-
 25
0






3var-NRRH




 


 292
GAAC
agCCTGTGGTTTTGGTCTTAG
17324
iSpyMacCas9
 25
0


 


 293
GG
AGCCTGTGGTTTTGGTCTTA
17325
SpyCas9-NG
 26
0


 


 294
GG
AGCCTGTGGTTTTGGTCTTA
17326
SpyCas9-xCas
 26
0


 


 295
GG
AGCCTGTGGTTTTGGTCTTA
17327
SpyCas9-
 26
0






xCas-NG




 


 296
GGA
AGCCTGTGGTTTTGGTCTTA
17328
SpyCas9-SpG
 26
0


 


 297
GGA
AGCCTGTGGTTTTGGTCTTA
17329
SpyCas9-
 26
0






SpRY




 


 298
GTC
TCAATCCTTTGGGTGTATGG
17330
SpyCas9-
 26
0






SpRY




 


 299
GGAACTTT
tcaaGCCTGTGGTTTTGGTCTTA
17331
BlatCas9
 26
0


 


 300
GGAAC
tcaaGCCTGTGGTTTTGGTCTTA
17332
BlatCas9
 26
0


 


 301
GGAACT
AAGCCTGTGGTTTTGGTCTTA
17333
cCas9-v16
 26
0


 


 302
GGAACT
AAGCCTGTGGTTTTGGTCTTA
17334
cCas9-v21
 26
0


 


 303
GGAACTT
CAAGCCTGTGGTTTTGGTCTTA
17335
CdiCas9
 26
0


 


 304
GGAA
AGCCTGTGGTTTTGGTCTTA
17336
SpyCas9-
 26
0






3var-NRRH




 


 305
GGAA
AGCCTGTGGTTTTGGTCTTA
17337
SpyCas9-
 26
0






VQR




 


 306
AGGAA
ctCAAGCCTGTGGTTTTGGTCTT
17338
SauCas9
 27
0


 


 307
AGGAA
CAAGCCTGTGGTTTTGGTCTT
17339
SauCas9KKH
 27
0


 


 308
AGG
AAGCCTGTGGTTTTGGTCTT
17340
ScaCas9
 27
0


 


 309
AGG
AAGCCTGTGGTTTTGGTCTT
17341
ScaCas9-
 27
0






HiFi-Sc++




 


 310
AGG
AAGCCTGTGGTTTTGGTCTT
17342
ScaCas9-Sc++
 27
0


 


 311
AGG
AAGCCTGTGGTTTTGGTCTT
17343
SpyCas9
 27
0


 


 312
AGG
AAGCCTGTGGTTTTGGTCTT
17344
SpyCas9-HF1
 27
0


 


 313
AGG
AAGCCTGTGGTTTTGGTCTT
17345
SpyCas9-SpG
 27
0


 


 314
AGG
AAGCCTGTGGTTTTGGTCTT
17346
SpyCas9-
 27
0






SpRY




 


 315
GG
CTCAATCCTTTGGGTGTATG
17347
SpyCas9-NG
 27
0


 


 316
GG
CTCAATCCTTTGGGTGTATG
17348
SpyCas9-xCas
 27
0


 


 317
GG
CTCAATCCTTTGGGTGTATG
17349
SpyCas9-
 27
0






xCas-NG




 


 318
AG
AAGCCTGTGGTTTTGGTCTT
17350
SpyCas9-NG
 27
0


 


 319
AG
AAGCCTGTGGTTTTGGTCTT
17351
SpyCas9-xCas
 27
0


 


 320
AG
AAGCCTGTGGTTTTGGTCTT
17352
SpyCas9-
 27
0






xCas-NG




 


 321
GGT
CTCAATCCTTTGGGTGTATG
17353
SpyCas9-SpG
 27
0


 


 322
GGT
CTCAATCCTTTGGGTGTATG
17354
SpyCas9-
 27
0






SpRY




 


 323
AGGAAC
CAAGCCTGTGGTTTTGGTCTT
17355
cCas9-v17
 27
0


 


 324
AGGAAC
CAAGCCTGTGGTTTTGGTCTT
17356
cCas9-v42
 27
0


 


 325
AGGAACT
TCAAGCCTGTGGTTTTGGTCTT
17357
CdiCas9
 27
0


 


 326
AGGA
AAGCCTGTGGTTTTGGTCTT
17358
SpyCas9-
 27
0






3var-NRRH




 


 327
GGTC
CTCAATCCTTTGGGTGTATG
17359
SpyCas9-
 27
0






3var-NRTH




 


 328
TAGGA
acTCAAGCCTGTGGTTTTGGTCT
17360
SauCas9
 28
0


 


 329
TAGGA
TCAAGCCTGTGGTTTTGGTCT
17361
SauCas9KKH
 28
0


 


 330
TAGG
TCAAGCCTGTGGTTTTGGTCT
17362
SauriCas9
 28
0


 


 331
TAGG
TCAAGCCTGTGGTTTTGGTCT
17363
SauriCas9-
 28
0






KKH




 


 332
GGG
CCTCAATCCTTTGGGTGTAT
17364
ScaCas9
 28
0


 


 333
GGG
CCTCAATCCTTTGGGTGTAT
17365
ScaCas9-
 28
0






HiFi-Sc++




 


 334
GGG
CCTCAATCCTTTGGGTGTAT
17366
ScaCas9-Sc++
 28
0


 


 335
GGG
CCTCAATCCTTTGGGTGTAT
17367
SpyCas9
 28
0


 


 336
GGG
CCTCAATCCTTTGGGTGTAT
17368
SpyCas9-HF1
 28
0


 


 337
GGG
CCTCAATCCTTTGGGTGTAT
17369
SpyCas9-SpG
 28
0


 


 338
GGG
CCTCAATCCTTTGGGTGTAT
17370
SpyCas9-
 28
0






SpRY




 


 339
TAG
CAAGCCTGTGGTTTTGGTCT
17371
ScaCas9
 28
0


 


 340
TAG
CAAGCCTGTGGTTTTGGTCT
17372
ScaCas9-
 28
0






HiFi-Sc++




 


 341
TAG
CAAGCCTGTGGTTTTGGTCT
17373
ScaCas9-Sc++
 28
0


 


 342
TAG
CAAGCCTGTGGTTTTGGTCT
17374
SpyCas9-
 28
0






SpRY




 


 343
GG
CCTCAATCCTTTGGGTGTAT
17375
SpyCas9-NG
 28
0


 


 344
GG
CCTCAATCCTTTGGGTGTAT
17376
SpyCas9-xCas
 28
0


 


 345
GG
CCTCAATCCTTTGGGTGTAT
17377
SpyCas9-
 28
0






xCas-NG




 


 346
GGGTCGTA
agacCTCAATCCTTTGGGTGTAT
17378
BlatCas9
 28
0


 


 347
GGGTC
agacCTCAATCCTTTGGGTGTAT
17379
BlatCas9
 28
0


 


 348
TAGGAA
TCAAGCCTGTGGTTTTGGTCT
17380
cCas9-v17
 28
0


 


 349
TAGGAA
TCAAGCCTGTGGTTTTGGTCT
17381
cCas9-v42
 28
0


 


 350
GGGT
CCTCAATCCTTTGGGTGTAT
17382
SpyCas9-
 28
0






3var-NRRH




 


 351
TTAGG
CTCAAGCCTGTGGTTTTGGTC
17383
SauCas9KKH
 29
0


 


 352
TGGG
GACCTCAATCCTTTGGGTGTA
17384
SauriCas9
 29
0


 


 353
TGGG
GACCTCAATCCTTTGGGTGTA
17385
SauriCas9-
 29
0






KKH




 


 354
TTAG
CTCAAGCCTGTGGTTTTGGTC
17386
SauriCas9-
 29
0






KKH




 


 355
TGG
ACCTCAATCCTTTGGGTGTA
17387
ScaCas9
 29
0


 


 356
TGG
ACCTCAATCCTTTGGGTGTA
17388
ScaCas9-
 29
0






HiFi-Sc++




 


 357
TGG
ACCTCAATCCTTTGGGTGTA
17389
ScaCas9-Sc++
 29
0


 


 358
TGG
ACCTCAATCCTTTGGGTGTA
17390
SpyCas9
 29
0


 


 359
TGG
ACCTCAATCCTTTGGGTGTA
17391
SpyCas9-HF1
 29
0


 


 360
TGG
ACCTCAATCCTTTGGGTGTA
17392
SpyCas9-SpG
 29
0


 


 361
TGG
ACCTCAATCCTTTGGGTGTA
17393
SpyCas9-
 29
0






SpRY




 


 362
TG
ACCTCAATCCTTTGGGTGTA
17394
SpyCas9-NG
 29
0


 


 363
TG
ACCTCAATCCTTTGGGTGTA
17395
SpyCas9-xCas
 29
0


 


 364
TG
ACCTCAATCCTTTGGGTGTA
17396
SpyCas9-
 29
0






xCas-NG




 


 365
TTA
TCAAGCCTGTGGTTTTGGTC
17397
SpyCas9-
 29
0






SpRY




 


 366
TTAGGAA
TCAAGCCTGTGGTTTTGGTC
17398
St1Cas9
 29
0


 


 367
TTAGGA
CTCAAGCCTGTGGTTTTGGTC
17399
cCas9-v17
 29
0


 


 368
TTAGGA
CTCAAGCCTGTGGTTTTGGTC
17400
cCas9-v42
 29
0


 


 369
ATGGG
caAGACCTCAATCCTTTGGGTGT
17401
SauCas9
 30
0


 


 370
ATGGG
AGACCTCAATCCTTTGGGTGT
17402
SauCas9KKH
 30
0


 


 371
ATGGGT
caAGACCTCAATCCTTTGGGTGT
17403
SauCas9
 30
0


 


 372
ATGGGT
AGACCTCAATCCTTTGGGTGT
17404
SauCas9KKH
 30
0


 


 373
ATGGGT
AGACCTCAATCCTTTGGGTGT
17405
cCas9-v17
 30
0


 


 374
ATGGGT
AGACCTCAATCCTTTGGGTGT
17406
cCas9-v42
 30
0


 


 375
CTTAG
ACTCAAGCCTGTGGTTTTGGT
17407
SauCas9KKH
 30
0


 


 376
ATGG
AGACCTCAATCCTTTGGGTGT
17408
SauriCas9
 30
0


 


 377
ATGG
AGACCTCAATCCTTTGGGTGT
17409
SauriCas9-
 30
0






KKH




 


 378
ATG
GACCTCAATCCTTTGGGTGT
17410
ScaCas9
 30
0


 


 379
ATG
GACCTCAATCCTTTGGGTGT
17411
ScaCas9-
 30
0






HiFi-Sc++




 


 380
ATG
GACCTCAATCCTTTGGGTGT
17412
ScaCas9-Sc++
 30
0


 


 381
ATG
GACCTCAATCCTTTGGGTGT
17413
SpyCas9-
 30
0






SpRY




 


 382
CTT
CTCAAGCCTGTGGTTTTGGT
17414
SpyCas9-
 30
0






SpRY




 


 383
TATGG
AAGACCTCAATCCTTTGGGTG
17415
SauCas9KKH
 31
0


 


 384
TAT
AGACCTCAATCCTTTGGGTG
17416
SpyCas9-
 31
0






SpRY




 


 385
TCT
ACTCAAGCCTGTGGTTTTGG
17417
SpyCas9-
 31
0






SpRY




 


 386
GTA
AAGACCTCAATCCTTTGGGT
17418
SpyCas9-
 32
0






SpRY




 


 387
GTC
CACTCAAGCCTGTGGTTTTG
17419
SpyCas9-
 32
0






SpRY




 


 388
TG
CAAGACCTCAATCCTTTGGG
17420
SpyCas9-NG
 33
0


 


 389
TG
CAAGACCTCAATCCTTTGGG
17421
SpyCas9-xCas
 33
0


 


 390
TG
CAAGACCTCAATCCTTTGGG
17422
SpyCas9-
 33
0






xCas-NG




 


 391
GG
TCACTCAAGCCTGTGGTTTT
17423
SpyCas9-NG
 33
0


 


 392
GG
TCACTCAAGCCTGTGGTTTT
17424
SpyCas9-xCas
 33
0


 


 393
GG
TCACTCAAGCCTGTGGTTTT
17425
SpyCas9-
 33
0






xCas-NG




 


 394
TGT
CAAGACCTCAATCCTTTGGG
17426
SpyCas9-SpG
 33
0


 


 395
TGT
CAAGACCTCAATCCTTTGGG
17427
SpyCas9-
 33
0






SpRY




 


 396
GGT
TCACTCAAGCCTGTGGTTTT
17428
SpyCas9-SpG
 33
0


 


 397
GGT
TCACTCAAGCCTGTGGTTTT
17429
SpyCas9-
 33
0






SpRY




 


 398
TGTA
CAAGACCTCAATCCTTTGGG
17430
SpyCas9-
 33
0






3var-NRTH




 


 399
GGTC
TCACTCAAGCCTGTGGTTTT
17431
SpyCas9-
 33
0






3var-NRTH




 


 400
GTG
CCAAGACCTCAATCCTTTGG
17432
ScaCas9
 34
0


 


 401
GTG
CCAAGACCTCAATCCTTTGG
17433
ScaCas9-
 34
0






HiFi-Sc++




 


 402
GTG
CCAAGACCTCAATCCTTTGG
17434
ScaCas9-Sc++
 34
0


 


 403
GTG
CCAAGACCTCAATCCTTTGG
17435
SpyCas9-
 34
0






SpRY




 


 404
TGG
TTCACTCAAGCCTGTGGTTT
17436
ScaCas9
 34
0


 


 405
TGG
TTCACTCAAGCCTGTGGTTT
17437
ScaCas9-
 34
0






HiFi-Sc++




 


 406
TGG
TTCACTCAAGCCTGTGGTTT
17438
ScaCas9-Sc++
 34
0


 


 407
TGG
TTCACTCAAGCCTGTGGTTT
17439
SpyCas9
 34
0


 


 408
TGG
TTCACTCAAGCCTGTGGTTT
17440
SpyCas9-HF1
 34
0


 


 409
TGG
TTCACTCAAGCCTGTGGTTT
17441
SpyCas9-SpG
 34
0


 


 410
TGG
TTCACTCAAGCCTGTGGTTT
17442
SpyCas9-
 34
0






SpRY




 


 411
TG
TTCACTCAAGCCTGTGGTTT
17443
SpyCas9-NG
 34
0


 


 412
TG
TTCACTCAAGCCTGTGGTTT
17444
SpyCas9-xCas
 34
0


 


 413
TG
TTCACTCAAGCCTGTGGTTT
17445
SpyCas9-
 34
0






xCas-NG




 


 414
TGGTCTTA
ccctTCACTCAAGCCTGTGGTTT
17446
BlatCas9
 34
0


 


 415
TGGTC
ccctTCACTCAAGCCTGTGGTTT
17447
BlatCas9
 34
0


 


 416
TGGTCTT
CCTTCACTCAAGCCTGTGGTTT
17448
CdiCas9
 34
0


 


 417
TGGT
TTCACTCAAGCCTGTGGTTT
17449
SpyCas9-
 34
0






3var-NRRH




 


 418
TTGG
CCTTCACTCAAGCCTGTGGTT
17450
SauriCas9
 35
0


 


 419
TTGG
CCTTCACTCAAGCCTGTGGTT
17451
SauriCas9-
 35
0






KKH




 


 420
TTG
CTTCACTCAAGCCTGTGGTT
17452
ScaCas9
 35
0


 


 421
TTG
CTTCACTCAAGCCTGTGGTT
17453
ScaCas9-
 35
0






HiFi-Sc++




 


 422
TTG
CTTCACTCAAGCCTGTGGTT
17454
ScaCas9-Sc++
 35
0


 


 423
TTG
CTTCACTCAAGCCTGTGGTT
17455
SpyCas9-
 35
0






SpRY




 


 424
GG
TCCAAGACCTCAATCCTTTG
17456
SpyCas9-NG
 35
0


 


 425
GG
TCCAAGACCTCAATCCTTTG
17457
SpyCas9-xCas
 35
0


 


 426
GG
TCCAAGACCTCAATCCTTTG
17458
SpyCas9-
 35
0






xCas-NG




 


 427
GGT
TCCAAGACCTCAATCCTTTG
17459
SpyCas9-SpG
 35
0


 


 428
GGT
TCCAAGACCTCAATCCTTTG
17460
SpyCas9-
 35
0






SpRY




 


 429
TTTGG
CCCTTCACTCAAGCCTGTGGT
17461
SauCas9KKH
 36
0


 


 430
TTTGGT
CCCTTCACTCAAGCCTGTGGT
17462
SauCas9KKH
 36
0


 


 431
GGG
GTCCAAGACCTCAATCCTTT
17463
ScaCas9
 36
0


 


 432
GGG
GTCCAAGACCTCAATCCTTT
17464
ScaCas9-
 36
0






HiFi-Sc++




 


 433
GGG
GTCCAAGACCTCAATCCTTT
17465
ScaCas9-Sc++
 36
0


 


 434
GGG
GTCCAAGACCTCAATCCTTT
17466
SpyCas9
 36
0


 


 435
GGG
GTCCAAGACCTCAATCCTTT
17467
SpyCas9-HF1
 36
0


 


 436
GGG
GTCCAAGACCTCAATCCTTT
17468
SpyCas9-SpG
 36
0


 


 437
GGG
GTCCAAGACCTCAATCCTTT
17469
SpyCas9-
 36
0






SpRY




 


 438
GG
GTCCAAGACCTCAATCCTTT
17470
SpyCas9-NG
 36
0


 


 439
GG
GTCCAAGACCTCAATCCTTT
17471
SpyCas9-xCas
 36
0


 


 440
GG
GTCCAAGACCTCAATCCTTT
17472
SpyCas9-
 36
0






xCas-NG




 


 441
TTT
CCTTCACTCAAGCCTGTGGT
17473
SpyCas9-
 36
0






SpRY




 


 442
TTTGGTCT
gtgcCCTTCACTCAAGCCTGTGGT
17474
NmeCas9
 36
0


 


 443
GGGT
GTCCAAGACCTCAATCCTTT
17475
SpyCas9-
 36
0






3var-NRRH




 


 444
TGGG
TTGTCCAAGACCTCAATCCTT
17476
SauriCas9
 37
0


 


 445
TGGG
TTGTCCAAGACCTCAATCCTT
17477
SauriCas9-
 37
0






KKH




 


 446
TGG
TGTCCAAGACCTCAATCCTT
17478
ScaCas9
 37
0


 


 447
TGG
TGTCCAAGACCTCAATCCTT
17479
ScaCas9-
 37
0






HiFi-Sc++




 


 448
TGG
TGTCCAAGACCTCAATCCTT
17480
ScaCas9-Sc++
 37
0


 


 449
TGG
TGTCCAAGACCTCAATCCTT
17481
SpyCas9
 37
0


 


 450
TGG
TGTCCAAGACCTCAATCCTT
17482
SpyCas9-HF1
 37
0


 


 451
TGG
TGTCCAAGACCTCAATCCTT
17483
SpyCas9-SpG
 37
0


 


 452
TGG
TGTCCAAGACCTCAATCCTT
17484
SpyCas9-
 37
0






SpRY




 


 453
TG
TGTCCAAGACCTCAATCCTT
17485
SpyCas9-NG
 37
0


 


 454
TG
TGTCCAAGACCTCAATCCTT
17486
SpyCas9-xCas
 37
0


 


 455
TG
TGTCCAAGACCTCAATCCTT
17487
SpyCas9-
 37
0






xCas-NG




 


 456
TTT
CCCTTCACTCAAGCCTGTGG
17488
SpyCas9-
 37
0






SpRY




 


 457
TGGGTG
TTGTCCAAGACCTCAATCCTT
17489
cCas9-v16
 37
0


 


 458
TGGGTG
TTGTCCAAGACCTCAATCCTT
17490
cCas9-v21
 37
0


 


 459
TTGGG
gtATTGTCCAAGACCTCAATCCT
17491
SauCas9
 38
0


 


 460
TTGGG
ATTGTCCAAGACCTCAATCCT
17492
SauCas9KKH
 38
0


 


 461
TTGGGT
gtATTGTCCAAGACCTCAATCCT
17493
SauCas9
 38
0


 


 462
TTGGGT
ATTGTCCAAGACCTCAATCCT
17494
SauCas9KKH
 38
0


 


 463
TTGGGT
ATTGTCCAAGACCTCAATCCT
17495
cCas9-v17
 38
0


 


 464
TTGGGT
ATTGTCCAAGACCTCAATCCT
17496
cCas9-v42
 38
0


 


 465
TTGG
ATTGTCCAAGACCTCAATCCT
17497
SauriCas9
 38
0


 


 466
TTGG
ATTGTCCAAGACCTCAATCCT
17498
SauriCas9-
 38
0






KKH




 


 467
TTG
TTGTCCAAGACCTCAATCCT
17499
ScaCas9
 38
0


 


 468
TTG
TTGTCCAAGACCTCAATCCT
17500
ScaCas9-
 38
0






HiFi-Sc++




 


 469
TTG
TTGTCCAAGACCTCAATCCT
17501
ScaCas9-Sc++
 38
0


 


 470
TTG
TTGTCCAAGACCTCAATCCT
17502
SpyCas9-
 38
0






SpRY




 


 471
GTT
GCCCTTCACTCAAGCCTGTG
17503
SpyCas9-
 38
0






SpRY




 


 472
TTTGG
TATTGTCCAAGACCTCAATCC
17504
SauCas9KKH
 39
0


 


 473
GG
TGCCCTTCACTCAAGCCTGT
17505
SpyCas9-NG
 39
0


 


 474
GG
TGCCCTTCACTCAAGCCTGT
17506
SpyCas9-xCas
 39
0


 


 475
GG
TGCCCTTCACTCAAGCCTGT
17507
SpyCas9-
 39
0






xCas-NG




 


 476
GGT
TGCCCTTCACTCAAGCCTGT
17508
SpyCas9-SpG
 39
0


 


 477
GGT
TGCCCTTCACTCAAGCCTGT
17509
SpyCas9-
 39
0






SpRY




 


 478
TTT
ATTGTCCAAGACCTCAATCC
17510
SpyCas9-
 39
0






SpRY




 


 479
GGTT
TGCCCTTCACTCAAGCCTGT
17511
SpyCas9-
 39
0






3var-NRTH




 


 480
TGG
GTGCCCTTCACTCAAGCCTG
17512
ScaCas9
 40
0


 


 481
TGG
GTGCCCTTCACTCAAGCCTG
17513
ScaCas9-
 40
0






HiFi-Sc++




 


 482
TGG
GTGCCCTTCACTCAAGCCTG
17514
ScaCas9-Sc++
 40
0


 


 483
TGG
GTGCCCTTCACTCAAGCCTG
17515
SpyCas9
 40
0


 


 484
TGG
GTGCCCTTCACTCAAGCCTG
17516
SpyCas9-HF1
 40
0


 


 485
TGG
GTGCCCTTCACTCAAGCCTG
17517
SpyCas9-SpG
 40
0


 


 486
TGG
GTGCCCTTCACTCAAGCCTG
17518
SpyCas9-
 40
0






SpRY




 


 487
TG
GTGCCCTTCACTCAAGCCTG
17519
SpyCas9-NG
 40
0


 


 488
TG
GTGCCCTTCACTCAAGCCTG
17520
SpyCas9-xCas
 40
0


 


 489
TG
GTGCCCTTCACTCAAGCCTG
17521
SpyCas9-
 40
0






xCas-NG




 


 490
CTT
TATTGTCCAAGACCTCAATC
17522
SpyCas9-
 40
0






SpRY




 


 491
TGGTTTT
TGGTGCCCTTCACTCAAGCCTG
17523
CdiCas9
 40
0


 


 492
TGGT
GTGCCCTTCACTCAAGCCTG
17524
SpyCas9-
 40
0






3var-NRRH




 


 493
GTGG
TGGTGCCCTTCACTCAAGCCT
17525
SauriCas9
 41
0


 


 494
GTGG
TGGTGCCCTTCACTCAAGCCT
17526
SauriCas9-
 41
0






KKH




 


 495
GTG
GGTGCCCTTCACTCAAGCCT
17527
ScaCas9
 41
0


 


 496
GTG
GGTGCCCTTCACTCAAGCCT
17528
ScaCas9-
 41
0






HiFi-Sc++




 


 497
GTG
GGTGCCCTTCACTCAAGCCT
17529
ScaCas9-Sc++
 41
0


 


 498
GTG
GGTGCCCTTCACTCAAGCCT
17530
SpyCas9-
 41
0






SpRY




 


 499
CCT
GTATTGTCCAAGACCTCAAT
17531
SpyCas9-
 41
0






SpRY




 


 500
GTGGTT
TGGTGCCCTTCACTCAAGCCT
17532
cCas9-v16
 41
0


 


 501
GTGGTT
TGGTGCCCTTCACTCAAGCCT
17533
cCas9-v21
 41
0


 


 502
TGTGGTT
caaATGGTGCCCTTCACTCAAGCC
17534
PpnCas9
 42
0


 


 503
TGTGG
ATGGTGCCCTTCACTCAAGCC
17535
SauCas9KKH
 42
0


 


 504
TGTGGT
ATGGTGCCCTTCACTCAAGCC
17536
SauCas9KKH
 42
0


 


 505
TG
TGGTGCCCTTCACTCAAGCC
17537
SpyCas9-NG
 42
0


 


 506
TG
TGGTGCCCTTCACTCAAGCC
17538
SpyCas9-xCas
 42
0


 


 507
TG
TGGTGCCCTTCACTCAAGCC
17539
SpyCas9-
 42
0






xCas-NG




 


 508
TGT
TGGTGCCCTTCACTCAAGCC
17540
SpyCas9-SpG
 42
0


 


 509
TGT
TGGTGCCCTTCACTCAAGCC
17541
SpyCas9-
 42
0






SpRY




 


 510
TCC
GGTATTGTCCAAGACCTCAA
17542
SpyCas9-
 42
0






SpRY




 


 511
TGTGGTTT
caaaTGGTGCCCTTCACTCAAGCC
17543
NmeCas9
 42
0


 


 512
CTG
ATGGTGCCCTTCACTCAAGC
17544
ScaCas9
 43
0


 


 513
CTG
ATGGTGCCCTTCACTCAAGC
17545
ScaCas9-
 43
0






HiFi-Sc++




 


 514
CTG
ATGGTGCCCTTCACTCAAGC
17546
ScaCas9-Sc++
 43
0


 


 515
CTG
ATGGTGCCCTTCACTCAAGC
17547
SpyCas9-
 43
0






SpRY




 


 516
ATC
GGGTATTGTCCAAGACCTCA
17548
SpyCas9-
 43
0






SpRY




 


 517
AAT
TGGGTATTGTCCAAGACCTC
17549
SpyCas9-
 44
0






SpRY




 


 518
CCT
AATGGTGCCCTTCACTCAAG
17550
SpyCas9-
 44
0






SpRY




 


 519
AATCCTTT
tgctGGGTATTGTCCAAGACCTC
17551
BlatCas9
 44
0


 


 520
AATCC
tgctGGGTATTGTCCAAGACCTC
17552
BlatCas9
 44
0


 


 521
AATC
TGGGTATTGTCCAAGACCTC
17553
SpyCas9-
 44
0






3var-NRTH




 


 522
CAATCC
gcTGCTGGGTATTGTCCAAGACCT
17554
Nme2Cas9
 45
0


 


 523
CAA
CTGGGTATTGTCCAAGACCT
17555
SpyCas9-
 45
0






SpRY




 


 524
GCC
AAATGGTGCCCTTCACTCAA
17556
SpyCas9-
 45
0






SpRY




 


 525
CAATCCTT
ctgcTGGGTATTGTCCAAGACCT
17557
BlatCas9
 45
0


 


 526
CAATC
ctgcTGGGTATTGTCCAAGACCT
17558
BlatCas9
 45
0


 


 527
CAATCCT
TGCTGGGTATTGTCCAAGACCT
17559
CdiCas9
 45
0


 


 528
CAAT
CTGGGTATTGTCCAAGACCT
17560
SpyCas9-
 45
0






3var-NRRH




 


 529
CAAT
gcTGGGTATTGTCCAAGACCT
17561
iSpyMacCas9
 45
0


 


 530
AG
CAAATGGTGCCCTTCACTCA
17562
SpyCas9-NG
 46
0


 


 531
AG
CAAATGGTGCCCTTCACTCA
17563
SpyCas9-xCas
 46
0


 


 532
AG
CAAATGGTGCCCTTCACTCA
17564
SpyCas9-
 46
0






xCas-NG




 


 533
AGC
CAAATGGTGCCCTTCACTCA
17565
SpyCas9-SpG
 46
0


 


 534
AGC
CAAATGGTGCCCTTCACTCA
17566
SpyCas9-
 46
0






SpRY




 


 535
TCA
GCTGGGTATTGTCCAAGACC
17567
SpyCas9-
 46
0






SpRY




 


 536
TCAATCC
CTGCTGGGTATTGTCCAAGACC
17568
CdiCas9
 46
0


 


 537
AGCC
CAAATGGTGCCCTTCACTCA
17569
SpyCas9-
 46
0






3var-NRCH




 


 538
CTCAA
CTGCTGGGTATTGTCCAAGAC
17570
SauCas9KKH
 47
0


 


 539
CTCAAT
CTGCTGGGTATTGTCCAAGAC
17571
SauCas9KKH
 47
0


 


 540
CTCAAT
CTGCTGGGTATTGTCCAAGAC
17572
cCas9-v17
 47
0


 


 541
CTCAAT
CTGCTGGGTATTGTCCAAGAC
17573
cCas9-v42
 47
0


 


 542
AAG
CCAAATGGTGCCCTTCACTC
17574
ScaCas9
 47
0


 


 543
AAG
CCAAATGGTGCCCTTCACTC
17575
ScaCas9-
 47
0






HiFi-Sc++




 


 544
AAG
CCAAATGGTGCCCTTCACTC
17576
ScaCas9-Sc++
 47
0


 


 545
AAG
CCAAATGGTGCCCTTCACTC
17577
SpyCas9-
 47
0






SpRY




 


 546
CTC
TGCTGGGTATTGTCCAAGAC
17578
SpyCas9-
 47
0






SpRY




 


 547
AAGCCTGT
tctcCAAATGGTGCCCTTCACTC
17579
BlatCas9
 47
0


 


 548
AAGCC
tctcCAAATGGTGCCCTTCACTC
17580
BlatCas9
 47
0


 


 549
AAGC
CCAAATGGTGCCCTTCACTC
17581
SpyCas9-
 47
0






3var-NRRH




 


 550
CAAGCC
ttTCTCCAAATGGTGCCCTTCACT
17582
Nme2Cas9
 48
0


 


 551
CAAG
CTCCAAATGGTGCCCTTCACT
17583
SauriCas9-
 48
0






KKH




 


 552
CAAG
TCCAAATGGTGCCCTTCACT
17584
SpyCas9-
 48
0






QQR1




 


 553
CAAG
ctCCAAATGGTGCCCTTCACT
17585
iSpy MacCas9
 48
0


 


 554
CAA
TCCAAATGGTGCCCTTCACT
17586
SpyCas9-
 48
0






SpRY




 


 555
CCT
CTGCTGGGTATTGTCCAAGA
17587
SpyCas9-
 48
0






SpRY




 


 556
CAAGCCTG
ttctCCAAATGGTGCCCTTCACT
17588
BlatCas9
 48
0


 


 557
CAAGC
ttctCCAAATGGTGCCCTTCACT
17589
BlatCas9
 48
0


 


 558
TCAAG
TCTCCAAATGGTGCCCTTCAC
17590
SauCas9KKH
 49
0


 


 559
ACC
GCTGCTGGGTATTGTCCAAG
17591
SpyCas9-
 49
0






SpRY




 


 560
TCA
CTCCAAATGGTGCCCTTCAC
17592
SpyCas9-
 49
0






SpRY




 


 561
ACCTCAAT
taagCTGCTGGGTATTGTCCAAG
17593
BlatCas9
 49
0


 


 562
ACCTC
taagCTGCTGGGTATTGTCCAAG
17594
BlatCas9
 49
0


 


 563
TCAAGC
TCTCCAAATGGTGCCCTTCAC
17595
cCas9-v17
 49
0


 


 564
TCAAGC
TCTCCAAATGGTGCCCTTCAC
17596
cCas9-v42
 49
0


 


 565
CTCAA
TTCTCCAAATGGTGCCCTTCA
17597
SauCas9KKH
 50
0


 


 566
GAC
AGCTGCTGGGTATTGTCCAA
17598
SpyCas9-
 50
0






SpRY




 


 567
CTC
TCTCCAAATGGTGCCCTTCA
17599
SpyCas9-
 50
0






SpRY




 


 568
CTCAAG
TTCTCCAAATGGTGCCCTTCA
17600
cCas9-v17
 50
0


 


 569
CTCAAG
TTCTCCAAATGGTGCCCTTCA
17601
cCas9-v42
 50
0


 


 570
GACC
AGCTGCTGGGTATTGTCCAA
17602
SpyCas9-
 50
0






3var-NRCH




 


 571
AG
AAGCTGCTGGGTATTGTCCA
17603
SpyCas9-NG
 51
0


 


 572
AG
AAGCTGCTGGGTATTGTCCA
17604
SpyCas9-xCas
 51
0


 


 573
AG
AAGCTGCTGGGTATTGTCCA
17605
SpyCas9-
 51
0






xCas-NG




 


 574
AGA
AAGCTGCTGGGTATTGTCCA
17606
SpyCas9-SpG
 51
0


 


 575
AGA
AAGCTGCTGGGTATTGTCCA
17607
SpyCas9-
 51
0






SpRY




 


 576
ACT
TTCTCCAAATGGTGCCCTTC
17608
SpyCas9-
 51
0






SpRY




 


 577
AGACC
cttaAGCTGCTGGGTATTGTCCA
17609
BlatCas9
 51
0


 


 578
AGACCTC
TTAAGCTGCTGGGTATTGTCCA
17610
CdiCas9
 51
0


 


 579
AGAC
AAGCTGCTGGGTATTGTCCA
17611
SpyCas9-
 51
0






3var-NRRH




 


 580
AGAC
AAGCTGCTGGGTATTGTCCA
17612
SpyCas9-
 51
0






VQR




 


 581
AAGACC
atCTTAAGCTGCTGGGTATTGTCC
17613
Nme2Cas9
 52
0


 


 582
AAG
TAAGCTGCTGGGTATTGTCC
17614
ScaCas9
 52
0


 


 583
AAG
TAAGCTGCTGGGTATTGTCC
17615
ScaCas9-
 52
0






HiFi-Sc++




 


 584
AAG
TAAGCTGCTGGGTATTGTCC
17616
ScaCas9-Sc++
 52
0


 


 585
AAG
TAAGCTGCTGGGTATTGTCC
17617
SpyCas9-
 52
0






SpRY




 


 586
CAC
TTTCTCCAAATGGTGCCCTT
17618
SpyCas9-
 52
0






SpRY




 


 587
CACTCAAG
acctTTCTCCAAATGGTGCCCTT
17619
BlatCas9
 52
0


 


 588
AAGAC
tcttAAGCTGCTGGGTATTGTCC
17620
BlatCas9
 52
0


 


 589
CACTC
acctTTCTCCAAATGGTGCCCTT
17621
BlatCas9
 52
0


 


 590
AAGACCT
CTTAAGCTGCTGGGTATTGTCC
17622
CdiCas9
 52
0


 


 591
AAGA
TAAGCTGCTGGGTATTGTCC
17623
SpyCas9-
 52
0






3var-NRRH




 


 592
CACT
TTTCTCCAAATGGTGCCCTT
17624
SpyCas9-
 52
0






3var-NRCH




 


 593
CAAGA
CTTAAGCTGCTGGGTATTGTC
17625
SauCas9KKH
 53
0


 


 594
CAAG
CTTAAGCTGCTGGGTATTGTC
17626
SauriCas9-
 53
0






KKH




 


 595
CAAG
TTAAGCTGCTGGGTATTGTC
17627
SpyCas9-
 53
0






QQR1




 


 596
CAAG
ctTAAGCTGCTGGGTATTGTC
17628
iSpyMacCas9
 53
0


 


 597
CAA
TTAAGCTGCTGGGTATTGTC
17629
SpyCas9-
 53
0






SpRY




 


 598
TCA
CTTTCTCCAAATGGTGCCCT
17630
SpyCas9-
 53
0






SpRY




 


 599
CAAGAC
CTTAAGCTGCTGGGTATTGTC
17631
cCas9-v17
 53
0


 


 600
CAAGAC
CTTAAGCTGCTGGGTATTGTC
17632
cCas9-v42
 53
0


 


 601
CCAAG
TCTTAAGCTGCTGGGTATTGT
17633
SauCas9KKH
 54
0


 


 602
CCA
CTTAAGCTGCTGGGTATTGT
17634
SpyCas9-
 54
0






SpRY




 


 603
TTC
CCTTTCTCCAAATGGTGCCC
17635
SpyCas9-
 54
0






SpRY




 


 604
TTCAC
ctacCTTTCTCCAAATGGTGCCC
17636
BlatCas9
 54
0


 


 605
TTCACT
ACCTTTCTCCAAATGGTGCCC
17637
cCas9-v16
 54
0


 


 606
TTCACT
ACCTTTCTCCAAATGGTGCCC
17638
cCas9-v21
 54
0


 


 607
CCAAGA
TCTTAAGCTGCTGGGTATTGT
17639
cCas9-v17
 54
0


 


 608
CCAAGA
TCTTAAGCTGCTGGGTATTGT
17640
cCas9-v42
 54
0


 


 609
TCCAA
ATCTTAAGCTGCTGGGTATTG
17641
SauCas9KKH
 55
0


 


 610
TCC
TCTTAAGCTGCTGGGTATTG
17642
SpyCas9-
 55
0






SpRY




 


 611
CTT
ACCTTTCTCCAAATGGTGCC
17643
SpyCas9-
 55
0






SpRY




 


 612
TCCAAG
ATCTTAAGCTGCTGGGTATTG
17644
cCas9-v17
 55
0


 


 613
TCCAAG
ATCTTAAGCTGCTGGGTATTG
17645
cCas9-v42
 55
0


 


 614
GTC
ATCTTAAGCTGCTGGGTATT
17646
SpyCas9-
 56
0






SpRY




 


 615
CCT
TACCTTTCTCCAAATGGTGC
17647
SpyCas9-
 56
0






SpRY




 


 616
CCTTC
gactACCTTTCTCCAAATGGTGC
17648
BlatCas9
 56
0


 


 617
TG
AATCTTAAGCTGCTGGGTAT
17649
SpyCas9-NG
 57
0


 


 618
TG
AATCTTAAGCTGCTGGGTAT
17650
SpyCas9-xCas
 57
0


 


 619
TG
AATCTTAAGCTGCTGGGTAT
17651
SpyCas9-
 57
0






xCas-NG




 


 620
TGT
AATCTTAAGCTGCTGGGTAT
17652
SpyCas9-SpG
 57
0


 


 621
TGT
AATCTTAAGCTGCTGGGTAT
17653
SpyCas9-
 57
0






SpRY




 


 622
CCC
CTACCTTTCTCCAAATGGTG
17654
SpyCas9-
 57
0






SpRY




 


 623
TGTCCAAG
caaaATCTTAAGCTGCTGGGTAT
17655
BlatCas9
 57
0


 


 624
TGTCC
caaaATCTTAAGCTGCTGGGTAT
17656
BlatCas9
 57
0


 


 625
TGTC
AATCTTAAGCTGCTGGGTAT
17657
SpyCas9-
 57
0






3var-NRTH




 


 626
TTGTCC
gcCAAAATCTTAAGCTGCTGGGTA
17658
Nme2Cas9
 58
0


 


 627
TTG
AAATCTTAAGCTGCTGGGTA
17659
ScaCas9
 58
0


 


 628
TTG
AAATCTTAAGCTGCTGGGTA
17660
ScaCas9-
 58
0






HiFi-Sc++




 


 629
TTG
AAATCTTAAGCTGCTGGGTA
17661
ScaCas9-Sc++
 58
0


 


 630
TTG
AAATCTTAAGCTGCTGGGTA
17662
SpyCas9-
 58
0






SpRY




 


 631
GCC
ACTACCTTTCTCCAAATGGT
17663
SpyCas9-
 58
0






SpRY




 


 632
TTGTCCAA
ccaaAATCTTAAGCTGCTGGGTA
17664
BlatCas9
 58
0


 


 633
TTGTCCAA
ccaaAATCTTAAGCTGCTGGGTA
17665
BlatCas9
 58
0


 


 634
TTGTC
ccaaAATCTTAAGCTGCTGGGTA
17666
BlatCas9
 58
0


 


 635
TG
GACTACCTTTCTCCAAATGG
17667
SpyCas9-NG
 59
0


 


 636
TG
GACTACCTTTCTCCAAATGG
17668
SpyCas9-xCas
 59
0


 


 637
TG
GACTACCTTTCTCCAAATGG
17669
SpyCas9-
 59
0






xCas-NG




 


 638
TGC
GACTACCTTTCTCCAAATGG
17670
SpyCas9-SpG
 59
0


 


 639
TGC
GACTACCTTTCTCCAAATGG
17671
SpyCas9-
 59
0






SpRY




 


 640
ATT
AAAATCTTAAGCTGCTGGGT
17672
SpyCas9-
 59
0






SpRY




 


 641
TGCCC
taagACTACCTTTCTCCAAATGG
17673
BlatCas9
 59
0


 


 642
TGCC
GACTACCTTTCTCCAAATGG
17674
SpyCas9-
 59
0






3var-NRCH




 


 643
GTGCCC
ctTAAGACTACCTTTCTCCAAATG
17675
Nme2Cas9
 60
0


 


 644
GTG
AGACTACCTTTCTCCAAATG
17676
ScaCas9
 60
0


 


 645
GTG
AGACTACCTTTCTCCAAATG
17677
ScaCas9-
 60
0






HiFi-Sc++




 


 646
GTG
AGACTACCTTTCTCCAAATG
17678
ScaCas9-Sc++
 60
0


 


 647
GTG
AGACTACCTTTCTCCAAATG
17679
SpyCas9-
 60
0






SpRY




 


 648
TAT
CAAAATCTTAAGCTGCTGGG
17680
SpyCas9-
 60
0






SpRY




 


 649
GTGCCCTT
ttaaGACTACCTTTCTCCAAATG
17681
BlatCas9
 60
0


 


 650
GTGCC
ttaaGACTACCTTTCTCCAAATG
17682
BlatCas9
 60
0


 


 651
GTGCCCT
TAAGACTACCTTTCTCCAAATG
17683
CdiCas9
 60
0


 


 652
TATT
CAAAATCTTAAGCTGCTGGG
17684
SpyCas9-
 60
0






3var-NRTH




 


 653
GGTGCC
tcTTAAGACTACCTTTCTCCAAAT
17685
Nme2Cas9
 61
0


 


 654
GG
AAGACTACCTTTCTCCAAAT
17686
SpyCas9-NG
 61
0


 


 655
GG
AAGACTACCTTTCTCCAAAT
17687
SpyCas9-xCas
 61
0


 


 656
GG
AAGACTACCTTTCTCCAAAT
17688
SpyCas9-
 61
0






xCas-NG




 


 657
GGT
AAGACTACCTTTCTCCAAAT
17689
SpyCas9-SpG
 61
0


 


 658
GGT
AAGACTACCTTTCTCCAAAT
17690
SpyCas9-
 61
0






SpRY




 


 659
GTA
CCAAAATCTTAAGCTGCTGG
17691
SpyCas9-
 61
0






SpRY




 


 660
GGTGC
cttaAGACTACCTTTCTCCAAAT
17692
BlatCas9
 61
0


 


 661
TGG
TAAGACTACCTTTCTCCAAA
17693
ScaCas9
 62
0


 


 662
TGG
TAAGACTACCTTTCTCCAAA
17694
ScaCas9-
 62
0






HiFi-Sc++




 


 663
TGG
TAAGACTACCTTTCTCCAAA
17695
ScaCas9-Sc++
 62
0


 


 664
TGG
TAAGACTACCTTTCTCCAAA
17696
SpyCas9
 62
0


 


 665
TGG
TAAGACTACCTTTCTCCAAA
17697
SpyCas9-HF1
 62
0


 


 666
TGG
TAAGACTACCTTTCTCCAAA
17698
SpyCas9-SpG
 62
0


 


 667
TGG
TAAGACTACCTTTCTCCAAA
17699
SpyCas9-
 62
0






SpRY




 


 668
GG
GCCAAAATCTTAAGCTGCTG
17700
SpyCas9-NG
 62
0


 


 669
GG
GCCAAAATCTTAAGCTGCTG
17701
SpyCas9-xCas
 62
0


 


 670
GG
GCCAAAATCTTAAGCTGCTG
17702
SpyCas9-
 62
0






xCas-NG




 


 671
TG
TAAGACTACCTTTCTCCAAA
17703
SpyCas9-NG
 62
0


 


 672
TG
TAAGACTACCTTTCTCCAAA
17704
SpyCas9-xCas
 62
0


 


 673
TG
TAAGACTACCTTTCTCCAAA
17705
SpyCas9-
 62
0






xCas-NG




 


 674
GGT
GCCAAAATCTTAAGCTGCTG
17706
SpyCas9-SpG
 62
0


 


 675
GGT
GCCAAAATCTTAAGCTGCTG
17707
SpyCas9-
 62
0






SpRY




 


 676
TGGT
TAAGACTACCTTTCTCCAAA
17708
SpyCas9-
 62
0






3var-NRRH




 


 677
GGTA
GCCAAAATCTTAAGCTGCTG
17709
SpyCas9-
 62
0






3var-NRTH




 


 678
GGGTATT
TCAGCCAAAATCTTAAGCTGCT
17710
CdiCas9
 63
0


 


 679
GGGTATT
aatCAGCCAAAATCTTAAGCTGCT
17711
PpnCas9
 63
0


 


 680
ATGG
CTTAAGACTACCTTTCTCCAA
17712
SauriCas9
 63
0


 


 681
ATGG
CTTAAGACTACCTTTCTCCAA
17713
SauriCas9-
 63
0






KKH




 


 682
GGG
AGCCAAAATCTTAAGCTGCT
17714
ScaCas9
 63
0


 


 683
GGG
AGCCAAAATCTTAAGCTGCT
17715
ScaCas9-
 63
0






HiFi-Sc++




 


 684
GGG
AGCCAAAATCTTAAGCTGCT
17716
ScaCas9-Sc++
 63
0


 


 685
GGG
AGCCAAAATCTTAAGCTGCT
17717
SpyCas9
 63
0


 


 686
GGG
AGCCAAAATCTTAAGCTGCT
17718
SpyCas9-HF1
 63
0


 


 687
GGG
AGCCAAAATCTTAAGCTGCT
17719
SpyCas9-SpG
 63
0


 


 688
GGG
AGCCAAAATCTTAAGCTGCT
17720
SpyCas9-
 63
0






SpRY




 


 689
ATG
TTAAGACTACCTTTCTCCAA
17721
ScaCas9
 63
0


 


 690
ATG
TTAAGACTACCTTTCTCCAA
17722
ScaCas9-
 63
0






HiFi-Sc++




 


 691
ATG
TTAAGACTACCTTTCTCCAA
17723
ScaCas9-Sc++
 63
0


 


 692
ATG
TTAAGACTACCTTTCTCCAA
17724
SpyCas9-
 63
0






SpRY




 


 693
GG
AGCCAAAATCTTAAGCTGCT
17725
SpyCas9-NG
 63
0


 


 694
GG
AGCCAAAATCTTAAGCTGCT
17726
SpyCas9-xCas
 63
0


 


 695
GG
AGCCAAAATCTTAAGCTGCT
17727
SpyCas9-
 63
0






xCas-NG




 


 696
ATGGTG
CTTAAGACTACCTTTCTCCAA
17728
cCas9-v16
 63
0


 


 697
ATGGTG
CTTAAGACTACCTTTCTCCAA
17729
cCas9-v21
 63
0


 


 698
GGGT
AGCCAAAATCTTAAGCTGCT
17730
SpyCas9-
 63
0






3var-NRRH




 


 699
AATGG
TCTTAAGACTACCTTTCTCCA
17731
SauCas9KKH
 64
0


 


 700
AATGGT
TCTTAAGACTACCTTTCTCCA
17732
SauCas9KKH
 64
0


 


 701
TGGG
TCAGCCAAAATCTTAAGCTGC
17733
SauriCas9
 64
0


 


 702
TGGG
TCAGCCAAAATCTTAAGCTGC
17734
SauriCas9-
 64
0






KKH




 


 703
TGG
CAGCCAAAATCTTAAGCTGC
17735
ScaCas9
 64
0


 


 704
TGG
CAGCCAAAATCTTAAGCTGC
17736
ScaCas9-
 64
0






HiFi-Sc++




 


 705
TGG
CAGCCAAAATCTTAAGCTGC
17737
ScaCas9-Sc++
 64
0


 


 706
TGG
CAGCCAAAATCTTAAGCTGC
17738
SpyCas9
 64
0


 


 707
TGG
CAGCCAAAATCTTAAGCTGC
17739
SpyCas9-HF1
 64
0


 


 708
TGG
CAGCCAAAATCTTAAGCTGC
17740
SpyCas9-SpG
 64
0


 


 709
TGG
CAGCCAAAATCTTAAGCTGC
17741
SpyCas9-
 64
0






SpRY




 


 710
TG
CAGCCAAAATCTTAAGCTGC
17742
SpyCas9-NG
 64
0


 


 711
TG
CAGCCAAAATCTTAAGCTGC
17743
SpyCas9-xCas
 64
0


 


 712
TG
CAGCCAAAATCTTAAGCTGC
17744
SpyCas9-
 64
0






xCas-NG




 


 713
AAT
CTTAAGACTACCTTTCTCCA
17745
SpyCas9-
 64
0






SpRY




 


 714
CTGGG
gaATCAGCCAAAATCTTAAGCTG
17746
SauCas9
 65
0


 


 715
CTGGG
ATCAGCCAAAATCTTAAGCTG
17747
SauCas9KKH
 65
0


 


 716
CTGGGT
gaATCAGCCAAAATCTTAAGCTG
17748
SauCas9
 65
0


 


 717
CTGGGT
ATCAGCCAAAATCTTAAGCTG
17749
SauCas9KKH
 65
0


 


 718
CTGGGT
ATCAGCCAAAATCTTAAGCTG
17750
cCas9-v17
 65
0


 


 719
CTGGGT
ATCAGCCAAAATCTTAAGCTG
17751
cCas9-v42
 65
0


 


 720
CTGG
ATCAGCCAAAATCTTAAGCTG
17752
SauriCas9
 65
0


 


 721
CTGG
ATCAGCCAAAATCTTAAGCTG
17753
SauriCas9-
 65
0






KKH




 


 722
CTG
TCAGCCAAAATCTTAAGCTG
17754
ScaCas9
 65
0


 


 723
CTG
TCAGCCAAAATCTTAAGCTG
17755
ScaCas9-
 65
0






HiFi-Sc++




 


 724
CTG
TCAGCCAAAATCTTAAGCTG
17756
ScaCas9-Sc++
 65
0


 


 725
CTG
TCAGCCAAAATCTTAAGCTG
17757
SpyCas9-
 65
0






SpRY




 


 726
AAA
TCTTAAGACTACCTTTCTCC
17758
SpyCas9-
 65
0






SpRY




 


 727
AAAT
TCTTAAGACTACCTTTCTCC
17759
SpyCas9-
 65
0






3var-NRRH




 


 728
AAAT
ctCTTAAGACTACCTTTCTCC
17760
iSpyMacCas9
 65
0


 


 729
GCTGG
AATCAGCCAAAATCTTAAGCT
17761
SauCas9KKH
 66
0


 


 730
CAA
CTCTTAAGACTACCTTTCTC
17762
SpyCas9-
 66
0






SpRY




 


 731
GCT
ATCAGCCAAAATCTTAAGCT
17763
SpyCas9-
 66
0






SpRY




 


 732
CAAA
CTCTTAAGACTACCTTTCTC
17764
SpyCas9-
 66
0






3var-NRRH




 


 733
CAAA
tcTCTTAAGACTACCTTTCTC
17765
iSpyMacCas9
 66
0


 


 734
CCAAA
CTCTCTTAAGACTACCTTTCT
17766
SauCas9KKH
 67
0


 


 735
CCAAAT
CTCTCTTAAGACTACCTTTCT
17767
SauCas9KKH
 67
0


 


 736
CCAAAT
CTCTCTTAAGACTACCTTTCT
17768
cCas9-v17
 67
0


 


 737
CCAAAT
CTCTCTTAAGACTACCTTTCT
17769
cCas9-v42
 67
0


 


 738
TG
AATCAGCCAAAATCTTAAGC
17770
SpyCas9-NG
 67
0


 


 739
TG
AATCAGCCAAAATCTTAAGC
17771
SpyCas9-xCas
 67
0


 


 740
TG
AATCAGCCAAAATCTTAAGC
17772
SpyCas9-
 67
0






xCas-NG




 


 741
TGC
AATCAGCCAAAATCTTAAGC
17773
SpyCas9-SpG
 67
0


 


 742
TGC
AATCAGCCAAAATCTTAAGC
17774
SpyCas9-
 67
0






SpRY




 


 743
CCA
TCTCTTAAGACTACCTTTCT
17775
SpyCas9-
 67
0






SpRY




 


 744
TGCT
AATCAGCCAAAATCTTAAGC
17776
SpyCas9-
 67
0






3var-NRCH




 


 745
TCCAA
ACTCTCTTAAGACTACCTTTC
17777
SauCas9KKH
 68
0


 


 746
CTG
GAATCAGCCAAAATCTTAAG
17778
ScaCas9
 68
0


 


 747
CTG
GAATCAGCCAAAATCTTAAG
17779
ScaCas9-
 68
0






HiFi-Sc++




 


 748
CTG
GAATCAGCCAAAATCTTAAG
17780
ScaCas9-Sc++
 68
0


 


 749
CTG
GAATCAGCCAAAATCTTAAG
17781
SpyCas9-
 68
0






SpRY




 


 750
TCC
CTCTCTTAAGACTACCTTTC
17782
SpyCas9-
 68
0






SpRY




 


 751
TCCAAA
ACTCTCTTAAGACTACCTTTC
17783
cCas9-v17
 68
0


 


 752
TCCAAA
ACTCTCTTAAGACTACCTTTC
17784
cCas9-v42
 68
0


 


 753
GCT
GGAATCAGCCAAAATCTTAA
17785
SpyCas9-
 69
0






SpRY




 


 754
CTC
ACTCTCTTAAGACTACCTTT
17786
SpyCas9-
 69
0






SpRY




 


 755
GCTGCTGG
aatgGAATCAGCCAAAATCTTAA
17787
BlatCas9
 69
0


 


 756
GCTGC
aatgGAATCAGCCAAAATCTTAA
17788
BlatCas9
 69
0


 


 757
AG
TGGAATCAGCCAAAATCTTA
17789
SpyCas9-NG
 70
0


 


 758
AG
TGGAATCAGCCAAAATCTTA
17790
SpyCas9-xCas
 70
0


 


 759
AG
TGGAATCAGCCAAAATCTTA
17791
SpyCas9-
 70
0






xCas-NG




 


 760
AGC
TGGAATCAGCCAAAATCTTA
17792
SpyCas9-SpG
 70
0


 


 761
AGC
TGGAATCAGCCAAAATCTTA
17793
SpyCas9-
 70
0






SpRY




 


 762
TCT
AACTCTCTTAAGACTACCTT
17794
SpyCas9-
 70
0






SpRY




 


 763
TCTCCAAA
gagaACTCTCTTAAGACTACCTT
17795
BlatCas9
 70
0


 


 764
TCTCCAAA
gagaACTCTCTTAAGACTACCTT
17796
BlatCas9
 70
0


 


 765
TCTCCAAA
gaGAACTCTCTTAAGACTACCTT
17797
GeoCas9
 70
0


 


 766
TCTCC
gagaACTCTCTTAAGACTACCTT
17798
BlatCas9
 70
0


 


 767
AGCT
TGGAATCAGCCAAAATCTTA
17799
SpyCas9-
 70
0






3var-NRCH




 


 768
TTCTCC
ctGAGAACTCTCTTAAGACTACCT
17800
Nme2Cas9
 71
0


 


 769
AAG
ATGGAATCAGCCAAAATCTT
17801
ScaCas9
 71
0


 


 770
AAG
ATGGAATCAGCCAAAATCTT
17802
ScaCas9-
 71
0






HiFi-Sc++




 


 771
AAG
ATGGAATCAGCCAAAATCTT
17803
ScaCas9-Sc++
 71
0


 


 772
AAG
ATGGAATCAGCCAAAATCTT
17804
SpyCas9-
 71
0






SpRY




 


 773
TTC
GAACTCTCTTAAGACTACCT
17805
SpyCas9-
 71
0






SpRY




 


 774
TTCTCCAA
tgagAACTCTCTTAAGACTACCT
17806
BlatCas9
 71
0


 


 775
TTCTCCAA
tgagAACTCTCTTAAGACTACCT
17807
BlatCas9
 71
0


 


 776
TTCTC
tgagAACTCTCTTAAGACTACCT
17808
BlatCas9
 71
0


 


 777
AAGC
ATGGAATCAGCCAAAATCTT
17809
SpyCas9-
 71
0






3var-NRRH




 


 778
TAAG
TAATGGAATCAGCCAAAATCT
17810
SauriCas9-
 72
0






KKH




 


 779
TAAG
AATGGAATCAGCCAAAATCT
17811
SpyCas9-
 72
0






QQR1




 


 780
TAAG
taATGGAATCAGCCAAAATCT
17812
iSpy MacCas9
 72
0


 


 781
TAA
AATGGAATCAGCCAAAATCT
17813
SpyCas9-
 72
0






SpRY




 


 782
TTT
AGAACTCTCTTAAGACTACC
17814
SpyCas9-
 72
0






SpRY




 


 783
TAAGC
gttaATGGAATCAGCCAAAATCT
17815
BlatCas9
 72
0


 


 784
TAAGCT
TAATGGAATCAGCCAAAATCT
17816
cCas9-v16
 72
0


 


 785
TAAGCT
TAATGGAATCAGCCAAAATCT
17817
cCas9-v21
 72
0


 


 786
TTAAG
TTAATGGAATCAGCCAAAATC
17818
SauCas9KKH
 73
0


 


 787
TTA
TAATGGAATCAGCCAAAATC
17819
SpyCas9-
 73
0






SpRY




 


 788
CTT
GAGAACTCTCTTAAGACTAC
17820
SpyCas9-
 73
0






SpRY




 


 789
CTTTC
actgAGAACTCTCTTAAGACTAC
17821
BlatCas9
 73
0


 


 790
TTAAGC
TTAATGGAATCAGCCAAAATC
17822
cCas9-v17
 73
0


 


 791
TTAAGC
TTAATGGAATCAGCCAAAATC
17823
cCas9-v42
 73
0


 


 792
CTTAA
GTTAATGGAATCAGCCAAAAT
17824
SauCas9KKH
 74
0


 


 793
CTT
TTAATGGAATCAGCCAAAAT
17825
SpyCas9-
 74
0






SpRY




 


 794
CCT
TGAGAACTCTCTTAAGACTA
17826
SpyCas9-
 74
0






SpRY




 


 795
TCT
GTTAATGGAATCAGCCAAAA
17827
SpyCas9-
 75
0






SpRY




 


 796
ACC
CTGAGAACTCTCTTAAGACT
17828
SpyCas9-
 75
0






SpRY




 


 797
TAC
ACTGAGAACTCTCTTAAGAC
17829
SpyCas9-
 76
0






SpRY




 


 798
ATC
TGTTAATGGAATCAGCCAAA
17830
SpyCas9-
 76
0






SpRY




 


 799
TACC
ACTGAGAACTCTCTTAAGAC
17831
SpyCas9-
 76
0






3var-NRCH




 


 800
AAT
CTGTTAATGGAATCAGCCAA
17832
SpyCas9-
 77
0






SpRY




 


 801
CTA
CACTGAGAACTCTCTTAAGA
17833
SpyCas9-
 77
0






SpRY




 


 802
CTACCTTT
tgccACTGAGAACTCTCTTAAGA
17834
BlatCas9
 77
0


 


 803
CTACC
tgccACTGAGAACTCTCTTAAGA
17835
BlatCas9
 77
0


 


 804
CTACCTT
GCCACTGAGAACTCTCTTAAGA
17836
CdiCas9
 77
0


 


 805
AATC
CTGTTAATGGAATCAGCCAA
17837
SpyCas9-
 77
0






3var-NRTH




 


 806
ACTACC
aaTGCCACTGAGAACTCTCTTAAG
17838
Nme2Cas9
 78
0


 


 807
AAA
ACTGTTAATGGAATCAGCCA
17839
SpyCas9-
 78
0






SpRY




 


 808
ACT
CCACTGAGAACTCTCTTAAG
17840
SpyCas9-
 78
0






SpRY




 


 809
AAATCTTA
cttaCTGTTAATGGAATCAGCCA
17841
BlatCas9
 78
0


 


 810
ACTACCTT
atgcCACTGAGAACTCTCTTAAG
17842
BlatCas9
 78
0


 


 811
AAATC
cttaCTGTTAATGGAATCAGCCA
17843
BlatCas9
 78
0


 


 812
ACTAC
atgcCACTGAGAACTCTCTTAAG
17844
BlatCas9
 78
0


 


 813
AAATCTT
TTACTGTTAATGGAATCAGCCA
17845
CdiCas9
 78
0


 


 814
AAAT
ACTGTTAATGGAATCAGCCA
17846
SpyCas9-
 78
0






3var-NRRH




 


 815
AAAT
taCTGTTAATGGAATCAGCCA
17847
iSpyMacCas9
 78
0


 


 816
AAA
TACTGTTAATGGAATCAGCC
17848
SpyCas9-
 79
0






SpRY




 


 817
GAC
GCCACTGAGAACTCTCTTAA
17849
SpyCas9-
 79
0






SpRY




 


 818
AAAATCT
CTTACTGTTAATGGAATCAGCC
17850
CdiCas9
 79
0


 


 819
AAAA
TACTGTTAATGGAATCAGCC
17851
SpyCas9-
 79
0






3var-NRRH




 


 820
AAAA
ttACTGTTAATGGAATCAGCC
17852
iSpy MacCas9
 79
0


 


 821
GACT
GCCACTGAGAACTCTCTTAA
17853
SpyCas9-
 79
0






3var-NRCH




 


 822
CAAAA
CTTACTGTTAATGGAATCAGC
17854
SauCas9KKH
 80
0


 


 823
CAAAAT
CTTACTGTTAATGGAATCAGC
17855
SauCas9KKH
 80
0


 


 824
CAAAAT
CTTACTGTTAATGGAATCAGC
17856
cCas9-v17
 80
0


 


 825
CAAAAT
CTTACTGTTAATGGAATCAGC
17857
cCas9-v42
 80
0


 


 826
AG
TGCCACTGAGAACTCTCTTA
17858
SpyCas9-NG
 80
0


 


 827
AG
TGCCACTGAGAACTCTCTTA
17859
SpyCas9-xCas
 80
0


 


 828
AG
TGCCACTGAGAACTCTCTTA
17860
SpyCas9-
 80
0






xCas-NG




 


 829
CAA
TTACTGTTAATGGAATCAGC
17861
SpyCas9-
 80
0






SpRY




 


 830
AGA
TGCCACTGAGAACTCTCTTA
17862
SpyCas9-SpG
 80
0


 


 831
AGA
TGCCACTGAGAACTCTCTTA
17863
SpyCas9-
 80
0






SpRY




 


 832
CAAAATC
ACTTACTGTTAATGGAATCAGC
17864
CdiCas9
 80
0


 


 833
AGACTAC
AATGCCACTGAGAACTCTCTTA
17865
CdiCas9
 80
0


 


 834
CAAA
TTACTGTTAATGGAATCAGC
17866
SpyCas9-
 80
0






3var-NRRH




 


 835
CAAA
ctTACTGTTAATGGAATCAGC
17867
iSpyMacCas9
 80
0


 


 836
AGAC
TGCCACTGAGAACTCTCTTA
17868
SpyCas9-
 80
0






3var-NRRH




 


 837
AGAC
TGCCACTGAGAACTCTCTTA
17869
SpyCas9-
 80
0






VQR




 


 838
CCAAA
ACTTACTGTTAATGGAATCAG
17870
SauCas9KKH
 81
0


 


 839
AAG
ATGCCACTGAGAACTCTCTT
17871
ScaCas9
 81
0


 


 840
AAG
ATGCCACTGAGAACTCTCTT
17872
ScaCas9-
 81
0






HiFi-Sc++




 


 841
AAG
ATGCCACTGAGAACTCTCTT
17873
ScaCas9-Sc++
 81
0


 


 842
AAG
ATGCCACTGAGAACTCTCTT
17874
SpyCas9-
 81
0






SpRY




 


 843
CCA
CTTACTGTTAATGGAATCAG
17875
SpyCas9-
 81
0






SpRY




 


 844
AAGAC
aaaaTGCCACTGAGAACTCTCTT
17876
BlatCas9
 81
0


 


 845
AAGACT
AATGCCACTGAGAACTCTCTT
17877
cCas9-v16
 81
0


 


 846
AAGACT
AATGCCACTGAGAACTCTCTT
17878
cCas9-v21
 81
0


 


 847
CCAAAA
CTTACTGTTAATGGAATCAG
17879
St1Cas9-
 81
0






MTH17CL396




 


 848
CCAAAA
ACTTACTGTTAATGGAATCAG
17880
cCas9-v17
 81
0


 


 849
CCAAAA
ACTTACTGTTAATGGAATCAG
17881
cCas9-v42
 81
0


 


 850
CCAAAAT
TACTTACTGTTAATGGAATCAG
17882
CdiCas9
 81
0


 


 851
CCAAAAT
TACTTACTGTTAATGGAATCAG
17883
CdiCas9
 81
0


 


 852
AAGA
ATGCCACTGAGAACTCTCTT
17884
SpyCas9-
 81
0






3var-NRRH




 


 853
GCCAA
TACTTACTGTTAATGGAATCA
17885
SauCas9KKH
 82
0


 


 854
TAAGA
AAATGCCACTGAGAACTCTCT
17886
SauCas9KKH
 82
0


 


 855
TAAG
AAATGCCACTGAGAACTCTCT
17887
SauriCas9-
 82
0






KKH




 


 856
TAAG
AATGCCACTGAGAACTCTCT
17888
SpyCas9-
 82
0






QQR1




 


 857
TAAG
aaATGCCACTGAGAACTCTCT
17889
iSpyMacCas9
 82
0


 


 858
TAA
AATGCCACTGAGAACTCTCT
17890
SpyCas9-
 82
0






SpRY




 


 859
GCC
ACTTACTGTTAATGGAATCA
17891
SpyCas9-
 82
0






SpRY




 


 860
GCCAAA
TACTTACTGTTAATGGAATCA
17892
cCas9-v17
 82
0


 


 861
GCCAAA
TACTTACTGTTAATGGAATCA
17893
cCas9-v42
 82
0


 


 862
TAAGAC
AAATGCCACTGAGAACTCTCT
17894
cCas9-v17
 82
0


 


 863
TAAGAC
AAATGCCACTGAGAACTCTCT
17895
cCas9-v42
 82
0


 


 864
TTAAG
AAAATGCCACTGAGAACTCTC
17896
SauCas9KKH
 83
0


 


 865
AG
TACTTACTGTTAATGGAATC
17897
SpyCas9-NG
 83
0


 


 866
AG
TACTTACTGTTAATGGAATC
17898
SpyCas9-xCas
 83
0


 


 867
AG
TACTTACTGTTAATGGAATC
17899
SpyCas9-
 83
0






xCas-NG




 


 868
AGC
TACTTACTGTTAATGGAATC
17900
SpyCas9-SpG
 83
0


 


 869
AGC
TACTTACTGTTAATGGAATC
17901
SpyCas9-
 83
0






SpRY




 


 870
TTA
AAATGCCACTGAGAACTCTC
17902
SpyCas9-
 83
0






SpRY




 


 871
TTAAGA
AAAATGCCACTGAGAACTCTC
17903
cCas9-v17
 83
0


 


 872
TTAAGA
AAAATGCCACTGAGAACTCTC
17904
cCas9-v42
 83
0


 


 873
TTAAGACT
agtaAAATGCCACTGAGAACTCTC
17905
NmeCas9
 83
0


 


 874
AGCC
TACTTACTGTTAATGGAATC
17906
SpyCas9-
 83
0






3var-NRCH




 


 875
CTTAA
TAAAATGCCACTGAGAACTCT
17907
SauCas9KKH
 84
0


 


 876
CAG
TTACTTACTGTTAATGGAAT
17908
ScaCas9
 84
0


 


 877
CAG
TTACTTACTGTTAATGGAAT
17909
ScaCas9-
 84
0






HiFi-Sc++




 


 878
CAG
TTACTTACTGTTAATGGAAT
17910
ScaCas9-Sc++
 84
0


 


 879
CAG
TTACTTACTGTTAATGGAAT
17911
SpyCas9-
 84
0






SpRY




 


 880
CTT
AAAATGCCACTGAGAACTCT
17912
SpyCas9-
 84
0






SpRY




 


 881
CAGCCAAA
aaatTACTTACTGTTAATGGAAT
17913
BlatCas9
 84
0


 


 882
CAGCCAAA
aaatTACTTACTGTTAATGGAAT
17914
BlatCas9
 84
0


 


 883
CAGCCAAA
aaATTACTTACTGTTAATGGAAT
17915
GeoCas9
 84
0


 


 884
CAGCC
aaatTACTTACTGTTAATGGAAT
17916
BlatCas9
 84
0


 


 885
CAGC
TTACTTACTGTTAATGGAAT
17917
SpyCas9-
 84
0






3var-NRRH




 


 886
TCAGCC
gtAAATTACTTACTGTTAATGGAA
17918
Nme2Cas9
 85
0


 


 887
TCAG
AATTACTTACTGTTAATGGAA
17919
SauriCas9-
 85
0






KKH




 


 888
TCA
ATTACTTACTGTTAATGGAA
17920
SpyCas9-
 85
0






SpRY




 


 889
TCT
TAAAATGCCACTGAGAACTC
17921
SpyCas9-
 85
0






SpRY




 


 890
TCAGCCAA
taaaTTACTTACTGTTAATGGAA
17922
BlatCas9
 85
0


 


 891
TCAGCCAA
taaaTTACTTACTGTTAATGGAA
17923
BlatCas9
 85
0


 


 892
TCAGC
taaaTTACTTACTGTTAATGGAA
17924
BlatCas9
 85
0


 


 893
ATCAG
AAATTACTTACTGTTAATGGA
17925
SauCas9KKH
 86
0


 


 894
ATC
AATTACTTACTGTTAATGGA
17926
SpyCas9-
 86
0






SpRY




 


 895
CTC
GTAAAATGCCACTGAGAACT
17927
SpyCas9-
 86
0






SpRY




 


 896
ATCAGC
AAATTACTTACTGTTAATGGA
17928
cCas9-v17
 86
0


 


 897
ATCAGC
AAATTACTTACTGTTAATGGA
17929
cCas9-v42
 86
0


 


 898
AAT
AAATTACTTACTGTTAATGG
17930
SpyCas9-
 87
0






SpRY




 


 899
TCT
AGTAAAATGCCACTGAGAAC
17931
SpyCas9-
 87
0






SpRY




 


 900
AATC
AAATTACTTACTGTTAATGG
17932
SpyCas9-
 87
0






3var-NRTH




 


 901
GAA
TAAATTACTTACTGTTAATG
17933
SpyCas9-
 88
0






SpRY




 


 902
GAA
TAAATTACTTACTGTTAATG
17934
SpyCas9-xCas
 88
0


 


 903
CTC
AAGTAAAATGCCACTGAGAA
17935
SpyCas9-
 88
0






SpRY




 


 904
CTCTCTTA
aagaAGTAAAATGCCACTGAGAA
17936
BlatCas9
 88
0


 


 905
GAATC
gtgtAAATTACTTACTGTTAATG
17937
BlatCas9
 88
0


 


 906
CTCTC
aagaAGTAAAATGCCACTGAGAA
17938
BlatCas9
 88
0


 


 907
GAAT
TAAATTACTTACTGTTAATG
17939
SpyCas9-
 88
0






3var-NRRH




 


 908
GAAT
gtAAATTACTTACTGTTAATG
17940
iSpyMacCas9
 88
0


 


 909
GG
GTAAATTACTTACTGTTAAT
17941
SpyCas9-NG
 89
0


 


 910
GG
GTAAATTACTTACTGTTAAT
17942
SpyCas9-xCas
 89
0


 


 911
GG
GTAAATTACTTACTGTTAAT
17943
SpyCas9-
 89
0






xCas-NG




 


 912
GGA
GTAAATTACTTACTGTTAAT
17944
SpyCas9-SpG
 89
0


 


 913
GGA
GTAAATTACTTACTGTTAAT
17945
SpyCas9-
 89
0






SpRY




 


 914
ACT
GAAGTAAAATGCCACTGAGA
17946
SpyCas9-
 89
0






SpRY




 


 915
GGAA
GTAAATTACTTACTGTTAAT
17947
SpyCas9-
 89
0






3var-NRRH




 


 916
GGAA
GTAAATTACTTACTGTTAAT
17948
SpyCas9-
 89
0






VQR




 


 917
TGGAA
agGTGTAAATTACTTACTGTTAA
17949
SauCas9
 90
0


 


 918
TGGAA
GTGTAAATTACTTACTGTTAA
17950
SauCas9KKH
 90
0


 


 919
TGGAAT
agGTGTAAATTACTTACTGTTAA
17951
SauCas9
 90
0


 


 920
TGGAAT
GTGTAAATTACTTACTGTTAA
17952
SauCas9KKH
 90
0


 


 921
TGGAAT
GTGTAAATTACTTACTGTTAA
17953
cCas9-v17
 90
0


 


 922
TGGAAT
GTGTAAATTACTTACTGTTAA
17954
cCas9-v42
 90
0


 


 923
TGG
TGTAAATTACTTACTGTTAA
17955
ScaCas9
 90
0


 


 924
TGG
TGTAAATTACTTACTGTTAA
17956
ScaCas9-
 90
0






HiFi-Sc++




 


 925
TGG
TGTAAATTACTTACTGTTAA
17957
ScaCas9-Sc++
 90
0


 


 926
TGG
TGTAAATTACTTACTGTTAA
17958
SpyCas9
 90
0


 


 927
TGG
TGTAAATTACTTACTGTTAA
17959
SpyCas9-HF1
 90
0


 


 928
TGG
TGTAAATTACTTACTGTTAA
17960
SpyCas9-SpG
 90
0


 


 929
TGG
TGTAAATTACTTACTGTTAA
17961
SpyCas9-
 90
0






SpRY




 


 930
TG
TGTAAATTACTTACTGTTAA
17962
SpyCas9-NG
 90
0


 


 931
TG
TGTAAATTACTTACTGTTAA
17963
SpyCas9-xCas
 90
0


 


 932
TG
TGTAAATTACTTACTGTTAA
17964
SpyCas9-
 90
0






xCas-NG




 


 933
AAC
AGAAGTAAAATGCCACTGAG
17965
SpyCas9-
 90
0






SpRY




 


 934
AACTC
aaaaGAAGTAAAATGCCACTGAG
17966
BlatCas9
 90
0


 


 935
TGGAATC
GGTGTAAATTACTTACTGTTAA
17967
CdiCas9
 90
0


 


 936
TGGA
TGTAAATTACTTACTGTTAA
17968
SpyCas9-
 90
0






3var-NRRH




 


 937
AACT
AGAAGTAAAATGCCACTGAG
17969
SpyCas9-
 90
0






3var-NRCH




 


 938
ATGGA
aaGGTGTAAATTACTTACTGTTA
17970
SauCas9
 91
0


 


 939
ATGGA
GGTGTAAATTACTTACTGTTA
17971
SauCas9KKH
 91
0


 


 940
ATGG
GGTGTAAATTACTTACTGTTA
17972
SauriCas9
 91
0


 


 941
ATGG
GGTGTAAATTACTTACTGTTA
17973
SauriCas9-
 91
0






KKH




 


 942
ATG
GTGTAAATTACTTACTGTTA
17974
ScaCas9
 91
0


 


 943
ATG
GTGTAAATTACTTACTGTTA
17975
ScaCas9-
 91
0






HiFi-Sc++




 


 944
ATG
GTGTAAATTACTTACTGTTA
17976
ScaCas9-Sc++
 91
0


 


 945
ATG
GTGTAAATTACTTACTGTTA
17977
SpyCas9-
 91
0






SpRY




 


 946
GAA
AAGAAGTAAAATGCCACTGA
17978
SpyCas9-
 91
0






SpRY




 


 947
GAA
AAGAAGTAAAATGCCACTGA
17979
SpyCas9-xCas
 91
0


 


 948
ATGGAAT
GTGTAAATTACTTACTGTTA
17980
St1Cas9
 91
0


 


 949
ATGGAA
GGTGTAAATTACTTACTGTTA
17981
cCas9-v17
 91
0


 


 950
ATGGAA
GGTGTAAATTACTTACTGTTA
17982
cCas9-v42
 91
0


 


 951
GAACTCT
AAAAGAAGTAAAATGCCACTGA
17983
CdiCas9
 91
0


 


 952
GAAC
AAGAAGTAAAATGCCACTGA
17984
SpyCas9-
 91
0






3var-NRRH




 


 953
GAAC
aaAGAAGTAAAATGCCACTGA
17985
iSpyMacCas9
 91
0


 


 954
AATGG
AGGTGTAAATTACTTACTGTT
17986
SauCas9KKH
 92
0


 


 955
AG
AAAGAAGTAAAATGCCACTG
17987
SpyCas9-NG
 92
0


 


 956
AG
AAAGAAGTAAAATGCCACTG
17988
SpyCas9-xCas
 92
0


 


 957
AG
AAAGAAGTAAAATGCCACTG
17989
SpyCas9-
 92
0






xCas-NG




 


 958
AAT
GGTGTAAATTACTTACTGTT
17990
SpyCas9-
 92
0






SpRY




 


 959
AGA
AAAGAAGTAAAATGCCACTG
17991
SpyCas9-SpG
 92
0


 


 960
AGA
AAAGAAGTAAAATGCCACTG
17992
SpyCas9-
 92
0






SpRY




 


 961
AGAAC
aaaaAAGAAGTAAAATGCCACTG
17993
BlatCas9
 92
0


 


 962
AGAACT
AAAAGAAGTAAAATGCCACTG
17994
cCas9-v16
 92
0


 


 963
AGAACT
AAAAGAAGTAAAATGCCACTG
17995
cCas9-v21
 92
0


 


 964
AGAACTC
AAAAAGAAGTAAAATGCCACTG
17996
CdiCas9
 92
0


 


 965
AGAA
AAAGAAGTAAAATGCCACTG
17997
SpyCas9-
 92
0






3var-NRRH




 


 966
AGAA
AAAGAAGTAAAATGCCACTG
17998
SpyCas9-
 92
0






VQR




 


 967
GAGAA
taAAAAAGAAGTAAAATGCCACT
17999
SauCas9
 93
0


 


 968
GAGAA
AAAAAGAAGTAAAATGCCACT
18000
SauCas9KKH
 93
0


 


 969
GAG
AAAAGAAGTAAAATGCCACT
18001
ScaCas9
 93
0


 


 970
GAG
AAAAGAAGTAAAATGCCACT
18002
ScaCas9-
 93
0






HiFi-Sc++




 


 971
GAG
AAAAGAAGTAAAATGCCACT
18003
ScaCas9-Sc++
 93
0


 


 972
GAG
AAAAGAAGTAAAATGCCACT
18004
SpyCas9-
 93
0






SpRY




 


 973
TAA
AGGTGTAAATTACTTACTGT
18005
SpyCas9-
 93
0






SpRY




 


 974
GAGAAC
AAAAAGAAGTAAAATGCCACT
18006
cCas9-v17
 93
0


 


 975
GAGAAC
AAAAAGAAGTAAAATGCCACT
18007
cCas9-v42
 93
0


 


 976
GAGAACT
AAAAAAGAAGTAAAATGCCACT
18008
CdiCas9
 93
0


 


 977
TAAT
AGGTGTAAATTACTTACTGT
18009
SpyCas9-
 93
0






3var-NRRH




 


 978
TAAT
aaGGTGTAAATTACTTACTGT
18010
iSpyMacCas9
 93
0


 


 979
GAGA
AAAAGAAGTAAAATGCCACT
18011
SpyCas9-
 93
0






3var-NRRH




 


 980
TGAGA
AAAAAAGAAGTAAAATGCCAC
18012
SauCas9KKH
 94
0


 


 981
TGAG
AAAAAAGAAGTAAAATGCCAC
18013
SauriCas9-
 94
0






KKH




 


 982
TGAG
AAAAAGAAGTAAAATGCCAC
18014
SpyCas9-
 94
0






VQR




 


 983
TG
AAAAAGAAGTAAAATGCCAC
18015
SpyCas9-NG
 94
0


 


 984
TG
AAAAAGAAGTAAAATGCCAC
18016
SpyCas9-xCas
 94
0


 


 985
TG
AAAAAGAAGTAAAATGCCAC
18017
SpyCas9-
 94
0






xCas-NG




 


 986
TGA
AAAAAGAAGTAAAATGCCAC
18018
SpyCas9-SpG
 94
0


 


 987
TGA
AAAAAGAAGTAAAATGCCAC
18019
SpyCas9-
 94
0






SpRY




 


 988
TTA
AAGGTGTAAATTACTTACTG
18020
SpyCas9-
 94
0






SpRY




 


 989
TGAGAA
AAAAAAGAAGTAAAATGCCAC
18021
cCas9-v17
 94
0


 


 990
TGAGAA
AAAAAAGAAGTAAAATGCCAC
18022
cCas9-v42
 94
0


 


 991
CTGAG
ccTAAAAAAGAAGTAAAATGCCA
18023
SauCas9
 95
0


 


 992
CTGAG
TAAAAAAGAAGTAAAATGCCA
18024
SauCas9KKH
 95
0


 


 993
GTTAA
GTAAGGTGTAAATTACTTACT
18025
SauCas9KKH
 95
0


 


 994
GTTAAT
GTAAGGTGTAAATTACTTACT
18026
SauCas9KKH
 95
0


 


 995
CTG
AAAAAAGAAGTAAAATGCCA
18027
ScaCas9
 95
0


 


 996
CTG
AAAAAAGAAGTAAAATGCCA
18028
ScaCas9-
 95
0






HiFi-Sc++




 


 997
CTG
AAAAAAGAAGTAAAATGCCA
18029
ScaCas9-Sc++
 95
0


 


 998
CTG
AAAAAAGAAGTAAAATGCCA
18030
SpyCas9-
 95
0






SpRY




 


 999
GTT
TAAGGTGTAAATTACTTACT
18031
SpyCas9-
 95
0






SpRY




 


1000
CTGAGA
TAAAAAAGAAGTAAAATGCCA
18032
cCas9-v17
 95
0


 


1001
CTGAGA
TAAAAAAGAAGTAAAATGCCA
18033
cCas9-v42
 95
0


 


1002
ACTGA
CTAAAAAAGAAGTAAAATGCC
18034
SauCas9KKH
 96
0


 


1003
TG
GTAAGGTGTAAATTACTTAC
18035
SpyCas9-NG
 96
0


 


1004
TG
GTAAGGTGTAAATTACTTAC
18036
SpyCas9-xCas
 96
0


 


1005
TG
GTAAGGTGTAAATTACTTAC
18037
SpyCas9-
 96
0






xCas-NG




 


1006
TGT
GTAAGGTGTAAATTACTTAC
18038
SpyCas9-SpG
 96
0


 


1007
TGT
GTAAGGTGTAAATTACTTAC
18039
SpyCas9-
 96
0






SpRY




 


1008
ACT
TAAAAAAGAAGTAAAATGCC
18040
SpyCas9-
 96
0






SpRY




 


1009
TGTT
GTAAGGTGTAAATTACTTAC
18041
SpyCas9-
 96
0






3var-NRTH




 


1010
CTG
CGTAAGGTGTAAATTACTTA
18042
ScaCas9
 97
0


 


1011
CTG
CGTAAGGTGTAAATTACTTA
18043
ScaCas9-
 97
0






HiFi-Sc++




 


1012
CTG
CGTAAGGTGTAAATTACTTA
18044
ScaCas9-Sc++
 97
0


 


1013
CTG
CGTAAGGTGTAAATTACTTA
18045
SpyCas9-
 97
0






SpRY




 


1014
CAC
CTAAAAAAGAAGTAAAATGC
18046
SpyCas9-
 97
0






SpRY




 


1015
CACT
CTAAAAAAGAAGTAAAATGC
18047
SpyCas9-
 97
0






3var-NRCH




 


1016
ACT
TCGTAAGGTGTAAATTACTT
18048
SpyCas9-
 98
0






SpRY




 


1017
CCA
CCTAAAAAAGAAGTAAAATG
18049
SpyCas9-
 98
0






SpRY




 


1018
TACTGTT
tggCCTCGTAAGGTGTAAATTACT
18050
PpnCas9
 99
0


 


1019
TAC
CTCGTAAGGTGTAAATTACT
18051
SpyCas9-
 99
0






SpRY




 


1020
GCC
TCCTAAAAAAGAAGTAAAAT
18052
SpyCas9-
 99
0






SpRY




 


1021
GCCACTGA
tgttCCTAAAAAAGAAGTAAAAT
18053
BlatCas9
 99
0


 


1022
GCCAC
tgttCCTAAAAAAGAAGTAAAAT
18054
BlatCas9
 99
0


 


1023
GCCACT
TTCCTAAAAAAGAAGTAAAAT
18055
cCas9-v16
 99
0


 


1024
GCCACT
TTCCTAAAAAAGAAGTAAAAT
18056
cCas9-v21
 99
0


 


1025
TACT
CTCGTAAGGTGTAAATTACT
18057
SpyCas9-
 99
0






3var-NRCH




 


1026
TG
TTCCTAAAAAAGAAGTAAAA
18058
SpyCas9-NG
100
0


 


1027
TG
TTCCTAAAAAAGAAGTAAAA
18059
SpyCas9-xCas
100
0


 


1028
TG
TTCCTAAAAAAGAAGTAAAA
18060
SpyCas9-
100
0






xCas-NG




 


1029
TGC
TTCCTAAAAAAGAAGTAAAA
18061
SpyCas9-SpG
100
0


 


1030
TGC
TTCCTAAAAAAGAAGTAAAA
18062
SpyCas9-
100
0






SpRY




 


1031
TTA
CCTCGTAAGGTGTAAATTAC
18063
SpyCas9-
100
0






SpRY




 


1032
TGCC
TTCCTAAAAAAGAAGTAAAA
18064
SpyCas9-
100
0






3var-NRCH
















TABLE 1B







Exemplary gRNA spacer Cas pairs for correcting the pathogenic R261Q


mutation


Table 1B provides a gRNA database for correcting the pathogenic R261Q mutation in PAH. List of


spacers, PAMs, and Cas variants for generating a nick at an appropriate position to enable


installation of a desired genomic edit with a gene modifying system. The spacers in this table


are designed to be used with a gene modifying polypeptide comprising a nickase variant of the


Cas species indicated in the table. Tables 2B, 3B, and 4B detail the other components of the


system and are organized such that the ID number shown here in Column 1 (“ID”) is meant to


correspond to the same ID number in Tables 2B, 2B, and 4B.
















SEQ






PAM

ID


Overlaps


ID
sequence
gRNA spacer
NO
Cas species
distance
mutation
















1
TCTTCC
tcTTGGGTGGCCTGGCCTTCCAA
19152
Nme2Cas9
0
0




G






 


2
TCT
GGGTGGCCTGGCCTTCCAAG
19153
SpyCas9-SpRY
0
0


 


3
TCTTC
cttgGGTGGCCTGGCCTTCCAAG
19154
BlatCas9
0
0


 


4
GAAGG
CTGTGTGCAGTGGAAGACTTG
19155
SauCas9KKH
1
0


 


5
GAAG
CTGTGTGCAGTGGAAGACTTG
19156
SauriCas9-KKH
1
0


 


6
GAAG
TGTGTGCAGTGGAAGACTTG
19157
SpyCas9-QQR1
1
0


 


7
GAAG
ctGTGTGCAGTGGAAGACTTG
19158
iSpy MacCas9
1
0


 


8
GAA
TGTGTGCAGTGGAAGACTTG
19159
SpyCas9-SpRY
1
0


 


9
GAA
TGTGTGCAGTGGAAGACTTG
19160
SpyCas9-xCas
1
0


 


10
GTC
TGGGTGGCCTGGCCTTCCAA
19161
SpyCas9-SpRY
1
0


 


11
GAAGGC
CTGTGTGCAGTGGAAGACTTG
19162
cCas9-v17
1
0


 


12
GAAGGC
CTGTGTGCAGTGGAAGACTTG
19163
cCas9-v42
1
0


 


13
GGAAG
ACTGTGTGCAGTGGAAGACTT
19164
SauCas9KKH
2
0


 


14
AG
TTGGGTGGCCTGGCCTTCCA
19165
SpyCas9-NG
2
0


 


15
AG
TTGGGTGGCCTGGCCTTCCA
19166
SpyCas9-xCas
2
0


 


16
AG
TTGGGTGGCCTGGCCTTCCA
19167
SpyCas9-xCas-
2
0






NG




 


17
GG
CTGTGTGCAGTGGAAGACTT
19168
SpyCas9-NG
2
0


 


18
GG
CTGTGTGCAGTGGAAGACTT
19169
SpyCas9-xCas
2
0


 


19
GG
CTGTGTGCAGTGGAAGACTT
19170
SpyCas9-xCas-
2
0






NG




 


20
AGT
TTGGGTGGCCTGGCCTTCCA
19171
SpyCas9-SpG
2
0


 


21
AGT
TTGGGTGGCCTGGCCTTCCA
19172
SpyCas9-SpRY
2
0


 


22
GGA
CTGTGTGCAGTGGAAGACTT
19173
SpyCas9-SpG
2
0


 


23
GGA
CTGTGTGCAGTGGAAGACTT
19174
SpyCas9-SpRY
2
0


 


24
GGAAGG
ACTGTGTGCAGTGGAAGACTT
19175
cCas9-v17
2
0


 


25
GGAAGG
ACTGTGTGCAGTGGAAGACTT
19176
cCas9-v42
2
0


 


26
GGAA
CTGTGTGCAGTGGAAGACTT
19177
SpyCas9-3var-
2
0






NRRH




 


27
GGAA
CTGTGTGCAGTGGAAGACTT
19178
SpyCas9-VQR
2
0


 


28
AGTC
TTGGGTGGCCTGGCCTTCCA
19179
SpyCas9-3var-
2
0






NRTH




 


29
tGGAA
tgTACTGTGTGCAGTGGAAGAC
19180
SauCas9
3
1




T






 


30
tGGAA
TACTGTGTGCAGTGGAAGACT
19181
SauCas9KKH
3
1


 


31
aAG
CTTGGGTGGCCTGGCCTTCC
19182
ScaCas9
3
1


 


32
aAG
CTTGGGTGGCCTGGCCTTCC
19183
ScaCas9-HiFi-
3
1






Sc++




 


33
aAG
CTTGGGTGGCCTGGCCTTCC
19184
ScaCas9-Sc++
3
1


 


34
aAG
CTTGGGTGGCCTGGCCTTCC
19185
SpyCas9-SpRY
3
1


 


35
tGG
ACTGTGTGCAGTGGAAGACT
19186
ScaCas9
3
1


 


36
tGG
ACTGTGTGCAGTGGAAGACT
19187
ScaCas9-HiFi-
3
1






Sc++




 


37
tGG
ACTGTGTGCAGTGGAAGACT
19188
ScaCas9-Sc++
3
1


 


38
tGG
ACTGTGTGCAGTGGAAGACT
19189
SpyCas9
3
1


 


39
tGG
ACTGTGTGCAGTGGAAGACT
19190
SpyCas9-HF1
3
1


 


40
tGG
ACTGTGTGCAGTGGAAGACT
19191
SpyCas9-SpG
3
1


 


41
tGG
ACTGTGTGCAGTGGAAGACT
19192
SpyCas9-SpRY
3
1


 


42
tG
ACTGTGTGCAGTGGAAGACT
19193
SpyCas9-NG
3
1


 


43
tG
ACTGTGTGCAGTGGAAGACT
19194
SpyCas9-xCas
3
1


 


44
tG
ACTGTGTGCAGTGGAAGACT
19195
SpyCas9-xCas-
3
1






NG




 


45
aAGTC
tttcTTGGGTGGCCTGGCCTTCC
19196
BlatCas9
3
1


 


46
tGGAAG
TACTGTGTGCAGTGGAAGACT
19197
cCas9-v17
3
1


 


47
tGGAAG
TACTGTGTGCAGTGGAAGACT
19198
cCas9-v42
3
1


 


48
aAGTCTT
TTCTTGGGTGGCCTGGCCTTCC
19199
CdiCas9
3
1


 


49
aAGT
CTTGGGTGGCCTGGCCTTCC
19200
SpyCas9-3var-
3
1






NRRH




 


50
tGGA
ACTGTGTGCAGTGGAAGACT
19201
SpyCas9-3var-
3
1






NRRH




 


51
TtGGA
atGTACTGTGTGCAGTGGAAGA
19202
SauCas9
4
1




C






 


52
TtGGA
GTACTGTGTGCAGTGGAAGAC
19203
SauCas9KKH
4
1


 


53
TtGG
GTACTGTGTGCAGTGGAAGAC
19204
SauriCas9
4
1


 


54
TtGG
GTACTGTGTGCAGTGGAAGAC
19205
SauriCas9-KKH
4
1


 


55
CaAG
TTCTTGGGTGGCCTGGCCTTC
19206
SauriCas9-KKH
4
1


 


56
CaAG
TCTTGGGTGGCCTGGCCTTC
19207
SpyCas9-QQR1
4
1


 


57
CaAG
ttCTTGGGTGGCCTGGCCTTC
19208
iSpyMacCas9
4
1


 


58
TtG
TACTGTGTGCAGTGGAAGAC
19209
ScaCas9
4
1


 


59
TtG
TACTGTGTGCAGTGGAAGAC
19210
ScaCas9-HiFi-
4
1






Sc++




 


60
TtG
TACTGTGTGCAGTGGAAGAC
19211
ScaCas9-Sc++
4
1


 


61
TtG
TACTGTGTGCAGTGGAAGAC
19212
SpyCas9-SpRY
4
1


 


62
CaA
TCTTGGGTGGCCTGGCCTTC
19213
SpyCas9-SpRY
4
1


 


63
TtGGAA
GTACTGTGTGCAGTGGAAGAC
19214
cCas9-v17
4
1


 


64
TtGGAA
GTACTGTGTGCAGTGGAAGAC
19215
cCas9-v42
4
1


 


65
CCaAG
TTTCTTGGGTGGCCTGGCCTT
19216
SauCas9KKH
5
1


 


66
CTtGG
TGTACTGTGTGCAGTGGAAGA
19217
SauCas9KKH
5
1


 


67
CCaAGT
TTTCTTGGGTGGCCTGGCCTT
19218
SauCas9KKH
5
1


 


68
CCaAGT
TTTCTTGGGTGGCCTGGCCTT
19219
cCas9-v17
5
1


 


69
CCaAGT
TTTCTTGGGTGGCCTGGCCTT
19220
cCas9-v42
5
1


 


70
CTt
GTACTGTGTGCAGTGGAAGA
19221
SpyCas9-SpRY
5
1


 


71
CCa
TTCTTGGGTGGCCTGGCCTT
19222
SpyCas9-SpRY
5
1


 


72
CCaAGTCT
ggatTTCTTGGGTGGCCTGGCCTT
19223
NmeCas9
5
1


 


73
TCCaA
ATTTCTTGGGTGGCCTGGCCT
19224
SauCas9KKH
6
1


 


74
ACT
TGTACTGTGTGCAGTGGAAG
19225
SpyCas9-SpRY
6
0


 


75
TCC
TTTCTTGGGTGGCCTGGCCT
19226
SpyCas9-SpRY
6
0


 


76
TCCaAG
ATTTCTTGGGTGGCCTGGCCT
19227
cCas9-v17
6
1


 


77
TCCaAG
ATTTCTTGGGTGGCCTGGCCT
19228
cCas9-v42
6
1


 


78
GAC
ATGTACTGTGTGCAGTGGAA
19229
SpyCas9-SpRY
7
0


 


79
TTC
ATTTCTTGGGTGGCCTGGCC
19230
Spy Cas9-SpRY
7
0


 


80
GACT
ATGTACTGTGTGCAGTGGAA
19231
SpyCas9-3var-
7
0






NRCH




 


81
AG
GATGTACTGTGTGCAGTGGA
19232
Spy Cas9-NG
8
0


 


82
AG
GATGTACTGTGTGCAGTGGA
19233
SpyCas9-xCas
8
0


 


83
AG
GATGTACTGTGTGCAGTGGA
19234
SpyCas9-xCas-
8
0






NG




 


84
AGA
GATGTACTGTGTGCAGTGGA
19235
SpyCas9-SpG
8
0


 


85
AGA
GATGTACTGTGTGCAGTGGA
19236
SpyCas9-SpRY
8
0


 


86
CTT
GATTTCTTGGGTGGCCTGGC
19237
SpyCas9-SpRY
8
0


 


87
CTTCCaAG
cgggATTTCTTGGGTGGCCTGGC
19238
BlatCas9
8
1


 


88
CTTCC
cgggATTTCTTGGGTGGCCTGGC
19239
BlatCas9
8
0


 


89
AGAC
GATGTACTGTGTGCAGTGGA
19240
SpyCas9-3var-
8
0






NRRH




 


90
AGAC
GATGTACTGTGTGCAGTGGA
19241
SpyCas9-VQR
8
0


 


91
CCTTCC
ctCGGGATTTCTTGGGTGGCCTG
19242
Nme2Cas9
9
0




G






 


92
AAG
TGATGTACTGTGTGCAGTGG
19243
ScaCas9
9
0


 


93
AAG
TGATGTACTGTGTGCAGTGG
19244
ScaCas9-HiFi-
9
0






Sc++




 


94
AAG
TGATGTACTGTGTGCAGTGG
19245
ScaCas9-Sc++
9
0


 


95
AAG
TGATGTACTGTGTGCAGTGG
19246
SpyCas9-SpRY
9
0


 


96
CCT
GGATTTCTTGGGTGGCCTGG
19247
SpyCas9-SpRY
9
0


 


97
AAGACTtG
gtctGATGTACTGTGTGCAGTGG
19248
BlatCas9
9
1


 


98
CCTTCCaA
tcggGATTTCTTGGGTGGCCTGG
19249
BlatCas9
9
1


 


99
CCTTCCaA
tcggGATTTCTTGGGTGGCCTGG
19250
BlatCas9
9
1


 


100
AAGAC
gtctGATGTACTGTGTGCAGTGG
19251
BlatCas9
9
0


 


101
CCTTC
tcggGATTTCTTGGGTGGCCTGG
19252
BlatCas9
9
0


 


102
AAGACT
CTGATGTACTGTGTGCAGTGG
19253
cCas9-v16
9
0


 


103
AAGACT
CTGATGTACTGTGTGCAGTGG
19254
cCas9-v21
9
0


 


104
AAGACTt
TCTGATGTACTGTGTGCAGTGG
19255
CdiCas9
9
1


 


105
AAGA
TGATGTACTGTGTGCAGTGG
19256
SpyCas9-3var-
9
0






NRRH




 


106
GAAGA
TCTGATGTACTGTGTGCAGTG
19257
SauCas9KKH
10
0


 


107
GAAG
TCTGATGTACTGTGTGCAGTG
19258
SauriCas9-KKH
10
0


 


108
GAAG
CTGATGTACTGTGTGCAGTG
19259
SpyCas9-QQR1
10
0


 


109
GAAG
tcTGATGTACTGTGTGCAGTG
19260
iSpyMacCas9
10
0


 


110
GAA
CTGATGTACTGTGTGCAGTG
19261
SpyCas9-SpRY
10
0


 


111
GAA
CTGATGTACTGTGTGCAGTG
19262
SpyCas9-xCas
10
0


 


112
GCC
GGGATTTCTTGGGTGGCCTG
19263
Spy Cas9-SpRY
10
0


 


113
GAAGAC
TCTGATGTACTGTGTGCAGTG
19264
cCas9-v17
10
0


 


114
GAAGAC
TCTGATGTACTGTGTGCAGTG
19265
cCas9-v42
10
0


 


115
GGAAG
GTCTGATGTACTGTGTGCAGT
19266
SauCas9KKH
11
0


 


116
GG
TCTGATGTACTGTGTGCAGT
19267
SpyCas9-NG
11
0


 


117
GG
TCTGATGTACTGTGTGCAGT
19268
SpyCas9-xCas
11
0


 


118
GG
TCTGATGTACTGTGTGCAGT
19269
SpyCas9-xCas-
11
0






NG




 


119
GG
CGGGATTTCTTGGGTGGCCT
19270
SpyCas9-NG
11
0


 


120
GG
CGGGATTTCTTGGGTGGCCT
19271
SpyCas9-xCas
11
0


 


121
GG
CGGGATTTCTTGGGTGGCCT
19272
SpyCas9-xCas-
11
0






NG




 


122
GGA
TCTGATGTACTGTGTGCAGT
19273
SpyCas9-SpG
11
0


 


123
GGA
TCTGATGTACTGTGTGCAGT
19274
SpyCas9-SpRY
11
0


 


124
GGC
CGGGATTTCTTGGGTGGCCT
19275
SpyCas9-SpG
11
0


 


125
GGC
CGGGATTTCTTGGGTGGCCT
19276
Spy Cas9-SpRY
11
0


 


126
GGAAGA
GTCTGATGTACTGTGTGCAGT
19277
cCas9-v17
11
0


 


127
GGAAGA
GTCTGATGTACTGTGTGCAGT
19278
cCas9-v42
11
0


 


128
GGAAGAC
catgTCTGATGTACTGTGTGCAG
19279
NmeCas9
11
0



T
T






 


129
GGAA
TCTGATGTACTGTGTGCAGT
19280
SpyCas9-3var-
11
0






NRRH




 


130
GGAA
TCTGATGTACTGTGTGCAGT
19281
SpyCas9-VQR
11
0


 


131
GGCC
CGGGATTTCTTGGGTGGCCT
19282
SpyCas9-3var-
11
0






NRCH




 


132
TGGAA
caTGTCTGATGTACTGTGTGCAG
19283
SauCas9
12
0


 


133
TGGAA
TGTCTGATGTACTGTGTGCAG
19284
SauCas9KKH
12
0


 


134
TGG
GTCTGATGTACTGTGTGCAG
19285
ScaCas9
12
0


 


135
TGG
GTCTGATGTACTGTGTGCAG
19286
ScaCas9-HiFi-
12
0






Sc++




 


136
TGG
GTCTGATGTACTGTGTGCAG
19287
ScaCas9-Sc++
12
0


 


137
TGG
GTCTGATGTACTGTGTGCAG
19288
SpyCas9
12
0


 


138
TGG
GTCTGATGTACTGTGTGCAG
19289
SpyCas9-HF1
12
0


 


139
TGG
GTCTGATGTACTGTGTGCAG
19290
SpyCas9-SpG
12
0


 


140
TGG
GTCTGATGTACTGTGTGCAG
19291
SpyCas9-SpRY
12
0


 


141
TGG
TCGGGATTTCTTGGGTGGCC
19292
ScaCas9
12
0


 


142
TGG
TCGGGATTTCTTGGGTGGCC
19293
ScaCas9-HiFi-
12
0






Sc++




 


143
TGG
TCGGGATTTCTTGGGTGGCC
19294
ScaCas9-Sc++
12
0


 


144
TGG
TCGGGATTTCTTGGGTGGCC
19295
SpyCas9
12
0


 


145
TGG
TCGGGATTTCTTGGGTGGCC
19296
SpyCas9-HF1
12
0


 


146
TGG
TCGGGATTTCTTGGGTGGCC
19297
SpyCas9-SpG
12
0


 


147
TGG
TCGGGATTTCTTGGGTGGCC
19298
SpyCas9-SpRY
12
0


 


148
TG
GTCTGATGTACTGTGTGCAG
19299
SpyCas9-NG
12
0


 


149
TG
GTCTGATGTACTGTGTGCAG
19300
SpyCas9-xCas
12
0


 


150
TG
GTCTGATGTACTGTGTGCAG
19301
SpyCas9-xCas-
12
0






NG




 


151
TG
TCGGGATTTCTTGGGTGGCC
19302
SpyCas9-NG
12
0


 


152
TG
TCGGGATTTCTTGGGTGGCC
19303
SpyCas9-xCas
12
0


 


153
TG
TCGGGATTTCTTGGGTGGCC
19304
SpyCas9-xCas-
12
0






NG




 


154
TGGCC
ctctCGGGATTTCTTGGGTGGCC
19305
BlatCas9
12
0


 


155
TGGAAG
TGTCTGATGTACTGTGTGCAG
19306
cCas9-v17
12
0


 


156
TGGAAG
TGTCTGATGTACTGTGTGCAG
19307
cCas9-v42
12
0


 


157
TGGCCTT
TCTCGGGATTTCTTGGGTGGCC
19308
CdiCas9
12
0


 


158
TGGA
GTCTGATGTACTGTGTGCAG
19309
SpyCas9-3var-
12
0






NRRH




 


159
TGGC
TCGGGATTTCTTGGGTGGCC
19310
SpyCas9-3var-
12
0






NRRH




 


160
CTGGCC
tcCTCTCGGGATTTCTTGGGTGG
19311
Nme2Cas9
13
0




C






 


161
GTGGA
ccATGTCTGATGTACTGTGTGCA
19312
SauCas9
13
0


 


162
GTGGA
ATGTCTGATGTACTGTGTGCA
19313
SauCas9KKH
13
0


 


163
GTGG
ATGTCTGATGTACTGTGTGCA
19314
SauriCas9
13
0


 


164
GTGG
ATGTCTGATGTACTGTGTGCA
19315
SauriCas9-KKH
13
0


 


165
CTGG
TCTCGGGATTTCTTGGGTGGC
19316
SauriCas9
13
0


 


166
CTGG
TCTCGGGATTTCTTGGGTGGC
19317
SauriCas9-KKH
13
0


 


167
GTG
TGTCTGATGTACTGTGTGCA
19318
ScaCas9
13
0


 


168
GTG
TGTCTGATGTACTGTGTGCA
19319
ScaCas9-HiFi-
13
0






Sc++




 


169
GTG
TGTCTGATGTACTGTGTGCA
19320
ScaCas9-Sc++
13
0


 


170
GTG
TGTCTGATGTACTGTGTGCA
19321
SpyCas9-SpRY
13
0


 


171
CTG
CTCGGGATTTCTTGGGTGGC
19322
ScaCas9
13
0


 


172
CTG
CTCGGGATTTCTTGGGTGGC
19323
ScaCas9-HiFi-
13
0






Sc++




 


173
CTG
CTCGGGATTTCTTGGGTGGC
19324
ScaCas9-Sc++
13
0


 


174
CTG
CTCGGGATTTCTTGGGTGGC
19325
SpyCas9-SpRY
13
0


 


175
CTGGCCTT
cctcTCGGGATTTCTTGGGTGGC
19326
BlatCas9
13
0


 


176
CTGGC
cctcTCGGGATTTCTTGGGTGGC
19327
BlatCas9
13
0


 


177
GTGGAA
ATGTCTGATGTACTGTGTGCA
19328
cCas9-v17
13
0


 


178
GTGGAA
ATGTCTGATGTACTGTGTGCA
19329
cCas9-v42
13
0


 


179
AGTGG
CATGTCTGATGTACTGTGTGC
19330
SauCas9KKH
14
0


 


180
CCTGG
CTCTCGGGATTTCTTGGGTGG
19331
SauCas9KKH
14
0


 


181
AG
ATGTCTGATGTACTGTGTGC
19332
SpyCas9-NG
14
0


 


182
AG
ATGTCTGATGTACTGTGTGC
19333
SpyCas9-xCas
14
0


 


183
AG
ATGTCTGATGTACTGTGTGC
19334
SpyCas9-xCas-
14
0






NG




 


184
AGT
ATGTCTGATGTACTGTGTGC
19335
SpyCas9-SpG
14
0


 


185
AGT
ATGTCTGATGTACTGTGTGC
19336
SpyCas9-SpRY
14
0


 


186
CCT
TCTCGGGATTTCTTGGGTGG
19337
Spy Cas9-SpRY
14
0


 


187
CAG
CATGTCTGATGTACTGTGTG
19338
ScaCas9
15
0


 


188
CAG
CATGTCTGATGTACTGTGTG
19339
ScaCas9-HiFi-
15
0






Sc++




 


189
CAG
CATGTCTGATGTACTGTGTG
19340
ScaCas9-Sc++
15
0


 


190
CAG
CATGTCTGATGTACTGTGTG
19341
SpyCas9-SpRY
15
0


 


191
GCC
CTCTCGGGATTTCTTGGGTG
19342
SpyCas9-SpRY
15
0


 


192
CAGT
CATGTCTGATGTACTGTGTG
19343
SpyCas9-3var-
15
0






NRRH




 


193
GCAG
TCCATGTCTGATGTACTGTGT
19344
SauriCas9-KKH
16
0


 


194
GG
CCTCTCGGGATTTCTTGGGT
19345
SpyCas9-NG
16
0


 


195
GG
CCTCTCGGGATTTCTTGGGT
19346
SpyCas9-xCas
16
0


 


196
GG
CCTCTCGGGATTTCTTGGGT
19347
SpyCas9-xCas-
16
0






NG




 


197
GGC
CCTCTCGGGATTTCTTGGGT
19348
SpyCas9-SpG
16
0


 


198
GGC
CCTCTCGGGATTTCTTGGGT
19349
SpyCas9-SpRY
16
0


 


199
GCA
CCATGTCTGATGTACTGTGT
19350
SpyCas9-SpRY
16
0


 


200
GCAGTG
TCCATGTCTGATGTACTGTGT
19351
cCas9-v16
16
0


 


201
GCAGTG
TCCATGTCTGATGTACTGTGT
19352
cCas9-v21
16
0


 


202
GGCC
CCTCTCGGGATTTCTTGGGT
19353
SpyCas9-3var-
16
0






NRCH




 


203
TGCAG
ATCCATGTCTGATGTACTGTG
19354
SauCas9KKH
17
0


 


204
TGCAGT
ATCCATGTCTGATGTACTGTG
19355
SauCas9KKH
17
0


 


205
TGCAGT
ATCCATGTCTGATGTACTGTG
19356
cCas9-v17
17
0


 


206
TGCAGT
ATCCATGTCTGATGTACTGTG
19357
cCas9-v42
17
0


 


207
TGG
TCCTCTCGGGATTTCTTGGG
19358
ScaCas9
17
0


 


208
TGG
TCCTCTCGGGATTTCTTGGG
19359
ScaCas9-HiFi-
17
0






Sc++




 


209
TGG
TCCTCTCGGGATTTCTTGGG
19360
ScaCas9-Sc++
17
0


 


210
TGG
TCCTCTCGGGATTTCTTGGG
19361
SpyCas9
17
0


 


211
TGG
TCCTCTCGGGATTTCTTGGG
19362
SpyCas9-HF1
17
0


 


212
TGG
TCCTCTCGGGATTTCTTGGG
19363
Spy Cas9-SpG
17
0


 


213
TGG
TCCTCTCGGGATTTCTTGGG
19364
SpyCas9-SpRY
17
0


 


214
TG
TCCATGTCTGATGTACTGTG
19365
SpyCas9-NG
17
0


 


215
TG
TCCATGTCTGATGTACTGTG
19366
SpyCas9-xCas
17
0


 


216
TG
TCCATGTCTGATGTACTGTG
19367
SpyCas9-xCas-
17
0






NG




 


217
TG
TCCTCTCGGGATTTCTTGGG
19368
SpyCas9-NG
17
0


 


218
TG
TCCTCTCGGGATTTCTTGGG
19369
SpyCas9-xCas
17
0


 


219
TG
TCCTCTCGGGATTTCTTGGG
19370
SpyCas9-xCas-
17
0






NG




 


220
TGC
TCCATGTCTGATGTACTGTG
19371
SpyCas9-SpG
17
0


 


221
TGC
TCCATGTCTGATGTACTGTG
19372
SpyCas9-SpRY
17
0


 


222
TGGCCTG
ctttCCTCTCGGGATTTCTTGGG
19373
BlatCas9
17
0



G







 


223
TGGCC
ctttCCTCTCGGGATTTCTTGGG
19374
BlatCas9
17
0


 


224
TGGC
TCCTCTCGGGATTTCTTGGG
19375
SpyCas9-3var-
17
0






NRRH




 


225
TGCA
TCCATGTCTGATGTACTGTG
19376
SpyCas9-3var-
17
0






NRCH




 


226
GTGGCC
tgCTTTCCTCTCGGGATTTCTTG
19377
Nme2Cas9
18
0




G






 


227
GTGG
TTTCCTCTCGGGATTTCTTGG
19378
SauriCas9
18
0


 


228
GTGG
TTTCCTCTCGGGATTTCTTGG
19379
SauriCas9-KKH
18
0


 


229
GTG
ATCCATGTCTGATGTACTGT
19380
ScaCas9
18
0


 


230
GTG
ATCCATGTCTGATGTACTGT
19381
ScaCas9-HiFi-
18
0






Sc++




 


231
GTG
ATCCATGTCTGATGTACTGT
19382
ScaCas9-Sc++
18
0


 


232
GTG
ATCCATGTCTGATGTACTGT
19383
SpyCas9-SpRY
18
0


 


233
GTG
TTCCTCTCGGGATTTCTTGG
19384
ScaCas9
18
0


 


234
GTG
TTCCTCTCGGGATTTCTTGG
19385
ScaCas9-HiFi-
18
0






Sc++




 


235
GTG
TTCCTCTCGGGATTTCTTGG
19386
ScaCas9-Sc++
18
0


 


236
GTG
TTCCTCTCGGGATTTCTTGG
19387
SpyCas9-SpRY
18
0


 


237
GTGGCCT
gcttTCCTCTCGGGATTTCTTGG
19388
BlatCas9
18
0



G







 


238
GTGGC
gcttTCCTCTCGGGATTTCTTGG
19389
BlatCas9
18
0


 


239
GGTGG
CTTTCCTCTCGGGATTTCTTG
19390
SauCas9KKH
19
0


 


240
TG
GATCCATGTCTGATGTACTG
19391
SpyCas9-NG
19
0


 


241
TG
GATCCATGTCTGATGTACTG
19392
SpyCas9-xCas
19
0


 


242
TG
GATCCATGTCTGATGTACTG
19393
SpyCas9-xCas-
19
0






NG




 


243
GG
TTTCCTCTCGGGATTTCTTG
19394
SpyCas9-NG
19
0


 


244
GG
TTTCCTCTCGGGATTTCTTG
19395
SpyCas9-xCas
19
0


 


245
GG
TTTCCTCTCGGGATTTCTTG
19396
SpyCas9-xCas-
19
0






NG




 


246
TGT
GATCCATGTCTGATGTACTG
19397
SpyCas9-SpG
19
0


 


247
TGT
GATCCATGTCTGATGTACTG
19398
SpyCas9-SpRY
19
0


 


248
GGT
TTTCCTCTCGGGATTTCTTG
19399
SpyCas9-SpG
19
0


 


249
GGT
TTTCCTCTCGGGATTTCTTG
19400
SpyCas9-SpRY
19
0


 


250
TGTGCAG
ttggATCCATGTCTGATGTACTG
19401
BlatCas9
19
0



T







 


251
TGTGC
ttggATCCATGTCTGATGTACTG
19402
BlatCas9
19
0


 


252
GTG
GGATCCATGTCTGATGTACT
19403
ScaCas9
20
0


 


253
GTG
GGATCCATGTCTGATGTACT
19404
ScaCas9-HiFi-
20
0






Sc++




 


254
GTG
GGATCCATGTCTGATGTACT
19405
ScaCas9-Sc++
20
0


 


255
GTG
GGATCCATGTCTGATGTACT
19406
SpyCas9-SpRY
20
0


 


256
GGG
CTTTCCTCTCGGGATTTCTT
19407
ScaCas9
20
0


 


257
GGG
CTTTCCTCTCGGGATTTCTT
19408
ScaCas9-HiFi-
20
0






Sc++




 


258
GGG
CTTTCCTCTCGGGATTTCTT
19409
ScaCas9-Sc++
20
0


 


259
GGG
CTTTCCTCTCGGGATTTCTT
19410
SpyCas9
20
0


 


260
GGG
CTTTCCTCTCGGGATTTCTT
19411
SpyCas9-HF1
20
0


 


261
GGG
CTTTCCTCTCGGGATTTCTT
19412
SpyCas9-SpG
20
0


 


262
GGG
CTTTCCTCTCGGGATTTCTT
19413
Spy Cas9-SpRY
20
0


 


263
GG
CTTTCCTCTCGGGATTTCTT
19414
SpyCas9-NG
20
0


 


264
GG
CTTTCCTCTCGGGATTTCTT
19415
SpyCas9-xCas
20
0


 


265
GG
CTTTCCTCTCGGGATTTCTT
19416
SpyCas9-xCas-
20
0






NG




 


266
GGGT
CTTTCCTCTCGGGATTTCTT
19417
SpyCas9-3var-
20
0






NRRH




 


267
TGGG
TGCTTTCCTCTCGGGATTTCT
19418
SauriCas9
21
0


 


268
TGGG
TGCTTTCCTCTCGGGATTTCT
19419
SauriCas9-KKH
21
0


 


269
TGG
GCTTTCCTCTCGGGATTTCT
19420
ScaCas9
21
0


 


270
TGG
GCTTTCCTCTCGGGATTTCT
19421
ScaCas9-HiFi-
21
0






Sc++




 


271
TGG
GCTTTCCTCTCGGGATTTCT
19422
ScaCas9-Sc++
21
0


 


272
TGG
GCTTTCCTCTCGGGATTTCT
19423
SpyCas9
21
0


 


273
TGG
GCTTTCCTCTCGGGATTTCT
19424
SpyCas9-HF1
21
0


 


274
TGG
GCTTTCCTCTCGGGATTTCT
19425
SpyCas9-SpG
21
0


 


275
TGG
GCTTTCCTCTCGGGATTTCT
19426
SpyCas9-SpRY
21
0


 


276
TG
TGGATCCATGTCTGATGTAC
19427
SpyCas9-NG
21
0


 


277
TG
TGGATCCATGTCTGATGTAC
19428
SpyCas9-xCas
21
0


 


278
TG
TGGATCCATGTCTGATGTAC
19429
SpyCas9-xCas-
21
0






NG




 


279
TG
GCTTTCCTCTCGGGATTTCT
19430
SpyCas9-NG
21
0


 


280
TG
GCTTTCCTCTCGGGATTTCT
19431
SpyCas9-xCas
21
0


 


281
TG
GCTTTCCTCTCGGGATTTCT
19432
SpyCas9-xCas-
21
0






NG




 


282
TGT
TGGATCCATGTCTGATGTAC
19433
SpyCas9-SpG
21
0


 


283
TGT
TGGATCCATGTCTGATGTAC
19434
SpyCas9-SpRY
21
0


 


284
TGGGTG
TGCTTTCCTCTCGGGATTTCT
19435
cCas9-v16
21
0


 


285
TGGGTG
TGCTTTCCTCTCGGGATTTCT
19436
cCas9-v21
21
0


 


286
TTGGG
gcCTGCTTTCCTCTCGGGATTTC
19437
SauCas9
22
0


 


287
TTGGG
CTGCTTTCCTCTCGGGATTTC
19438
SauCas9KKH
22
0


 


288
TTGGGT
gcCTGCTTTCCTCTCGGGATTTC
19439
SauCas9
22
0


 


289
TTGGGT
CTGCTTTCCTCTCGGGATTTC
19440
SauCas9KKH
22
0


 


290
TTGGGT
CTGCTTTCCTCTCGGGATTTC
19441
cCas9-v17
22
0


 


291
TTGGGT
CTGCTTTCCTCTCGGGATTTC
19442
cCas9-v42
22
0


 


292
TTGG
CTGCTTTCCTCTCGGGATTTC
19443
SauriCas9
22
0


 


293
TTGG
CTGCTTTCCTCTCGGGATTTC
19444
SauriCas9-KKH
22
0


 


294
CTG
TTGGATCCATGTCTGATGTA
19445
ScaCas9
22
0


 


295
CTG
TTGGATCCATGTCTGATGTA
19446
ScaCas9-HiFi-
22
0






Sc++




 


296
CTG
TTGGATCCATGTCTGATGTA
19447
ScaCas9-Sc++
22
0


 


297
CTG
TTGGATCCATGTCTGATGTA
19448
SpyCas9-SpRY
22
0


 


298
TTG
TGCTTTCCTCTCGGGATTTC
19449
ScaCas9
22
0


 


299
TTG
TGCTTTCCTCTCGGGATTTC
19450
ScaCas9-HiFi-
22
0






Sc++




 


300
TTG
TGCTTTCCTCTCGGGATTTC
19451
ScaCas9-Sc++
22
0


 


301
TTG
TGCTTTCCTCTCGGGATTTC
19452
SpyCas9-SpRY
22
0


 


302
CTTGG
CCTGCTTTCCTCTCGGGATTT
19453
SauCas9KKH
23
0


 


303
ACT
CTTGGATCCATGTCTGATGT
19454
SpyCas9-SpRY
23
0


 


304
CTT
CTGCTTTCCTCTCGGGATTT
19455
Spy Cas9-SpRY
23
0


 


305
TAC
GCTTGGATCCATGTCTGATG
19456
SpyCas9-SpRY
24
0


 


306
TCT
CCTGCTTTCCTCTCGGGATT
19457
SpyCas9-SpRY
24
0


 


307
TACT
GCTTGGATCCATGTCTGATG
19458
SpyCas9-3var-
24
0






NRCH




 


308
GTA
GGCTTGGATCCATGTCTGAT
19459
SpyCas9-SpRY
25
0


 


309
TTC
GCCTGCTTTCCTCTCGGGAT
19460
SpyCas9-SpRY
25
0


 


310
TG
GGGCTTGGATCCATGTCTGA
19461
SpyCas9-NG
26
0


 


311
TG
GGGCTTGGATCCATGTCTGA
19462
SpyCas9-xCas
26
0


 


312
TG
GGGCTTGGATCCATGTCTGA
19463
SpyCas9-xCas-
26
0






NG




 


313
TGT
GGGCTTGGATCCATGTCTGA
19464
SpyCas9-SpG
26
0


 


314
TGT
GGGCTTGGATCCATGTCTGA
19465
Spy Cas9-SpRY
26
0


 


315
TTT
GGCCTGCTTTCCTCTCGGGA
19466
SpyCas9-SpRY
26
0


 


316
TGTACTGT
catgGGCTTGGATCCATGTCTGA
19467
BlatCas9
26
0


 


317
TGTAC
catgGGCTTGGATCCATGTCTGA
19468
BlatCas9
26
0


 


318
TGTA
GGGCTTGGATCCATGTCTGA
19469
SpyCas9-3var-
26
0






NRTH




 


319
ATG
TGGGCTTGGATCCATGTCTG
19470
ScaCas9
27
0


 


320
ATG
TGGGCTTGGATCCATGTCTG
19471
ScaCas9-HiFi-
27
0






Sc++




 


321
ATG
TGGGCTTGGATCCATGTCTG
19472
ScaCas9-Sc++
27
0


 


322
ATG
TGGGCTTGGATCCATGTCTG
19473
SpyCas9-SpRY
27
0


 


323
ATT
TGGCCTGCTTTCCTCTCGGG
19474
SpyCas9-SpRY
27
0


 


324
ATTTCTTG
ggctGGCCTGCTTTCCTCTCGGG
19475
BlatCas9
27
0


 


325
ATTTC
ggctGGCCTGCTTTCCTCTCGGG
19476
BlatCas9
27
0


 


326
ATGTACT
CATGGGCTTGGATCCATGTCTG
19477
CdiCas9
27
0


 


327
GAT
ATGGGCTTGGATCCATGTCT
19478
SpyCas9-SpRY
28
0


 


328
GAT
ATGGGCTTGGATCCATGTCT
19479
SpyCas9-xCas
28
0


 


329
GAT
CTGGCCTGCTTTCCTCTCGG
19480
SpyCas9-SpRY
28
0


 


330
GAT
CTGGCCTGCTTTCCTCTCGG
19481
SpyCas9-xCas
28
0


 


331
GATT
CTGGCCTGCTTTCCTCTCGG
19482
SpyCas9-3var-
28
0






NRTH




 


332
TG
CATGGGCTTGGATCCATGTC
19483
SpyCas9-NG
29
0


 


333
TG
CATGGGCTTGGATCCATGTC
19484
SpyCas9-xCas
29
0


 


334
TG
CATGGGCTTGGATCCATGTC
19485
SpyCas9-xCas-
29
0






NG




 


335
GG
GCTGGCCTGCTTTCCTCTCG
19486
SpyCas9-NG
29
0


 


336
GG
GCTGGCCTGCTTTCCTCTCG
19487
SpyCas9-xCas
29
0


 


337
GG
GCTGGCCTGCTTTCCTCTCG
19488
SpyCas9-xCas-
29
0






NG




 


338
TGA
CATGGGCTTGGATCCATGTC
19489
SpyCas9-SpG
29
0


 


339
TGA
CATGGGCTTGGATCCATGTC
19490
SpyCas9-SpRY
29
0


 


340
GGA
GCTGGCCTGCTTTCCTCTCG
19491
SpyCas9-SpG
29
0


 


341
GGA
GCTGGCCTGCTTTCCTCTCG
19492
SpyCas9-SpRY
29
0


 


342
GGATTTC
TGGCTGGCCTGCTTTCCTCTCG
19493
CdiCas9
29
0


 


343
TGATGTA
atACATGGGCTTGGATCCATGTC
19494
CjeCas9
29
0



C







 


344
TGAT
CATGGGCTTGGATCCATGTC
19495
SpyCas9-3var-
29
0






NRRH




 


345
TGAT
CATGGGCTTGGATCCATGTC
19496
SpyCas9-VQR
29
0


 


346
GGAT
GCTGGCCTGCTTTCCTCTCG
19497
SpyCas9-3var-
29
0






NRRH




 


347
GGAT
GCTGGCCTGCTTTCCTCTCG
19498
SpyCas9-VQR
29
0


 


348
CTG
ACATGGGCTTGGATCCATGT
19499
ScaCas9
30
0


 


349
CTG
ACATGGGCTTGGATCCATGT
19500
ScaCas9-HiFi-
30
0






Sc++




 


350
CTG
ACATGGGCTTGGATCCATGT
19501
ScaCas9-Sc++
30
0


 


351
CTG
ACATGGGCTTGGATCCATGT
19502
SpyCas9-SpRY
30
0


 


352
GGG
GGCTGGCCTGCTTTCCTCTC
19503
ScaCas9
30
0


 


353
GGG
GGCTGGCCTGCTTTCCTCTC
19504
ScaCas9-HiFi-
30
0






Sc++




 


354
GGG
GGCTGGCCTGCTTTCCTCTC
19505
ScaCas9-Sc++
30
0


 


355
GGG
GGCTGGCCTGCTTTCCTCTC
19506
SpyCas9
30
0


 


356
GGG
GGCTGGCCTGCTTTCCTCTC
19507
SpyCas9-HF1
30
0


 


357
GGG
GGCTGGCCTGCTTTCCTCTC
19508
SpyCas9-SpG
30
0


 


358
GGG
GGCTGGCCTGCTTTCCTCTC
19509
SpyCas9-SpRY
30
0


 


359
GG
GGCTGGCCTGCTTTCCTCTC
19510
SpyCas9-NG
30
0


 


360
GG
GGCTGGCCTGCTTTCCTCTC
19511
SpyCas9-xCas
30
0


 


361
GG
GGCTGGCCTGCTTTCCTCTC
19512
SpyCas9-xCas-
30
0






NG




 


362
GGGATT
TGGCTGGCCTGCTTTCCTCTC
19513
cCas9-v16
30
0


 


363
GGGATT
TGGCTGGCCTGCTTTCCTCTC
19514
cCas9-v21
30
0


 


364
GGGATTT
GTGGCTGGCCTGCTTTCCTCTC
19515
CdiCas9
30
0


 


365
GGGA
GGCTGGCCTGCTTTCCTCTC
19516
SpyCas9-3var-
30
0






NRRH




 


366
CGGGATT
cctGTGGCTGGCCTGCTTTCCTC
19517
PpnCas9
31
0




T






 


367
CGGGA
ctGTGGCTGGCCTGCTTTCCTCT
19518
SauCas9
31
0


 


368
CGGGA
GTGGCTGGCCTGCTTTCCTCT
19519
SauCas9KKH
31
0


 


369
CGGGAT
ctGTGGCTGGCCTGCTTTCCTCT
19520
SauCas9
31
0


 


370
CGGGAT
GTGGCTGGCCTGCTTTCCTCT
19521
SauCas9KKH
31
0


 


371
CGGGAT
GTGGCTGGCCTGCTTTCCTCT
19522
cCas9-v17
31
0


 


372
CGGGAT
GTGGCTGGCCTGCTTTCCTCT
19523
cCas9-v42
31
0


 


373
TCTGA
ATACATGGGCTTGGATCCATG
19524
SauCas9KKH
31
0


 


374
TCTGAT
ATACATGGGCTTGGATCCATG
19525
SauCas9KKH
31
0


 


375
CGGG
GTGGCTGGCCTGCTTTCCTCT
19526
SauriCas9
31
0


 


376
CGGG
GTGGCTGGCCTGCTTTCCTCT
19527
SauriCas9-KKH
31
0


 


377
CGG
TGGCTGGCCTGCTTTCCTCT
19528
ScaCas9
31
0


 


378
CGG
TGGCTGGCCTGCTTTCCTCT
19529
ScaCas9-HiFi-
31
0






Sc++




 


379
CGG
TGGCTGGCCTGCTTTCCTCT
19530
ScaCas9-Sc++
31
0


 


380
CGG
TGGCTGGCCTGCTTTCCTCT
19531
SpyCas9
31
0


 


381
CGG
TGGCTGGCCTGCTTTCCTCT
19532
SpyCas9-HF1
31
0


 


382
CGG
TGGCTGGCCTGCTTTCCTCT
19533
SpyCas9-SpG
31
0


 


383
CGG
TGGCTGGCCTGCTTTCCTCT
19534
SpyCas9-SpRY
31
0


 


384
CG
TGGCTGGCCTGCTTTCCTCT
19535
SpyCas9-NG
31
0


 


385
CG
TGGCTGGCCTGCTTTCCTCT
19536
SpyCas9-xCas
31
0


 


386
CG
TGGCTGGCCTGCTTTCCTCT
19537
SpyCas9-xCas-
31
0






NG




 


387
TCT
TACATGGGCTTGGATCCATG
19538
SpyCas9-SpRY
31
0


 


388
TCGGG
ccTGTGGCTGGCCTGCTTTCCTC
19539
SauCas9
32
0


 


389
TCGGG
TGTGGCTGGCCTGCTTTCCTC
19540
SauCas9KKH
32
0


 


390
TCGG
TGTGGCTGGCCTGCTTTCCTC
19541
SauriCas9
32
0


 


391
TCGG
TGTGGCTGGCCTGCTTTCCTC
19542
SauriCas9-KKH
32
0


 


392
TCG
GTGGCTGGCCTGCTTTCCTC
19543
ScaCas9
32
0


 


393
TCG
GTGGCTGGCCTGCTTTCCTC
19544
ScaCas9-HiFi-
32
0






Sc++




 


394
TCG
GTGGCTGGCCTGCTTTCCTC
19545
ScaCas9-Sc++
32
0


 


395
TCG
GTGGCTGGCCTGCTTTCCTC
19546
SpyCas9-SpRY
32
0


 


396
GTC
ATACATGGGCTTGGATCCAT
19547
SpyCas9-SpRY
32
0


 


397
TCGGGA
TGTGGCTGGCCTGCTTTCCTC
19548
cCas9-v17
32
0


 


398
TCGGGA
TGTGGCTGGCCTGCTTTCCTC
19549
cCas9-v42
32
0


 


399
TCGGGAT
acctGTGGCTGGCCTGCTTTCCTC
19550
NmeCas9
32
0



T







 


400
CTCGG
CTGTGGCTGGCCTGCTTTCCT
19551
SauCas9KKH
33
0


 


401
TG
TATACATGGGCTTGGATCCA
19552
Spy Cas9-NG
33
0


 


402
TG
TATACATGGGCTTGGATCCA
19553
SpyCas9-xCas
33
0


 


403
TG
TATACATGGGCTTGGATCCA
19554
SpyCas9-xCas-
33
0






NG




 


404
TGT
TATACATGGGCTTGGATCCA
19555
SpyCas9-SpG
33
0


 


405
TGT
TATACATGGGCTTGGATCCA
19556
SpyCas9-SpRY
33
0


 


406
CTC
TGTGGCTGGCCTGCTTTCCT
19557
SpyCas9-SpRY
33
0


 


407
CTCGGG
CTGTGGCTGGCCTGCTTTCCT
19558
cCas9-v17
33
0


 


408
CTCGGG
CTGTGGCTGGCCTGCTTTCCT
19559
cCas9-v42
33
0


 


409
TGTC
TATACATGGGCTTGGATCCA
19560
SpyCas9-3var-
33
0






NRTH




 


410
ATG
GTATACATGGGCTTGGATCC
19561
ScaCas9
34
0


 


411
ATG
GTATACATGGGCTTGGATCC
19562
ScaCas9-HiFi-
34
0






Sc++




 


412
ATG
GTATACATGGGCTTGGATCC
19563
ScaCas9-Sc++
34
0


 


413
ATG
GTATACATGGGCTTGGATCC
19564
SpyCas9-SpRY
34
0


 


414
TCT
CTGTGGCTGGCCTGCTTTCC
19565
SpyCas9-SpRY
34
0


 


415
ATGTCTG
ggggTATACATGGGCTTGGATCC
19566
BlatCas9
34
0



A







 


416
ATGTC
ggggTATACATGGGCTTGGATCC
19567
BlatCas9
34
0


 


417
CAT
GGTATACATGGGCTTGGATC
19568
SpyCas9-SpRY
35
0


 


418
CTC
CCTGTGGCTGGCCTGCTTTC
19569
SpyCas9-SpRY
35
0


 


419
CTCTCGG
cgacCTGTGGCTGGCCTGCTTTC
19570
BlatCas9
35
0



G







 


420
CTCTC
cgacCTGTGGCTGGCCTGCTTTC
19571
BlatCas9
35
0


 


421
CCA
GGGTATACATGGGCTTGGAT
19572
SpyCas9-SpRY
36
0)


 


422
CCT
ACCTGTGGCTGGCCTGCTTT
19573
SpyCas9-SpRY
36
0


 


423
CCATGTCT
tcggGGGTATACATGGGCTTGGA
19574
NmeCas9
36
0




T






 


424
TCC
GGGGTATACATGGGCTTGGA
19575
SpyCas9-SpRY
37
0


 


425
TCC
GACCTGTGGCTGGCCTGCTT
19576
SpyCas9-SpRY
37
0


 


426
TCCTC
tccgACCTGTGGCTGGCCTGCTT
19577
BlatCas9
37
0


 


427
ATC
GGGGGTATACATGGGCTTGG
19578
SpyCas9-SpRY
38
0


 


428
TTC
CGACCTGTGGCTGGCCTGCT
19579
SpyCas9-SpRY
38
0


 


429
GAT
CGGGGGTATACATGGGCTTG
19580
SpyCas9-SpRY
39
0


 


430
GAT
CGGGGGTATACATGGGCTTG
19581
SpyCas9-xCas
39
0


 


431
TTT
CCGACCTGTGGCTGGCCTGC
19582
Spy Cas9-SpRY
39
0


 


432
GATCCAT
gttcGGGGGTATACATGGGCTTG
19583
BlatCas9
39
0



G







 


433
GATCC
gttcGGGGGTATACATGGGCTTG
19584
BlatCas9
39
0


 


434
TTTCC
cctcCGACCTGTGGCTGGCCTGC
19585
BlatCas9
39
0


 


435
GATC
CGGGGGTATACATGGGCTTG
19586
SpyCas9-3var-
39
0






NRTH




 


436
GGATCC
cgGTTCGGGGGTATACATGGGC
19587
Nme2Cas9
40
0




TT






 


437
CTTTCC
cgCCTCCGACCTGTGGCTGGCC
19588
Nme2Cas9
40
0




TG






 


438
GG
TCGGGGGTATACATGGGCTT
19589
SpyCas9-NG
40
0


 


439
GG
TCGGGGGTATACATGGGCTT
19590
SpyCas9-xCas
40
0


 


440
GG
TCGGGGGTATACATGGGCTT
19591
SpyCas9-xCas-
40
0






NG




 


441
GGA
TCGGGGGTATACATGGGCTT
19592
SpyCas9-SpG
40
0


 


442
GGA
TCGGGGGTATACATGGGCTT
19593
SpyCas9-SpRY
40
0


 


443
CTT
TCCGACCTGTGGCTGGCCTG
19594
SpyCas9-SpRY
40
0


 


444
GGATCCA
ggttCGGGGGTATACATGGGCTT
19595
BlatCas9
40
0



T







 


445
GGATC
ggttCGGGGGTATACATGGGCTT
19596
BlatCas9
40
0


 


446
CTTTC
gcctCCGACCTGTGGCTGGCCTG
19597
BlatCas9
40
0


 


447
GGAT
TCGGGGGTATACATGGGCTT
19598
SpyCas9-3var-
40
0






NRRH




 


448
GGAT
TCGGGGGTATACATGGGCTT
19599
SpyCas9-VQR
40
0


 


449
TGG
TTCGGGGGTATACATGGGCT
19600
ScaCas9
41
0


 


450
TGG
TTCGGGGGTATACATGGGCT
19601
ScaCas9-HiFi-
41
0






Sc++




 


451
TGG
TTCGGGGGTATACATGGGCT
19602
ScaCas9-Sc++
41
0


 


452
TGG
TTCGGGGGTATACATGGGCT
19603
SpyCas9
41
0


 


453
TGG
TTCGGGGGTATACATGGGCT
19604
SpyCas9-HF1
41
0


 


454
TGG
TTCGGGGGTATACATGGGCT
19605
SpyCas9-SpG
41
0


 


455
TGG
TTCGGGGGTATACATGGGCT
19606
SpyCas9-SpRY
41
0


 


456
TG
TTCGGGGGTATACATGGGCT
19607
SpyCas9-NG
41
0


 


457
TG
TTCGGGGGTATACATGGGCT
19608
SpyCas9-xCas
41
0


 


458
TG
TTCGGGGGTATACATGGGCT
19609
SpyCas9-xCas-
41
0






NG




 


459
GCT
CTCCGACCTGTGGCTGGCCT
19610
SpyCas9-SpRY
41
0


 


460
TGGATCC
GGTTCGGGGGTATACATGGGC
19611
CdiCas9
41
0




T






 


461
TGGA
TTCGGGGGTATACATGGGCT
19612
SpyCas9-3var-
41
0






NRRH




 


462
TTGGA
acGGTTCGGGGGTATACATGGG
19613
SauCas9
42
0




C






 


463
TTGGA
GGTTCGGGGGTATACATGGGC
19614
SauCas9KKH
42
0


 


464
TTGGAT
acGGTTCGGGGGTATACATGGG
19615
SauCas9
42
0




C






 


465
TTGGAT
GGTTCGGGGGTATACATGGGC
19616
SauCas9KKH
42
0


 


466
TTGGAT
GGTTCGGGGGTATACATGGGC
19617
cCas9-v17
42
0


 


467
TTGGAT
GGTTCGGGGGTATACATGGGC
19618
cCas9-v42
42
0


 


468
TTGG
GGTTCGGGGGTATACATGGGC
19619
SauriCas9
42
0


 


469
TTGG
GGTTCGGGGGTATACATGGGC
19620
SauriCas9-KKH
42
0


 


470
TTG
GTTCGGGGGTATACATGGGC
19621
ScaCas9
42
0


 


471
TTG
GTTCGGGGGTATACATGGGC
19622
ScaCas9-HiFi-
42
0






Sc++




 


472
TTG
GTTCGGGGGTATACATGGGC
19623
ScaCas9-Sc++
42
0


 


473
TTG
GTTCGGGGGTATACATGGGC
19624
SpyCas9-SpRY
42
0


 


474
TG
CCTCCGACCTGTGGCTGGCC
19625
SpyCas9-NG
42
0


 


475
TG
CCTCCGACCTGTGGCTGGCC
19626
SpyCas9-xCas
42
0


 


476
TG
CCTCCGACCTGTGGCTGGCC
19627
SpyCas9-xCas-
42
0






NG




 


477
TGC
CCTCCGACCTGTGGCTGGCC
19628
SpyCas9-SpG
42
0


 


478
TGC
CCTCCGACCTGTGGCTGGCC
19629
SpyCas9-SpRY
42
0


 


479
TGCT
CCTCCGACCTGTGGCTGGCC
19630
SpyCas9-3var-
42
0






NRCH




 


480
CTTGG
CGGTTCGGGGGTATACATGGG
19631
SauCas9KKH
43
0


 


481
CTG
GCCTCCGACCTGTGGCTGGC
19632
ScaCas9
43
0


 


482
CTG
GCCTCCGACCTGTGGCTGGC
19633
ScaCas9-HiFi-
43
0






Sc++




 


483
CTG
GCCTCCGACCTGTGGCTGGC
19634
ScaCas9-Sc++
43
0


 


484
CTG
GCCTCCGACCTGTGGCTGGC
19635
SpyCas9-SpRY
43
0


 


485
CTT
GGTTCGGGGGTATACATGGG
19636
SpyCas9-SpRY
43
0


 


486
CTGCTTT
CCGCCTCCGACCTGTGGCTGGC
19637
CdiCas9
43
0


 


487
GCT
CGGTTCGGGGGTATACATGG
19638
SpyCas9-SpRY
44
0


 


488
CCT
CGCCTCCGACCTGTGGCTGG
19639
SpyCas9-SpRY
44
0


 


489
CCTGCTTT
ttccGCCTCCGACCTGTGGCTGG
19640
BlatCas9
44
0


 


490
CCTGC
ttccGCCTCCGACCTGTGGCTGG
19641
BlatCas9
44
0


 


491
GG
ACGGTTCGGGGGTATACATG
19642
SpyCas9-NG
45
0


 


492
GG
ACGGTTCGGGGGTATACATG
19643
SpyCas9-xCas
45
0


 


493
GG
ACGGTTCGGGGGTATACATG
19644
SpyCas9-xCas-
45
0






NG




 


494
GGC
ACGGTTCGGGGGTATACATG
19645
SpyCas9-SpG
45
0


 


495
GGC
ACGGTTCGGGGGTATACATG
19646
SpyCas9-SpRY
45
0


 


496
GCC
CCGCCTCCGACCTGTGGCTG
19647
SpyCas9-SpRY
45
0


 


497
GCCTGCTT
gtttCCGCCTCCGACCTGTGGCTG
19648
NmeCas9
45
0


 


498
GGCT
ACGGTTCGGGGGTATACATG
19649
SpyCas9-3var-
45
0






NRCH




 


499
GGG
CACGGTTCGGGGGTATACAT
19650
ScaCas9
46
0


 


500
GGG
CACGGTTCGGGGGTATACAT
19651
ScaCas9-HiFi-
46
0






Sc++




 


501
GGG
CACGGTTCGGGGGTATACAT
19652
ScaCas9-Sc++
46
0


 


502
GGG
CACGGTTCGGGGGTATACAT
19653
SpyCas9
46
0


 


503
GGG
CACGGTTCGGGGGTATACAT
19654
SpyCas9-HF1
46
0


 


504
GGG
CACGGTTCGGGGGTATACAT
19655
SpyCas9-SpG
46
0


 


505
GGG
CACGGTTCGGGGGTATACAT
19656
SpyCas9-SpRY
46
0


 


506
GG
CACGGTTCGGGGGTATACAT
19657
SpyCas9-NG
46
0


 


507
GG
CACGGTTCGGGGGTATACAT
19658
SpyCas9-xCas
46
0


 


508
GG
CACGGTTCGGGGGTATACAT
19659
SpyCas9-xCas-
46
0






NG




 


509
GG
TCCGCCTCCGACCTGTGGCT
19660
SpyCas9-NG
46
0


 


510
GG
TCCGCCTCCGACCTGTGGCT
19661
SpyCas9-xCas
46
0


 


511
GG
TCCGCCTCCGACCTGTGGCT
19662
SpyCas9-xCas-
46
0






NG




 


512
GGC
TCCGCCTCCGACCTGTGGCT
19663
SpyCas9-SpG
46
0


 


513
GGC
TCCGCCTCCGACCTGTGGCT
19664
SpyCas9-SpRY
46
0


 


514
GGGC
CACGGTTCGGGGGTATACAT
19665
SpyCas9-3var-
46
0






NRRH




 


515
GGCC
TCCGCCTCCGACCTGTGGCT
19666
SpyCas9-3var-
46
0






NRCH




 


516
TGGG
CTCACGGTTCGGGGGTATACA
19667
SauriCas9
47
0


 


517
TGGG
CTCACGGTTCGGGGGTATACA
19668
SauriCas9-KKH
47
0


 


518
TGG
TCACGGTTCGGGGGTATACA
19669
ScaCas9
47
0


 


519
TGG
TCACGGTTCGGGGGTATACA
19670
ScaCas9-HiFi-
47
0






Sc++




 


520
TGG
TCACGGTTCGGGGGTATACA
19671
ScaCas9-Sc++
47
0


 


521
TGG
TCACGGTTCGGGGGTATACA
19672
SpyCas9
47
0


 


522
TGG
TCACGGTTCGGGGGTATACA
19673
SpyCas9-HF1
47
0


 


523
TGG
TCACGGTTCGGGGGTATACA
19674
SpyCas9-SpG
47
0


 


524
TGG
TCACGGTTCGGGGGTATACA
19675
SpyCas9-SpRY
47
0


 


525
TGG
TTCCGCCTCCGACCTGTGGC
19676
ScaCas9
47
0


 


526
TGG
TTCCGCCTCCGACCTGTGGC
19677
ScaCas9-HiFi-
47
0






Sc++




 


527
TGG
TTCCGCCTCCGACCTGTGGC
19678
ScaCas9-Sc++
47
0


 


528
TGG
TTCCGCCTCCGACCTGTGGC
19679
SpyCas9
47
0


 


529
TGG
TTCCGCCTCCGACCTGTGGC
19680
SpyCas9-HF1
47
0


 


530
TGG
TTCCGCCTCCGACCTGTGGC
19681
SpyCas9-SpG
47
0


 


531
TGG
TTCCGCCTCCGACCTGTGGC
19682
SpyCas9-SpRY
47
0


 


532
TG
TCACGGTTCGGGGGTATACA
19683
SpyCas9-NG
47
0


 


533
TG
TCACGGTTCGGGGGTATACA
19684
SpyCas9-xCas
47
0


 


534
TG
TCACGGTTCGGGGGTATACA
19685
SpyCas9-xCas-
47
0






NG




 


535
TG
TTCCGCCTCCGACCTGTGGC
19686
SpyCas9-NG
47
0


 


536
TG
TTCCGCCTCCGACCTGTGGC
19687
SpyCas9-xCas
47
0


 


537
TG
TTCCGCCTCCGACCTGTGGC
19688
SpyCas9-xCas-
47
0






NG




 


538
TGGGCTT
tactCACGGTTCGGGGGTATACA
19689
BlatCas9
47
0



G







 


539
TGGGC
tactCACGGTTCGGGGGTATACA
19690
BlatCas9
47
0


 


540
TGGCC
ggttTCCGCCTCCGACCTGTGGC
19691
BlatCas9
47
0


 


541
TGGGCT
CTCACGGTTCGGGGGTATACA
19692
cCas9-v16
47
0


 


542
TGGGCT
CTCACGGTTCGGGGGTATACA
19693
cCas9-v21
47
0


 


543
TGGC
TTCCGCCTCCGACCTGTGGC
19694
SpyCas9-3var-
47
0






NRRH




 


544
CTGGCC
ctGGTTTCCGCCTCCGACCTGTG
19695
Nme2Cas9
48
0




G






 


545
ATGGG
gtACTCACGGTTCGGGGGTATA
19696
SauCas9
48
0




C






 


546
ATGGG
ACTCACGGTTCGGGGGTATAC
19697
SauCas9KKH
48
0


 


547
ATGG
ACTCACGGTTCGGGGGTATAC
19698
SauriCas9
48
0


 


548
ATGG
ACTCACGGTTCGGGGGTATAC
19699
SauriCas9-KKH
48
0


 


549
CTGG
GTTTCCGCCTCCGACCTGTGG
19700
SauriCas9
48
0


 


550
CTGG
GTTTCCGCCTCCGACCTGTGG
19701
SauriCas9-KKH
48
0


 


551
ATG
CTCACGGTTCGGGGGTATAC
19702
ScaCas9
48
0


 


552
ATG
CTCACGGTTCGGGGGTATAC
19703
ScaCas9-HiFi-
48
0






Sc++




 


553
ATG
CTCACGGTTCGGGGGTATAC
19704
ScaCas9-Sc++
48
0


 


554
ATG
CTCACGGTTCGGGGGTATAC
19705
SpyCas9-SpRY
48
0


 


555
CTG
TTTCCGCCTCCGACCTGTGG
19706
ScaCas9
48
0


 


556
CTG
TTTCCGCCTCCGACCTGTGG
19707
ScaCas9-HiFi-
48
0






Sc++




 


557
CTG
TTTCCGCCTCCGACCTGTGG
19708
ScaCas9-Sc++
48
0


 


558
CTG
TTTCCGCCTCCGACCTGTGG
19709
SpyCas9-SpRY
48
0


 


559
CTGGCCT
tggtTTCCGCCTCCGACCTGTGG
19710
BlatCas9
48
0



G







 


560
CTGGC
tggtTTCCGCCTCCGACCTGTGG
19711
BlatCas9
48
0


 


561
ATGGGC
ACTCACGGTTCGGGGGTATAC
19712
cCas9-v17
48
0


 


562
ATGGGC
ACTCACGGTTCGGGGGTATAC
19713
cCas9-v42
48
0


 


563
ATGGGCT
agtaCTCACGGTTCGGGGGTATA
19714
NmeCas9
48
0



T
C






 


564
CATGG
TACTCACGGTTCGGGGGTATA
19715
SauCas9KKH
49
0


 


565
GCTGG
GGTTTCCGCCTCCGACCTGTG
19716
SauCas9KKH
49
0


 


566
CAT
ACTCACGGTTCGGGGGTATA
19717
SpyCas9-SpRY
49
0


 


567
GCT
GTTTCCGCCTCCGACCTGTG
19718
SpyCas9-SpRY
49
0


 


568
GG
GGTTTCCGCCTCCGACCTGT
19719
SpyCas9-NG
50
0


 


569
GG
GGTTTCCGCCTCCGACCTGT
19720
SpyCas9-xCas
50
0


 


570
GG
GGTTTCCGCCTCCGACCTGT
19721
SpyCas9-xCas-
50
0






NG




 


571
GGC
GGTTTCCGCCTCCGACCTGT
19722
SpyCas9-SpG
50
0


 


572
GGC
GGTTTCCGCCTCCGACCTGT
19723
SpyCas9-SpRY
50
0


 


573
ACA
TACTCACGGTTCGGGGGTAT
19724
Spy Cas9-SpRY
50
0


 


574
GGCT
GGTTTCCGCCTCCGACCTGT
19725
SpyCas9-3var-
50
0






NRCH




 


575
TGG
TGGTTTCCGCCTCCGACCTG
19726
ScaCas9
51
0


 


576
TGG
TGGTTTCCGCCTCCGACCTG
19727
ScaCas9-HiFi-
51
0






Sc++




 


577
TGG
TGGTTTCCGCCTCCGACCTG
19728
ScaCas9-Sc++
51
0


 


578
TGG
TGGTTTCCGCCTCCGACCTG
19729
SpyCas9
51
0


 


579
TGG
TGGTTTCCGCCTCCGACCTG
19730
SpyCas9-HF1
51
0


 


580
TGG
TGGTTTCCGCCTCCGACCTG
19731
SpyCas9-SpG
51
0


 


581
TGG
TGGTTTCCGCCTCCGACCTG
19732
SpyCas9-SpRY
51
0


 


582
TG
TGGTTTCCGCCTCCGACCTG
19733
SpyCas9-NG
51
0


 


583
TG
TGGTTTCCGCCTCCGACCTG
19734
SpyCas9-xCas
51
0


 


584
TG
TGGTTTCCGCCTCCGACCTG
19735
SpyCas9-xCas-
51
0






NG




 


585
TAC
GTACTCACGGTTCGGGGGTA
19736
SpyCas9-SpRY
51
0


 


586
TGGC
TGGTTTCCGCCTCCGACCTG
19737
SpyCas9-3var-
51
0






NRRH




 


587
TACA
GTACTCACGGTTCGGGGGTA
19738
SpyCas9-3var-
51
0






NRCH




 


588
GTGG
ACTGGTTTCCGCCTCCGACCT
19739
SauriCas9
52
0


 


589
GTGG
ACTGGTTTCCGCCTCCGACCT
19740
SauriCas9-KKH
52
0


 


590
GTG
CTGGTTTCCGCCTCCGACCT
19741
ScaCas9
52
0


 


591
GTG
CTGGTTTCCGCCTCCGACCT
19742
ScaCas9-HiFi-
52
0






Sc++




 


592
GTG
CTGGTTTCCGCCTCCGACCT
19743
ScaCas9-Sc++
52
0


 


593
GTG
CTGGTTTCCGCCTCCGACCT
19744
SpyCas9-SpRY
52
0


 


594
ATA
AGTACTCACGGTTCGGGGGT
19745
SpyCas9-SpRY
52
0


 


595
GTGGCTG
gcacTGGTTTCCGCCTCCGACCT
19746
BlatCas9
52
0



G







 


596
GTGGC
gcacTGGTTTCCGCCTCCGACCT
19747
BlatCas9
52
0


 


597
GTGGCT
ACTGGTTTCCGCCTCCGACCT
19748
cCas9-v16
52
0


 


598
GTGGCT
ACTGGTTTCCGCCTCCGACCT
19749
cCas9-v21
52
0


 


599
TGTGG
CACTGGTTTCCGCCTCCGACC
19750
SauCas9KKH
53
0


 


600
TG
ACTGGTTTCCGCCTCCGACC
19751
SpyCas9-NG
53
0


 


601
TG
ACTGGTTTCCGCCTCCGACC
19752
SpyCas9-xCas
53
0


 


602
TG
ACTGGTTTCCGCCTCCGACC
19753
SpyCas9-xCas-
53
0






NG




 


603
TAT
CAGTACTCACGGTTCGGGGG
19754
SpyCas9-SpRY
53
0


 


604
TGT
ACTGGTTTCCGCCTCCGACC
19755
SpyCas9-SpG
53
0


 


605
TGT
ACTGGTTTCCGCCTCCGACC
19756
SpyCas9-SpRY
53
0


 


606
TATACAT
ggacAGTACTCACGGTTCGGGGG
19757
BlatCas9
53
0



G







 


607
TATAC
ggacAGTACTCACGGTTCGGGGG
19758
BlatCas9
53
0


 


608
TATA
CAGTACTCACGGTTCGGGGG
19759
SpyCas9-3var-
53
0






NRTH




 


609
CTG
CACTGGTTTCCGCCTCCGAC
19760
ScaCas9
54
0


 


610
CTG
CACTGGTTTCCGCCTCCGAC
19761
ScaCas9-HiFi-
54
0






Sc++




 


611
CTG
CACTGGTTTCCGCCTCCGAC
19762
ScaCas9-Sc++
54
0


 


612
CTG
CACTGGTTTCCGCCTCCGAC
19763
SpyCas9-SpRY
54
0


 


613
GTA
ACAGTACTCACGGTTCGGGG
19764
SpyCas9-SpRY
54
0


 


614
GG
GACAGTACTCACGGTTCGGG
19765
SpyCas9-NG
55
0


 


615
GG
GACAGTACTCACGGTTCGGG
19766
SpyCas9-xCas
55
0


 


616
GG
GACAGTACTCACGGTTCGGG
19767
SpyCas9-xCas-
55
0






NG




 


617
GGT
GACAGTACTCACGGTTCGGG
19768
SpyCas9-SpG
55
0


 


618
GGT
GACAGTACTCACGGTTCGGG
19769
SpyCas9-SpRY
55
0


 


619
CCT
GCACTGGTTTCCGCCTCCGA
19770
SpyCas9-SpRY
55
0


 


620
GGTA
GACAGTACTCACGGTTCGGG
19771
SpyCas9-3var-
55
0






NRTH




 


621
GGG
GGACAGTACTCACGGTTCGG
19772
ScaCas9
56
0


 


622
GGG
GGACAGTACTCACGGTTCGG
19773
ScaCas9-HiFi-
56
0






Sc++




 


623
GGG
GGACAGTACTCACGGTTCGG
19774
ScaCas9-Sc++
56
0


 


624
GGG
GGACAGTACTCACGGTTCGG
19775
SpyCas9
56
0


 


625
GGG
GGACAGTACTCACGGTTCGG
19776
SpyCas9-HF1
56
0


 


626
GGG
GGACAGTACTCACGGTTCGG
19777
SpyCas9-SpG
56
0


 


627
GGG
GGACAGTACTCACGGTTCGG
19778
SpyCas9-SpRY
56
0


 


628
GG
GGACAGTACTCACGGTTCGG
19779
SpyCas9-NG
56
0


 


629
GG
GGACAGTACTCACGGTTCGG
19780
SpyCas9-xCas
56
0


 


630
GG
GGACAGTACTCACGGTTCGG
19781
SpyCas9-xCas-
56
0






NG




 


631
ACC
TGCACTGGTTTCCGCCTCCG
19782
SpyCas9-SpRY
56
0


 


632
GGGTATA
ggAGGACAGTACTCACGGTTCG
19783
CjeCas9
56
0



C
G






 


633
GGGT
GGACAGTACTCACGGTTCGG
19784
SpyCas9-3var-
56
0






NRRH




 


634
GGGG
GAGGACAGTACTCACGGTTCG
19785
SauriCas9
57
0


 


635
GGGG
GAGGACAGTACTCACGGTTCG
19786
SauriCas9-KKH
57
0


 


636
GGG
AGGACAGTACTCACGGTTCG
19787
ScaCas9
57
0


 


637
GGG
AGGACAGTACTCACGGTTCG
19788
ScaCas9-HiFi-
57
0






Sc++




 


638
GGG
AGGACAGTACTCACGGTTCG
19789
ScaCas9-Sc++
57
0


 


639
GGG
AGGACAGTACTCACGGTTCG
19790
SpyCas9
57
0


 


640
GGG
AGGACAGTACTCACGGTTCG
19791
SpyCas9-HF1
57
0


 


641
GGG
AGGACAGTACTCACGGTTCG
19792
SpyCas9-SpG
57
0


 


642
GGG
AGGACAGTACTCACGGTTCG
19793
SpyCas9-SpRY
57
0


 


643
GG
AGGACAGTACTCACGGTTCG
19794
SpyCas9-NG
57
0


 


644
GG
AGGACAGTACTCACGGTTCG
19795
SpyCas9-xCas
57
0


 


645
GG
AGGACAGTACTCACGGTTCG
19796
SpyCas9-xCas-
57
0






NG




 


646
GAC
TTGCACTGGTTTCCGCCTCC
19797
SpyCas9-SpRY
57
0


 


647
GACC
TTGCACTGGTTTCCGCCTCC
19798
SpyCas9-3var-
57
0






NRCH




 


648
GGGGG
ctGGAGGACAGTACTCACGGTT
19799
SauCas9
58
0




C






 


649
GGGGG
GGAGGACAGTACTCACGGTTC
19800
SauCas9KKH
58
0


 


650
GGGGGT
ctGGAGGACAGTACTCACGGTT
19801
SauCas9
58
0




C






 


651
GGGGGT
GGAGGACAGTACTCACGGTTC
19802
SauCas9KKH
58
0


 


652
GGGGGT
GGAGGACAGTACTCACGGTTC
19803
cCas9-v17
58
0


 


653
GGGGGT
GGAGGACAGTACTCACGGTTC
19804
cCas9-v42
58
0


 


654
GGGG
GGAGGACAGTACTCACGGTTC
19805
SauriCas9
58
0


 


655
GGGG
GGAGGACAGTACTCACGGTTC
19806
SauriCas9-KKH
58
0


 


656
GGG
GAGGACAGTACTCACGGTTC
19807
ScaCas9
58
0


 


657
GGG
GAGGACAGTACTCACGGTTC
19808
ScaCas9-HiFi-
58
0






Sc++




 


658
GGG
GAGGACAGTACTCACGGTTC
19809
ScaCas9-Sc++
58
0


 


659
GGG
GAGGACAGTACTCACGGTTC
19810
SpyCas9
58
0


 


660
GGG
GAGGACAGTACTCACGGTTC
19811
SpyCas9-HF1
58
0


 


661
GGG
GAGGACAGTACTCACGGTTC
19812
SpyCas9-SpG
58
0


 


662
GGG
GAGGACAGTACTCACGGTTC
19813
SpyCas9-SpRY
58
0


 


663
GG
GAGGACAGTACTCACGGTTC
19814
SpyCas9-NG
58
0


 


664
GG
GAGGACAGTACTCACGGTTC
19815
SpyCas9-xCas
58
0


 


665
GG
GAGGACAGTACTCACGGTTC
19816
SpyCas9-xCas-
58
0






NG




 


666
CG
CTTGCACTGGTTTCCGCCTC
19817
SpyCas9-NG
58
0


 


667
CG
CTTGCACTGGTTTCCGCCTC
19818
SpyCas9-xCas
58
0


 


668
CG
CTTGCACTGGTTTCCGCCTC
19819
SpyCas9-xCas-
58
0






NG




 


669
CGA
CTTGCACTGGTTTCCGCCTC
19820
SpyCas9-SpG
58
0


 


670
CGA
CTTGCACTGGTTTCCGCCTC
19821
SpyCas9-SpRY
58
0


 


671
CGACCTG
cagcTTGCACTGGTTTCCGCCTC
19822
BlatCas9
58
0



T







 


672
CGACC
cagcTTGCACTGGTTTCCGCCTC
19823
BlatCas9
58
0


 


673
CGAC
CTTGCACTGGTTTCCGCCTC
19824
SpyCas9-3var-
58
0






NRRH




 


674
CGAC
CTTGCACTGGTTTCCGCCTC
19825
SpyCas9-VQR
58
0


 


675
CCGACC
ccCAGCTTGCACTGGTTTCCGCC
19826
Nme2Cas9
59
0




T






 


676
CGGGG
gcTGGAGGACAGTACTCACGGT
19827
SauCas9
59
0




T






 


677
CGGGG
TGGAGGACAGTACTCACGGTT
19828
SauCas9KKH
59
0


 


678
CGGG
TGGAGGACAGTACTCACGGTT
19829
SauriCas9
59
0


 


679
CGGG
TGGAGGACAGTACTCACGGTT
19830
SauriCas9-KKH
59
0


 


680
CGG
GGAGGACAGTACTCACGGTT
19831
ScaCas9
59
0


 


681
CGG
GGAGGACAGTACTCACGGTT
19832
ScaCas9-HiFi-
59
0






Sc++




 


682
CGG
GGAGGACAGTACTCACGGTT
19833
ScaCas9-Sc++
59
0


 


683
CGG
GGAGGACAGTACTCACGGTT
19834
SpyCas9
59
0


 


684
CGG
GGAGGACAGTACTCACGGTT
19835
SpyCas9-HF1
59
0


 


685
CGG
GGAGGACAGTACTCACGGTT
19836
SpyCas9-SpG
59
0


 


686
CGG
GGAGGACAGTACTCACGGTT
19837
SpyCas9-SpRY
59
0


 


687
CCG
GCTTGCACTGGTTTCCGCCT
19838
ScaCas9
59
0


 


688
CCG
GCTTGCACTGGTTTCCGCCT
19839
ScaCas9-HiFi-
59
0






Sc++




 


689
CCG
GCTTGCACTGGTTTCCGCCT
19840
ScaCas9-Sc++
59
0


 


690
CCG
GCTTGCACTGGTTTCCGCCT
19841
SpyCas9-SpRY
59
0


 


691
CG
GGAGGACAGTACTCACGGTT
19842
SpyCas9-NG
59
0


 


692
CG
GGAGGACAGTACTCACGGTT
19843
SpyCas9-xCas
59
0


 


693
CG
GGAGGACAGTACTCACGGTT
19844
SpyCas9-xCas-
59
0






NG




 


694
CCGACCT
ccagCTTGCACTGGTTTCCGCCT
19845
BlatCas9
59
0



G







 


695
CCGAC
ccagCTTGCACTGGTTTCCGCCT
19846
BlatCas9
59
0


 


696
CGGGGG
TGGAGGACAGTACTCACGGTT
19847
cCas9-v17
59
0


 


697
CGGGGG
TGGAGGACAGTACTCACGGTT
19848
cCas9-v42
59
0


 


698
CCGACCT
CAGCTTGCACTGGTTTCCGCCT
19849
CdiCas9
59
0


 


699
TCGGG
agCTGGAGGACAGTACTCACGG
19850
SauCas9
60
0




T






 


700
TCGGG
CTGGAGGACAGTACTCACGGT
19851
SauCas9KKH
60
0


 


701
TCCGA
CAGCTTGCACTGGTTTCCGCC
19852
SauCas9KKH
60
0


 


702
TCGG
CTGGAGGACAGTACTCACGGT
19853
SauriCas9
60
0


 


703
TCGG
CTGGAGGACAGTACTCACGGT
19854
SauriCas9-KKH
60
0


 


704
TCG
TGGAGGACAGTACTCACGGT
19855
ScaCas9
60
0


 


705
TCG
TGGAGGACAGTACTCACGGT
19856
ScaCas9-HiFi-
60
0






Sc++




 


706
TCG
TGGAGGACAGTACTCACGGT
19857
ScaCas9-Sc++
60
0


 


707
TCG
TGGAGGACAGTACTCACGGT
19858
SpyCas9-SpRY
60
0


 


708
TCC
AGCTTGCACTGGTTTCCGCC
19859
SpyCas9-SpRY
60
0


 


709
TCGGGG
CTGGAGGACAGTACTCACGGT
19860
cCas9-v17
60
0


 


710
TCGGGG
CTGGAGGACAGTACTCACGGT
19861
cCas9-v42
60
0


 


711
TCCGAC
CAGCTTGCACTGGTTTCCGCC
19862
cCas9-v17
60
0


 


712
TCCGAC
CAGCTTGCACTGGTTTCCGCC
19863
cCas9-v42
60
0


 


713
TTCGG
GCTGGAGGACAGTACTCACGG
19864
SauCas9KKH
61
0


 


714
TTC
CTGGAGGACAGTACTCACGG
19865
SpyCas9-SpRY
61
0


 


715
CTC
CAGCTTGCACTGGTTTCCGC
19866
SpyCas9-SpRY
61
0


 


716
TTCGGG
GCTGGAGGACAGTACTCACGG
19867
cCas9-v17
61
0


 


717
TTCGGG
GCTGGAGGACAGTACTCACGG
19868
cCas9-v42
61
0


 


718
GTT
GCTGGAGGACAGTACTCACG
19869
SpyCas9-SpRY
62
0


 


719
CCT
CCAGCTTGCACTGGTTTCCG
19870
SpyCas9-SpRY
62
0


 


720
CCTCC
atccCAGCTTGCACTGGTTTCCG
19871
BlatCas9
62
0


 


721
GCCTCC
tcATCCCAGCTTGCACTGGTTTC
19872
Nme2Cas9
63
0




C






 


722
GG
AGCTGGAGGACAGTACTCAC
19873
SpyCas9-NG
63
0


 


723
GG
AGCTGGAGGACAGTACTCAC
19874
SpyCas9-xCas
63
0


 


724
GG
AGCTGGAGGACAGTACTCAC
19875
SpyCas9-xCas-
63
0






NG




 


725
GGT
AGCTGGAGGACAGTACTCAC
19876
SpyCas9-SpG
63
0


 


726
GGT
AGCTGGAGGACAGTACTCAC
19877
SpyCas9-SpRY
63
0


 


727
GCC
CCCAGCTTGCACTGGTTTCC
19878
SpyCas9-SpRY
63
0


 


728
GGTTCGG
ggtaGCTGGAGGACAGTACTCAC
19879
BlatCas9
63
0



G







 


729
GCCTCCG
catcCCAGCTTGCACTGGTTTCC
19880
BlatCas9
63
0



A







 


730
GGTTC
ggtaGCTGGAGGACAGTACTCAC
19881
BlatCas9
63
0


 


731
GCCTC
catcCCAGCTTGCACTGGTTTCC
19882
BlatCas9
63
0


 


732
GGTT
AGCTGGAGGACAGTACTCAC
19883
SpyCas9-3var-
63
0






NRTH




 


733
CGG
TAGCTGGAGGACAGTACTCA
19884
ScaCas9
64
0


 


734
CGG
TAGCTGGAGGACAGTACTCA
19885
ScaCas9-HiFi-
64
0






Sc++




 


735
CGG
TAGCTGGAGGACAGTACTCA
19886
ScaCas9-Sc++
64
0


 


736
CGG
TAGCTGGAGGACAGTACTCA
19887
Spy Cas9
64
0


 


737
CGG
TAGCTGGAGGACAGTACTCA
19888
SpyCas9-HF1
64
0


 


738
CGG
TAGCTGGAGGACAGTACTCA
19889
SpyCas9-SpG
64
0


 


739
CGG
TAGCTGGAGGACAGTACTCA
19890
SpyCas9-SpRY
64
0


 


740
CG
TAGCTGGAGGACAGTACTCA
19891
SpyCas9-NG
64
0


 


741
CG
TAGCTGGAGGACAGTACTCA
19892
SpyCas9-xCas
64
0


 


742
CG
TAGCTGGAGGACAGTACTCA
19893
SpyCas9-xCas-
64
0






NG




 


743
CG
TCCCAGCTTGCACTGGTTTC
19894
SpyCas9-NG
64
0


 


744
CG
TCCCAGCTTGCACTGGTTTC
19895
SpyCas9-xCas
64
0


 


745
CG
TCCCAGCTTGCACTGGTTTC
19896
SpyCas9-xCas-
64
0






NG




 


746
CGC
TCCCAGCTTGCACTGGTTTC
19897
SpyCas9-SpG
64
0


 


747
CGC
TCCCAGCTTGCACTGGTTTC
19898
SpyCas9-SpRY
64
0


 


748
CGGT
TAGCTGGAGGACAGTACTCA
19899
SpyCas9-3var-
64
0






NRRH




 


749
CGCC
TCCCAGCTTGCACTGGTTTC
19900
SpyCas9-3var-
64
0






NRCH




 


750
ACGG
GGTAGCTGGAGGACAGTACTC
19901
SauriCas9
65
0


 


751
ACGG
GGTAGCTGGAGGACAGTACTC
19902
SauriCas9-KKH
65
0


 


752
ACG
GTAGCTGGAGGACAGTACTC
19903
ScaCas9
65
0


 


753
ACG
GTAGCTGGAGGACAGTACTC
19904
ScaCas9-HiFi-
65
0






Sc++




 


754
ACG
GTAGCTGGAGGACAGTACTC
19905
ScaCas9-Sc++
65
0


 


755
ACG
GTAGCTGGAGGACAGTACTC
19906
SpyCas9-SpRY
65
0


 


756
CCG
ATCCCAGCTTGCACTGGTTT
19907
ScaCas9
65
0


 


757
CCG
ATCCCAGCTTGCACTGGTTT
19908
ScaCas9-HiFi-
65
0






Sc++




 


758
CCG
ATCCCAGCTTGCACTGGTTT
19909
ScaCas9-Sc++
65
0


 


759
CCG
ATCCCAGCTTGCACTGGTTT
19910
SpyCas9-SpRY
65
0


 


760
CCGCC
ttcaTCCCAGCTTGCACTGGTTT
19911
BlatCas9
65
0


 


761
ACGGTT
GGTAGCTGGAGGACAGTACTC
19912
cCas9-v16
65
0


 


762
ACGGTT
GGTAGCTGGAGGACAGTACTC
19913
cCas9-v21
65
0


 


763
CCGCCTC
TCATCCCAGCTTGCACTGGTTT
19914
CdiCas9
65
0


 


764
TCCGCC
ttTTCATCCCAGCTTGCACTGGT
19915
Nme2Cas9
66
0




T






 


765
CACGGTT
aacTGGTAGCTGGAGGACAGTA
19916
PpnCas9
66
0




CT






 


766
CACGG
TGGTAGCTGGAGGACAGTACT
19917
SauCas9KKH
66
0


 


767
CACGGT
TGGTAGCTGGAGGACAGTACT
19918
SauCas9KKH
66
0


 


768
CACGGT
TGGTAGCTGGAGGACAGTACT
19919
cCas9-v17
66
0


 


769
CACGGT
TGGTAGCTGGAGGACAGTACT
19920
cCas9-v42
66
0


 


770
CAC
GGTAGCTGGAGGACAGTACT
19921
SpyCas9-SpRY
66
0


 


771
TCC
CATCCCAGCTTGCACTGGTT
19922
SpyCas9-SpRY
66
0


 


772
TCCGC
tttcATCCCAGCTTGCACTGGTT
19923
BlatCas9
66
0


 


773
TCA
TGGTAGCTGGAGGACAGTAC
19924
SpyCas9-SpRY
67
0


 


774
TTC
TCATCCCAGCTTGCACTGGT
19925
SpyCas9-SpRY
67
0


 


775
CTC
CTGGTAGCTGGAGGACAGTA
19926
SpyCas9-SpRY
68
0


 


776
TTT
TTCATCCCAGCTTGCACTGG
19927
SpyCas9-SpRY
68
0


 


777
CTCACGG
caacTGGTAGCTGGAGGACAGTA
19928
BlatCas9
68
0



T







 


778
CTCAC
caacTGGTAGCTGGAGGACAGTA
19929
BlatCas9
68
0


 


779
TTTCC
ctttTCATCCCAGCTTGCACTGG
19930
BlatCas9
68
0


 


780
GTTTCC
ttCTTTTCATCCCAGCTTGCACT
19931
Nme2Cas9
69
0




G






 


781
ACT
ACTGGTAGCTGGAGGACAGT
19932
SpyCas9-SpRY
69
0


 


782
GTT
TTTCATCCCAGCTTGCACTG
19933
SpyCas9-SpRY
69
0


 


783
GTTTC
tcttTTCATCCCAGCTTGCACTG
19934
BlatCas9
69
0


 


784
GG
TTTTCATCCCAGCTTGCACT
19935
SpyCas9-NG
70
0


 


785
GG
TTTTCATCCCAGCTTGCACT
19936
SpyCas9-xCas
70
0


 


786
GG
TTTTCATCCCAGCTTGCACT
19937
SpyCas9-xCas-
70
0






NG




 


787
TAC
AACTGGTAGCTGGAGGACAG
19938
SpyCas9-SpRY
70
0


 


788
GGT
TTTTCATCCCAGCTTGCACT
19939
SpyCas9-SpG
70
0


 


789
GGT
TTTTCATCCCAGCTTGCACT
19940
SpyCas9-SpRY
70
0


 


790
TACTC
ggcaACTGGTAGCTGGAGGACA
19941
BlatCas9
70
0




G






 


791
GGTT
TTTTCATCCCAGCTTGCACT
19942
SpyCas9-3var-
70
0






NRTH




 


792
TACT
AACTGGTAGCTGGAGGACAG
19943
SpyCas9-3var-
70
0






NRCH




 


793
TGG
CTTTTCATCCCAGCTTGCAC
19944
ScaCas9
71
0


 


794
TGG
CTTTTCATCCCAGCTTGCAC
19945
ScaCas9-HiFi-
71
0






Sc++




 


795
TGG
CTTTTCATCCCAGCTTGCAC
19946
ScaCas9-Sc++
71
0


 


796
TGG
CTTTTCATCCCAGCTTGCAC
19947
SpyCas9
71
0


 


797
TGG
CTTTTCATCCCAGCTTGCAC
19948
SpyCas9-HF1
71
0


 


798
TGG
CTTTTCATCCCAGCTTGCAC
19949
SpyCas9-SpG
71
0


 


799
TGG
CTTTTCATCCCAGCTTGCAC
19950
Spy Cas9-SpRY
71
0


 


800
TG
CTTTTCATCCCAGCTTGCAC
19951
SpyCas9-NG
71
0


 


801
TG
CTTTTCATCCCAGCTTGCAC
19952
SpyCas9-xCas
71
0


 


802
TG
CTTTTCATCCCAGCTTGCAC
19953
SpyCas9-xCas-
71
0






NG




 


803
GTA
CAACTGGTAGCTGGAGGACA
19954
SpyCas9-SpRY
71
0


 


804
TGGTTTC
TTCTTTTCATCCCAGCTTGCAC
19955
CdiCas9
71
0


 


805
TGGT
CTTTTCATCCCAGCTTGCAC
19956
SpyCas9-3var-
71
0






NRRH




 


806
CTGG
TTCTTTTCATCCCAGCTTGCA
19957
SauriCas9
72
0


 


807
CTGG
TTCTTTTCATCCCAGCTTGCA
19958
SauriCas9-KKH
72
0


 


808
CTG
TCTTTTCATCCCAGCTTGCA
19959
ScaCas9
72
0


 


809
CTG
TCTTTTCATCCCAGCTTGCA
19960
ScaCas9-HiFi-
72
0






Sc++




 


810
CTG
TCTTTTCATCCCAGCTTGCA
19961
ScaCas9-Sc++
72
0


 


811
CTG
TCTTTTCATCCCAGCTTGCA
19962
SpyCas9-SpRY
72
0


 


812
AG
GCAACTGGTAGCTGGAGGAC
19963
SpyCas9-NG
72
0


 


813
AG
GCAACTGGTAGCTGGAGGAC
19964
SpyCas9-xCas
72
0


 


814
AG
GCAACTGGTAGCTGGAGGAC
19965
SpyCas9-xCas-
72
0






NG




 


815
AGT
GCAACTGGTAGCTGGAGGAC
19966
SpyCas9-SpG
72
0


 


816
AGT
GCAACTGGTAGCTGGAGGAC
19967
SpyCas9-SpRY
72
0


 


817
AGTAC
ctggCAACTGGTAGCTGGAGGAC
19968
BlatCas9
72
0


 


818
CTGGTT
TTCTTTTCATCCCAGCTTGCA
19969
cCas9-v16
72
0


 


819
CTGGTT
TTCTTTTCATCCCAGCTTGCA
19970
cCas9-v21
72
0


 


820
AGTA
GCAACTGGTAGCTGGAGGAC
19971
SpyCas9-3var-
72
0






NRTH




 


821
ACTGGTT
tttCTTCTTTTCATCCCAGCTTGC
19972
PpnCas9
73
0


 


822
ACTGG
CTTCTTTTCATCCCAGCTTGC
19973
SauCas9KKH
73
0


 


823
ACTGGT
CTTCTTTTCATCCCAGCTTGC
19974
SauCas9KKH
73
0


 


824
CAG
GGCAACTGGTAGCTGGAGGA
19975
ScaCas9
73
0


 


825
CAG
GGCAACTGGTAGCTGGAGGA
19976
ScaCas9-HiFi-
73
0






Sc++




 


826
CAG
GGCAACTGGTAGCTGGAGGA
19977
ScaCas9-Sc++
73
0


 


827
CAG
GGCAACTGGTAGCTGGAGGA
19978
SpyCas9-SpRY
73
0


 


828
ACT
TTCTTTTCATCCCAGCTTGC
19979
SpyCas9-SpRY
73
0


 


829
CAGTACT
CTGGCAACTGGTAGCTGGAGG
19980
CdiCas9
73
0




A






 


830
ACTGGTTT
tttcTTCTTTTCATCCCAGCTTGC
19981
NmeCas9
73
0


 


831
CAGT
GGCAACTGGTAGCTGGAGGA
19982
SpyCas9-3var-
73
0






NRRH




 


832
ACAG
CTGGCAACTGGTAGCTGGAGG
19983
SauriCas9-KKH
74
0


 


833
CAC
CTTCTTTTCATCCCAGCTTG
19984
SpyCas9-SpRY
74
0


 


834
ACA
TGGCAACTGGTAGCTGGAGG
19985
SpyCas9-SpRY
74
0


 


835
CACT
CTTCTTTTCATCCCAGCTTG
19986
SpyCas9-3var-
74
0






NRCH




 


836
GACAG
CCTGGCAACTGGTAGCTGGAG
19987
SauCas9KKH
75
0


 


837
GACAGT
CCTGGCAACTGGTAGCTGGAG
19988
SauCas9KKH
75
0


 


838
GACAGT
CCTGGCAACTGGTAGCTGGAG
19989
cCas9-v17
75
0


 


839
GACAGT
CCTGGCAACTGGTAGCTGGAG
19990
cCas9-v42
75
0


 


840
GAC
CTGGCAACTGGTAGCTGGAG
19991
SpyCas9-SpRY
75
0


 


841
GCA
TCTTCTTTTCATCCCAGCTT
19992
SpyCas9-SpRY
75
0


 


842
GACAGTA
tgCCTGGCAACTGGTAGCTGGA
19993
CjeCas9
75
0



C
G






 


843
GACA
CTGGCAACTGGTAGCTGGAG
19994
SpyCas9-3var-
75
0






NRCH




 


844
GG
CCTGGCAACTGGTAGCTGGA
19995
SpyCas9-NG
76
0


 


845
GG
CCTGGCAACTGGTAGCTGGA
19996
SpyCas9-xCas
76
0


 


846
GG
CCTGGCAACTGGTAGCTGGA
19997
SpyCas9-xCas-
76
0






NG




 


847
TG
TTCTTCTTTTCATCCCAGCT
19998
SpyCas9-NG
76
0


 


848
TG
TTCTTCTTTTCATCCCAGCT
19999
SpyCas9-xCas
76
0


 


849
TG
TTCTTCTTTTCATCCCAGCT
20000
SpyCas9-xCas-
76
0






NG




 


850
GGA
CCTGGCAACTGGTAGCTGGA
20001
SpyCas9-SpG
76
0


 


851
GGA
CCTGGCAACTGGTAGCTGGA
20002
SpyCas9-SpRY
76
0


 


852
TGC
TTCTTCTTTTCATCCCAGCT
20003
SpyCas9-SpG
76
0


 


853
TGC
TTCTTCTTTTCATCCCAGCT
20004
SpyCas9-SpRY
76
0


 


854
TGCACTG
tcttTCTTCTTTTCATCCCAGCT
20005
BlatCas9
76
0



G







 


855
TGCAC
tcttTCTTCTTTTCATCCCAGCT
20006
BlatCas9
76
0


 


856
TGCACT
TTTCTTCTTTTCATCCCAGCT
20007
cCas9-v16
76
0


 


857
TGCACT
TTTCTTCTTTTCATCCCAGCT
20008
cCas9-v21
76
0


 


858
GGAC
CCTGGCAACTGGTAGCTGGA
20009
SpyCas9-3var-
76
0






NRRH




 


859
GGAC
CCTGGCAACTGGTAGCTGGA
20010
SpyCas9-VQR
76
0


 


860
TGCA
TTCTTCTTTTCATCCCAGCT
20011
SpyCas9-3var-
76
0






NRCH




 


861
AGG
GCCTGGCAACTGGTAGCTGG
20012
ScaCas9
77
0


 


862
AGG
GCCTGGCAACTGGTAGCTGG
20013
ScaCas9-HiFi-
77
0






Sc++




 


863
AGG
GCCTGGCAACTGGTAGCTGG
20014
ScaCas9-Sc++
77
0


 


864
AGG
GCCTGGCAACTGGTAGCTGG
20015
SpyCas9
77
0


 


865
AGG
GCCTGGCAACTGGTAGCTGG
20016
SpyCas9-HF1
77
0


 


866
AGG
GCCTGGCAACTGGTAGCTGG
20017
SpyCas9-SpG
77
0


 


867
AGG
GCCTGGCAACTGGTAGCTGG
20018
SpyCas9-SpRY
77
0


 


868
TTG
TTTCTTCTTTTCATCCCAGC
20019
ScaCas9
77
0


 


869
TTG
TTTCTTCTTTTCATCCCAGC
20020
ScaCas9-HiFi-
77
0






Sc++




 


870
TTG
TTTCTTCTTTTCATCCCAGC
20021
ScaCas9-Sc++
77
0


 


871
TTG
TTTCTTCTTTTCATCCCAGC
20022
SpyCas9-SpRY
77
0


 


872
AG
GCCTGGCAACTGGTAGCTGG
20023
SpyCas9-NG
77
0


 


873
AG
GCCTGGCAACTGGTAGCTGG
20024
SpyCas9-xCas
77
0


 


874
AG
GCCTGGCAACTGGTAGCTGG
20025
SpyCas9-xCas-
77
0






NG




 


875
AGGACAG
tgtgCCTGGCAACTGGTAGCTGG
20026
BlatCas9
77
0



T







 


876
AGGAC
tgtgCCTGGCAACTGGTAGCTGG
20027
BlatCas9
77
0


 


877
TTGCACT
TCTTTCTTCTTTTCATCCCAGC
20028
CdiCas9
77
0


 


878
AGGA
GCCTGGCAACTGGTAGCTGG
20029
SpyCas9-3var-
77
0






NRRH




 


879
GAGGA
ttGTGCCTGGCAACTGGTAGCTG
20030
SauCas9
78
0


 


880
GAGGA
GTGCCTGGCAACTGGTAGCTG
20031
SauCas9KKH
78
0


 


881
GAGG
GTGCCTGGCAACTGGTAGCTG
20032
SauriCas9
78
0


 


882
GAGG
GTGCCTGGCAACTGGTAGCTG
20033
SauriCas9-KKH
78
0


 


883
GAG
TGCCTGGCAACTGGTAGCTG
20034
ScaCas9
78
0


 


884
GAG
TGCCTGGCAACTGGTAGCTG
20035
ScaCas9-HiFi-
78
0






Sc++




 


885
GAG
TGCCTGGCAACTGGTAGCTG
20036
ScaCas9-Sc++
78
0


 


886
GAG
TGCCTGGCAACTGGTAGCTG
20037
SpyCas9-SpRY
78
0


 


887
CTT
CTTTCTTCTTTTCATCCCAG
20038
SpyCas9-SpRY
78
0


 


888
CTTGC
tttcTTTCTTCTTTTCATCCCAG
20039
BlatCas9
78
0


 


889
GAGGAC
GTGCCTGGCAACTGGTAGCTG
20040
cCas9-v17
78
0


 


890
GAGGAC
GTGCCTGGCAACTGGTAGCTG
20041
cCas9-v42
78
0


 


891
GGAGG
TGTGCCTGGCAACTGGTAGCT
20042
SauCas9KKH
79
0


 


892
GGAG
TGTGCCTGGCAACTGGTAGCT
20043
SauriCas9-KKH
79
0


 


893
GGAG
GTGCCTGGCAACTGGTAGCT
20044
SpyCas9-VQR
79
0


 


894
GG
GTGCCTGGCAACTGGTAGCT
20045
SpyCas9-NG
79
0


 


895
GG
GTGCCTGGCAACTGGTAGCT
20046
SpyCas9-xCas
79
0


 


896
GG
GTGCCTGGCAACTGGTAGCT
20047
SpyCas9-xCas-
79
0






NG




 


897
GGA
GTGCCTGGCAACTGGTAGCT
20048
SpyCas9-SpG
79
0


 


898
GGA
GTGCCTGGCAACTGGTAGCT
20049
SpyCas9-SpRY
79
0


 


899
GCT
TCTTTCTTCTTTTCATCCCA
20050
SpyCas9-SpRY
79
0


 


900
GGAGGA
TGTGCCTGGCAACTGGTAGCT
20051
cCas9-v17
79
0


 


901
GGAGGA
TGTGCCTGGCAACTGGTAGCT
20052
cCas9-v42
79
0


 


902
GCTTGCA
ttTTCTTTCTTCTTTTCATCCCA
20053
CjeCas9
79
0



C







 


903
GGAGGAC
cattGTGCCTGGCAACTGGTAGC
20054
NmeCas9
79
0



A
T






 


904
TGGAG
caTTGTGCCTGGCAACTGGTAG
20055
SauCas9
80
0




C






 


905
TGGAG
TTGTGCCTGGCAACTGGTAGC
20056
SauCas9KKH
80
0


 


906
TGG
TGTGCCTGGCAACTGGTAGC
20057
ScaCas9
80
0


 


907
TGG
TGTGCCTGGCAACTGGTAGC
20058
ScaCas9-HiFi-
80
0






Sc++




 


908
TGG
TGTGCCTGGCAACTGGTAGC
20059
ScaCas9-Sc++
80
0


 


909
TGG
TGTGCCTGGCAACTGGTAGC
20060
SpyCas9
80
0


 


910
TGG
TGTGCCTGGCAACTGGTAGC
20061
SpyCas9-HF1
80
0


 


911
TGG
TGTGCCTGGCAACTGGTAGC
20062
SpyCas9-SpG
80
0


 


912
TGG
TGTGCCTGGCAACTGGTAGC
20063
SpyCas9-SpRY
80
0


 


913
TG
TGTGCCTGGCAACTGGTAGC
20064
SpyCas9-NG
80
0


 


914
TG
TGTGCCTGGCAACTGGTAGC
20065
SpyCas9-xCas
80
0


 


915
TG
TGTGCCTGGCAACTGGTAGC
20066
SpyCas9-xCas-
80
0






NG




 


916
AG
TTCTTTCTTCTTTTCATCCC
20067
SpyCas9-NG
80
0


 


917
AG
TTCTTTCTTCTTTTCATCCC
20068
SpyCas9-xCas
80
0


 


918
AG
TTCTTTCTTCTTTTCATCCC
20069
SpyCas9-xCas-
80
0






NG




 


919
AGC
TTCTTTCTTCTTTTCATCCC
20070
SpyCas9-SpG
80
0


 


920
AGC
TTCTTTCTTCTTTTCATCCC
20071
SpyCas9-SpRY
80
0


 


921
TGGAGG
TTGTGCCTGGCAACTGGTAGC
20072
cCas9-v17
80
0


 


922
TGGAGG
TTGTGCCTGGCAACTGGTAGC
20073
cCas9-v42
80
0


 


923
TGGA
TGTGCCTGGCAACTGGTAGC
20074
SpyCas9-3var-
80
0






NRRH




 


924
AGCT
TTCTTTCTTCTTTTCATCCC
20075
SpyCas9-3var-
80
0






NRCH




 


925
CTGGA
tcATTGTGCCTGGCAACTGGTAG
20076
SauCas9
81
0


 


926
CTGGA
ATTGTGCCTGGCAACTGGTAG
20077
SauCas9KKH
81
0


 


927
CTGG
ATTGTGCCTGGCAACTGGTAG
20078
SauriCas9
81
0


 


928
CTGG
ATTGTGCCTGGCAACTGGTAG
20079
SauriCas9-KKH
81
0


 


929
CTG
TTGTGCCTGGCAACTGGTAG
20080
ScaCas9
81
0


 


930
CTG
TTGTGCCTGGCAACTGGTAG
20081
ScaCas9-HiFi-
81
0






Sc++




 


931
CTG
TTGTGCCTGGCAACTGGTAG
20082
ScaCas9-Sc++
81
0


 


932
CTG
TTGTGCCTGGCAACTGGTAG
20083
SpyCas9-SpRY
81
0


 


933
CAG
TTTCTTTCTTCTTTTCATCC
20084
ScaCas9
81
0


 


934
CAG
TTTCTTTCTTCTTTTCATCC
20085
ScaCas9-HiFi-
81
0






Sc++




 


935
CAG
TTTCTTTCTTCTTTTCATCC
20086
ScaCas9-Sc++
81
0


 


936
CAG
TTTCTTTCTTCTTTTCATCC
20087
SpyCas9-SpRY
81
0


 


937
CTGGAG
ATTGTGCCTGGCAACTGGTAG
20088
cCas9-v17
81
0


 


938
CTGGAG
ATTGTGCCTGGCAACTGGTAG
20089
cCas9-v42
81
0


 


939
CAGC
TTTCTTTCTTCTTTTCATCC
20090
SpyCas9-3var-
81
0






NRRH




 


940
GCTGG
CATTGTGCCTGGCAACTGGTA
20091
SauCas9KKH
82
0


 


941
CCAG
GTTTTCTTTCTTCTTTTCATC
20092
SauriCas9-KKH
82
0


 


942
GCT
ATTGTGCCTGGCAACTGGTA
20093
SpyCas9-SpRY
82
0


 


943
CCA
TTTTCTTTCTTCTTTTCATC
20094
SpyCas9-SpRY
82
0


 


944
CCAGCTT
gagtTTTCTTTCTTCTTTTCATC
20095
BlatCas9
82
0



G







 


945
CCAGC
gagtTTTCTTTCTTCTTTTCATC
20096
BlatCas9
82
0


 


946
CCAGCT
GTTTTCTTTCTTCTTTTCATC
20097
cCas9-v16
82
0


 


947
CCAGCT
GTTTTCTTTCTTCTTTTCATC
20098
cCas9-v21
82
0


 


948
CCCAG
AGTTTTCTTTCTTCTTTTCAT
20099
SauCas9KKH
83
0


 


949
AG
CATTGTGCCTGGCAACTGGT
20100
SpyCas9-NG
83
0


 


950
AG
CATTGTGCCTGGCAACTGGT
20101
SpyCas9-xCas
83
0


 


951
AG
CATTGTGCCTGGCAACTGGT
20102
SpyCas9-xCas-
83
0






NG




 


952
AGC
CATTGTGCCTGGCAACTGGT
20103
SpyCas9-SpG
83
0


 


953
AGC
CATTGTGCCTGGCAACTGGT
20104
SpyCas9-SpRY
83
0


 


954
CCC
GTTTTCTTTCTTCTTTTCAT
20105
SpyCas9-SpRY
83
0


 


955
CCCAGC
AGTTTTCTTTCTTCTTTTCAT
20106
cCas9-v17
83
0


 


956
CCCAGC
AGTTTTCTTTCTTCTTTTCAT
20107
cCas9-v42
83
0


 


957
CCCAGCT
ttgaGTTTTCTTTCTTCTTTTCAT
20108
NmeCas9
83
0



T







 


958
AGCT
CATTGTGCCTGGCAACTGGT
20109
SpyCas9-3var-
83
0






NRCH




 


959
TAG
TCATTGTGCCTGGCAACTGG
20110
ScaCas9
84
0


 


960
TAG
TCATTGTGCCTGGCAACTGG
20111
ScaCas9-HiFi-
84
0






Sc++




 


961
TAG
TCATTGTGCCTGGCAACTGG
20112
ScaCas9-Sc++
84
0


 


962
TAG
TCATTGTGCCTGGCAACTGG
20113
SpyCas9-SpRY
84
0


 


963
TCC
AGTTTTCTTTCTTCTTTTCA
20114
SpyCas9-SpRY
84
0


 


964
TAGC
TCATTGTGCCTGGCAACTGG
20115
SpyCas9-3var-
84
0






NRRH




 


965
GTAG
GCTCATTGTGCCTGGCAACTG
20116
SauriCas9-KKH
85
0


 


966
GTA
CTCATTGTGCCTGGCAACTG
20117
SpyCas9-SpRY
85
0


 


967
ATC
GAGTTTTCTTTCTTCTTTTC
20118
Spy Cas9-SpRY
85
0


 


968
GTAGCTG
gcgcTCATTGTGCCTGGCAACTG
20119
BlatCas9
85
0



G







 


969
GTAGC
gcgcTCATTGTGCCTGGCAACTG
20120
BlatCas9
85
0


 


970
ATCCC
tttgAGTTTTCTTTCTTCTTTTC
20121
BlatCas9
85
0


 


971
GTAGCT
GCTCATTGTGCCTGGCAACTG
20122
cCas9-v16
85
0


 


972
GTAGCT
GCTCATTGTGCCTGGCAACTG
20123
cCas9-v21
85
0


 


973
CATCCC
gcTTTGAGTTTTCTTTCTTCTTTT
20124
Nme2Cas9
86
0


 


974
GGTAG
CGCTCATTGTGCCTGGCAACT
20125
SauCas9KKH
86
0


 


975
GG
GCTCATTGTGCCTGGCAACT
20126
SpyCas9-NG
86
0


 


976
GG
GCTCATTGTGCCTGGCAACT
20127
SpyCas9-xCas
86
0


 


977
GG
GCTCATTGTGCCTGGCAACT
20128
SpyCas9-xCas-
86
0






NG




 


978
GGT
GCTCATTGTGCCTGGCAACT
20129
SpyCas9-SpG
86
0


 


979
GGT
GCTCATTGTGCCTGGCAACT
20130
SpyCas9-SpRY
86
0


 


980
CAT
TGAGTTTTCTTTCTTCTTTT
20131
SpyCas9-SpRY
86
0


 


981
CATCCCA
ctttGAGTTTTCTTTCTTCTTTT
20132
BlatCas9
86
0



G







 


982
CATCC
ctttGAGTTTTCTTTCTTCTTTT
20133
BlatCas9
86
0


 


983
GGTA
GCTCATTGTGCCTGGCAACT
20134
SpyCas9-3var-
86
0






NRTH




 


984
CATC
TGAGTTTTCTTTCTTCTTTT
20135
SpyCas9-3var-
86
0






NRTH




 


985
TCATCC
agCTTTGAGTTTTCTTTCTTCTTT
20136
Nme2Cas9
87
0


 


986
TGG
CGCTCATTGTGCCTGGCAAC
20137
ScaCas9
87
0


 


987
TGG
CGCTCATTGTGCCTGGCAAC
20138
ScaCas9-HiFi-
87
0






Sc++




 


988
TGG
CGCTCATTGTGCCTGGCAAC
20139
ScaCas9-Sc++
87
0


 


989
TGG
CGCTCATTGTGCCTGGCAAC
20140
SpyCas9
87
0


 


990
TGG
CGCTCATTGTGCCTGGCAAC
20141
SpyCas9-HF1
87
0


 


991
TGG
CGCTCATTGTGCCTGGCAAC
20142
SpyCas9-SpG
87
0


 


992
TGG
CGCTCATTGTGCCTGGCAAC
20143
SpyCas9-SpRY
87
0


 


993
TG
CGCTCATTGTGCCTGGCAAC
20144
SpyCas9-NG
87
0


 


994
TG
CGCTCATTGTGCCTGGCAAC
20145
SpyCas9-xCas
87
0


 


995
TG
CGCTCATTGTGCCTGGCAAC
20146
SpyCas9-xCas-
87
0






NG




 


996
TCA
TTGAGTTTTCTTTCTTCTTT
20147
SpyCas9-SpRY
87
0


 


997
TCATC
gcttTGAGTTTTCTTTCTTCTTT
20148
BlatCas9
87
0


 


998
TCATCCC
CTTTGAGTTTTCTTTCTTCTTT
20149
CdiCas9
87
0


 


999
TGGT
CGCTCATTGTGCCTGGCAAC
20150
SpyCas9-3var-
87
0






NRRH




 


1000
CTGG
GGCGCTCATTGTGCCTGGCAA
20151
SauriCas9
88
0


 


1001
CTGG
GGCGCTCATTGTGCCTGGCAA
20152
SauriCas9-KKH
88
0


 


1002
CTG
GCGCTCATTGTGCCTGGCAA
20153
ScaCas9
88
0


 


1003
CTG
GCGCTCATTGTGCCTGGCAA
20154
ScaCas9-HiFi-
88
0






Sc++




 


1004
CTG
GCGCTCATTGTGCCTGGCAA
20155
ScaCas9-Sc++
88
0


 


1005
CTG
GCGCTCATTGTGCCTGGCAA
20156
SpyCas9-SpRY
88
0


 


1006
TTC
TTTGAGTTTTCTTTCTTCTT
20157
SpyCas9-SpRY
88
0


 


1007
ACTGG
TGGCGCTCATTGTGCCTGGCA
20158
SauCas9KKH
89
0


 


1008
ACTGGT
TGGCGCTCATTGTGCCTGGCA
20159
SauCas9KKH
89
0


 


1009
ACT
GGCGCTCATTGTGCCTGGCA
20160
SpyCas9-SpRY
89
0


 


1010
TTT
CTTTGAGTTTTCTTTCTTCT
20161
SpyCas9-SpRY
89
0


 


1011
AAC
TGGCGCTCATTGTGCCTGGC
20162
SpyCas9-SpRY
90
0


 


1012
TTT
GCTTTGAGTTTTCTTTCTTC
20163
SpyCas9-SpRY
90
0


 


1013
TTTTC
tgagCTTTGAGTTTTCTTTCTTC
20164
BlatCas9
90
0


 


1014
AACT
TGGCGCTCATTGTGCCTGGC
20165
SpyCas9-3var-
90
0






NRCH




 


1015
CAA
ATGGCGCTCATTGTGCCTGG
20166
SpyCas9-SpRY
91
0


 


1016
CTT
AGCTTTGAGTTTTCTTTCTT
20167
SpyCas9-SpRY
91
0


 


1017
CAAC
ATGGCGCTCATTGTGCCTGG
20168
SpyCas9-3var-
91
0






NRRH




 


1018
CAAC
gaTGGCGCTCATTGTGCCTGG
20169
iSpyMacCas9
91
0


 


1019
GCA
GATGGCGCTCATTGTGCCTG
20170
SpyCas9-SpRY
92
0


 


1020
TCT
GAGCTTTGAGTTTTCTTTCT
20171
SpyCas9-SpRY
92
0


 


1021
GCAACTG
aaagATGGCGCTCATTGTGCCTG
20172
BlatCas9
92
0



G







 


1022
GCAAC
aaagATGGCGCTCATTGTGCCTG
20173
BlatCas9
92
0


 


1023
GCAACT
AGATGGCGCTCATTGTGCCTG
20174
cCas9-v16
92
0


 


1024
GCAACT
AGATGGCGCTCATTGTGCCTG
20175
cCas9-v21
92
0


 


1025
GGCAA
AAGATGGCGCTCATTGTGCCT
20176
SauCas9KKH
93
0


 


1026
GG
AGATGGCGCTCATTGTGCCT
20177
SpyCas9-NG
93
0


 


1027
GG
AGATGGCGCTCATTGTGCCT
20178
SpyCas9-xCas
93
0


 


1028
GG
AGATGGCGCTCATTGTGCCT
20179
SpyCas9-xCas-
93
0






NG




 


1029
GGC
AGATGGCGCTCATTGTGCCT
20180
SpyCas9-SpG
93
0


 


1030
GGC
AGATGGCGCTCATTGTGCCT
20181
SpyCas9-SpRY
93
0


 


1031
TTC
TGAGCTTTGAGTTTTCTTTC
20182
SpyCas9-SpRY
93
0


 


1032
GGCAAC
AAGATGGCGCTCATTGTGCCT
20183
cCas9-v17
93
0


 


1033
GGCAAC
AAGATGGCGCTCATTGTGCCT
20184
cCas9-v42
93
0


 


1034
GGCA
AGATGGCGCTCATTGTGCCT
20185
SpyCas9-3var-
93
0






NRCH




 


1035
TGG
AAGATGGCGCTCATTGTGCC
20186
ScaCas9
94
0


 


1036
TGG
AAGATGGCGCTCATTGTGCC
20187
ScaCas9-HiFi-
94
0






Sc++




 


1037
TGG
AAGATGGCGCTCATTGTGCC
20188
ScaCas9-Sc++
94
0


 


1038
TGG
AAGATGGCGCTCATTGTGCC
20189
SpyCas9
94
0


 


1039
TGG
AAGATGGCGCTCATTGTGCC
20190
SpyCas9-HF1
94
0


 


1040
TGG
AAGATGGCGCTCATTGTGCC
20191
SpyCas9-SpG
94
0


 


1041
TGG
AAGATGGCGCTCATTGTGCC
20192
SpyCas9-SpRY
94
0


 


1042
TG
AAGATGGCGCTCATTGTGCC
20193
SpyCas9-NG
94
0


 


1043
TG
AAGATGGCGCTCATTGTGCC
20194
SpyCas9-xCas
94
0


 


1044
TG
AAGATGGCGCTCATTGTGCC
20195
SpyCas9-xCas-
94
0






NG




 


1045
CTT
ATGAGCTTTGAGTTTTCTTT
20196
SpyCas9-SpRY
94
0


 


1046
TGGCAAC
AAAAGATGGCGCTCATTGTGC
20197
CdiCas9
94
0




C






 


1047
TGGC
AAGATGGCGCTCATTGTGCC
20198
SpyCas9-3var-
94
0






NRRH




 


1048
TGGCAA
AAGATGGCGCTCATTGTGCC
20199
St1Cas9-
94
0






LMG1831




 


1049
CTGG
AAAAGATGGCGCTCATTGTGC
20200
SauriCas9
95
0


 


1050
CTGG
AAAAGATGGCGCTCATTGTGC
20201
SauriCas9-KKH
95
0


 


1051
CTG
AAAGATGGCGCTCATTGTGC
20202
ScaCas9
95
0


 


1052
CTG
AAAGATGGCGCTCATTGTGC
20203
ScaCas9-HiFi-
95
0






Sc++




 


1053
CTG
AAAGATGGCGCTCATTGTGC
20204
ScaCas9-Sc++
95
0


 


1054
CTG
AAAGATGGCGCTCATTGTGC
20205
SpyCas9-SpRY
95
0


 


1055
TCT
GATGAGCTTTGAGTTTTCTT
20206
SpyCas9-SpRY
95
0


 


1056
TCTTCTTT
ggtgATGAGCTTTGAGTTTTCTT
20207
BlatCas9
95
0


 


1057
CTGGC
ggaaAAGATGGCGCTCATTGTGC
20208
BlatCas9
95
0


 


1058
TCTTC
ggtgATGAGCTTTGAGTTTTCTT
20209
BlatCas9
95
0


 


1059
CCTGG
GAAAAGATGGCGCTCATTGTG
20210
SauCas9KKH
96
0


 


1060
CCT
AAAAGATGGCGCTCATTGTG
20211
SpyCas9-SpRY
96
0


 


1061
TTC
TGATGAGCTTTGAGTTTTCT
20212
SpyCas9-SpRY
96
0


 


1062
GCC
GAAAAGATGGCGCTCATTGT
20213
SpyCas9-SpRY
97
0


 


1063
TTT
GTGATGAGCTTTGAGTTTTC
20214
SpyCas9-SpRY
97
0


 


1064
TG
GGAAAAGATGGCGCTCATTG
20215
SpyCas9-NG
98
0


 


1065
TG
GGAAAAGATGGCGCTCATTG
20216
SpyCas9-xCas
98
0


 


1066
TG
GGAAAAGATGGCGCTCATTG
20217
SpyCas9-xCas-
98
0






NG




 


1067
TGC
GGAAAAGATGGCGCTCATTG
20218
SpyCas9-SpG
98
0


 


1068
TGC
GGAAAAGATGGCGCTCATTG
20219
SpyCas9-SpRY
98
0


 


1069
CTT
GGTGATGAGCTTTGAGTTTT
20220
SpyCas9-SpRY
98
0


 


1070
CTTTC
agtgGTGATGAGCTTTGAGTTTT
20221
BlatCas9
98
0


 


1071
TGCC
GGAAAAGATGGCGCTCATTG
20222
SpyCas9-3var-
98
0






NRCH




 


1072
GTG
AGGAAAAGATGGCGCTCATT
20223
ScaCas9
99
0


 


1073
GTG
AGGAAAAGATGGCGCTCATT
20224
ScaCas9-HiFi-
99
0






Sc++




 


1074
GTG
AGGAAAAGATGGCGCTCATT
20225
ScaCas9-Sc++
99
0


 


1075
GTG
AGGAAAAGATGGCGCTCATT
20226
SpyCas9-SpRY
99
0


 


1076
TCT
TGGTGATGAGCTTTGAGTTT
20227
SpyCas9-SpRY
99
0


 


1077
GTGCCTG
agcaGGAAAAGATGGCGCTCATT
20228
BlatCas9
99
0



G







 


1078
GTGCC
agcaGGAAAAGATGGCGCTCATT
20229
BlatCas9
99
0


 


1079
TGTGCC
gcAGCAGGAAAAGATGGCGCTC
20230
Nme2Cas9
100
0




AT






 


1080
TG
CAGGAAAAGATGGCGCTCAT
20231
SpyCas9-NG
100
0


 


1081
TG
CAGGAAAAGATGGCGCTCAT
20232
SpyCas9-xCas
100
0


 


1082
TG
CAGGAAAAGATGGCGCTCAT
20233
SpyCas9-xCas-
100
0






NG




 


1083
TGT
CAGGAAAAGATGGCGCTCAT
20234
SpyCas9-SpG
100
0


 


1084
TGT
CAGGAAAAGATGGCGCTCAT
20235
SpyCas9-SpRY
100
0


 


1085
TTC
GTGGTGATGAGCTTTGAGTT
20236
SpyCas9-SpRY
100
0


 


1086
TGTGCCT
cagcAGGAAAAGATGGCGCTCA
20237
BlatCas9
100
0



G
T






 


1087
TGTGC
cagcAGGAAAAGATGGCGCTCA
20238
BlatCas9
100
0




T
















TABLE 1C







Exemplary gRNA spacer Cas pairs for correcting the pathogenic R243Q


mutation


Table 1C provides a gRNA database for correcting the pathogenic R243Q mutation in PAH. List of


spacers, PAMs, and Cas variants for generating a nick at an appropriate position to enable


installation of a desired genomic edit with a gene modifying system. The spacers in this table are


designed to be used with a gene modifying polypeptide comprising a nickase variant of the Cas


species indicated in the table. Tables 2C, 3C, and 4C detail the other components of the system and


are organized such that the ID number shown here in Column 1 (“ID”) is meant to correspond to the


same ID number in Tables 2C, 2C, and 4C.
















SEQ






PAM

ID


Overlaps


ID
sequence
gRNA spacer
NO
Cas species
distance
mutation
















1
CTG
CACTGGTTTCCGCCTCCAAC
21312
ScaCas9
0
0


 


2
CTG
CACTGGTTTCCGCCTCCAAC
21313
ScaCas9-
0
0






HiFi-Sc++




 


3
CTG
CACTGGTTTCCGCCTCCAAC
21314
ScaCas9-
0
0






Sc++




 


4
CTG
CACTGGTTTCCGCCTCCAAC
21315
SpyCas9-
0
0






SpRY




 


5
GAGG
AAGCAGGCCAGCCACAGGTTG
21316
SauriCas9
1
0


 


6
GAGG
AAGCAGGCCAGCCACAGGTTG
21317
SauriCas9-
1
0






KKH




 


7
GAG
AGCAGGCCAGCCACAGGTTG
21318
ScaCas9
1
0


 


8
GAG
AGCAGGCCAGCCACAGGTTG
21319
ScaCas9-
1
0






HiFi-Sc++




 


9
GAG
AGCAGGCCAGCCACAGGTTG
21320
ScaCas9-
1
0






Sc++




 


10
GAG
AGCAGGCCAGCCACAGGTTG
21321
SpyCas9-
1
0






SpRY




 


11
CCT
GCACTGGTTTCCGCCTCCAA
21322
SpyCas9-
1
0






SpRY




 


12
GAGGCGG
gaaaGCAGGCCAGCCACAGGTT
21323
BlatCas9
1
0



A
G






 


13
GAGGC
gaaaGCAGGCCAGCCACAGGTT
21324
BlatCas9
1
0




G






 


14
GGAGG
AAAGCAGGCCAGCCACAGGTT
21325
SauCas9KKH
2
0


 


15
GGAG
AAAGCAGGCCAGCCACAGGTT
21326
SauriCas9-
2
0






KKH




 


16
GGAG
AAGCAGGCCAGCCACAGGTT
21327
SpyCas9-
2
0






VQR




 


17
GG
AAGCAGGCCAGCCACAGGTT
21328
SpyCas9-
2
0






NG




 


18
GG
AAGCAGGCCAGCCACAGGTT
21329
SpyCas9-
2
0






xCas




 


19
GG
AAGCAGGCCAGCCACAGGTT
21330
SpyCas9-
2
0






xCas-NG




 


20
GGA
AAGCAGGCCAGCCACAGGTT
21331
SpyCas9-
2
0






SpG




 


21
GGA
AAGCAGGCCAGCCACAGGTT
21332
SpyCas9-
2
0






SpRY




 


22
ACC
TGCACTGGTTTCCGCCTCCA
21333
SpyCas9-
2
0






SpRY




 


23
GGAGGC
AAAGCAGGCCAGCCACAGGTT
21334
cCas9-v17
2
0


 


24
GGAGGC
AAAGCAGGCCAGCCACAGGTT
21335
cCas9-v42
2
0


 


25
tGGAG
agGAAAGCAGGCCAGCCACAG
21336
SauCas9
3
1




GT






 


26
tGGAG
GAAAGCAGGCCAGCCACAGG
21337
SauCas9KKH
3
1




T






 


27
tGG
AAAGCAGGCCAGCCACAGGT
21338
ScaCas9
3
1


 


28
tGG
AAAGCAGGCCAGCCACAGGT
21339
ScaCas9-
3
1






HiFi-Sc++




 


29
tGG
AAAGCAGGCCAGCCACAGGT
21340
ScaCas9-
3
1






Sc++




 


30
tGG
AAAGCAGGCCAGCCACAGGT
21341
SpyCas9
3
1


 


31
tGG
AAAGCAGGCCAGCCACAGGT
21342
SpyCas9-
3
1






HF1




 


32
tGG
AAAGCAGGCCAGCCACAGGT
21343
SpyCas9-
3
1






SpG




 


33
tGG
AAAGCAGGCCAGCCACAGGT
21344
SpyCas9-
3
1






SpRY




 


34
tG
AAAGCAGGCCAGCCACAGGT
21345
SpyCas9-
3
1






NG




 


35
tG
AAAGCAGGCCAGCCACAGGT
21346
SpyCas9-
3
1






xCas




 


36
tG
AAAGCAGGCCAGCCACAGGT
21347
SpyCas9-
3
1






xCas-NG




 


37
aAC
TTGCACTGGTTTCCGCCTCC
21348
SpyCas9-
3
1






SpRY




 


38
tGGAGG
GAAAGCAGGCCAGCCACAGG
21349
cCas9-v17
3
1




T






 


39
tGGAGG
GAAAGCAGGCCAGCCACAGG
21350
cCas9-v42
3
1




T






 


40
tGGA
AAAGCAGGCCAGCCACAGGT
21351
SpyCas9-
3
1






3var-NRRH




 


41
aACC
TTGCACTGGTTTCCGCCTCC
21352
SpyCas9-
3
1






3var-NRCH




 


42
TtGGA
gaGGAAAGCAGGCCAGCCACA
21353
SauCas9
4
1




GG






 


43
TtGGA
GGAAAGCAGGCCAGCCACAG
21354
SauCas9KKH
4
1




G






 


44
TtGG
GGAAAGCAGGCCAGCCACAG
21355
SauriCas9
4
1




G






 


45
TtGG
GGAAAGCAGGCCAGCCACAG
21356
SauriCas9-
4
1




G

KKH




 


46
TtG
GAAAGCAGGCCAGCCACAGG
21357
ScaCas9
4
1


 


47
TtG
GAAAGCAGGCCAGCCACAGG
21358
ScaCas9-
4
1






HiFi-Sc++




 


48
TtG
GAAAGCAGGCCAGCCACAGG
21359
ScaCas9-
4
1






Sc++




 


49
TtG
GAAAGCAGGCCAGCCACAGG
21360
SpyCas9-
4
1






SpRY




 


50
CaA
CTTGCACTGGTTTCCGCCTC
21361
SpyCas9-
4
1






SpRY




 


51
CaACCTG
cagcTTGCACTGGTTTCCGCCTC
21362
BlatCas9
4
1



T







 


52
CaACC
cagcTTGCACTGGTTTCCGCCTC
21363
BlatCas9
4
1


 


53
TtGGAG
GGAAAGCAGGCCAGCCACAG
21364
cCas9-v17
4
1




G






 


54
TtGGAG
GGAAAGCAGGCCAGCCACAG
21365
cCas9-v42
4
1




G






 


55
CaAC
CTTGCACTGGTTTCCGCCTC
21366
SpyCas9-
4
1






3var-NRRH




 


56
CaAC
gcTTGCACTGGTTTCCGCCTC
21367
iSpyMacCas9
4
1


 


57
CCaACC
ccCAGCTTGCACTGGTTTCCGC
21368
Nme2Cas9
5
1




CT






 


58
GTtGG
AGGAAAGCAGGCCAGCCACA
21369
SauCas9KKH
5
1




G






 


59
GTt
GGAAAGCAGGCCAGCCACAG
21370
SpyCas9-
5
1






SpRY




 


60
CCa
GCTTGCACTGGTTTCCGCCT
21371
SpyCas9-
5
1






SpRY




 


61
CCaACCT
ccagCTTGCACTGGTTTCCGCCT
21372
BlatCas9
5
1



G







 


62
CCaAC
ccagCTTGCACTGGTTTCCGCCT
21373
BlatCas9
5
1


 


63
CCaACCT
CAGCTTGCACTGGTTTCCGCC
21374
CdiCas9
5
1




T






 


64
TCCaA
CAGCTTGCACTGGTTTCCGCC
21375
SauCas9KKH
6
1


 


65
GG
AGGAAAGCAGGCCAGCCACA
21376
SpyCas9-
6
0






NG




 


66
GG
AGGAAAGCAGGCCAGCCACA
21377
SpyCas9-
6
0






xCas




 


67
GG
AGGAAAGCAGGCCAGCCACA
21378
SpyCas9-
6
0






xCas-NG




 


68
GGT
AGGAAAGCAGGCCAGCCACA
21379
SpyCas9-
6
0






SpG




 


69
GGT
AGGAAAGCAGGCCAGCCACA
21380
SpyCas9-
6
0






SpRY




 


70
TCC
AGCTTGCACTGGTTTCCGCC
21381
SpyCas9-
6
0






SpRY




 


71
TCCaAC
CAGCTTGCACTGGTTTCCGCC
21382
cCas9-v17
6
1


 


72
TCCaAC
CAGCTTGCACTGGTTTCCGCC
21383
cCas9-v42
6
1


 


73
GGTt
AGGAAAGCAGGCCAGCCACA
21384
SpyCas9-
6
1






3var-NRTH




 


74
AGG
GAGGAAAGCAGGCCAGCCAC
21385
ScaCas9
7
0


 


75
AGG
GAGGAAAGCAGGCCAGCCAC
21386
ScaCas9-
7
0






HiFi-Sc++




 


76
AGG
GAGGAAAGCAGGCCAGCCAC
21387
ScaCas9-
7
0






Sc++




 


77
AGG
GAGGAAAGCAGGCCAGCCAC
21388
SpyCas9
7
0


 


78
AGG
GAGGAAAGCAGGCCAGCCAC
21389
SpyCas9-
7
0






HF1




 


79
AGG
GAGGAAAGCAGGCCAGCCAC
21390
SpyCas9-
7
0






SpG




 


80
AGG
GAGGAAAGCAGGCCAGCCAC
21391
SpyCas9-
7
0






SpRY




 


81
AG
GAGGAAAGCAGGCCAGCCAC
21392
SpyCas9-
7
0






NG




 


82
AG
GAGGAAAGCAGGCCAGCCAC
21393
SpyCas9-
7
0






xCas




 


83
AG
GAGGAAAGCAGGCCAGCCAC
21394
SpyCas9-
7
0






xCas-NG




 


84
CTC
CAGCTTGCACTGGTTTCCGC
21395
SpyCas9-
7
0






SpRY




 


85
AGGT
GAGGAAAGCAGGCCAGCCAC
21396
SpyCas9-
7
0






3var-NRRH




 


86
CAGG
GAGAGGAAAGCAGGCCAGCC
21397
SauriCas9
8
0




A






 


87
CAGG
GAGAGGAAAGCAGGCCAGCC
21398
SauriCas9-
8
0




A

KKH




 


88
CAG
AGAGGAAAGCAGGCCAGCCA
21399
ScaCas9
8
0


 


89
CAG
AGAGGAAAGCAGGCCAGCCA
21400
ScaCas9-
8
0






HiFi-Sc++




 


90
CAG
AGAGGAAAGCAGGCCAGCCA
21401
ScaCas9-
8
0






Sc++




 


91
CAG
AGAGGAAAGCAGGCCAGCCA
21402
SpyCas9-
8
0






SpRY




 


92
CCT
CCAGCTTGCACTGGTTTCCG
21403
SpyCas9-
8
0






SpRY




 


93
CCTCC
atccCAGCTTGCACTGGTTTCCG
21404
BlatCas9
8
0


 


94
CAGGTt
GAGAGGAAAGCAGGCCAGCC
21405
cCas9-v16
8
1




A






 


95
CAGGTt
GAGAGGAAAGCAGGCCAGCC
21406
cCas9-v21
8
1




A






 


96
GCCTCC
tcATCCCAGCTTGCACTGGTTT
21407
Nme2Cas9
9
0




CC






 


97
ACAGGTt
tccCGAGAGGAAAGCAGGCCA
21408
PpnCas9
9
1




GCC






 


98
ACAGG
CGAGAGGAAAGCAGGCCAGC
21409
SauCas9KKH
9
0




C






 


99
ACAGGT
CGAGAGGAAAGCAGGCCAGC
21410
SauCas9KKH
9
0




C






 


100
ACAGGT
CGAGAGGAAAGCAGGCCAGC
21411
cCas9-v17
9
0




C






 


101
ACAGGT
CGAGAGGAAAGCAGGCCAGC
21412
cCas9-v42
9
0




C






 


102
ACAG
CGAGAGGAAAGCAGGCCAGC
21413
SauriCas9-
9
0




C

KKH




 


103
ACA
GAGAGGAAAGCAGGCCAGCC
21414
SpyCas9-
9
0






SpRY




 


104
GCC
CCCAGCTTGCACTGGTTTCC
21415
SpyCas9-
9
0






SpRY




 


105
GCCTCCa
catcCCAGCTTGCACTGGTTTCC
21416
BlatCas9
9
1



A







 


106
GCCTCCa
catcCCAGCTTGCACTGGTTTCC
21417
BlatCas9
9
1



A







 


107
GCCTC
catcCCAGCTTGCACTGGTTTCC
21418
BlatCas9
9
0


 


108
CACAG
CCGAGAGGAAAGCAGGCCAG
21419
SauCas9KK
10
0




C

H




 


109
CG
TCCCAGCTTGCACTGGTTTC
21420
SpyCas9-
10
0






NG




 


110
CG
TCCCAGCTTGCACTGGTTTC
21421
SpyCas9-
10
0






xCas




 


111
CG
TCCCAGCTTGCACTGGTTTC
21422
SpyCas9-
10
0






xCas-NG




 


112
CAC
CGAGAGGAAAGCAGGCCAGC
21423
SpyCas9-
10
0






SpRY




 


113
CGC
TCCCAGCTTGCACTGGTTTC
21424
SpyCas9-
10
0






SpG




 


114
CGC
TCCCAGCTTGCACTGGTTTC
21425
SpyCas9-
10
0






SpRY




 


115
CACAGG
CCGAGAGGAAAGCAGGCCAG
21426
cCas9-v17
10
0




C






 


116
CACAGG
CCGAGAGGAAAGCAGGCCAG
21427
cCas9-v42
10
0




C






 


117
CACA
CGAGAGGAAAGCAGGCCAGC
21428
SpyCas9-
10
0






3var-NRCH




 


118
CGCC
TCCCAGCTTGCACTGGTTTC
21429
SpyCas9-
10
0






3var-NRCH




 


119
CCG
ATCCCAGCTTGCACTGGTTT
21430
ScaCas9
11
0


 


120
CCG
ATCCCAGCTTGCACTGGTTT
21431
ScaCas9-
11
0






HiFi-Sc++




 


121
CCG
ATCCCAGCTTGCACTGGTTT
21432
ScaCas9-
11
0






Sc++




 


122
CCG
ATCCCAGCTTGCACTGGTTT
21433
SpyCas9-
11
0






SpRY




 


123
CCA
CCGAGAGGAAAGCAGGCCAG
21434
SpyCas9-
11
0






SpRY




 


124
CCGCC
ttcaTCCCAGCTTGCACTGGTTT
21435
BlatCas9
11
0


 


125
CCGCCTC
TCATCCCAGCTTGCACTGGTTT
21436
CdiCas9
11
0


 


126
TCCGCC
ttTTCATCCCAGCTTGCACTGGT
21437
Nme2Cas9
12
0




T






 


127
GCC
CCCGAGAGGAAAGCAGGCCA
21438
SpyCas9-
12
0






SpRY




 


128
TCC
CATCCCAGCTTGCACTGGTT
21439
SpyCas9-
12
0






SpRY




 


129
GCCACAG
aatcCCGAGAGGAAAGCAGGCC
21440
BlatCas9
12
0



G
A






 


130
GCCAC
aatcCCGAGAGGAAAGCAGGCC
21441
BlatCas9
12
0




A






 


131
TCCGC
tttcATCCCAGCTTGCACTGGTT
21442
BlatCas9
12
0


 


132
AG
TCCCGAGAGGAAAGCAGGCC
21443
SpyCas9-
13
0






NG




 


133
AG
TCCCGAGAGGAAAGCAGGCC
21444
SpyCas9-
13
0






xCas




 


134
AG
TCCCGAGAGGAAAGCAGGCC
21445
SpyCas9-
13
0






xCas-NG




 


135
AGC
TCCCGAGAGGAAAGCAGGCC
21446
SpyCas9-
13
0






SpG




 


136
AGC
TCCCGAGAGGAAAGCAGGCC
21447
SpyCas9-
13
0






SpRY




 


137
TTC
TCATCCCAGCTTGCACTGGT
21448
SpyCas9-
13
0






SpRY




 


138
AGCC
TCCCGAGAGGAAAGCAGGCC
21449
SpyCas9-
13
0






3var-NRCH




 


139
CAG
ATCCCGAGAGGAAAGCAGGC
21450
ScaCas9
14
0


 


140
CAG
ATCCCGAGAGGAAAGCAGGC
21451
ScaCas9-
14
0






HiFi-Sc++




 


141
CAG
ATCCCGAGAGGAAAGCAGGC
21452
ScaCas9-
14
0






Sc++




 


142
CAG
ATCCCGAGAGGAAAGCAGGC
21453
SpyCas9-
14
0






SpRY




 


143
TTT
TTCATCCCAGCTTGCACTGG
21454
SpyCas9-
14
0






SpRY




 


144
CAGCC
gaaaTCCCGAGAGGAAAGCAGG
21455
BlatCas9
14
0




C






 


145
TTTCC
ctttTCATCCCAGCTTGCACTGG
21456
BlatCas9
14
0


 


146
CAGCCAC
AAATCCCGAGAGGAAAGCAG
21457
CdiCas9
14
0




GC






 


147
CAGC
ATCCCGAGAGGAAAGCAGGC
21458
SpyCas9-
14
0






3var-NRRH




 


148
CCAGCC
aaGAAATCCCGAGAGGAAAGC
21459
Nme2Cas9
15
0




AGG






 


149
GTTTCC
ttCTTTTCATCCCAGCTTGCACT
21460
Nme2Cas9
15
0




G






 


150
CCAG
AAATCCCGAGAGGAAAGCAG
21461
SauriCas9-
15
0




G

KKH




 


151
CCA
AATCCCGAGAGGAAAGCAGG
21462
SpyCas9-
15
0






SpRY




 


152
GTT
TTTCATCCCAGCTTGCACTG
21463
SpyCas9-
15
0






SpRY




 


153
CCAGC
agaaATCCCGAGAGGAAAGCAG
21464
BlatCas9
15
0




G






 


154
GTTTC
tcttTTCATCCCAGCTTGCACTG
21465
BlatCas9
15
0


 


155
GCCAG
GAAATCCCGAGAGGAAAGCA
21466
SauCas9KKH
16
0




G






 


156
GG
TTTTCATCCCAGCTTGCACT
21467
SpyCas9-
16
0






NG




 


157
GG
TTTTCATCCCAGCTTGCACT
21468
SpyCas9-
16
0






xCas




 


158
GG
TTTTCATCCCAGCTTGCACT
21469
SpyCas9-
16
0






xCas-NG




 


159
GGT
TTTTCATCCCAGCTTGCACT
21470
SpyCas9-
16
0






SpG




 


160
GGT
TTTTCATCCCAGCTTGCACT
21471
SpyCas9-
16
0






SpRY




 


161
GCC
AAATCCCGAGAGGAAAGCAG
21472
SpyCas9-
16
0






SpRY




 


162
GCCAGC
GAAATCCCGAGAGGAAAGCA
21473
cCas9-v17
16
0




G






 


163
GCCAGC
GAAATCCCGAGAGGAAAGCA
21474
cCas9-v42
16
0




G






 


164
GGTT
TTTTCATCCCAGCTTGCACT
21475
SpyCas9-
16
0






3var-NRTH




 


165
TGG
CTTTTCATCCCAGCTTGCAC
21476
ScaCas9
17
0


 


166
TGG
CTTTTCATCCCAGCTTGCAC
21477
ScaCas9-
17
0






HiFi-Sc++




 


167
TGG
CTTTTCATCCCAGCTTGCAC
21478
ScaCas9-
17
0






Sc++




 


168
TGG
CTTTTCATCCCAGCTTGCAC
21479
SpyCas9
17
0


 


169
TGG
CTTTTCATCCCAGCTTGCAC
21480
SpyCas9-
17
0






HF1




 


170
TGG
CTTTTCATCCCAGCTTGCAC
21481
SpyCas9-
17
0






SpG




 


171
TGG
CTTTTCATCCCAGCTTGCAC
21482
SpyCas9-
17
0






SpRY




 


172
GG
GAAATCCCGAGAGGAAAGCA
21483
SpyCas9-
17
0






NG




 


173
GG
GAAATCCCGAGAGGAAAGCA
21484
SpyCas9-
17
0






xCas




 


174
GG
GAAATCCCGAGAGGAAAGCA
21485
SpyCas9-
17
0






xCas-NG




 


175
TG
CTTTTCATCCCAGCTTGCAC
21486
SpyCas9-
17
0






NG




 


176
TG
CTTTTCATCCCAGCTTGCAC
21487
SpyCas9-
17
0






xCas




 


177
TG
CTTTTCATCCCAGCTTGCAC
21488
SpyCas9-
17
0






xCas-NG




 


178
GGC
GAAATCCCGAGAGGAAAGCA
21489
SpyCas9-
17
0






SpG




 


179
GGC
GAAATCCCGAGAGGAAAGCA
21490
SpyCas9-
17
0






SpRY




 


180
TGGTTTC
TTCTTTTCATCCCAGCTTGCAC
21491
CdiCas9
17
0


 


181
TGGT
CTTTTCATCCCAGCTTGCAC
21492
SpyCas9-
17
0






3var-NRRH




 


182
GGCC
GAAATCCCGAGAGGAAAGCA
21493
SpyCas9-
17
0






3var-NRCH




 


183
CTGG
TTCTTTTCATCCCAGCTTGCA
21494
SauriCas9
18
0


 


184
CTGG
TTCTTTTCATCCCAGCTTGCA
21495
SauriCas9-
18
0






KKH




 


185
AGG
AGAAATCCCGAGAGGAAAGC
21496
ScaCas9
18
0


 


186
AGG
AGAAATCCCGAGAGGAAAGC
21497
ScaCas9-
18
0






HiFi-Sc++




 


187
AGG
AGAAATCCCGAGAGGAAAGC
21498
ScaCas9-
18
0






Sc++




 


188
AGG
AGAAATCCCGAGAGGAAAGC
21499
SpyCas9
18
0


 


189
AGG
AGAAATCCCGAGAGGAAAGC
21500
SpyCas9-
18
0






HF1




 


190
AGG
AGAAATCCCGAGAGGAAAGC
21501
SpyCas9-
18
0






SpG




 


191
AGG
AGAAATCCCGAGAGGAAAGC
21502
SpyCas9-
18
0






SpRY




 


192
CTG
TCTTTTCATCCCAGCTTGCA
21503
ScaCas9
18
0


 


193
CTG
TCTTTTCATCCCAGCTTGCA
21504
ScaCas9-
18
0






HiFi-Sc++




 


194
CTG
TCTTTTCATCCCAGCTTGCA
21505
ScaCas9-
18
0






Sc++




 


195
CTG
TCTTTTCATCCCAGCTTGCA
21506
SpyCas9-
18
0






SpRY




 


196
AG
AGAAATCCCGAGAGGAAAGC
21507
SpyCas9-
18
0






NG




 


197
AG
AGAAATCCCGAGAGGAAAGC
21508
SpyCas9-
18
0






xCas




 


198
AG
AGAAATCCCGAGAGGAAAGC
21509
SpyCas9-
18
0






xCas-NG




 


199
AGGCC
ccaaGAAATCCCGAGAGGAAAG
21510
BlatCas9
18
0




C






 


200
CTGGTT
TTCTTTTCATCCCAGCTTGCA
21511
cCas9-v16
18
0


 


201
CTGGTT
TTCTTTTCATCCCAGCTTGCA
21512
cCas9-v21
18
0


 


202
AGGC
AGAAATCCCGAGAGGAAAGC
21513
SpyCas9-
18
0






3var-NRRH




 


203
CAGGCC
acCCAAGAAATCCCGAGAGGA
21514
Nme2Cas9
19
0




AAG






 


204
ACTGGTT
tttCTTCTTTTCATCCCAGCTTGC
21515
PpnCas9
19
0


 


205
ACTGG
CTTCTTTTCATCCCAGCTTGC
21516
SauCas9KKH
19
0


 


206
ACTGGT
CTTCTTTTCATCCCAGCTTGC
21517
SauCas9KKH
19
0


 


207
CAGG
CAAGAAATCCCGAGAGGAAA
21518
SauriCas9
19
0




G






 


208
CAGG
CAAGAAATCCCGAGAGGAAA
21519
SauriCas9-
19
0




G

KKH




 


209
CAG
AAGAAATCCCGAGAGGAAAG
21520
ScaCas9
19
0


 


210
CAG
AAGAAATCCCGAGAGGAAAG
21521
ScaCas9-
19
0






HiFi-Sc++




 


211
CAG
AAGAAATCCCGAGAGGAAAG
21522
ScaCas9-
19
0






Sc++




 


212
CAG
AAGAAATCCCGAGAGGAAAG
21523
SpyCas9-
19
0






SpRY




 


213
ACT
TTCTTTTCATCCCAGCTTGC
21524
SpyCas9-
19
0






SpRY




 


214
CAGGCCA
cccaAGAAATCCCGAGAGGAAA
21525
BlatCas9
19
0



G
G






 


215
CAGGC
cccaAGAAATCCCGAGAGGAAA
21526
BlatCas9
19
0




G






 


216
ACTGGTT
tttcTTCTTTTCATCCCAGCTTGC
21527
NmeCas9
19
0



T







 


217
GCAGG
CCAAGAAATCCCGAGAGGAA
21528
SauCas9KKH
20
0




A






 


218
GCAG
CCAAGAAATCCCGAGAGGAA
21529
SauriCas9-
20
0




A

KKH




 


219
CAC
CTTCTTTTCATCCCAGCTTG
21530
SpyCas9-
20
0






SpRY




 


220
GCA
CAAGAAATCCCGAGAGGAAA
21531
SpyCas9-
20
0






SpRY




 


221
GCAGGC
CCAAGAAATCCCGAGAGGAA
21532
cCas9-v17
20
0




A






 


222
GCAGGC
CCAAGAAATCCCGAGAGGAA
21533
cCas9-v42
20
0




A






 


223
CACT
CTTCTTTTCATCCCAGCTTG
21534
SpyCas9-
20
0






3var-NRCH




 


224
AGCAG
CCCAAGAAATCCCGAGAGGA
21535
SauCas9KKH
21
0




A






 


225
AG
CCAAGAAATCCCGAGAGGAA
21536
SpyCas9-
21
0






NG




 


226
AG
CCAAGAAATCCCGAGAGGAA
21537
SpyCas9-
21
0






xCas




 


227
AG
CCAAGAAATCCCGAGAGGAA
21538
SpyCas9-
21
0






xCas-NG




 


228
AGC
CCAAGAAATCCCGAGAGGAA
21539
SpyCas9-
21
0






SpG




 


229
AGC
CCAAGAAATCCCGAGAGGAA
21540
SpyCas9-
21
0






SpRY




 


230
GCA
TCTTCTTTTCATCCCAGCTT
21541
SpyCas9-
21
0






SpRY




 


231
AGCAGG
CCCAAGAAATCCCGAGAGGA
21542
cCas9-v17
21
0




A






 


232
AGCAGG
CCCAAGAAATCCCGAGAGGA
21543
cCas9-v42
21
0




A






 


233
AGCA
CCAAGAAATCCCGAGAGGAA
21544
SpyCas9-
21
0






3var-NRCH




 


234
AAG
CCCAAGAAATCCCGAGAGGA
21545
ScaCas9
22
0


 


235
AAG
CCCAAGAAATCCCGAGAGGA
21546
ScaCas9-
22
0






HiFi-Sc++




 


236
AAG
CCCAAGAAATCCCGAGAGGA
21547
ScaCas9-
22
0






Sc++




 


237
AAG
CCCAAGAAATCCCGAGAGGA
21548
SpyCas9-
22
0






SpRY




 


238
TG
TTCTTCTTTTCATCCCAGCT
21549
SpyCas9-
22
0






NG




 


239
TG
TTCTTCTTTTCATCCCAGCT
21550
SpyCas9-
22
0






xCas




 


240
TG
TTCTTCTTTTCATCCCAGCT
21551
SpyCas9-
22
0






xCas-NG




 


241
TGC
TTCTTCTTTTCATCCCAGCT
21552
SpyCas9-
22
0






SpG




 


242
TGC
TTCTTCTTTTCATCCCAGCT
21553
SpyCas9-
22
0






SpRY




 


243
TGCACTG
tcttTCTTCTTTTCATCCCAGCT
21554
BlatCas9
22
0



G







 


244
TGCAC
tcttTCTTCTTTTCATCCCAGCT
21555
BlatCas9
22
0


 


245
TGCACT
TTTCTTCTTTTCATCCCAGCT
21556
cCas9-v16
22
0


 


246
TGCACT
TTTCTTCTTTTCATCCCAGCT
21557
cCas9-v21
22
0


 


247
AAGC
CCCAAGAAATCCCGAGAGGA
21558
SpyCas9-
22
0






3var-NRRH




 


248
TGCA
TTCTTCTTTTCATCCCAGCT
21559
SpyCas9-
22
0






3var-NRCH




 


249
AAAG
CACCCAAGAAATCCCGAGAG
21560
SauriCas9-
23
0




G

KKH




 


250
AAAG
ACCCAAGAAATCCCGAGAGG
21561
SpyCas9-
23
0






QQR1




 


251
AAAG
caCCCAAGAAATCCCGAGAGG
21562
iSpyMacCas9
23
0


 


252
TTG
TTTCTTCTTTTCATCCCAGC
21563
ScaCas9
23
0


 


253
TTG
TTTCTTCTTTTCATCCCAGC
21564
ScaCas9-
23
0






HiFi-Sc++




 


254
TTG
TTTCTTCTTTTCATCCCAGC
21565
ScaCas9-
23
0






Sc++




 


255
TTG
TTTCTTCTTTTCATCCCAGC
21566
SpyCas9-
23
0






SpRY




 


256
AAA
ACCCAAGAAATCCCGAGAGG
21567
SpyCas9-
23
0






SpRY




 


257
AAAGCAG
gccaCCCAAGAAATCCCGAGAG
21568
BlatCas9
23
0



G
G






 


258
AAAGC
gccaCCCAAGAAATCCCGAGAG
21569
BlatCas9
23
0




G






 


259
TTGCACT
TCTTTCTTCTTTTCATCCCAGC
21570
CdiCas9
23
0


 


260
GAAAG
CCACCCAAGAAATCCCGAGAG
21571
SauCas9KKH
24
0


 


261
GAA
CACCCAAGAAATCCCGAGAG
21572
SpyCas9-
24
0






SpRY




 


262
GAA
CACCCAAGAAATCCCGAGAG
21573
SpyCas9-
24
0






xCas




 


263
CTT
CTTTCTTCTTTTCATCCCAG
21574
SpyCas9-
24
0






SpRY




 


264
CTTGC
tttcTTTCTTCTTTTCATCCCAG
21575
BlatCas9
24
0


 


265
GAAAGC
CCACCCAAGAAATCCCGAGAG
21576
cCas9-v17
24
0


 


266
GAAAGC
CCACCCAAGAAATCCCGAGAG
21577
cCas9-v42
24
0


 


267
GAAA
CACCCAAGAAATCCCGAGAG
21578
SpyCas9-
24
0






3var-NRRH




 


268
GAAA
ccACCCAAGAAATCCCGAGAG
21579
iSpyMacCas9
24
0


 


269
GGAAA
GCCACCCAAGAAATCCCGAGA
21580
SauCas9KKH
25
0


 


270
GG
CCACCCAAGAAATCCCGAGA
21581
SpyCas9-
25
0






NG




 


271
GG
CCACCCAAGAAATCCCGAGA
21582
SpyCas9-
25
0






xCas




 


272
GG
CCACCCAAGAAATCCCGAGA
21583
SpyCas9-
25
0






xCas-NG




 


273
GGA
CCACCCAAGAAATCCCGAGA
21584
SpyCas9-
25
0






SpG




 


274
GGA
CCACCCAAGAAATCCCGAGA
21585
SpyCas9-
25
0






SpRY




 


275
GCT
TCTTTCTTCTTTTCATCCCA
21586
SpyCas9-
25
0






SpRY




 


276
GGAAAG
GCCACCCAAGAAATCCCGAGA
21587
cCas9-v17
25
0


 


277
GGAAAG
GCCACCCAAGAAATCCCGAGA
21588
cCas9-v42
25
0


 


278
GCTTGCA
ttTTCTTTCTTCTTTTCATCCCA
21589
CjeCas9
25
0



C







 


279
GGAA
CCACCCAAGAAATCCCGAGA
21590
SpyCas9-
25
0






3var-NRRH




 


280
GGAA
CCACCCAAGAAATCCCGAGA
21591
SpyCas9-
25
0






VQR




 


281
AGGAA
caGGCCACCCAAGAAATCCCG
21592
SauCas9
26
0




AG






 


282
AGGAA
GGCCACCCAAGAAATCCCGAG
21593
SauCas9KKH
26
0


 


283
AGG
GCCACCCAAGAAATCCCGAG
21594
ScaCas9
26
0


 


284
AGG
GCCACCCAAGAAATCCCGAG
21595
ScaCas9-
26
0






HiFi-Sc++




 


285
AGG
GCCACCCAAGAAATCCCGAG
21596
ScaCas9-
26
0






Sc++




 


286
AGG
GCCACCCAAGAAATCCCGAG
21597
SpyCas9
26
0


 


287
AGG
GCCACCCAAGAAATCCCGAG
21598
SpyCas9-
26
0






HF1




 


288
AGG
GCCACCCAAGAAATCCCGAG
21599
SpyCas9-
26
0






SpG




 


289
AGG
GCCACCCAAGAAATCCCGAG
21600
SpyCas9-
26
0






SpRY




 


290
AG
GCCACCCAAGAAATCCCGAG
21601
SpyCas9-
26
0






NG




 


291
AG
GCCACCCAAGAAATCCCGAG
21602
SpyCas9-
26
0






xCas




 


292
AG
GCCACCCAAGAAATCCCGAG
21603
SpyCas9-
26
0






xCas-NG




 


293
AG
TTCTTTCTTCTTTTCATCCC
21604
SpyCas9-
26
0






NG




 


294
AG
TTCTTTCTTCTTTTCATCCC
21605
SpyCas9-
26
0






xCas




 


295
AG
TTCTTTCTTCTTTTCATCCC
21606
SpyCas9-
26
0






xCas-NG




 


296
AGC
TTCTTTCTTCTTTTCATCCC
21607
SpyCas9-
26
0






SpG




 


297
AGC
TTCTTTCTTCTTTTCATCCC
21608
SpyCas9-
26
0






SpRY




 


298
AGGAAA
GCCACCCAAGAAATCCCGAG
21609
St1Cas9-
26
0






TH1477




 


299
AGGAAA
GGCCACCCAAGAAATCCCGAG
21610
cCas9-v17
26
0


 


300
AGGAAA
GGCCACCCAAGAAATCCCGAG
21611
cCas9-v42
26
0


 


301
AGGA
GCCACCCAAGAAATCCCGAG
21612
SpyCas9-
26
0






3var-NRRH




 


302
AGCT
TTCTTTCTTCTTTTCATCCC
21613
SpyCas9-
26
0






3var-NRCH




 


303
GAGGA
ccAGGCCACCCAAGAAATCCC
21614
SauCas9
27
0




GA






 


304
GAGGA
AGGCCACCCAAGAAATCCCGA
21615
SauCas9KKH
27
0


 


305
GAGG
AGGCCACCCAAGAAATCCCGA
21616
SauriCas9
27
0


 


306
GAGG
AGGCCACCCAAGAAATCCCGA
21617
SauriCas9-
27
0






KKH




 


307
GAG
GGCCACCCAAGAAATCCCGA
21618
ScaCas9
27
0


 


308
GAG
GGCCACCCAAGAAATCCCGA
21619
ScaCas9-
27
0






HiFi-Sc++




 


309
GAG
GGCCACCCAAGAAATCCCGA
21620
ScaCas9-
27
0






Sc++




 


310
GAG
GGCCACCCAAGAAATCCCGA
21621
SpyCas9-
27
0






SpRY




 


311
CAG
TTTCTTTCTTCTTTTCATCC
21622
ScaCas9
27
0


 


312
CAG
TTTCTTTCTTCTTTTCATCC
21623
ScaCas9-
27
0






HiFi-Sc++




 


313
CAG
TTTCTTTCTTCTTTTCATCC
21624
ScaCas9-
27
0






Sc++




 


314
CAG
TTTCTTTCTTCTTTTCATCC
21625
SpyCas9-
27
0






SpRY




 


315
GAGGAAA
GGCCACCCAAGAAATCCCGA
21626
St1Cas9
27
0


 


316
GAGGAA
AGGCCACCCAAGAAATCCCGA
21627
cCas9-v17
27
0


 


317
GAGGAA
AGGCCACCCAAGAAATCCCGA
21628
cCas9-v42
27
0


 


318
CAGC
TTTCTTTCTTCTTTTCATCC
21629
SpyCas9-
27
0






3var-NRRH




 


319
AGAGG
CAGGCCACCCAAGAAATCCCG
21630
SauCas9KKH
28
0


 


320
AGAG
CAGGCCACCCAAGAAATCCCG
21631
SauriCas9-
28
0






KKH




 


321
AGAG
AGGCCACCCAAGAAATCCCG
21632
SpyCas9-
28
0






VQR




 


322
CCAG
GTTTTCTTTCTTCTTTTCATC
21633
SauriCas9-
28
0






KKH




 


323
AG
AGGCCACCCAAGAAATCCCG
21634
SpyCas9-
28
0






NG




 


324
AG
AGGCCACCCAAGAAATCCCG
21635
SpyCas9-
28
0






xCas




 


325
AG
AGGCCACCCAAGAAATCCCG
21636
SpyCas9-
28
0






xCas-NG




 


326
AGA
AGGCCACCCAAGAAATCCCG
21637
SpyCas9-
28
0






SpG




 


327
AGA
AGGCCACCCAAGAAATCCCG
21638
SpyCas9-
28
0






SpRY




 


328
CCA
TTTTCTTTCTTCTTTTCATC
21639
SpyCas9-
28
0






SpRY




 


329
AGAGGAA
AGGCCACCCAAGAAATCCCG
21640
St1Cas9
28
0


 


330
CCAGCTT
gagtTTTCTTTCTTCTTTTCATC
21641
BlatCas9
28
0



G







 


331
CCAGC
gagtTTTCTTTCTTCTTTTCATC
21642
BlatCas9
28
0


 


332
CCAGCT
GTTTTCTTTCTTCTTTTCATC
21643
cCas9-v16
28
0


 


333
CCAGCT
GTTTTCTTTCTTCTTTTCATC
21644
cCas9-v21
28
0


 


334
AGAGGA
CAGGCCACCCAAGAAATCCCG
21645
cCas9-v17
28
0


 


335
AGAGGA
CAGGCCACCCAAGAAATCCCG
21646
cCas9-v42
28
0


 


336
GAGAG
ggCCAGGCCACCCAAGAAATC
21647
SauCas9
29
0




CC






 


337
GAGAG
CCAGGCCACCCAAGAAATCCC
21648
SauCas9KKH
29
0


 


338
CCCAG
AGTTTTCTTTCTTCTTTTCAT
21649
SauCas9KKH
29
0


 


339
GAG
CAGGCCACCCAAGAAATCCC
21650
ScaCas9
29
0


 


340
GAG
CAGGCCACCCAAGAAATCCC
21651
ScaCas9-
29
0






HiFi-Sc++




 


341
GAG
CAGGCCACCCAAGAAATCCC
21652
ScaCas9-
29
0






Sc++




 


342
GAG
CAGGCCACCCAAGAAATCCC
21653
SpyCas9-
29
0






SpRY




 


343
CCC
GTTTTCTTTCTTCTTTTCAT
21654
SpyCas9-
29
0






SpRY




 


344
GAGAGG
CCAGGCCACCCAAGAAATCCC
21655
cCas9-v17
29
0


 


345
GAGAGG
CCAGGCCACCCAAGAAATCCC
21656
cCas9-v42
29
0


 


346
CCCAGC
AGTTTTCTTTCTTCTTTTCAT
21657
cCas9-v17
29
0


 


347
CCCAGC
AGTTTTCTTTCTTCTTTTCAT
21658
cCas9-v42
29
0


 


348
CCCAGCT
ttgaGTTTTCTTTCTTCTTTTCAT
21659
NmeCas9
29
0



T







 


349
GAGA
CAGGCCACCCAAGAAATCCC
21660
SpyCas9-
29
0






3var-NRRH




 


350
CGAGA
GCCAGGCCACCCAAGAAATCC
21661
SauCas9KKH
30
0


 


351
CGAG
GCCAGGCCACCCAAGAAATCC
21662
SauriCas9-
30
0






KKH




 


352
CGAG
CCAGGCCACCCAAGAAATCC
21663
SpyCas9-
30
0






VQR




 


353
CG
CCAGGCCACCCAAGAAATCC
21664
SpyCas9-
30
0






NG




 


354
CG
CCAGGCCACCCAAGAAATCC
21665
SpyCas9-
30
0






xCas




 


355
CG
CCAGGCCACCCAAGAAATCC
21666
SpyCas9-
30
0






xCas-NG




 


356
CGA
CCAGGCCACCCAAGAAATCC
21667
SpyCas9-
30
0






SpG




 


357
CGA
CCAGGCCACCCAAGAAATCC
21668
SpyCas9-
30
0






SpRY




 


358
TCC
AGTTTTCTTTCTTCTTTTCA
21669
SpyCas9-
30
0






SpRY




 


359
CGAGAG
GCCAGGCCACCCAAGAAATCC
21670
cCas9-v17
30
0


 


360
CGAGAG
GCCAGGCCACCCAAGAAATCC
21671
cCas9-v42
30
0


 


361
CCGAG
aaGGCCAGGCCACCCAAGAAA
21672
SauCas9
31
0




TC






 


362
CCGAG
GGCCAGGCCACCCAAGAAATC
21673
SauCas9KKH
31
0


 


363
CCG
GCCAGGCCACCCAAGAAATC
21674
ScaCas9
31
0


 


364
CCG
GCCAGGCCACCCAAGAAATC
21675
ScaCas9-
31
0






HiFi-Sc++




 


365
CCG
GCCAGGCCACCCAAGAAATC
21676
ScaCas9-
31
0






Sc++




 


366
CCG
GCCAGGCCACCCAAGAAATC
21677
SpyCas9-
31
0






SpRY




 


367
ATC
GAGTTTTCTTTCTTCTTTTC
21678
SpyCas9-
31
0






SpRY




 


368
ATCCC
tttgAGTTTTCTTTCTTCTTTTC
21679
BlatCas9
31
0


 


369
CCGAGA
GGCCAGGCCACCCAAGAAATC
21680
cCas9-v17
31
0


 


370
CCGAGA
GGCCAGGCCACCCAAGAAATC
21681
cCas9-v42
31
0


 


371
CATCCC
gcTTTGAGTTTTCTTTCTTCTTT
21682
Nme2Cas9
32
0




T






 


372
CCCGA
AGGCCAGGCCACCCAAGAAA
21683
SauCas9KKH
32
0




T






 


373
CAT
TGAGTTTTCTTTCTTCTTTT
21684
SpyCas9-
32
0






SpRY




 


374
CCC
GGCCAGGCCACCCAAGAAAT
21685
SpyCas9-
32
0






SpRY




 


375
CATCCCA
ctttGAGTTTTCTTTCTTCTTTT
21686
BlatCas9
32
0



G







 


376
CATCC
ctttGAGTTTTCTTTCTTCTTTT
21687
BlatCas9
32
0


 


377
CCCGAG
AGGCCAGGCCACCCAAGAAA
21688
cCas9-v17
32
0




T






 


378
CCCGAG
AGGCCAGGCCACCCAAGAAA
21689
cCas9-v42
32
0




T






 


379
CATC
TGAGTTTTCTTTCTTCTTTT
21690
SpyCas9-
32
0






3var-NRTH




 


380
TCATCC
agCTTTGAGTTTTCTTTCTTCTT
21691
Nme2Cas9
33
0




T






 


381
TCC
AGGCCAGGCCACCCAAGAAA
21692
SpyCas9-
33
0






SpRY




 


382
TCA
TTGAGTTTTCTTTCTTCTTT
21693
SpyCas9-
33
0






SpRY




 


383
TCATC
gcttTGAGTTTTCTTTCTTCTTT
21694
BlatCas9
33
0


 


384
TCATCCC
CTTTGAGTTTTCTTTCTTCTTT
21695
CdiCas9
33
0


 


385
ATC
AAGGCCAGGCCACCCAAGAA
21696
SpyCas9-
34
0






SpRY




 


386
TTC
TTTGAGTTTTCTTTCTTCTT
21697
SpyCas9-
34
0






SpRY




 


387
ATCCCGA
cggaAGGCCAGGCCACCCAAGA
21698
BlatCas9
34
0



G
A






 


388
ATCCC
cggaAGGCCAGGCCACCCAAGA
21699
BlatCas9
34
0




A






 


389
AATCCC
ctCGGAAGGCCAGGCCACCCA
21700
Nme2Cas9
35
0




AGA






 


390
AAT
GAAGGCCAGGCCACCCAAGA
21701
SpyCas9-
35
0






SpRY




 


391
TTT
CTTTGAGTTTTCTTTCTTCT
21702
SpyCas9-
35
0






SpRY




 


392
AATCCCG
tcggAAGGCCAGGCCACCCAAG
21703
BlatCas9
35
0



A
A






 


393
AATCC
tcggAAGGCCAGGCCACCCAAG
21704
BlatCas9
35
0




A






 


394
AATC
GAAGGCCAGGCCACCCAAGA
21705
SpyCas9-
35
0






3var-NRTH




 


395
AAATCC
acTCGGAAGGCCAGGCCACCC
21706
Nme2Cas9
36
0




AAG






 


396
AAA
GGAAGGCCAGGCCACCCAAG
21707
SpyCas9-
36
0






SpRY




 


397
TTT
GCTTTGAGTTTTCTTTCTTC
21708
SpyCas9-
36
0






SpRY




 


398
AAATC
ctcgGAAGGCCAGGCCACCCAA
21709
BlatCas9
36
0




G






 


399
TTTTC
tgagCTTTGAGTTTTCTTTCTTC
21710
BlatCas9
36
0


 


400
AAATCCC
TCGGAAGGCCAGGCCACCCAA
21711
CdiCas9
36
0




G






 


401
AAAT
GGAAGGCCAGGCCACCCAAG
21712
SpyCas9-
36
0






3var-NRRH




 


402
AAAT
cgGAAGGCCAGGCCACCCAAG
21713
iSpyMacCas9
36
0


 


403
GAA
CGGAAGGCCAGGCCACCCAA
21714
SpyCas9-
37
0






SpRY




 


404
GAA
CGGAAGGCCAGGCCACCCAA
21715
SpyCas9-
37
0






xCas




 


405
CTT
AGCTTTGAGTTTTCTTTCTT
21716
SpyCas9-
37
0






SpRY




 


406
GAAATCC
CTCGGAAGGCCAGGCCACCCA
21717
CdiCas9
37
0




A






 


407
GAAA
CGGAAGGCCAGGCCACCCAA
21718
SpyCas9-
37
0






3var-NRRH




 


408
GAAA
tcGGAAGGCCAGGCCACCCAA
21719
iSpyMacCas9
37
0


 


409
AGAAA
CTCGGAAGGCCAGGCCACCCA
21720
SauCas9KKH
38
0


 


410
AGAAAT
CTCGGAAGGCCAGGCCACCCA
21721
SauCas9KKH
38
0


 


411
AGAAAT
CTCGGAAGGCCAGGCCACCCA
21722
cCas9-v17
38
0


 


412
AGAAAT
CTCGGAAGGCCAGGCCACCCA
21723
cCas9-v42
38
0


 


413
AG
TCGGAAGGCCAGGCCACCCA
21724
SpyCas9-
38
0






NG




 


414
AG
TCGGAAGGCCAGGCCACCCA
21725
SpyCas9-
38
0






xCas




 


415
AG
TCGGAAGGCCAGGCCACCCA
21726
SpyCas9-
38
0






xCas-NG




 


416
AGA
TCGGAAGGCCAGGCCACCCA
21727
SpyCas9-
38
0






SpG




 


417
AGA
TCGGAAGGCCAGGCCACCCA
21728
SpyCas9-
38
0






SpRY




 


418
TCT
GAGCTTTGAGTTTTCTTTCT
21729
SpyCas9-
38
0






SpRY




 


419
AGAAATC
ACTCGGAAGGCCAGGCCACCC
21730
CdiCas9
38
0




A






 


420
AGAA
TCGGAAGGCCAGGCCACCCA
21731
SpyCas9-
38
0






3var-NRRH




 


421
AGAA
TCGGAAGGCCAGGCCACCCA
21732
SpyCas9-
38
0






VQR




 


422
AAGAA
agACTCGGAAGGCCAGGCCAC
21733
SauCas9
39
0




CC






 


423
AAGAA
ACTCGGAAGGCCAGGCCACCC
21734
SauCas9KKH
39
0


 


424
AAG
CTCGGAAGGCCAGGCCACCC
21735
ScaCas9
39
0


 


425
AAG
CTCGGAAGGCCAGGCCACCC
21736
ScaCas9-
39
0






HiFi-Sc++




 


426
AAG
CTCGGAAGGCCAGGCCACCC
21737
ScaCas9-
39
0






Sc++




 


427
AAG
CTCGGAAGGCCAGGCCACCC
21738
SpyCas9-
39
0






SpRY




 


428
TTC
TGAGCTTTGAGTTTTCTTTC
21739
SpyCas9-
39
0






SpRY




 


429
AAGAAA
CTCGGAAGGCCAGGCCACCC
21740
St1Cas9-
39
0






TH1477




 


430
AAGAAA
ACTCGGAAGGCCAGGCCACCC
21741
cCas9-v17
39
0


 


431
AAGAAA
ACTCGGAAGGCCAGGCCACCC
21742
cCas9-v42
39
0


 


432
AAGAAAT
GACTCGGAAGGCCAGGCCACC
21743
CdiCas9
39
0




C






 


433
AAGAAAT
GACTCGGAAGGCCAGGCCACC
21744
CdiCas9
39
0




C






 


434
AAGA
CTCGGAAGGCCAGGCCACCC
21745
SpyCas9-
39
0






3var-NRRH




 


435
CAAGA
GACTCGGAAGGCCAGGCCACC
21746
SauCas9KKH
40
0


 


436
CAAG
GACTCGGAAGGCCAGGCCACC
21747
SauriCas9-
40
0






KKH




 


437
CAAG
ACTCGGAAGGCCAGGCCACC
21748
SpyCas9-
40
0






QQR1




 


438
CAAG
gaCTCGGAAGGCCAGGCCACC
21749
iSpyMacCa
40
0






s9




 


439
CAA
ACTCGGAAGGCCAGGCCACC
21750
SpyCas9-
40
0






SpRY




 


440
CTT
ATGAGCTTTGAGTTTTCTTT
21751
SpyCas9-
40
0






SpRY




 


441
CAAGAAA
ACTCGGAAGGCCAGGCCACC
21752
St1Cas9
40
0


 


442
CAAGAA
GACTCGGAAGGCCAGGCCACC
21753
cCas9-v17
40
0


 


443
CAAGAA
GACTCGGAAGGCCAGGCCACC
21754
cCas9-v42
40
0


 


444
CCAAG
AGACTCGGAAGGCCAGGCCA
21755
SauCas9KKH
41
0




C






 


445
CCA
GACTCGGAAGGCCAGGCCAC
21756
SpyCas9-
41
0






SpRY




 


446
TCT
GATGAGCTTTGAGTTTTCTT
21757
SpyCas9-
41
0






SpRY




 


447
TCTTCTTT
ggtgATGAGCTTTGAGTTTTCTT
21758
BlatCas9
41
0


 


448
TCTTC
ggtgATGAGCTTTGAGTTTTCTT
21759
BlatCas9
41
0


 


449
CCAAGA
AGACTCGGAAGGCCAGGCCA
21760
cCas9-v17
41
0




C






 


450
CCAAGA
AGACTCGGAAGGCCAGGCCA
21761
cCas9-v42
41
0




C






 


451
CCCAA
AAGACTCGGAAGGCCAGGCC
21762
SauCas9KKH
42
0




A






 


452
CCC
AGACTCGGAAGGCCAGGCCA
21763
SpyCas9-
42
0






SpRY




 


453
TTC
TGATGAGCTTTGAGTTTTCT
21764
SpyCas9-
42
0






SpRY




 


454
CCCAAG
AAGACTCGGAAGGCCAGGCC
21765
cCas9-v17
42
0




A






 


455
CCCAAG
AAGACTCGGAAGGCCAGGCC
21766
cCas9-v42
42
0




A






 


456
ACC
AAGACTCGGAAGGCCAGGCC
21767
SpyCas9-
43
0






SpRY




 


457
TTT
GTGATGAGCTTTGAGTTTTC
21768
SpyCas9-
43
0






SpRY




 


458
CAC
GAAGACTCGGAAGGCCAGGC
21769
SpyCas9-
44
0






SpRY




 


459
CTT
GGTGATGAGCTTTGAGTTTT
21770
SpyCas9-
44
0






SpRY




 


460
CACCCAA
gtggAAGACTCGGAAGGCCAGG
21771
BlatCas9
44
0



G
C






 


461
CACCC
gtggAAGACTCGGAAGGCCAGG
21772
BlatCas9
44
0




C






 


462
CTTTC
agtgGTGATGAGCTTTGAGTTTT
21773
BlatCas9
44
0


 


463
CACC
GAAGACTCGGAAGGCCAGGC
21774
SpyCas9-
44
0






3var-NRCH




 


464
CCACCC
caGTGGAAGACTCGGAAGGCC
21775
Nme2Cas9
45
0




AGG






 


465
CCA
GGAAGACTCGGAAGGCCAGG
21776
SpyCas9-
45
0






SpRY




 


466
TCT
TGGTGATGAGCTTTGAGTTT
21777
SpyCas9-
45
0






SpRY




 


467
CCACCCA
agtgGAAGACTCGGAAGGCCAG
21778
BlatCas9
45
0



A
G






 


468
CCACCCA
agtgGAAGACTCGGAAGGCCAG
21779
BlatCas9
45
0



A
G






 


469
CCACC
agtgGAAGACTCGGAAGGCCAG
21780
BlatCas9
45
0




G






 


470
GCCACC
gcAGTGGAAGACTCGGAAGGC
21781
Nme2Cas9
46
0




CAG






 


471
GCC
TGGAAGACTCGGAAGGCCAG
21782
SpyCas9-
46
0






SpRY




 


472
TTC
GTGGTGATGAGCTTTGAGTT
21783
SpyCas9-
46
0






SpRY




 


473
GCCAC
cagtGGAAGACTCGGAAGGCCA
21784
BlatCas9
46
0




G






 


474
GG
GTGGAAGACTCGGAAGGCCA
21785
SpyCas9-
47
0






NG




 


475
GG
GTGGAAGACTCGGAAGGCCA
21786
SpyCas9-
47
0






xCas




 


476
GG
GTGGAAGACTCGGAAGGCCA
21787
SpyCas9-
47
0






xCas-NG




 


477
GGC
GTGGAAGACTCGGAAGGCCA
21788
SpyCas9-
47
0






SpG




 


478
GGC
GTGGAAGACTCGGAAGGCCA
21789
SpyCas9-
47
0






SpRY




 


479
TTT
AGTGGTGATGAGCTTTGAGT
21790
SpyCas9-
47
0






SpRY




 


480
GGCC
GTGGAAGACTCGGAAGGCCA
21791
SpyCas9-
47
0






3var-NRCH




 


481
AGG
AGTGGAAGACTCGGAAGGCC
21792
ScaCas9
48
0


 


482
AGG
AGTGGAAGACTCGGAAGGCC
21793
ScaCas9-
48
0






HiFi-Sc++




 


483
AGG
AGTGGAAGACTCGGAAGGCC
21794
ScaCas9-
48
0






Sc++




 


484
AGG
AGTGGAAGACTCGGAAGGCC
21795
SpyCas9
48
0


 


485
AGG
AGTGGAAGACTCGGAAGGCC
21796
SpyCas9-
48
0






HF1




 


486
AGG
AGTGGAAGACTCGGAAGGCC
21797
SpyCas9-
48
0






SpG




 


487
AGG
AGTGGAAGACTCGGAAGGCC
21798
SpyCas9-
48
0






SpRY




 


488
AG
AGTGGAAGACTCGGAAGGCC
21799
SpyCas9-
48
0






NG




 


489
AG
AGTGGAAGACTCGGAAGGCC
21800
SpyCas9-
48
0






xCas




 


490
AG
AGTGGAAGACTCGGAAGGCC
21801
SpyCas9-
48
0






xCas-NG




 


491
TTT
CAGTGGTGATGAGCTTTGAG
21802
SpyCas9-
48
0






SpRY




 


492
TTTTCTTT
actcAGTGGTGATGAGCTTTGAG
21803
BlatCas9
48
0


 


493
AGGCC
tgcaGTGGAAGACTCGGAAGGC
21804
BlatCas9
48
0




C






 


494
TTTTC
actcAGTGGTGATGAGCTTTGAG
21805
BlatCas9
48
0


 


495
AGGCCAC
GCAGTGGAAGACTCGGAAGG
21806
CdiCas9
48
0




CC






 


496
AGGC
AGTGGAAGACTCGGAAGGCC
21807
SpyCas9-
48
0






3var-NRRH




 


497
CAGGCC
tgTGCAGTGGAAGACTCGGAA
21808
Nme2Cas9
49
0




GGC






 


498
CAGG
GCAGTGGAAGACTCGGAAGG
21809
SauriCas9
49
0




C






 


499
CAGG
GCAGTGGAAGACTCGGAAGG
21810
SauriCas9-
49
0




C

KKH




 


500
CAG
CAGTGGAAGACTCGGAAGGC
21811
ScaCas9
49
0


 


501
CAG
CAGTGGAAGACTCGGAAGGC
21812
ScaCas9-
49
0






HiFi-Sc++




 


502
CAG
CAGTGGAAGACTCGGAAGGC
21813
ScaCas9-
49
0






Sc++




 


503
CAG
CAGTGGAAGACTCGGAAGGC
21814
SpyCas9-
49
0






SpRY




 


504
GTT
TCAGTGGTGATGAGCTTTGA
21815
SpyCas9-
49
0






SpRY




 


505
CAGGC
gtgcAGTGGAAGACTCGGAAGG
21816
BlatCas9
49
0




C






 


506
CCAGG
TGCAGTGGAAGACTCGGAAG
21817
SauCas9KKH
50
0




G






 


507
CCAG
TGCAGTGGAAGACTCGGAAG
21818
SauriCas9-
50
0




G

KKH




 


508
AG
CTCAGTGGTGATGAGCTTTG
21819
SpyCas9-
50
0






NG




 


509
AG
CTCAGTGGTGATGAGCTTTG
21820
SpyCas9-
50
0






xCas




 


510
AG
CTCAGTGGTGATGAGCTTTG
21821
SpyCas9-
50
0






xCas-NG




 


511
AGT
CTCAGTGGTGATGAGCTTTG
21822
SpyCas9-
50
0






SpG




 


512
AGT
CTCAGTGGTGATGAGCTTTG
21823
SpyCas9-
50
0






SpRY




 


513
CCA
GCAGTGGAAGACTCGGAAGG
21824
SpyCas9-
50
0






SpRY




 


514
CCAGGC
TGCAGTGGAAGACTCGGAAG
21825
cCas9-v17
50
0




G






 


515
CCAGGC
TGCAGTGGAAGACTCGGAAG
21826
cCas9-v42
50
0




G






 


516
AGTT
CTCAGTGGTGATGAGCTTTG
21827
SpyCas9-
50
0






3var-NRTH




 


517
GCCAG
GTGCAGTGGAAGACTCGGAA
21828
SauCas9KKH
51
0




G






 


518
GAG
ACTCAGTGGTGATGAGCTTT
21829
ScaCas9
51
0


 


519
GAG
ACTCAGTGGTGATGAGCTTT
21830
ScaCas9-
51
0






HiFi-Sc++




 


520
GAG
ACTCAGTGGTGATGAGCTTT
21831
ScaCas9-
51
0






Sc++




 


521
GAG
ACTCAGTGGTGATGAGCTTT
21832
SpyCas9-
51
0






SpRY




 


522
GCC
TGCAGTGGAAGACTCGGAAG
21833
SpyCas9-
51
0






SpRY




 


523
GCCAGG
GTGCAGTGGAAGACTCGGAA
21834
cCas9-v17
51
0




G






 


524
GCCAGG
GTGCAGTGGAAGACTCGGAA
21835
cCas9-v42
51
0




G






 


525
GAGTTTT
TGACTCAGTGGTGATGAGCTT
21836
CdiCas9
51
0




T






 


526
GAGT
ACTCAGTGGTGATGAGCTTT
21837
SpyCas9-
51
0






3var-NRRH




 


527
TGAG
TGACTCAGTGGTGATGAGCTT
21838
SauriCas9-
52
0






KKH




 


528
TGAG
GACTCAGTGGTGATGAGCTT
21839
SpyCas9-
52
0






VQR




 


529
GG
GTGCAGTGGAAGACTCGGAA
21840
SpyCas9-
52
0






NG




 


530
GG
GTGCAGTGGAAGACTCGGAA
21841
SpyCas9-
52
0






xCas




 


531
GG
GTGCAGTGGAAGACTCGGAA
21842
SpyCas9-
52
0






xCas-NG




 


532
TG
GACTCAGTGGTGATGAGCTT
21843
SpyCas9-
52
0






NG




 


533
TG
GACTCAGTGGTGATGAGCTT
21844
SpyCas9-
52
0






xCas




 


534
TG
GACTCAGTGGTGATGAGCTT
21845
SpyCas9-
52
0






xCas-NG




 


535
GGC
GTGCAGTGGAAGACTCGGAA
21846
SpyCas9-
52
0






SpG




 


536
GGC
GTGCAGTGGAAGACTCGGAA
21847
SpyCas9-
52
0






SpRY




 


537
TGA
GACTCAGTGGTGATGAGCTT
21848
SpyCas9-
52
0






SpG




 


538
TGA
GACTCAGTGGTGATGAGCTT
21849
SpyCas9-
52
0






SpRY




 


539
TGAGTT
TGACTCAGTGGTGATGAGCTT
21850
cCas9-v16
52
0


 


540
TGAGTT
TGACTCAGTGGTGATGAGCTT
21851
cCas9-v21
52
0


 


541
GGCC
GTGCAGTGGAAGACTCGGAA
21852
SpyCas9-
52
0






3var-NRCH




 


542
TTGAGTT
cctCTGACTCAGTGGTGATGAG
21853
PpnCas9
53
0




CT






 


543
TTGAG
ctCTGACTCAGTGGTGATGAGC
21854
SauCas9
53
0




T






 


544
TTGAG
CTGACTCAGTGGTGATGAGCT
21855
SauCas9KKH
53
0


 


545
TTGAGT
ctCTGACTCAGTGGTGATGAGC
21856
SauCas9
53
0




T






 


546
TTGAGT
CTGACTCAGTGGTGATGAGCT
21857
SauCas9KKH
53
0


 


547
TTGAGT
CTGACTCAGTGGTGATGAGCT
21858
cCas9-v17
53
0


 


548
TTGAGT
CTGACTCAGTGGTGATGAGCT
21859
cCas9-v42
53
0


 


549
AGG
TGTGCAGTGGAAGACTCGGA
21860
ScaCas9
53
0


 


550
AGG
TGTGCAGTGGAAGACTCGGA
21861
ScaCas9-
53
0






HiFi-Sc++




 


551
AGG
TGTGCAGTGGAAGACTCGGA
21862
ScaCas9-
53
0






Sc++




 


552
AGG
TGTGCAGTGGAAGACTCGGA
21863
SpyCas9
53
0


 


553
AGG
TGTGCAGTGGAAGACTCGGA
21864
SpyCas9-
53
0






HF1




 


554
AGG
TGTGCAGTGGAAGACTCGGA
21865
SpyCas9-
53
0






SpG




 


555
AGG
TGTGCAGTGGAAGACTCGGA
21866
SpyCas9-
53
0






SpRY




 


556
TTG
TGACTCAGTGGTGATGAGCT
21867
ScaCas9
53
0


 


557
TTG
TGACTCAGTGGTGATGAGCT
21868
ScaCas9-
53
0






HiFi-Sc++




 


558
TTG
TGACTCAGTGGTGATGAGCT
21869
ScaCas9-
53
0






Sc++




 


559
TTG
TGACTCAGTGGTGATGAGCT
21870
SpyCas9-
53
0






SpRY




 


560
AG
TGTGCAGTGGAAGACTCGGA
21871
SpyCas9-
53
0






NG




 


561
AG
TGTGCAGTGGAAGACTCGGA
21872
SpyCas9-
53
0






xCas




 


562
AG
TGTGCAGTGGAAGACTCGGA
21873
SpyCas9-
53
0






xCas-NG




 


563
AGGCCAG
ctgtGTGCAGTGGAAGACTCGG
21874
BlatCas9
53
0



G
A






 


564
AGGCC
ctgtGTGCAGTGGAAGACTCGG
21875
BlatCas9
53
0




A






 


565
TTGAGTT
cctcTGACTCAGTGGTGATGAGC
21876
NmeCas9
53
0



T
T






 


566
AGGC
TGTGCAGTGGAAGACTCGGA
21877
SpyCas9-
53
0






3var-NRRH




 


567
AAGGCC
taCTGTGTGCAGTGGAAGACTC
21878
Nme2Cas9
54
0




GG






 


568
TTTGA
TCTGACTCAGTGGTGATGAGC
21879
SauCas9KKH
54
0


 


569
AAGG
TGTGTGCAGTGGAAGACTCGG
21880
SauriCas9
54
0


 


570
AAGG
TGTGTGCAGTGGAAGACTCGG
21881
SauriCas9-
54
0






KKH




 


571
AAG
GTGTGCAGTGGAAGACTCGG
21882
ScaCas9
54
0


 


572
AAG
GTGTGCAGTGGAAGACTCGG
21883
ScaCas9-
54
0






HiFi-Sc++




 


573
AAG
GTGTGCAGTGGAAGACTCGG
21884
ScaCas9-
54
0






Sc++




 


574
AAG
GTGTGCAGTGGAAGACTCGG
21885
SpyCas9-
54
0






SpRY




 


575
TTT
CTGACTCAGTGGTGATGAGC
21886
SpyCas9-
54
0






SpRY




 


576
AAGGCCA
actgTGTGCAGTGGAAGACTCG
21887
BlatCas9
54
0



G
G






 


577
AAGGC
actgTGTGCAGTGGAAGACTCG
21888
BlatCas9
54
0




G






 


578
GAAGG
CTGTGTGCAGTGGAAGACTCG
21889
SauCas9KKH
55
0


 


579
GAAG
CTGTGTGCAGTGGAAGACTCG
21890
SauriCas9-
55
0






KKH




 


580
GAAG
TGTGTGCAGTGGAAGACTCG
21891
SpyCas9-
55
0






QQR1




 


581
GAAG
ctGTGTGCAGTGGAAGACTCG
21892
iSpyMacCas9
55
0


 


582
GAA
TGTGTGCAGTGGAAGACTCG
21893
SpyCas9-
55
0






SpRY




 


583
GAA
TGTGTGCAGTGGAAGACTCG
21894
SpyCas9-
55
0






xCas




 


584
CTT
TCTGACTCAGTGGTGATGAG
21895
SpyCas9-
55
0






SpRY




 


585
GAAGGC
CTGTGTGCAGTGGAAGACTCG
21896
cCas9-v17
55
0


 


586
GAAGGC
CTGTGTGCAGTGGAAGACTCG
21897
cCas9-v42
55
0


 


587
GGAAG
ACTGTGTGCAGTGGAAGACTC
21898
SauCas9KKH
56
0


 


588
GG
CTGTGTGCAGTGGAAGACTC
21899
SpyCas9-
56
0






NG




 


589
GG
CTGTGTGCAGTGGAAGACTC
21900
SpyCas9-
56
0






xCas




 


590
GG
CTGTGTGCAGTGGAAGACTC
21901
SpyCas9-
56
0






xCas-NG




 


591
GGA
CTGTGTGCAGTGGAAGACTC
21902
SpyCas9-
56
0






SpG




 


592
GGA
CTGTGTGCAGTGGAAGACTC
21903
SpyCas9-
56
0






SpRY




 


593
GCT
CTCTGACTCAGTGGTGATGA
21904
SpyCas9-
56
0






SpRY




 


594
GGAAGG
ACTGTGTGCAGTGGAAGACTC
21905
cCas9-v17
56
0


 


595
GGAAGG
ACTGTGTGCAGTGGAAGACTC
21906
cCas9-v42
56
0


 


596
GGAA
CTGTGTGCAGTGGAAGACTC
21907
SpyCas9-
56
0






3var-NRRH




 


597
GGAA
CTGTGTGCAGTGGAAGACTC
21908
SpyCas9-
56
0






VQR




 


598
CGGAA
tgTACTGTGTGCAGTGGAAGAC
21909
SauCas9
57
0




T






 


599
CGGAA
TACTGTGTGCAGTGGAAGACT
21910
SauCas9KKH
57
0


 


600
CGG
ACTGTGTGCAGTGGAAGACT
21911
ScaCas9
57
0


 


601
CGG
ACTGTGTGCAGTGGAAGACT
21912
ScaCas9-
57
0






HiFi-Sc++




 


602
CGG
ACTGTGTGCAGTGGAAGACT
21913
ScaCas9-
57
0






Sc++




 


603
CGG
ACTGTGTGCAGTGGAAGACT
21914
SpyCas9
57
0


 


604
CGG
ACTGTGTGCAGTGGAAGACT
21915
SpyCas9-
57
0






HF1




 


605
CGG
ACTGTGTGCAGTGGAAGACT
21916
SpyCas9-
57
0






SpG




 


606
CGG
ACTGTGTGCAGTGGAAGACT
21917
SpyCas9-
57
0






SpRY




 


607
CG
ACTGTGTGCAGTGGAAGACT
21918
SpyCas9-
57
0






NG




 


608
CG
ACTGTGTGCAGTGGAAGACT
21919
SpyCas9-
57
0






xCas




 


609
CG
ACTGTGTGCAGTGGAAGACT
21920
SpyCas9-
57
0






xCas-NG




 


610
AG
CCTCTGACTCAGTGGTGATG
21921
SpyCas9-
57
0






NG




 


611
AG
CCTCTGACTCAGTGGTGATG
21922
SpyCas9-
57
0






xCas




 


612
AG
CCTCTGACTCAGTGGTGATG
21923
SpyCas9-
57
0






xCas-NG




 


613
AGC
CCTCTGACTCAGTGGTGATG
21924
SpyCas9-
57
0






SpG




 


614
AGC
CCTCTGACTCAGTGGTGATG
21925
SpyCas9-
57
0






SpRY




 


615
CGGAAG
TACTGTGTGCAGTGGAAGACT
21926
cCas9-v17
57
0


 


616
CGGAAG
TACTGTGTGCAGTGGAAGACT
21927
cCas9-v42
57
0


 


617
CGGA
ACTGTGTGCAGTGGAAGACT
21928
SpyCas9-
57
0






3var-NRRH




 


618
AGCT
CCTCTGACTCAGTGGTGATG
21929
SpyCas9-
57
0






3var-NRCH




 


619
TCGGA
atGTACTGTGTGCAGTGGAAGA
21930
SauCas9
58
0




C






 


620
TCGGA
GTACTGTGTGCAGTGGAAGAC
21931
SauCas9KKH
58
0


 


621
TCGG
GTACTGTGTGCAGTGGAAGAC
21932
SauriCas9
58
0


 


622
TCGG
GTACTGTGTGCAGTGGAAGAC
21933
SauriCas9-
58
0






KKH




 


623
TCG
TACTGTGTGCAGTGGAAGAC
21934
ScaCas9
58
0


 


624
TCG
TACTGTGTGCAGTGGAAGAC
21935
ScaCas9-
58
0






HiFi-Sc++




 


625
TCG
TACTGTGTGCAGTGGAAGAC
21936
ScaCas9-
58
0






Sc++




 


626
TCG
TACTGTGTGCAGTGGAAGAC
21937
SpyCas9-
58
0






SpRY




 


627
GAG
GCCTCTGACTCAGTGGTGAT
21938
ScaCas9
58
0


 


628
GAG
GCCTCTGACTCAGTGGTGAT
21939
ScaCas9-
58
0






HiFi-Sc++




 


629
GAG
GCCTCTGACTCAGTGGTGAT
21940
ScaCas9-
58
0






Sc++




 


630
GAG
GCCTCTGACTCAGTGGTGAT
21941
SpyCas9-
58
0






SpRY




 


631
TCGGAA
GTACTGTGTGCAGTGGAAGAC
21942
cCas9-v17
58
0


 


632
TCGGAA
GTACTGTGTGCAGTGGAAGAC
21943
cCas9-v42
58
0


 


633
GAGCTTT
GTGCCTCTGACTCAGTGGTGA
21944
CdiCas9
58
0




T






 


634
GAGC
GCCTCTGACTCAGTGGTGAT
21945
SpyCas9-
58
0






3var-NRRH




 


635
CTCGG
TGTACTGTGTGCAGTGGAAGA
21946
SauCas9KKH
59
0


 


636
TGAG
GTGCCTCTGACTCAGTGGTGA
21947
SauriCas9-
59
0






KKH




 


637
TGAG
TGCCTCTGACTCAGTGGTGA
21948
SpyCas9-
59
0






VQR




 


638
TG
TGCCTCTGACTCAGTGGTGA
21949
SpyCas9-
59
0






NG




 


639
TG
TGCCTCTGACTCAGTGGTGA
21950
SpyCas9-
59
0






xCas




 


640
TG
TGCCTCTGACTCAGTGGTGA
21951
SpyCas9-
59
0






xCas-NG




 


641
TGA
TGCCTCTGACTCAGTGGTGA
21952
SpyCas9-
59
0






SpG




 


642
TGA
TGCCTCTGACTCAGTGGTGA
21953
SpyCas9-
59
0






SpRY




 


643
CTC
GTACTGTGTGCAGTGGAAGA
21954
SpyCas9-
59
0






SpRY




 


644
TGAGCTT
tagtGCCTCTGACTCAGTGGTGA
21955
BlatCas9
59
0



T







 


645
TGAGC
tagtGCCTCTGACTCAGTGGTGA
21956
BlatCas9
59
0


 


646
TGAGCT
GTGCCTCTGACTCAGTGGTGA
21957
cCas9-v16
59
0


 


647
TGAGCT
GTGCCTCTGACTCAGTGGTGA
21958
cCas9-v21
59
0


 


648
CTCGGA
TGTACTGTGTGCAGTGGAAGA
21959
cCas9-v17
59
0


 


649
CTCGGA
TGTACTGTGTGCAGTGGAAGA
21960
cCas9-v42
59
0


 


650
ATGAG
ctAGTGCCTCTGACTCAGTGGT
21961
SauCas9
60
0




G






 


651
ATGAG
AGTGCCTCTGACTCAGTGGTG
21962
SauCas9KKH
60
0


 


652
ATG
GTGCCTCTGACTCAGTGGTG
21963
ScaCas9
60
0


 


653
ATG
GTGCCTCTGACTCAGTGGTG
21964
ScaCas9-
60
0






HiFi-Sc++




 


654
ATG
GTGCCTCTGACTCAGTGGTG
21965
ScaCas9-
60
0






Sc++




 


655
ATG
GTGCCTCTGACTCAGTGGTG
21966
SpyCas9-
60
0






SpRY




 


656
ACT
TGTACTGTGTGCAGTGGAAG
21967
SpyCas9-
60
0






SpRY




 


657
ATGAGC
AGTGCCTCTGACTCAGTGGTG
21968
cCas9-v17
60
0


 


658
ATGAGC
AGTGCCTCTGACTCAGTGGTG
21969
cCas9-v42
60
0


 


659
ATGAGCT
cctaGTGCCTCTGACTCAGTGGT
21970
NmeCas9
60
0



T
G






 


660
GATGA
TAGTGCCTCTGACTCAGTGGT
21971
SauCas9KKH
61
0


 


661
GAC
ATGTACTGTGTGCAGTGGAA
21972
SpyCas9-
61
0






SpRY




 


662
GAT
AGTGCCTCTGACTCAGTGGT
21973
SpyCas9-
61
0






SpRY




 


663
GAT
AGTGCCTCTGACTCAGTGGT
21974
SpyCas9-
61
0






xCas




 


664
GACTCGG
ctgaTGTACTGTGTGCAGTGGAA
21975
BlatCas9
61
0



A







 


665
GACTC
ctgaTGTACTGTGTGCAGTGGAA
21976
BlatCas9
61
0


 


666
GACT
ATGTACTGTGTGCAGTGGAA
21977
SpyCas9-
61
0






3var-NRCH




 


667
AG
GATGTACTGTGTGCAGTGGA
21978
SpyCas9-
62
0






NG




 


668
AG
GATGTACTGTGTGCAGTGGA
21979
SpyCas9-
62
0






xCas




 


669
AG
GATGTACTGTGTGCAGTGGA
21980
SpyCas9-
62
0






xCas-NG




 


670
TG
TAGTGCCTCTGACTCAGTGG
21981
SpyCas9-
62
0






NG




 


671
TG
TAGTGCCTCTGACTCAGTGG
21982
SpyCas9-
62
0






xCas




 


672
TG
TAGTGCCTCTGACTCAGTGG
21983
SpyCas9-
62
0






xCas-NG




 


673
AGA
GATGTACTGTGTGCAGTGGA
21984
SpyCas9-
62
0






SpG




 


674
AGA
GATGTACTGTGTGCAGTGGA
21985
SpyCas9-
62
0






SpRY




 


675
TGA
TAGTGCCTCTGACTCAGTGG
21986
SpyCas9-
62
0






SpG




 


676
TGA
TAGTGCCTCTGACTCAGTGG
21987
SpyCas9-
62
0






SpRY




 


677
AGAC
GATGTACTGTGTGCAGTGGA
21988
SpyCas9-
62
0






3var-NRRH




 


678
AGAC
GATGTACTGTGTGCAGTGGA
21989
SpyCas9-
62
0






VQR




 


679
TGAT
TAGTGCCTCTGACTCAGTGG
21990
SpyCas9-
62
0






3var-NRRH




 


680
TGAT
TAGTGCCTCTGACTCAGTGG
21991
SpyCas9-
62
0






VQR




 


681
AAG
TGATGTACTGTGTGCAGTGG
21992
ScaCas9
63
0


 


682
AAG
TGATGTACTGTGTGCAGTGG
21993
ScaCas9-
63
0






HiFi-Sc++




 


683
AAG
TGATGTACTGTGTGCAGTGG
21994
ScaCas9-
63
0






Sc++




 


684
AAG
TGATGTACTGTGTGCAGTGG
21995
SpyCas9-
63
0






SpRY




 


685
GTG
CTAGTGCCTCTGACTCAGTG
21996
ScaCas9
63
0


 


686
GTG
CTAGTGCCTCTGACTCAGTG
21997
ScaCas9-
63
0






HiFi-Sc++




 


687
GTG
CTAGTGCCTCTGACTCAGTG
21998
ScaCas9-
63
0






Sc++




 


688
GTG
CTAGTGCCTCTGACTCAGTG
21999
SpyCas9-
63
0






SpRY




 


689
AAGAC
gtctGATGTACTGTGTGCAGTGG
22000
BlatCas9
63
0


 


690
AAGACT
CTGATGTACTGTGTGCAGTGG
22001
cCas9-v16
63
0


 


691
AAGACT
CTGATGTACTGTGTGCAGTGG
22002
cCas9-v21
63
0


 


692
GTGATGA
CCTAGTGCCTCTGACTCAGTG
22003
cCas9-v16
63
0


 


693
GTGATGA
CCTAGTGCCTCTGACTCAGTG
22004
cCas9-v21
63
0


 


694
AAGACTC
TCTGATGTACTGTGTGCAGTG
22005
CdiCas9
63
0




G






 


695
AAGA
TGATGTACTGTGTGCAGTGG
22006
SpyCas9-
63
0






3var-NRRH




 


696
GAAGA
TCTGATGTACTGTGTGCAGTG
22007
SauCas9KKH
64
0


 


697
GGTGA
TCCTAGTGCCTCTGACTCAGT
22008
SauCas9KKH
64
0


 


698
GGTGAT
TCCTAGTGCCTCTGACTCAGT
22009
SauCas9KKH
64
0


 


699
GAAG
TCTGATGTACTGTGTGCAGTG
22010
SauriCas9-
64
0






KKH




 


700
GAAG
CTGATGTACTGTGTGCAGTG
22011
SpyCas9-
64
0






QQR1




 


701
GAAG
tcTGATGTACTGTGTGCAGTG
22012
iSpyMacCas9
64
0


 


702
GG
CCTAGTGCCTCTGACTCAGT
22013
SpyCas9-
64
0






NG




 


703
GG
CCTAGTGCCTCTGACTCAGT
22014
SpyCas9-
64
0






xCas




 


704
GG
CCTAGTGCCTCTGACTCAGT
22015
SpyCas9-
64
0






xCas-NG




 


705
GAA
CTGATGTACTGTGTGCAGTG
22016
SpyCas9-
64
0






SpRY




 


706
GAA
CTGATGTACTGTGTGCAGTG
22017
SpyCas9-
64
0






xCas




 


707
GGT
CCTAGTGCCTCTGACTCAGT
22018
SpyCas9-
64
0






SpG




 


708
GGT
CCTAGTGCCTCTGACTCAGT
22019
SpyCas9-
64
0






SpRY




 


709
GAAGAC
TCTGATGTACTGTGTGCAGTG
22020
cCas9-v17
64
0


 


710
GAAGAC
TCTGATGTACTGTGTGCAGTG
22021
cCas9-v42
64
0


 


711
GGAAG
GTCTGATGTACTGTGTGCAGT
22022
SauCas9KKH
65
0


 


712
TGG
TCCTAGTGCCTCTGACTCAG
22023
ScaCas9
65
0


 


713
TGG
TCCTAGTGCCTCTGACTCAG
22024
ScaCas9-
65
0






HiFi-Sc++




 


714
TGG
TCCTAGTGCCTCTGACTCAG
22025
ScaCas9-
65
0






Sc++




 


715
TGG
TCCTAGTGCCTCTGACTCAG
22026
SpyCas9
65
0


 


716
TGG
TCCTAGTGCCTCTGACTCAG
22027
SpyCas9-
65
0






HF1




 


717
TGG
TCCTAGTGCCTCTGACTCAG
22028
SpyCas9-
65
0






SpG




 


718
TGG
TCCTAGTGCCTCTGACTCAG
22029
SpyCas9-
65
0






SpRY




 


719
GG
TCTGATGTACTGTGTGCAGT
22030
SpyCas9-
65
0






NG




 


720
GG
TCTGATGTACTGTGTGCAGT
22031
SpyCas9-
65
0






xCas




 


721
GG
TCTGATGTACTGTGTGCAGT
22032
SpyCas9-
65
0






xCas-NG




 


722
TG
TCCTAGTGCCTCTGACTCAG
22033
SpyCas9-
65
0






NG




 


723
TG
TCCTAGTGCCTCTGACTCAG
22034
SpyCas9-
65
0






xCas




 


724
TG
TCCTAGTGCCTCTGACTCAG
22035
SpyCas9-
65
0






xCas-NG




 


725
GGA
TCTGATGTACTGTGTGCAGT
22036
SpyCas9-
65
0






SpG




 


726
GGA
TCTGATGTACTGTGTGCAGT
22037
SpyCas9-
65
0






SpRY




 


727
GGAAGA
GTCTGATGTACTGTGTGCAGT
22038
cCas9-v17
65
0


 


728
GGAAGA
GTCTGATGTACTGTGTGCAGT
22039
cCas9-v42
65
0


 


729
GGAAGAC
catgTCTGATGTACTGTGTGCAG
22040
NmeCas9
65
0



T
T






 


730
GGAA
TCTGATGTACTGTGTGCAGT
22041
SpyCas9-
65
0






3var-NRRH




 


731
GGAA
TCTGATGTACTGTGTGCAGT
22042
SpyCas9-
65
0






VQR




 


732
TGGT
TCCTAGTGCCTCTGACTCAG
22043
SpyCas9-
65
0






3var-NRRH




 


733
TGGAA
caTGTCTGATGTACTGTGTGCA
22044
SauCas9
66
0




G






 


734
TGGAA
TGTCTGATGTACTGTGTGCAG
22045
SauCas9KKH
66
0


 


735
GTGG
TCTCCTAGTGCCTCTGACTCA
22046
SauriCas9
66
0


 


736
GTGG
TCTCCTAGTGCCTCTGACTCA
22047
SauriCas9-
66
0






KKH




 


737
TGG
GTCTGATGTACTGTGTGCAG
22048
ScaCas9
66
0


 


738
TGG
GTCTGATGTACTGTGTGCAG
22049
ScaCas9-
66
0






HiFi-Sc++




 


739
TGG
GTCTGATGTACTGTGTGCAG
22050
ScaCas9-
66
0






Sc++




 


740
TGG
GTCTGATGTACTGTGTGCAG
22051
SpyCas9
66
0


 


741
TGG
GTCTGATGTACTGTGTGCAG
22052
SpyCas9-
66
0






HF1




 


742
TGG
GTCTGATGTACTGTGTGCAG
22053
SpyCas9-
66
0






SpG




 


743
TGG
GTCTGATGTACTGTGTGCAG
22054
SpyCas9-
66
0






SpRY




 


744
GTG
CTCCTAGTGCCTCTGACTCA
22055
ScaCas9
66
0


 


745
GTG
CTCCTAGTGCCTCTGACTCA
22056
ScaCas9-
66
0






HiFi-Sc++




 


746
GTG
CTCCTAGTGCCTCTGACTCA
22057
ScaCas9-
66
0






Sc++




 


747
GTG
CTCCTAGTGCCTCTGACTCA
22058
SpyCas9-
66
0






SpRY




 


748
TG
GTCTGATGTACTGTGTGCAG
22059
SpyCas9-
66
0






NG




 


749
TG
GTCTGATGTACTGTGTGCAG
22060
SpyCas9-
66
0






xCas




 


750
TG
GTCTGATGTACTGTGTGCAG
22061
SpyCas9-
66
0






xCas-NG




 


751
GTGGTG
TCTCCTAGTGCCTCTGACTCA
22062
cCas9-v16
66
0


 


752
GTGGTG
TCTCCTAGTGCCTCTGACTCA
22063
cCas9-v21
66
0


 


753
TGGAAG
TGTCTGATGTACTGTGTGCAG
22064
cCas9-v17
66
0


 


754
TGGAAG
TGTCTGATGTACTGTGTGCAG
22065
cCas9-v42
66
0


 


755
TGGA
GTCTGATGTACTGTGTGCAG
22066
SpyCas9-
66
0






3var-NRRH




 


756
GTGGA
ccATGTCTGATGTACTGTGTGC
22067
SauCas9
67
0




A






 


757
GTGGA
ATGTCTGATGTACTGTGTGCA
22068
SauCas9KKH
67
0


 


758
AGTGG
GTCTCCTAGTGCCTCTGACTC
22069
SauCas9KKH
67
0


 


759
AGTGGT
GTCTCCTAGTGCCTCTGACTC
22070
SauCas9KKH
67
0


 


760
GTGG
ATGTCTGATGTACTGTGTGCA
22071
SauriCas9
67
0


 


761
GTGG
ATGTCTGATGTACTGTGTGCA
22072
SauriCas9-
67
0






KKH




 


762
GTG
TGTCTGATGTACTGTGTGCA
22073
ScaCas9
67
0


 


763
GTG
TGTCTGATGTACTGTGTGCA
22074
ScaCas9-
67
0






HiFi-Sc++




 


764
GTG
TGTCTGATGTACTGTGTGCA
22075
ScaCas9-
67
0






Sc++




 


765
GTG
TGTCTGATGTACTGTGTGCA
22076
SpyCas9-
67
0






SpRY




 


766
AG
TCTCCTAGTGCCTCTGACTC
22077
SpyCas9-
67
0






NG




 


767
AG
TCTCCTAGTGCCTCTGACTC
22078
SpyCas9-
67
0






xCas




 


768
AG
TCTCCTAGTGCCTCTGACTC
22079
SpyCas9-
67
0






xCas-NG




 


769
AGT
TCTCCTAGTGCCTCTGACTC
22080
SpyCas9-
67
0






SpG




 


770
AGT
TCTCCTAGTGCCTCTGACTC
22081
SpyCas9-
67
0






SpRY




 


771
GTGGAA
ATGTCTGATGTACTGTGTGCA
22082
cCas9-v17
67
0


 


772
GTGGAA
ATGTCTGATGTACTGTGTGCA
22083
cCas9-v42
67
0


 


773
AGTGG
CATGTCTGATGTACTGTGTGC
22084
SauCas9KKH
68
0


 


774
CAG
GTCTCCTAGTGCCTCTGACT
22085
ScaCas9
68
0


 


775
CAG
GTCTCCTAGTGCCTCTGACT
22086
ScaCas9-
68
0






HiFi-Sc++




 


776
CAG
GTCTCCTAGTGCCTCTGACT
22087
ScaCas9-
68
0






Sc++




 


777
CAG
GTCTCCTAGTGCCTCTGACT
22088
SpyCas9-
68
0






SpRY




 


778
AG
ATGTCTGATGTACTGTGTGC
22089
SpyCas9-
68
0






NG




 


779
AG
ATGTCTGATGTACTGTGTGC
22090
SpyCas9-
68
0






xCas




 


780
AG
ATGTCTGATGTACTGTGTGC
22091
SpyCas9-
68
0






xCas-NG




 


781
AGT
ATGTCTGATGTACTGTGTGC
22092
SpyCas9-
68
0






SpG




 


782
AGT
ATGTCTGATGTACTGTGTGC
22093
SpyCas9-
68
0






SpRY




 


783
CAGT
GTCTCCTAGTGCCTCTGACT
22094
SpyCas9-
68
0






3var-NRRH




 


784
TCAG
AGGTCTCCTAGTGCCTCTGAC
22095
SauriCas9-
69
0






KKH




 


785
CAG
CATGTCTGATGTACTGTGTG
22096
ScaCas9
69
0


 


786
CAG
CATGTCTGATGTACTGTGTG
22097
ScaCas9-
69
0






HiFi-Sc++




 


787
CAG
CATGTCTGATGTACTGTGTG
22098
ScaCas9-
69
0






Sc++




 


788
CAG
CATGTCTGATGTACTGTGTG
22099
SpyCas9-
69
0






SpRY




 


789
TCA
GGTCTCCTAGTGCCTCTGAC
22100
SpyCas9-
69
0






SpRY




 


790
TCAGTG
AGGTCTCCTAGTGCCTCTGAC
22101
cCas9-v16
69
0


 


791
TCAGTG
AGGTCTCCTAGTGCCTCTGAC
22102
cCas9-v21
69
0


 


792
CAGT
CATGTCTGATGTACTGTGTG
22103
SpyCas9-
69
0






3var-NRRH




 


793
CTCAG
AAGGTCTCCTAGTGCCTCTGA
22104
SauCas9KKH
70
0


 


794
CTCAGT
AAGGTCTCCTAGTGCCTCTGA
22105
SauCas9KKH
70
0


 


795
CTCAGT
AAGGTCTCCTAGTGCCTCTGA
22106
cCas9-v17
70
0


 


796
CTCAGT
AAGGTCTCCTAGTGCCTCTGA
22107
cCas9-v42
70
0


 


797
GCAG
TCCATGTCTGATGTACTGTGT
22108
SauriCas9-
70
0






KKH




 


798
GCA
CCATGTCTGATGTACTGTGT
22109
SpyCas9-
70
0






SpRY




 


799
CTC
AGGTCTCCTAGTGCCTCTGA
22110
SpyCas9-
70
0






SpRY




 


800
GCAGTG
TCCATGTCTGATGTACTGTGT
22111
cCas9-v16
70
0


 


801
GCAGTG
TCCATGTCTGATGTACTGTGT
22112
cCas9-v21
70
0


 


802
TGCAG
ATCCATGTCTGATGTACTGTG
22113
SauCas9KKH
71
0


 


803
TGCAGT
ATCCATGTCTGATGTACTGTG
22114
SauCas9KKH
71
0


 


804
TGCAGT
ATCCATGTCTGATGTACTGTG
22115
cCas9-v17
71
0


 


805
TGCAGT
ATCCATGTCTGATGTACTGTG
22116
cCas9-v42
71
0


 


806
TG
TCCATGTCTGATGTACTGTG
22117
SpyCas9-
71
0






NG




 


807
TG
TCCATGTCTGATGTACTGTG
22118
SpyCas9-
71
0






xCas




 


808
TG
TCCATGTCTGATGTACTGTG
22119
SpyCas9-
71
0






xCas-NG




 


809
TGC
TCCATGTCTGATGTACTGTG
22120
SpyCas9-
71
0






SpG




 


810
TGC
TCCATGTCTGATGTACTGTG
22121
SpyCas9-
71
0






SpRY




 


811
ACT
AAGGTCTCCTAGTGCCTCTG
22122
SpyCas9-
71
0






SpRY




 


812
TGCA
TCCATGTCTGATGTACTGTG
22123
SpyCas9-
71
0






3var-NRCH




 


813
GTG
ATCCATGTCTGATGTACTGT
22124
ScaCas9
72
0


 


814
GTG
ATCCATGTCTGATGTACTGT
22125
ScaCas9-
72
0






HiFi-Sc++




 


815
GTG
ATCCATGTCTGATGTACTGT
22126
ScaCas9-
72
0






Sc++




 


816
GTG
ATCCATGTCTGATGTACTGT
22127
SpyCas9-
72
0






SpRY




 


817
GAC
AAAGGTCTCCTAGTGCCTCT
22128
SpyCas9-
72
0






SpRY




 


818
GACTCAG
cctaAAGGTCTCCTAGTGCCTCT
22129
BlatCas9
72
0



T







 


819
GACTC
cctaAAGGTCTCCTAGTGCCTCT
22130
BlatCas9
72
0


 


820
GACT
AAAGGTCTCCTAGTGCCTCT
22131
SpyCas9-
72
0






3var-NRCH




 


821
TG
GATCCATGTCTGATGTACTG
22132
SpyCas9-
73
0






NG




 


822
TG
GATCCATGTCTGATGTACTG
22133
SpyCas9-
73
0






xCas




 


823
TG
GATCCATGTCTGATGTACTG
22134
SpyCas9-
73
0






xCas-NG




 


824
TG
TAAAGGTCTCCTAGTGCCTC
22135
SpyCas9-
73
0






NG




 


825
TG
TAAAGGTCTCCTAGTGCCTC
22136
SpyCas9-
73
0






xCas




 


826
TG
TAAAGGTCTCCTAGTGCCTC
22137
SpyCas9-
73
0






xCas-NG




 


827
TGT
GATCCATGTCTGATGTACTG
22138
SpyCas9-
73
0






SpG




 


828
TGT
GATCCATGTCTGATGTACTG
22139
SpyCas9-
73
0






SpRY




 


829
TGA
TAAAGGTCTCCTAGTGCCTC
22140
SpyCas9-
73
0






SpG




 


830
TGA
TAAAGGTCTCCTAGTGCCTC
22141
SpyCas9-
73
0






SpRY




 


831
TGTGCAG
ttggATCCATGTCTGATGTACTG
22142
BlatCas9
73
0



T







 


832
TGTGC
ttggATCCATGTCTGATGTACTG
22143
BlatCas9
73
0


 


833
TGAC
TAAAGGTCTCCTAGTGCCTC
22144
SpyCas9-
73
0






3var-NRRH




 


834
TGAC
TAAAGGTCTCCTAGTGCCTC
22145
SpyCas9-
73
0






VQR




 


835
GTG
GGATCCATGTCTGATGTACT
22146
ScaCas9
74
0


 


836
GTG
GGATCCATGTCTGATGTACT
22147
ScaCas9-
74
0






HiFi-Sc++




 


837
GTG
GGATCCATGTCTGATGTACT
22148
ScaCas9-
74
0






Sc++




 


838
GTG
GGATCCATGTCTGATGTACT
22149
SpyCas9-
74
0






SpRY




 


839
CTG
CTAAAGGTCTCCTAGTGCCT
22150
ScaCas9
74
0


 


840
CTG
CTAAAGGTCTCCTAGTGCCT
22151
ScaCas9-
74
0






HiFi-Sc++




 


841
CTG
CTAAAGGTCTCCTAGTGCCT
22152
ScaCas9-
74
0






Sc++




 


842
CTG
CTAAAGGTCTCCTAGTGCCT
22153
SpyCas9-
74
0






SpRY




 


843
CTGAC
taccTAAAGGTCTCCTAGTGCCT
22154
BlatCas9
74
0


 


844
CTGACT
CCTAAAGGTCTCCTAGTGCCT
22155
cCas9-v16
74
0


 


845
CTGACT
CCTAAAGGTCTCCTAGTGCCT
22156
cCas9-v21
74
0


 


846
CTGACTC
ACCTAAAGGTCTCCTAGTGCC
22157
CdiCas9
74
0




T






 


847
TCTGA
ACCTAAAGGTCTCCTAGTGCC
22158
SauCas9KKH
75
0


 


848
TG
TGGATCCATGTCTGATGTAC
22159
SpyCas9-
75
0






NG




 


849
TG
TGGATCCATGTCTGATGTAC
22160
SpyCas9-
75
0






xCas




 


850
TG
TGGATCCATGTCTGATGTAC
22161
SpyCas9-
75
0






xCas-NG




 


851
TGT
TGGATCCATGTCTGATGTAC
22162
SpyCas9-
75
0






SpG




 


852
TGT
TGGATCCATGTCTGATGTAC
22163
SpyCas9-
75
0






SpRY




 


853
TCT
CCTAAAGGTCTCCTAGTGCC
22164
SpyCas9-
75
0






SpRY




 


854
CTG
TTGGATCCATGTCTGATGTA
22165
ScaCas9
76
0


 


855
CTG
TTGGATCCATGTCTGATGTA
22166
ScaCas9-
76
0






HiFi-Sc++




 


856
CTG
TTGGATCCATGTCTGATGTA
22167
ScaCas9-
76
0






Sc++




 


857
CTG
TTGGATCCATGTCTGATGTA
22168
SpyCas9-
76
0






SpRY




 


858
CTC
ACCTAAAGGTCTCCTAGTGC
22169
SpyCas9-
76
0






SpRY




 


859
CTCTGAC
cactACCTAAAGGTCTCCTAGTG
22170
NmeCas9
76
0



T
C






 


860
ACT
CTTGGATCCATGTCTGATGT
22171
SpyCas9-
77
0






SpRY




 


861
CCT
TACCTAAAGGTCTCCTAGTG
22172
SpyCas9-
77
0






SpRY




 


862
TAC
GCTTGGATCCATGTCTGATG
22173
SpyCas9-
78
0






SpRY




 


863
GCC
CTACCTAAAGGTCTCCTAGT
22174
SpyCas9-
78
0






SpRY




 


864
GCCTCTG
ccacTACCTAAAGGTCTCCTAGT
22175
BlatCas9
78
0



A







 


865
GCCTC
ccacTACCTAAAGGTCTCCTAGT
22176
BlatCas9
78
0


 


866
TACT
GCTTGGATCCATGTCTGATG
22177
SpyCas9-
78
0






3var-NRCH




 


867
TG
ACTACCTAAAGGTCTCCTAG
22178
SpyCas9-
79
0






NG




 


868
TG
ACTACCTAAAGGTCTCCTAG
22179
SpyCas9-
79
0






xCas




 


869
TG
ACTACCTAAAGGTCTCCTAG
22180
SpyCas9-
79
0






xCas-NG




 


870
TGC
ACTACCTAAAGGTCTCCTAG
22181
SpyCas9-
79
0






SpG




 


871
TGC
ACTACCTAAAGGTCTCCTAG
22182
SpyCas9-
79
0






SpRY




 


872
GTA
GGCTTGGATCCATGTCTGAT
22183
SpyCas9-
79
0






SpRY




 


873
TGCC
ACTACCTAAAGGTCTCCTAG
22184
SpyCas9-
79
0






3var-NRCH




 


874
GTG
CACTACCTAAAGGTCTCCTA
22185
ScaCas9
80
0


 


875
GTG
CACTACCTAAAGGTCTCCTA
22186
ScaCas9-
80
0






HiFi-Sc++




 


876
GTG
CACTACCTAAAGGTCTCCTA
22187
ScaCas9-
80
0






Sc++




 


877
GTG
CACTACCTAAAGGTCTCCTA
22188
SpyCas9-
80
0






SpRY




 


878
TG
GGGCTTGGATCCATGTCTGA
22189
SpyCas9-
80
0






NG




 


879
TG
GGGCTTGGATCCATGTCTGA
22190
SpyCas9-
80
0






xCas




 


880
TG
GGGCTTGGATCCATGTCTGA
22191
SpyCas9-
80
0






xCas-NG




 


881
TGT
GGGCTTGGATCCATGTCTGA
22192
SpyCas9-
80
0






SpG




 


882
TGT
GGGCTTGGATCCATGTCTGA
22193
SpyCas9-
80
0






SpRY




 


883
TGTACTG
catgGGCTTGGATCCATGTCTGA
22194
BlatCas9
80
0



T







 


884
TGTAC
catgGGCTTGGATCCATGTCTGA
22195
BlatCas9
80
0


 


885
GTGCC
ctccACTACCTAAAGGTCTCCTA
22196
BlatCas9
80
0


 


886
GTGCCTC
TCCACTACCTAAAGGTCTCCT
22197
CdiCas9
80
0




A






 


887
TGTA
GGGCTTGGATCCATGTCTGA
22198
SpyCas9-
80
0






3var-NRTH




 


888
AGTGCC
taCTCCACTACCTAAAGGTCTC
22199
Nme2Cas9
81
0




CT






 


889
ATG
TGGGCTTGGATCCATGTCTG
22200
ScaCas9
81
0


 


890
ATG
TGGGCTTGGATCCATGTCTG
22201
ScaCas9-
81
0






HiFi-Sc++




 


891
ATG
TGGGCTTGGATCCATGTCTG
22202
ScaCas9-
81
0






Sc++




 


892
ATG
TGGGCTTGGATCCATGTCTG
22203
SpyCas9-
81
0






SpRY




 


893
AG
CCACTACCTAAAGGTCTCCT
22204
SpyCas9-
81
0






NG




 


894
AG
CCACTACCTAAAGGTCTCCT
22205
SpyCas9-
81
0






xCas




 


895
AG
CCACTACCTAAAGGTCTCCT
22206
SpyCas9-
81
0






xCas-NG




 


896
AGT
CCACTACCTAAAGGTCTCCT
22207
SpyCas9-
81
0






SpG




 


897
AGT
CCACTACCTAAAGGTCTCCT
22208
SpyCas9-
81
0






SpRY




 


898
AGTGC
actcCACTACCTAAAGGTCTCCT
22209
BlatCas9
81
0


 


899
ATGTACT
CATGGGCTTGGATCCATGTCT
22210
CdiCas9
81
0




G






 


900
TAG
TCCACTACCTAAAGGTCTCC
22211
ScaCas9
82
0


 


901
TAG
TCCACTACCTAAAGGTCTCC
22212
ScaCas9-
82
0






HiFi-Sc++




 


902
TAG
TCCACTACCTAAAGGTCTCC
22213
ScaCas9-
82
0






Sc++




 


903
TAG
TCCACTACCTAAAGGTCTCC
22214
SpyCas9-
82
0






SpRY




 


904
GAT
ATGGGCTTGGATCCATGTCT
22215
SpyCas9-
82
0






SpRY




 


905
GAT
ATGGGCTTGGATCCATGTCT
22216
SpyCas9-
82
0






xCas




 


906
TAGT
TCCACTACCTAAAGGTCTCC
22217
SpyCas9-
82
0






3var-NRRH




 


907
CTAG
ACTCCACTACCTAAAGGTCTC
22218
SauriCas9-
83
0






KKH




 


908
TG
CATGGGCTTGGATCCATGTC
22219
SpyCas9-
83
0






NG




 


909
TG
CATGGGCTTGGATCCATGTC
22220
SpyCas9-
83
0






xCas




 


910
TG
CATGGGCTTGGATCCATGTC
22221
SpyCas9-
83
0






xCas-NG




 


911
TGA
CATGGGCTTGGATCCATGTC
22222
SpyCas9-
83
0






SpG




 


912
TGA
CATGGGCTTGGATCCATGTC
22223
SpyCas9-
83
0






SpRY




 


913
CTA
CTCCACTACCTAAAGGTCTC
22224
SpyCas9-
83
0






SpRY




 


914
CTAGTG
ACTCCACTACCTAAAGGTCTC
22225
cCas9-v16
83
0


 


915
CTAGTG
ACTCCACTACCTAAAGGTCTC
22226
cCas9-v21
83
0


 


916
TGATGTA
atACATGGGCTTGGATCCATGT
22227
CjeCas9
83
0



C
C






 


917
TGAT
CATGGGCTTGGATCCATGTC
22228
SpyCas9-
83
0






3var-NRRH




 


918
TGAT
CATGGGCTTGGATCCATGTC
22229
SpyCas9-
83
0






VQR




 


919
CCTAG
TACTCCACTACCTAAAGGTCT
22230
SauCas9KKH
84
0


 


920
CCTAGT
TACTCCACTACCTAAAGGTCT
22231
SauCas9KKH
84
0


 


921
CTG
ACATGGGCTTGGATCCATGT
22232
ScaCas9
84
0


 


922
CTG
ACATGGGCTTGGATCCATGT
22233
ScaCas9-
84
0






HiFi-Sc++




 


923
CTG
ACATGGGCTTGGATCCATGT
22234
ScaCas9-
84
0






Sc++




 


924
CTG
ACATGGGCTTGGATCCATGT
22235
SpyCas9-
84
0






SpRY




 


925
CCT
ACTCCACTACCTAAAGGTCT
22236
SpyCas9-
84
0






SpRY




 


926
TCTGA
ATACATGGGCTTGGATCCATG
22237
SauCas9KKH
85
0


 


927
TCTGAT
ATACATGGGCTTGGATCCATG
22238
SauCas9KKH
85
0


 


928
TCT
TACATGGGCTTGGATCCATG
22239
SpyCas9-
85
0






SpRY




 


929
TCC
TACTCCACTACCTAAAGGTC
22240
SpyCas9-
85
0






SpRY




 


930
GTC
ATACATGGGCTTGGATCCAT
22241
SpyCas9-
86
0






SpRY




 


931
CTC
CTACTCCACTACCTAAAGGT
22242
SpyCas9-
86
0






SpRY




 


932
TG
TATACATGGGCTTGGATCCA
22243
SpyCas9-
87
0






NG




 


933
TG
TATACATGGGCTTGGATCCA
22244
SpyCas9-
87
0






xCas




 


934
TG
TATACATGGGCTTGGATCCA
22245
SpyCas9-
87
0






xCas-NG




 


935
TGT
TATACATGGGCTTGGATCCA
22246
SpyCas9-
87
0






SpG




 


936
TGT
TATACATGGGCTTGGATCCA
22247
SpyCas9-
87
0






SpRY




 


937
TCT
ACTACTCCACTACCTAAAGG
22248
SpyCas9-
87
0






SpRY




 


938
TCTCCTA
tgtaCTACTCCACTACCTAAAGG
22249
BlatCas9
87
0



G







 


939
TCTCC
tgtaCTACTCCACTACCTAAAGG
22250
BlatCas9
87
0


 


940
TGTC
TATACATGGGCTTGGATCCA
22251
SpyCas9-
87
0






3var-NRTH




 


941
GTCTCC
tgTGTACTACTCCACTACCTAA
22252
Nme2Cas9
88
0




AG






 


942
ATG
GTATACATGGGCTTGGATCC
22253
ScaCas9
88
0


 


943
ATG
GTATACATGGGCTTGGATCC
22254
ScaCas9-
88
0






HiFi-Sc++




 


944
ATG
GTATACATGGGCTTGGATCC
22255
ScaCas9-
88
0






Sc++




 


945
ATG
GTATACATGGGCTTGGATCC
22256
SpyCas9-
88
0






SpRY




 


946
GTC
TACTACTCCACTACCTAAAG
22257
SpyCas9-
88
0






SpRY




 


947
ATGTCTG
ggggTATACATGGGCTTGGATC
22258
BlatCas9
88
0



A
C






 


948
GTCTCCT
gtgtACTACTCCACTACCTAAAG
22259
BlatCas9
88
0



A







 


949
ATGTC
ggggTATACATGGGCTTGGATC
22260
BlatCas9
88
0




C






 


950
GTCTC
gtgtACTACTCCACTACCTAAAG
22261
BlatCas9
88
0


 


951
GG
GTACTACTCCACTACCTAAA
22262
SpyCas9-
89
0






NG




 


952
GG
GTACTACTCCACTACCTAAA
22263
SpyCas9-
89
0






xCas




 


953
GG
GTACTACTCCACTACCTAAA
22264
SpyCas9-
89
0






xCas-NG




 


954
CAT
GGTATACATGGGCTTGGATC
22265
SpyCas9-
89
0






SpRY




 


955
GGT
GTACTACTCCACTACCTAAA
22266
SpyCas9-
89
0






SpG




 


956
GGT
GTACTACTCCACTACCTAAA
22267
SpyCas9-
89
0






SpRY




 


957
GGTC
GTACTACTCCACTACCTAAA
22268
SpyCas9-
89
0






3var-NRTH




 


958
AGG
TGTACTACTCCACTACCTAA
22269
ScaCas9
90
0


 


959
AGG
TGTACTACTCCACTACCTAA
22270
ScaCas9-
90
0






HiFi-Sc++




 


960
AGG
TGTACTACTCCACTACCTAA
22271
ScaCas9-
90
0






Sc++




 


961
AGG
TGTACTACTCCACTACCTAA
22272
SpyCas9
90
0


 


962
AGG
TGTACTACTCCACTACCTAA
22273
SpyCas9-
90
0






HF1




 


963
AGG
TGTACTACTCCACTACCTAA
22274
SpyCas9-
90
0






SpG




 


964
AGG
TGTACTACTCCACTACCTAA
22275
SpyCas9-
90
0






SpRY




 


965
AG
TGTACTACTCCACTACCTAA
22276
SpyCas9-
90
0






NG




 


966
AG
TGTACTACTCCACTACCTAA
22277
SpyCas9-
90
0






xCas




 


967
AG
TGTACTACTCCACTACCTAA
22278
SpyCas9-
90
0






xCas-NG




 


968
CCA
GGGTATACATGGGCTTGGAT
22279
SpyCas9-
90
0






SpRY




 


969
AGGTC
atgtGTACTACTCCACTACCTAA
22280
BlatCas9
90
0


 


970
AGGTCTC
TGTGTACTACTCCACTACCTA
22281
CdiCas9
90
0




A






 


971
CCATGTC
tcggGGGTATACATGGGCTTGG
22282
NmeCas9
90
0



T
AT






 


972
AGGT
TGTACTACTCCACTACCTAA
22283
SpyCas9-
90
0






3var-NRRH




 


973
AAGG
TGTGTACTACTCCACTACCTA
22284
SauriCas9
91
0


 


974
AAGG
TGTGTACTACTCCACTACCTA
22285
SauriCas9-
91
0






KKH




 


975
AAG
GTGTACTACTCCACTACCTA
22286
ScaCas9
91
0


 


976
AAG
GTGTACTACTCCACTACCTA
22287
ScaCas9-
91
0






HiFi-Sc++




 


977
AAG
GTGTACTACTCCACTACCTA
22288
ScaCas9-
91
0






Sc++




 


978
AAG
GTGTACTACTCCACTACCTA
22289
SpyCas9-
91
0






SpRY




 


979
TCC
GGGGTATACATGGGCTTGGA
22290
SpyCas9-
91
0






SpRY




 


980
AAAGG
ATGTGTACTACTCCACTACCT
22291
SauCas9KK
92
0






H




 


981
AAAGGT
ATGTGTACTACTCCACTACCT
22292
SauCas9KKH
92
0


 


982
AAAGGT
ATGTGTACTACTCCACTACCT
22293
cCas9-v17
92
0


 


983
AAAGGT
ATGTGTACTACTCCACTACCT
22294
cCas9-v42
92
0


 


984
AAAG
ATGTGTACTACTCCACTACCT
22295
SauriCas9-
92
0






KKH




 


985
AAAG
TGTGTACTACTCCACTACCT
22296
SpyCas9-
92
0






QQR1




 


986
AAAG
atGTGTACTACTCCACTACCT
22297
iSpyMacCas9
92
0


 


987
AAA
TGTGTACTACTCCACTACCT
22298
SpyCas9-
92
0






SpRY




 


988
ATC
GGGGGTATACATGGGCTTGG
22299
SpyCas9-
92
0






SpRY




 


989
AAAGGTC
gttaTGTGTACTACTCCACTACC
22300
NmeCas9
92
0



T
T






 


990
TAAAG
TATGTGTACTACTCCACTACC
22301
SauCas9KKH
93
0


 


991
GAT
CGGGGGTATACATGGGCTTG
22302
SpyCas9-
93
0






SpRY




 


992
GAT
CGGGGGTATACATGGGCTTG
22303
SpyCas9-
93
0






xCas




 


993
TAA
ATGTGTACTACTCCACTACC
22304
SpyCas9-
93
0






SpRY




 


994
GATCCAT
gttcGGGGGTATACATGGGCTTG
22305
BlatCas9
93
0



G







 


995
GATCC
gttcGGGGGTATACATGGGCTTG
22306
BlatCas9
93
0


 


996
TAAAGG
TATGTGTACTACTCCACTACC
22307
cCas9-v17
93
0


 


997
TAAAGG
TATGTGTACTACTCCACTACC
22308
cCas9-v42
93
0


 


998
TAAA
ATGTGTACTACTCCACTACC
22309
SpyCas9-
93
0






3var-NRRH




 


999
TAAA
taTGTGTACTACTCCACTACC
22310
iSpyMacCas9
93
0


 


1000
GATC
CGGGGGTATACATGGGCTTG
22311
SpyCas9-
93
0






3var-NRTH




 


1001
GGATCC
cgGTTCGGGGGTATACATGGG
22312
Nme2Cas9
94
0




CTT






 


1002
CTAAA
TTATGTGTACTACTCCACTAC
22313
SauCas9KKH
94
0


 


1003
GG
TCGGGGGTATACATGGGCTT
22314
SpyCas9-
94
0






NG




 


1004
GG
TCGGGGGTATACATGGGCTT
22315
SpyCas9-
94
0






xCas




 


1005
GG
TCGGGGGTATACATGGGCTT
22316
SpyCas9-
94
0






xCas-NG




 


1006
GGA
TCGGGGGTATACATGGGCTT
22317
SpyCas9-
94
0






SpG




 


1007
GGA
TCGGGGGTATACATGGGCTT
22318
SpyCas9-
94
0






SpRY




 


1008
CTA
TATGTGTACTACTCCACTAC
22319
SpyCas9-
94
0






SpRY




 


1009
GGATCCA
ggttCGGGGGTATACATGGGCTT
22320
BlatCas9
94
0



T







 


1010
GGATC
ggttCGGGGGTATACATGGGCTT
22321
BlatCas9
94
0


 


1011
CTAAAG
TTATGTGTACTACTCCACTAC
22322
cCas9-v17
94
0


 


1012
CTAAAG
TTATGTGTACTACTCCACTAC
22323
cCas9-v42
94
0


 


1013
GGAT
TCGGGGGTATACATGGGCTT
22324
SpyCas9-
94
0






3var-NRRH




 


1014
GGAT
TCGGGGGTATACATGGGCTT
22325
SpyCas9-
94
0






VQR




 


1015
CCTAA
GTTATGTGTACTACTCCACTA
22326
SauCas9KKH
95
0


 


1016
TGG
TTCGGGGGTATACATGGGCT
22327
ScaCas9
95
0


 


1017
TGG
TTCGGGGGTATACATGGGCT
22328
ScaCas9-
95
0






HiFi-Sc++




 


1018
TGG
TTCGGGGGTATACATGGGCT
22329
ScaCas9-
95
0






Sc++




 


1019
TGG
TTCGGGGGTATACATGGGCT
22330
SpyCas9
95
0


 


1020
TGG
TTCGGGGGTATACATGGGCT
22331
SpyCas9-
95
0






HF1




 


1021
TGG
TTCGGGGGTATACATGGGCT
22332
SpyCas9-
95
0






SpG




 


1022
TGG
TTCGGGGGTATACATGGGCT
22333
SpyCas9-
95
0






SpRY




 


1023
TG
TTCGGGGGTATACATGGGCT
22334
SpyCas9-
95
0






NG




 


1024
TG
TTCGGGGGTATACATGGGCT
22335
SpyCas9-
95
0






xCas




 


1025
TG
TTCGGGGGTATACATGGGCT
22336
SpyCas9-
95
0






xCas-NG




 


1026
CCT
TTATGTGTACTACTCCACTA
22337
SpyCas9-
95
0






SpRY




 


1027
TGGATCC
GGTTCGGGGGTATACATGGGC
22338
CdiCas9
95
0




T






 


1028
TGGA
TTCGGGGGTATACATGGGCT
22339
SpyCas9-
95
0






3var-NRRH




 


1029
TTGGA
acGGTTCGGGGGTATACATGG
22340
SauCas9
96
0




GC






 


1030
TTGGA
GGTTCGGGGGTATACATGGGC
22341
SauCas9KKH
96
0


 


1031
TTGGAT
acGGTTCGGGGGTATACATGG
22342
SauCas9
96
0




GC






 


1032
TTGGAT
GGTTCGGGGGTATACATGGGC
22343
SauCas9KKH
96
0


 


1033
TTGGAT
GGTTCGGGGGTATACATGGGC
22344
cCas9-v17
96
0


 


1034
TTGGAT
GGTTCGGGGGTATACATGGGC
22345
cCas9-v42
96
0


 


1035
TTGG
GGTTCGGGGGTATACATGGGC
22346
SauriCas9
96
0


 


1036
TTGG
GGTTCGGGGGTATACATGGGC
22347
SauriCas9-
96
0






KKH




 


1037
TTG
GTTCGGGGGTATACATGGGC
22348
ScaCas9
96
0


 


1038
TTG
GTTCGGGGGTATACATGGGC
22349
ScaCas9-
96
0






HiFi-Sc++




 


1039
TTG
GTTCGGGGGTATACATGGGC
22350
ScaCas9-
96
0






Sc++




 


1040
TTG
GTTCGGGGGTATACATGGGC
22351
SpyCas9-
96
0






SpRY




 


1041
ACC
GTTATGTGTACTACTCCACT
22352
SpyCas9-
96
0






SpRY




 


1042
CTTGG
CGGTTCGGGGGTATACATGGG
22353
SauCas9KKH
97
0


 


1043
TAC
AGTTATGTGTACTACTCCAC
22354
SpyCas9-
97
0






SpRY




 


1044
CTT
GGTTCGGGGGTATACATGGG
22355
SpyCas9-
97
0






SpRY




 


1045
TACC
AGTTATGTGTACTACTCCAC
22356
SpyCas9-
97
0






3var-NRCH




 


1046
GCT
CGGTTCGGGGGTATACATGG
22357
SpyCas9-
98
0






SpRY




 


1047
CTA
CAGTTATGTGTACTACTCCA
22358
SpyCas9-
98
0






SpRY




 


1048
CTACCTA
gggcAGTTATGTGTACTACTCC
22359
BlatCas9
98
0



A
A






 


1049
CTACCTA
gggcAGTTATGTGTACTACTCC
22360
BlatCas9
98
0



A
A






 


1050
CTACC
gggcAGTTATGTGTACTACTCC
22361
BlatCas9
98
0




A






 


1051
ACTACC
ctGGGCAGTTATGTGTACTACT
22362
Nme2Cas9
99
0




CC






 


1052
GG
ACGGTTCGGGGGTATACATG
22363
SpyCas9-
99
0






NG




 


1053
GG
ACGGTTCGGGGGTATACATG
22364
SpyCas9-
99
0






xCas




 


1054
GG
ACGGTTCGGGGGTATACATG
22365
SpyCas9-
99
0






xCas-NG




 


1055
GGC
ACGGTTCGGGGGTATACATG
22366
SpyCas9-
99
0






SpG




 


1056
GGC
ACGGTTCGGGGGTATACATG
22367
SpyCas9-
99
0






SpRY




 


1057
ACT
GCAGTTATGTGTACTACTCC
22368
SpyCas9-
99
0






SpRY




 


1058
ACTACCT
tgggCAGTTATGTGTACTACTCC
22369
BlatCas9
99
0



A







 


1059
ACTAC
tgggCAGTTATGTGTACTACTCC
22370
BlatCas9
99
0


 


1060
GGCT
ACGGTTCGGGGGTATACATG
22371
SpyCas9-
99
0






3var-NRCH




 


1061
GGG
CACGGTTCGGGGGTATACAT
22372
ScaCas9
100
0


 


1062
GGG
CACGGTTCGGGGGTATACAT
22373
ScaCas9-
100
0






HiFi-Sc++




 


1063
GGG
CACGGTTCGGGGGTATACAT
22374
ScaCas9-
100
0






Sc++




 


1064
GGG
CACGGTTCGGGGGTATACAT
22375
SpyCas9
100
0


 


1065
GGG
CACGGTTCGGGGGTATACAT
22376
SpyCas9-
100
0






HF1




 


1066
GGG
CACGGTTCGGGGGTATACAT
22377
SpyCas9-
100
0






SpG




 


1067
GGG
CACGGTTCGGGGGTATACAT
22378
SpyCas9-
100
0






SpRY




 


1068
GG
CACGGTTCGGGGGTATACAT
22379
SpyCas9-
100
0






NG




 


1069
GG
CACGGTTCGGGGGTATACAT
22380
SpyCas9-
100
0






xCas




 


1070
GG
CACGGTTCGGGGGTATACAT
22381
SpyCas9-
100
0






xCas-NG




 


1071
CAC
GGCAGTTATGTGTACTACTC
22382
SpyCas9-
100
0






SpRY




 


1072
GGGC
CACGGTTCGGGGGTATACAT
22383
SpyCas9-
100
0






3var-NRRH




 


1073
CACT
GGCAGTTATGTGTACTACTC
22384
SpyCas9-
100
0






3var-NRCH
















TABLE 1D







Exemplary gRNA spacer Cas pairs for correcting the pathogenic IVS10-11G > A


mutation


Table 1D provides a gRNA database for correcting the pathogenic IVS10-11G > A mutation in


PAH. List of spacers, PAMs, and Cas variants for generating a nick at an appropriate


position to enable installation of a desired genomic edit with a gene modifying system.


The spacers in this table are designed to be used with a gene modifying polypeptide


comprising a nickase variant of the Cas species indicated in the table. Tables 2D, 3D, and


4D detail the other components of the system and are organized such that the ID number


shown here in Column 1 (″ID″) is meant to correspond to the same ID number in Tables 2D,


2D, and 4D.














PAM

SEQ
Cas

Overlaps


ID
sequence
gRNA spacer
ID NO
species
distance
mutation
















1
GCC
ATAATAACTTTTCACTTAGG
23599
SpyCas9-
0
0






SpRY




 


2
AGTGA
TAAGCAGTACTGTAGGCCCTA
23600
SauCas9KKH
1
0


 


3
GG
GATAATAACTTTTCACTTAG
23601
SpyCas9-NG
1
0


 


4
GG
GATAATAACTTTTCACTTAG
23602
SpyCas9-
1
0






xCas




 


5
GG
GATAATAACTTTTCACTTAG
23603
SpyCas9-
1
0






xCas-NG




 


6
AG
AAGCAGTACTGTAGGCCCTA
23604
SpyCas9-NG
1
0


 


7
AG
AAGCAGTACTGTAGGCCCTA
23605
SpyCas9-
1
0






xCas




 


8
AG
AAGCAGTACTGTAGGCCCTA
23606
SpyCas9-
1
0






xCas-NG




 


9
GGC
GATAATAACTTTTCACTTAG
23607
SpyCas9-
1
0






SpG




 


10
GGC
GATAATAACTTTTCACTTAG
23608
SpyCas9-
1
0






SpRY




 


11
AGT
AAGCAGTACTGTAGGCCCTA
23609
SpyCas9-
1
0






SpG




 


12
AGT
AAGCAGTACTGTAGGCCCTA
23610
SpyCas9-
1
0






SpRY




 


13
GGCC
GATAATAACTTTTCACTTAG
23611
SpyCas9-
1
0






3var-NRCH




 


14
GGG
TGATAATAACTTTTCACTTA
23612
ScaCas9
2
0


 


15
GGG
TGATAATAACTTTTCACTTA
23613
ScaCas9-
2
0






HiFi-Sc++




 


16
GGG
TGATAATAACTTTTCACTTA
23614
ScaCas9-
2
0






Sc++




 


17
GGG
TGATAATAACTTTTCACTTA
23615
SpyCas9
2
0


 


18
GGG
TGATAATAACTTTTCACTTA
23616
SpyCas9-
2
0






HF1




 


19
GGG
TGATAATAACTTTTCACTTA
23617
SpyCas9-
2
0






SpG




 


20
GGG
TGATAATAACTTTTCACTTA
23618
SpyCas9-
2
0






SpRY




 


21
AAG
TAAGCAGTACTGTAGGCCCT
23619
ScaCas9
2
0


 


22
AAG
TAAGCAGTACTGTAGGCCCT
23620
ScaCas9-
2
0






HiFi-Sc++




 


23
AAG
TAAGCAGTACTGTAGGCCCT
23621
ScaCas9-
2
0






Sc++




 


24
AAG
TAAGCAGTACTGTAGGCCCT
23622
SpyCas9-
2
0






SpRY




 


25
GG
TGATAATAACTTTTCACTTA
23623
SpyCas9-NG
2
0


 


26
GG
TGATAATAACTTTTCACTTA
23624
SpyCas9-
2
0






xCas




 


27
GG
TGATAATAACTTTTCACTTA
23625
SpyCas9-
2
0






xCas-NG




 


28
GGGCC
cagtGATAATAACTTTTCACTTA
23626
BlatCas9
2
0


 


29
GGGC
TGATAATAACTTTTCACTTA
23627
SpyCas9-
2
0






3var-NRRH




 


30
AAGT
TAAGCAGTACTGTAGGCCCT
23628
SpyCas9-
2
0






3var-NRRH




 


31
aGGGCC
aaCAGTGATAATAACTTTTCACTT
23629
Nme2Cas9
3
1


 


32
aGGG
AGTGATAATAACTTTTCACTT
23630
SauriCas9
3
1


 


33
aGGG
AGTGATAATAACTTTTCACTT
23631
SauriCas9-
3
1






KKH




 


34
tAAG
GATAAGCAGTACTGTAGGCCC
23632
SauriCas9-
3
1






KKH




 


35
tAAG
ATAAGCAGTACTGTAGGCCC
23633
SpyCas9-
3
1






QQR1




 


36
tAAG
gaTAAGCAGTACTGTAGGCCC
23634
iSpyMacCas9
3
1


 


37
aGG
GTGATAATAACTTTTCACTT
23635
ScaCas9
3
1


 


38
aGG
GTGATAATAACTTTTCACTT
23636
ScaCas9-
3
1






HiFi-Sc++




 


39
aGG
GTGATAATAACTTTTCACTT
23637
ScaCas9-
3
1






Sc++




 


40
aGG
GTGATAATAACTTTTCACTT
23638
SpyCas9
3
1


 


41
aGG
GTGATAATAACTTTTCACTT
23639
SpyCas9-
3
1






HF1




 


42
aGG
GTGATAATAACTTTTCACTT
23640
SpyCas9-
3
1






SpG




 


43
aGG
GTGATAATAACTTTTCACTT
23641
SpyCas9-
3
1






SpRY




 


44
aG
GTGATAATAACTTTTCACTT
23642
SpyCas9-NG
3
1


 


45
aG
GTGATAATAACTTTTCACTT
23643
SpyCas9-
3
1






xCas




 


46
aG
GTGATAATAACTTTTCACTT
23644
SpyCas9-
3
1






xCas-NG




 


47
tAA
ATAAGCAGTACTGTAGGCCC
23645
SpyCas9-
3
1






SpRY




 


48
aGGGCCTA
acagTGATAATAACTTTTCACTT
23646
BlatCas9
3
1


 


49
aGGGC
acagTGATAATAACTTTTCACTT
23647
BlatCas9
3
1


 


50
tAAGTG
GATAAGCAGTACTGTAGGCCC
23648
cCas9-v16
3
1


 


51
tAAGTG
GATAAGCAGTACTGTAGGCCC
23649
cCas9-v21
3
1


 


52
TaGGG
aaCAGTGATAATAACTTTTCACT
23650
SauCas9
4
1


 


53
TaGGG
CAGTGATAATAACTTTTCACT
23651
SauCas9KKH
4
1


 


54
CtAAG
TGATAAGCAGTACTGTAGGCC
23652
SauCas9KKH
4
1


 


55
CtAAGT
TGATAAGCAGTACTGTAGGCC
23653
SauCas9KKH
4
1


 


56
CtAAGT
TGATAAGCAGTACTGTAGGCC
23654
cCas9-v17
4
1


 


57
CtAAGT
TGATAAGCAGTACTGTAGGCC
23655
cCas9-v42
4
1


 


58
TaGG
CAGTGATAATAACTTTTCACT
23656
SauriCas9
4
1


 


59
TaGG
CAGTGATAATAACTTTTCACT
23657
SauriCas9-
4
1






KKH




 


60
TaG
AGTGATAATAACTTTTCACT
23658
ScaCas9
4
1


 


61
TaG
AGTGATAATAACTTTTCACT
23659
ScaCas9-
4
1






HiFi-Sc++




 


62
TaG
AGTGATAATAACTTTTCACT
23660
ScaCas9-
4
1






Sc++




 


63
TaG
AGTGATAATAACTTTTCACT
23661
SpyCas9-
4
1






SpRY




 


64
CtA
GATAAGCAGTACTGTAGGCC
23662
SpyCas9-
4
1






SpRY




 


65
TaGGGC
CAGTGATAATAACTTTTCACT
23663
cCas9-v17
4
1


 


66
TaGGGC
CAGTGATAATAACTTTTCACT
23664
cCas9-v42
4
1


 


67
CCtAA
CTGATAAGCAGTACTGTAGGC
23665
SauCas9KKH
5
1


 


68
TTaGG
ACAGTGATAATAACTTTTCAC
23666
SauCas9KKH
5
1


 


69
TTaG
ACAGTGATAATAACTTTTCAC
23667
SauriCas9-
5
1






KKH




 


70
CCt
TGATAAGCAGTACTGTAGGC
23668
SpyCas9-
5
1






SpRY




 


71
TTa
CAGTGATAATAACTTTTCAC
23669
SpyCas9-
5
1






SpRY




 


72
TTaGGG
ACAGTGATAATAACTTTTCAC
23670
cCas9-v17
5
1


 


73
TTaGGG
ACAGTGATAATAACTTTTCAC
23671
cCas9-v42
5
1


 


74
CTTaG
AACAGTGATAATAACTTTTCA
23672
SauCas9KKH
6
1


 


75
CCC
CTGATAAGCAGTACTGTAGG
23673
SpyCas9-
6
0






SpRY




 


76
CTT
ACAGTGATAATAACTTTTCA
23674
SpyCas9-
6
0






SpRY




 


77
GCC
TCTGATAAGCAGTACTGTAG
23675
SpyCas9-
7
0






SpRY




 


78
ACT
AACAGTGATAATAACTTTTC
23676
SpyCas9-
7
0






SpRY




 


79
GG
CTCTGATAAGCAGTACTGTA
23677
SpyCas9-NG
8
0


 


80
GG
CTCTGATAAGCAGTACTGTA
23678
SpyCas9-
8
0






xCas




 


81
GG
CTCTGATAAGCAGTACTGTA
23679
SpyCas9-
8
0






xCas-NG




 


82
GGC
CTCTGATAAGCAGTACTGTA
23680
SpyCas9-
8
0






SpG




 


83
GGC
CTCTGATAAGCAGTACTGTA
23681
SpyCas9-
8
0






SpRY




 


84
CAC
TAACAGTGATAATAACTTTT
23682
SpyCas9-
8
0






SpRY




 


85
GGCCCtAA
cttcTCTGATAAGCAGTACTGTA
23683
BlatCas9
8
1


 


86
GGCCCtAA
cttcTCTGATAAGCAGTACTGTA
23684
BlatCas9
8
1


 


87
GGCCC
cttcTCTGATAAGCAGTACTGTA
23685
BlatCas9
8
0


 


88
GGCC
CTCTGATAAGCAGTACTGTA
23686
SpyCas9-
8
0






3var-NRCH




 


89
CACT
TAACAGTGATAATAACTTTT
23687
SpyCas9-
8
0






3var-NRCH




 


90
AGGCCC
ggCTTCTCTGATAAGCAGTACTG
23688
Nme2Cas9
9
0




T






 


91
AGG
TCTCTGATAAGCAGTACTGT
23689
ScaCas9
9
0


 


92
AGG
TCTCTGATAAGCAGTACTGT
23690
ScaCas9-
9
0






HiFi-Sc++




 


93
AGG
TCTCTGATAAGCAGTACTGT
23691
ScaCas9-
9
0






Sc++




 


94
AGG
TCTCTGATAAGCAGTACTGT
23692
SpyCas9
9
0


 


95
AGG
TCTCTGATAAGCAGTACTGT
23693
SpyCas9-
9
0






HF1




 


96
AGG
TCTCTGATAAGCAGTACTGT
23694
SpyCas9-
9
0






SpG




 


97
AGG
TCTCTGATAAGCAGTACTGT
23695
SpyCas9-
9
0






SpRY




 


98
AG
TCTCTGATAAGCAGTACTGT
23696
SpyCas9-NG
9
0


 


99
AG
TCTCTGATAAGCAGTACTGT
23697
SpyCas9-
9
0






xCas




 


100
AG
TCTCTGATAAGCAGTACTGT
23698
SpyCas9-
9
0






xCas-NG




 


101
TCA
TTAACAGTGATAATAACTTT
23699
SpyCas9-
9
0






SpRY




 


102
AGGCCCtA
gcttCTCTGATAAGCAGTACTGT
23700
BlatCas9
9
1


 


103
AGGCC
gcttCTCTGATAAGCAGTACTGT
23701
BlatCas9
9
0


 


104
AGGCCCt
CTTCTCTGATAAGCAGTACTGT
23702
CdiCas9
9
1


 


105
AGGC
TCTCTGATAAGCAGTACTGT
23703
SpyCas9-
9
0






3var-NRRH




 


106
TAGGCC
tgGCTTCTCTGATAAGCAGTACTG
23704
Nme2Cas9
10
0


 


107
TAGG
CTTCTCTGATAAGCAGTACTG
23705
SauriCas9
10
0


 


108
TAGG
CTTCTCTGATAAGCAGTACTG
23706
SauriCas9-
10
0






KKH




 


109
TAG
TTCTCTGATAAGCAGTACTG
23707
ScaCas9
10
0


 


110
TAG
TTCTCTGATAAGCAGTACTG
23708
ScaCas9-
10
0






HiFi-Sc++




 


111
TAG
TTCTCTGATAAGCAGTACTG
23709
ScaCas9-
10
0






Sc++




 


112
TAG
TTCTCTGATAAGCAGTACTG
23710
SpyCas9-
10
0






SpRY




 


113
TTC
TTTAACAGTGATAATAACTT
23711
SpyCas9-
10
0






SpRY




 


114
TTCACTTa
tgatTTAACAGTGATAATAACTT
23712
BlatCas9
10
1


 


115
TAGGC
ggctTCTCTGATAAGCAGTACTG
23713
BlatCas9
10
0


 


116
TTCAC
tgatTTAACAGTGATAATAACTT
23714
BlatCas9
10
0


 


117
TTCACT
ATTTAACAGTGATAATAACTT
23715
cCas9-v16
10
0


 


118
TTCACT
ATTTAACAGTGATAATAACTT
23716
cCas9-v21
10
0


 


119
GTAGG
GCTTCTCTGATAAGCAGTACT
23717
SauCas9KKH
11
0


 


120
GTAG
GCTTCTCTGATAAGCAGTACT
23718
SauriCas9-
11
0






KKH




 


121
GTA
CTTCTCTGATAAGCAGTACT
23719
SpyCas9-
11
0






SpRY




 


122
TTT
ATTTAACAGTGATAATAACT
23720
SpyCas9-
11
0






SpRY




 


123
GTAGGC
GCTTCTCTGATAAGCAGTACT
23721
cCas9-v17
11
0


 


124
GTAGGC
GCTTCTCTGATAAGCAGTACT
23722
cCas9-v42
11
0


 


125
TGTAG
GGCTTCTCTGATAAGCAGTAC
23723
SauCas9KKH
12
0


 


126
TG
GCTTCTCTGATAAGCAGTAC
23724
SpyCas9-NG
12
0


 


127
TG
GCTTCTCTGATAAGCAGTAC
23725
SpyCas9-
12
0






xCas




 


128
TG
GCTTCTCTGATAAGCAGTAC
23726
SpyCas9-
12
0






xCas-NG




 


129
TGT
GCTTCTCTGATAAGCAGTAC
23727
SpyCas9-
12
0






SpG




 


130
TGT
GCTTCTCTGATAAGCAGTAC
23728
SpyCas9-
12
0






SpRY




 


131
TTT
GATTTAACAGTGATAATAAC
23729
SpyCas9-
12
0






SpRY




 


132
TTTTC
cctgATTTAACAGTGATAATAAC
23730
BlatCas9
12
0


 


133
TGTA
GCTTCTCTGATAAGCAGTAC
23731
SpyCas9-
12
0






3var-NRTH




 


134
CTG
GGCTTCTCTGATAAGCAGTA
23732
ScaCas9
13
0


 


135
CTG
GGCTTCTCTGATAAGCAGTA
23733
ScaCas9-
13
0






HiFi-Sc++




 


136
CTG
GGCTTCTCTGATAAGCAGTA
23734
ScaCas9-
13
0






Sc++




 


137
CTG
GGCTTCTCTGATAAGCAGTA
23735
SpyCas9-
13
0






SpRY




 


138
CTT
TGATTTAACAGTGATAATAA
23736
SpyCas9-
13
0






SpRY




 


139
ACT
TGGCTTCTCTGATAAGCAGT
23737
SpyCas9-
14
0






SpRY




 


140
ACT
CTGATTTAACAGTGATAATA
23738
SpyCas9-
14
0






SpRY




 


141
TAC
TTGGCTTCTCTGATAAGCAG
23739
SpyCas9-
15
0






SpRY




 


142
AAC
CCTGATTTAACAGTGATAAT
23740
SpyCas9-
15
0






SpRY




 


143
TACT
TTGGCTTCTCTGATAAGCAG
23741
SpyCas9-
15
0






3var-NRCH




 


144
AACT
CCTGATTTAACAGTGATAAT
23742
SpyCas9-
15
0






3var-NRCH




 


145
TAA
TCCTGATTTAACAGTGATAA
23743
SpyCas9-
16
0






SpRY




 


146
GTA
TTTGGCTTCTCTGATAAGCA
23744
SpyCas9-
16
0






SpRY




 


147
TAACTTT
GATCCTGATTTAACAGTGATAA
23745
CdiCas9
16
0


 


148
TAAC
TCCTGATTTAACAGTGATAA
23746
SpyCas9-
16
0






3var-NRRH




 


149
TAAC
atCCTGATTTAACAGTGATAA
23747
iSpyMacCas9
16
0


 


150
AG
CTTTGGCTTCTCTGATAAGC
23748
SpyCas9-NG
17
0


 


151
AG
CTTTGGCTTCTCTGATAAGC
23749
SpyCas9-
17
0






xCas




 


152
AG
CTTTGGCTTCTCTGATAAGC
23750
SpyCas9-
17
0






xCas-NG




 


153
AGT
CTTTGGCTTCTCTGATAAGC
23751
SpyCas9-
17
0






SpG




 


154
AGT
CTTTGGCTTCTCTGATAAGC
23752
SpyCas9-
17
0






SpRY




 


155
ATA
ATCCTGATTTAACAGTGATA
23753
SpyCas9-
17
0






SpRY




 


156
AGTACTG
aagcTTTGGCTTCTCTGATAAGC
23754
BlatCas9
17
0



T







 


157
ATAACTTT
ctgaTCCTGATTTAACAGTGATA
23755
BlatCas9
17
0


 


158
AGTAC
aagcTTTGGCTTCTCTGATAAGC
23756
BlatCas9
17
0


 


159
ATAAC
ctgaTCCTGATTTAACAGTGATA
23757
BlatCas9
17
0


 


160
ATAACT
GATCCTGATTTAACAGTGATA
23758
cCas9-v16
17
0


 


161
ATAACT
GATCCTGATTTAACAGTGATA
23759
cCas9-v21
17
0


 


162
ATAACTT
TGATCCTGATTTAACAGTGATA
23760
CdiCas9
17
0


 


163
AGTA
CTTTGGCTTCTCTGATAAGC
23761
SpyCas9-
17
0






3var-NRTH




 


164
AATAA
TGATCCTGATTTAACAGTGAT
23762
SauCas9KKH
18
0


 


165
CAG
GCTTTGGCTTCTCTGATAAG
23763
ScaCas9
18
0


 


166
CAG
GCTTTGGCTTCTCTGATAAG
23764
ScaCas9-
18
0






HiFi-Sc++




 


167
CAG
GCTTTGGCTTCTCTGATAAG
23765
ScaCas9-
18
0






Sc++




 


168
CAG
GCTTTGGCTTCTCTGATAAG
23766
SpyCas9-
18
0






SpRY




 


169
AAT
GATCCTGATTTAACAGTGAT
23767
SpyCas9-
18
0






SpRY




 


170
CAGTACT
AAGCTTTGGCTTCTCTGATAAG
23768
CdiCas9
18
0


 


171
CAGT
GCTTTGGCTTCTCTGATAAG
23769
SpyCas9-
18
0






3var-NRRH




 


172
AATA
GATCCTGATTTAACAGTGAT
23770
SpyCas9-
18
0






3var-NRTH




 


173
GCAG
AAGCTTTGGCTTCTCTGATAA
23771
SauriCas9-
19
0






KKH




 


174
TAA
TGATCCTGATTTAACAGTGA
23772
SpyCas9-
19
0






SpRY




 


175
GCA
AGCTTTGGCTTCTCTGATAA
23773
SpyCas9-
19
0






SpRY




 


176
TAATAAC
ACTGATCCTGATTTAACAGTGA
23774
CdiCas9
19
0


 


177
TAAT
TGATCCTGATTTAACAGTGA
23775
SpyCas9-
19
0






3var-NRRH




 


178
TAAT
ctGATCCTGATTTAACAGTGA
23776
iSpyMacCas9
19
0


 


179
AGCAG
GAAGCTTTGGCTTCTCTGATA
23777
SauCas9KKH
20
0


 


180
AGCAGT
GAAGCTTTGGCTTCTCTGATA
23778
SauCas9KKH
20
0


 


181
AGCAGT
GAAGCTTTGGCTTCTCTGATA
23779
cCas9-v17
20
0


 


182
AGCAGT
GAAGCTTTGGCTTCTCTGATA
23780
cCas9-v42
20
0


 


183
AG
AAGCTTTGGCTTCTCTGATA
23781
SpyCas9-NG
20
0


 


184
AG
AAGCTTTGGCTTCTCTGATA
23782
SpyCas9-
20
0






xCas




 


185
AG
AAGCTTTGGCTTCTCTGATA
23783
SpyCas9-
20
0






xCas-NG




 


186
AGC
AAGCTTTGGCTTCTCTGATA
23784
SpyCas9-
20
0






SpG




 


187
AGC
AAGCTTTGGCTTCTCTGATA
23785
SpyCas9-
20
0






SpRY




 


188
ATA
CTGATCCTGATTTAACAGTG
23786
SpyCas9-
20
0






SpRY




 


189
AGCAGTA
gaGAAGCTTTGGCTTCTCTGATA
23787
CjeCas9
20
0



C







 


190
AGCA
AAGCTTTGGCTTCTCTGATA
23788
SpyCas9-
20
0






3var-NRCH




 


191
GATAA
TACTGATCCTGATTTAACAGT
23789
SauCas9KKH
21
0


 


192
GATAAT
TACTGATCCTGATTTAACAGT
23790
SauCas9KKH
21
0


 


193
AAG
GAAGCTTTGGCTTCTCTGAT
23791
ScaCas9
21
0


 


194
AAG
GAAGCTTTGGCTTCTCTGAT
23792
ScaCas9-
21
0






HiFi-Sc++




 


195
AAG
GAAGCTTTGGCTTCTCTGAT
23793
ScaCas9-
21
0






Sc++




 


196
AAG
GAAGCTTTGGCTTCTCTGAT
23794
SpyCas9-
21
0






SpRY




 


197
GAT
ACTGATCCTGATTTAACAGT
23795
SpyCas9-
21
0






SpRY




 


198
GAT
ACTGATCCTGATTTAACAGT
23796
SpyCas9-
21
0






xCas




 


199
AAGC
GAAGCTTTGGCTTCTCTGAT
23797
SpyCas9-
21
0






3var-NRRH




 


200
GATA
ACTGATCCTGATTTAACAGT
23798
SpyCas9-
21
0






3var-NRTH




 


201
TAAG
GAGAAGCTTTGGCTTCTCTGA
23799
SauriCas9-
22
0






KKH




 


202
TAAG
AGAAGCTTTGGCTTCTCTGA
23800
SpyCas9-
22
0






QQR1




 


203
TAAG
gaGAAGCTTTGGCTTCTCTGA
23801
iSpyMacCas9
22
0


 


204
TG
TACTGATCCTGATTTAACAG
23802
SpyCas9-NG
22
0


 


205
TG
TACTGATCCTGATTTAACAG
23803
SpyCas9-
22
0






xCas




 


206
TG
TACTGATCCTGATTTAACAG
23804
SpyCas9-
22
0






xCas-NG




 


207
TAA
AGAAGCTTTGGCTTCTCTGA
23805
SpyCas9-
22
0






SpRY




 


208
TGA
TACTGATCCTGATTTAACAG
23806
SpyCas9-
22
0






SpG




 


209
TGA
TACTGATCCTGATTTAACAG
23807
SpyCas9-
22
0






SpRY




 


210
TAAGCAG
gggaGAAGCTTTGGCTTCTCTGA
23808
BlatCas9
22
0



T







 


211
TAAGC
gggaGAAGCTTTGGCTTCTCTGA
23809
BlatCas9
22
0


 


212
TGATAAT
AATACTGATCCTGATTTAACAG
23810
CdiCas9
22
0


 


213
TGAT
TACTGATCCTGATTTAACAG
23811
SpyCas9-
22
0






3var-NRRH




 


214
TGAT
TACTGATCCTGATTTAACAG
23812
SpyCas9-
22
0






VQR




 


215
ATAAG
GGAGAAGCTTTGGCTTCTCTG
23813
SauCas9KKH
23
0


 


216
GTG
ATACTGATCCTGATTTAACA
23814
ScaCas9
23
0


 


217
GTG
ATACTGATCCTGATTTAACA
23815
ScaCas9-
23
0






HiFi-Sc++




 


218
GTG
ATACTGATCCTGATTTAACA
23816
ScaCas9-
23
0






Sc++




 


219
GTG
ATACTGATCCTGATTTAACA
23817
SpyCas9-
23
0






SpRY




 


220
ATA
GAGAAGCTTTGGCTTCTCTG
23818
SpyCas9-
23
0






SpRY




 


221
ATAAGC
GGAGAAGCTTTGGCTTCTCTG
23819
cCas9-v17
23
0


 


222
ATAAGC
GGAGAAGCTTTGGCTTCTCTG
23820
cCas9-v42
23
0


 


223
GATAA
GGGAGAAGCTTTGGCTTCTCT
23821
SauCas9KKH
24
0


 


224
AGTGA
GAATACTGATCCTGATTTAAC
23822
SauCas9KKH
24
0


 


225
AGTGAT
GAATACTGATCCTGATTTAAC
23823
SauCas9KKH
24
0


 


226
AG
AATACTGATCCTGATTTAAC
23824
SpyCas9-NG
24
0


 


227
AG
AATACTGATCCTGATTTAAC
23825
SpyCas9-
24
0






xCas




 


228
AG
AATACTGATCCTGATTTAAC
23826
SpyCas9-
24
0






xCas-NG




 


229
GAT
GGAGAAGCTTTGGCTTCTCT
23827
SpyCas9-
24
0






SpRY




 


230
GAT
GGAGAAGCTTTGGCTTCTCT
23828
SpyCas9-
24
0






xCas




 


231
AGT
AATACTGATCCTGATTTAAC
23829
SpyCas9-
24
0






SpG




 


232
AGT
AATACTGATCCTGATTTAAC
23830
SpyCas9-
24
0






SpRY




 


233
GATA
GGAGAAGCTTTGGCTTCTCT
23831
SpyCas9-
24
0






3var-NRTH




 


234
CAG
GAATACTGATCCTGATTTAA
23832
ScaCas9
25
0


 


235
CAG
GAATACTGATCCTGATTTAA
23833
ScaCas9-
25
0






HiFi-Sc++




 


236
CAG
GAATACTGATCCTGATTTAA
23834
ScaCas9-
25
0






Sc++




 


237
CAG
GAATACTGATCCTGATTTAA
23835
SpyCas9-
25
0






SpRY




 


238
TG
GGGAGAAGCTTTGGCTTCTC
23836
SpyCas9-NG
25
0


 


239
TG
GGGAGAAGCTTTGGCTTCTC
23837
SpyCas9-
25
0






xCas




 


240
TG
GGGAGAAGCTTTGGCTTCTC
23838
SpyCas9-
25
0






xCas-NG




 


241
TGA
GGGAGAAGCTTTGGCTTCTC
23839
SpyCas9-
25
0






SpG




 


242
TGA
GGGAGAAGCTTTGGCTTCTC
23840
SpyCas9-
25
0






SpRY




 


243
CAGTGAT
caggGAATACTGATCCTGATTTAA
23841
NmeCas9
25
0



A







 


244
TGAT
GGGAGAAGCTTTGGCTTCTC
23842
SpyCas9-
25
0






3var-NRRH




 


245
TGAT
GGGAGAAGCTTTGGCTTCTC
23843
SpyCas9-
25
0






VQR




 


246
CAGT
GAATACTGATCCTGATTTAA
23844
SpyCas9-
25
0






3var-NRRH




 


247
ACAG
GGGAATACTGATCCTGATTTA
23845
SauriCas9-
26
0






KKH




 


248
CTG
GGGGAGAAGCTTTGGCTTCT
23846
ScaCas9
26
0


 


249
CTG
GGGGAGAAGCTTTGGCTTCT
23847
ScaCas9-
26
0






HiFi-Sc++




 


250
CTG
GGGGAGAAGCTTTGGCTTCT
23848
ScaCas9-
26
0






Sc++




 


251
CTG
GGGGAGAAGCTTTGGCTTCT
23849
SpyCas9-
26
0






SpRY




 


252
ACA
GGAATACTGATCCTGATTTA
23850
SpyCas9-
26
0






SpRY




 


253
ACAGTG
GGGAATACTGATCCTGATTTA
23851
cCas9-v16
26
0


 


254
ACAGTG
GGGAATACTGATCCTGATTTA
23852
cCas9-v21
26
0


 


255
TCTGA
AGGGGGAGAAGCTTTGGCTTC
23853
SauCas9KKH
27
0


 


256
AACAG
AGGGAATACTGATCCTGATTT
23854
SauCas9KKH
27
0


 


257
TCTGAT
AGGGGGAGAAGCTTTGGCTTC
23855
SauCas9KKH
27
0


 


258
AACAGT
AGGGAATACTGATCCTGATTT
23856
SauCas9KKH
27
0


 


259
AACAGT
AGGGAATACTGATCCTGATTT
23857
cCas9-v17
27
0


 


260
AACAGT
AGGGAATACTGATCCTGATTT
23858
cCas9-v42
27
0


 


261
AAC
GGGAATACTGATCCTGATTT
23859
SpyCas9-
27
0






SpRY




 


262
TCT
GGGGGAGAAGCTTTGGCTTC
23860
SpyCas9-
27
0






SpRY




 


263
AACA
GGGAATACTGATCCTGATTT
23861
SpyCas9-
27
0






3var-NRCH




 


264
TAA
AGGGAATACTGATCCTGATT
23862
SpyCas9-
28
0






SpRY




 


265
CTC
AGGGGGAGAAGCTTTGGCTT
23863
SpyCas9-
28
0






SpRY




 


266
TAAC
AGGGAATACTGATCCTGATT
23864
SpyCas9-
28
0






3var-NRRH




 


267
TAAC
caGGGAATACTGATCCTGATT
23865
iSpyMacCas9
28
0


 


268
CTCTGATA
ctccAGGGGGAGAAGCTTTGGCTT
23866
NmeCas9
28
0


 


269
TCT
CAGGGGGAGAAGCTTTGGCT
23867
SpyCas9-
29
0






SpRY




 


270
TTA
CAGGGAATACTGATCCTGAT
23868
SpyCas9-
29
0






SpRY




 


271
TTAACAG
cagcAGGGAATACTGATCCTGAT
23869
BlatCas9
29
0



T







 


272
TTAAC
cagcAGGGAATACTGATCCTGAT
23870
BlatCas9
29
0


 


273
TTTAA
AGCAGGGAATACTGATCCTGA
23871
SauCas9KKH
30
0


 


274
TTC
CCAGGGGGAGAAGCTTTGGC
23872
SpyCas9-
30
0






SpRY




 


275
TTT
GCAGGGAATACTGATCCTGA
23873
SpyCas9-
30
0






SpRY




 


276
TTCTCTGA
gctcCAGGGGGAGAAGCTTTGGC
23874
BlatCas9
30
0


 


277
TTCTC
gctcCAGGGGGAGAAGCTTTGGC
23875
BlatCas9
30
0


 


278
CTT
TCCAGGGGGAGAAGCTTTGG
23876
SpyCas9-
31
0






SpRY




 


279
ATT
AGCAGGGAATACTGATCCTG
23877
SpyCas9-
31
0






SpRY




 


280
GAT
CAGCAGGGAATACTGATCCT
23878
SpyCas9-
32
0






SpRY




 


281
GAT
CAGCAGGGAATACTGATCCT
23879
SpyCas9-
32
0






xCas




 


282
GCT
CTCCAGGGGGAGAAGCTTTG
23880
SpyCas9-
32
0






SpRY




 


283
GCTTC
cagcTCCAGGGGGAGAAGCTTTG
23881
BlatCas9
32
0


 


284
GATT
CAGCAGGGAATACTGATCCT
23882
SpyCas9-
32
0






3var-NRTH




 


285
GG
GCTCCAGGGGGAGAAGCTTT
23883
SpyCas9-NG
33
0


 


286
GG
GCTCCAGGGGGAGAAGCTTT
23884
SpyCas9-
33
0






xCas




 


287
GG
GCTCCAGGGGGAGAAGCTTT
23885
SpyCas9-
33
0






xCas-NG




 


288
TG
GCAGCAGGGAATACTGATCC
23886
SpyCas9-NG
33
0


 


289
TG
GCAGCAGGGAATACTGATCC
23887
SpyCas9-
33
0






xCas




 


290
TG
GCAGCAGGGAATACTGATCC
23888
SpyCas9-
33
0






xCas-NG




 


291
GGC
GCTCCAGGGGGAGAAGCTTT
23889
SpyCas9-
33
0






SpG




 


292
GGC
GCTCCAGGGGGAGAAGCTTT
23890
SpyCas9-
33
0






SpRY




 


293
TGA
GCAGCAGGGAATACTGATCC
23891
SpyCas9-
33
0






SpG




 


294
TGA
GCAGCAGGGAATACTGATCC
23892
SpyCas9-
33
0






SpRY




 


295
TGAT
GCAGCAGGGAATACTGATCC
23893
SpyCas9-
33
0






3var-NRRH




 


296
TGAT
GCAGCAGGGAATACTGATCC
23894
SpyCas9-
33
0






VQR




 


297
GGCT
GCTCCAGGGGGAGAAGCTTT
23895
SpyCas9-
33
0






3var-NRCH




 


298
TGG
AGCTCCAGGGGGAGAAGCTT
23896
ScaCas9
34
0


 


299
TGG
AGCTCCAGGGGGAGAAGCTT
23897
ScaCas9-
34
0






HiFi-Sc++




 


300
TGG
AGCTCCAGGGGGAGAAGCTT
23898
ScaCas9-
34
0






Sc++




 


301
TGG
AGCTCCAGGGGGAGAAGCTT
23899
SpyCas9
34
0


 


302
TGG
AGCTCCAGGGGGAGAAGCTT
23900
SpyCas9-
34
0






HF1




 


303
TGG
AGCTCCAGGGGGAGAAGCTT
23901
SpyCas9-
34
0






SpG




 


304
TGG
AGCTCCAGGGGGAGAAGCTT
23902
SpyCas9-
34
0






SpRY




 


305
CTG
TGCAGCAGGGAATACTGATC
23903
ScaCas9
34
0


 


306
CTG
TGCAGCAGGGAATACTGATC
23904
ScaCas9-
34
0






HiFi-Sc++




 


307
CTG
TGCAGCAGGGAATACTGATC
23905
ScaCas9-
34
0






Sc++




 


308
CTG
TGCAGCAGGGAATACTGATC
23906
SpyCas9-
34
0






SpRY




 


309
TG
AGCTCCAGGGGGAGAAGCTT
23907
SpyCas9-NG
34
0


 


310
TG
AGCTCCAGGGGGAGAAGCTT
23908
SpyCas9-
34
0






xCas




 


311
TG
AGCTCCAGGGGGAGAAGCTT
23909
SpyCas9-
34
0






xCas-NG




 


312
CTGATT
ATGCAGCAGGGAATACTGATC
23910
cCas9-v16
34
0


 


313
CTGATT
ATGCAGCAGGGAATACTGATC
23911
cCas9-v21
34
0


 


314
TGGCTTC
CCAGCTCCAGGGGGAGAAGCTT
23912
CdiCas9
34
0


 


315
CTGATTT
GATGCAGCAGGGAATACTGATC
23913
CdiCas9
34
0


 


316
TGGC
AGCTCCAGGGGGAGAAGCTT
23914
SpyCas9-
34
0






3var-NRRH




 


317
CCTGATT
tggGATGCAGCAGGGAATACTGA
23915
PpnCas9
35
0




T






 


318
CCTGA
GATGCAGCAGGGAATACTGAT
23916
SauCas9KKH
35
0


 


319
CCTGAT
GATGCAGCAGGGAATACTGAT
23917
SauCas9KKH
35
0


 


320
TTGG
CCAGCTCCAGGGGGAGAAGCT
23918
SauriCas9
35
0


 


321
TTGG
CCAGCTCCAGGGGGAGAAGCT
23919
SauriCas9-
35
0






KKH




 


322
TTG
CAGCTCCAGGGGGAGAAGCT
23920
ScaCas9
35
0


 


323
TTG
CAGCTCCAGGGGGAGAAGCT
23921
ScaCas9-
35
0






HiFi-Sc++




 


324
TTG
CAGCTCCAGGGGGAGAAGCT
23922
ScaCas9-
35
0






Sc++




 


325
TTG
CAGCTCCAGGGGGAGAAGCT
23923
SpyCas9-
35
0






SpRY




 


326
CCT
ATGCAGCAGGGAATACTGAT
23924
SpyCas9-
35
0






SpRY




 


327
TTGGC
ctccAGCTCCAGGGGGAGAAGCT
23925
BlatCas9
35
0


 


328
TTGGCT
CCAGCTCCAGGGGGAGAAGCT
23926
cCas9-v16
35
0


 


329
TTGGCT
CCAGCTCCAGGGGGAGAAGCT
23927
cCas9-v21
35
0


 


330
TTTGG
TCCAGCTCCAGGGGGAGAAGC
23928
SauCas9KKH
36
0


 


331
TTT
CCAGCTCCAGGGGGAGAAGC
23929
SpyCas9-
36
0






SpRY




 


332
TCC
GATGCAGCAGGGAATACTGA
23930
SpyCas9-
36
0






SpRY




 


333
TCCTGATT
atggGATGCAGCAGGGAATACTGA
23931
NmeCas9
36
0


 


334
TTTGGCTT
ttctCCAGCTCCAGGGGGAGAAGC
23932
NmeCas9
36
0


 


335
CTT
TCCAGCTCCAGGGGGAGAAG
23933
SpyCas9-
37
0






SpRY




 


336
ATC
GGATGCAGCAGGGAATACTG
23934
SpyCas9-
37
0






SpRY




 


337
GAT
GGGATGCAGCAGGGAATACT
23935
SpyCas9-
38
0






SpRY




 


338
GAT
GGGATGCAGCAGGGAATACT
23936
SpyCas9-
38
0






xCas




 


339
GCT
CTCCAGCTCCAGGGGGAGAA
23937
SpyCas9-
38
0






SpRY




 


340
GATCCTG
tatgGGATGCAGCAGGGAATACT
23938
BlatCas9
38
0



A







 


341
GATCC
tatgGGATGCAGCAGGGAATACT
23939
BlatCas9
38
0


 


342
GATC
GGGATGCAGCAGGGAATACT
23940
SpyCas9-
38
0






3var-NRTH




 


343
TGATCC
ccTATGGGATGCAGCAGGGAATA
23941
Nme2Cas9
39
0




C






 


344
AG
TCTCCAGCTCCAGGGGGAGA
23942
SpyCas9-NG
39
0


 


345
AG
TCTCCAGCTCCAGGGGGAGA
23943
SpyCas9-
39
0






xCas




 


346
AG
TCTCCAGCTCCAGGGGGAGA
23944
SpyCas9-
39
0






xCas-NG




 


347
TG
TGGGATGCAGCAGGGAATAC
23945
SpyCas9-NG
39
0


 


348
TG
TGGGATGCAGCAGGGAATAC
23946
SpyCas9-
39
0






xCas




 


349
TG
TGGGATGCAGCAGGGAATAC
23947
SpyCas9-
39
0






xCas-NG




 


350
AGC
TCTCCAGCTCCAGGGGGAGA
23948
SpyCas9-
39
0






SpG




 


351
AGC
TCTCCAGCTCCAGGGGGAGA
23949
SpyCas9-
39
0






SpRY




 


352
TGA
TGGGATGCAGCAGGGAATAC
23950
SpyCas9-
39
0






SpG




 


353
TGA
TGGGATGCAGCAGGGAATAC
23951
SpyCas9-
39
0






SpRY




 


354
TGATCCTG
ctatGGGATGCAGCAGGGAATAC
23952
BlatCas9
39
0


 


355
TGATC
ctatGGGATGCAGCAGGGAATAC
23953
BlatCas9
39
0


 


356
TGATCCT
TATGGGATGCAGCAGGGAATAC
23954
CdiCas9
39
0


 


357
TGAT
TGGGATGCAGCAGGGAATAC
23955
SpyCas9-
39
0






3var-NRRH




 


358
TGAT
TGGGATGCAGCAGGGAATAC
23956
SpyCas9-
39
0






VQR




 


359
AGCT
TCTCCAGCTCCAGGGGGAGA
23957
SpyCas9-
39
0






3var-NRCH




 


360
AAG
TTCTCCAGCTCCAGGGGGAG
23958
ScaCas9
40
0


 


361
AAG
TTCTCCAGCTCCAGGGGGAG
23959
ScaCas9-
40
0






HiFi-Sc++




 


362
AAG
TTCTCCAGCTCCAGGGGGAG
23960
ScaCas9-
40
0






Sc++




 


363
AAG
TTCTCCAGCTCCAGGGGGAG
23961
SpyCas9-
40
0






SpRY




 


364
CTG
ATGGGATGCAGCAGGGAATA
23962
ScaCas9
40
0


 


365
CTG
ATGGGATGCAGCAGGGAATA
23963
ScaCas9-
40
0






HiFi-Sc++




 


366
CTG
ATGGGATGCAGCAGGGAATA
23964
ScaCas9-
40
0






Sc++




 


367
CTG
ATGGGATGCAGCAGGGAATA
23965
SpyCas9-
40
0






SpRY




 


368
AAGCTTT
TCTTCTCCAGCTCCAGGGGGAG
23966
CdiCas9
40
0


 


369
CTGATCC
CTATGGGATGCAGCAGGGAATA
23967
CdiCas9
40
0


 


370
AAGC
TTCTCCAGCTCCAGGGGGAG
23968
SpyCas9-
40
0






3var-NRRH




 


371
ACTGA
CTATGGGATGCAGCAGGGAAT
23969
SauCas9KKH
41
0


 


372
ACTGAT
CTATGGGATGCAGCAGGGAAT
23970
SauCas9KKH
41
0


 


373
GAAG
TCTTCTCCAGCTCCAGGGGGA
23971
SauriCas9-
41
0






KKH




 


374
GAAG
CTTCTCCAGCTCCAGGGGGA
23972
SpyCas9-
41
0






QQR1




 


375
GAAG
tcTTCTCCAGCTCCAGGGGGA
23973
iSpyMacCas9
41
0


 


376
GAA
CTTCTCCAGCTCCAGGGGGA
23974
SpyCas9-
41
0






SpRY




 


377
GAA
CTTCTCCAGCTCCAGGGGGA
23975
SpyCas9-
41
0






xCas




 


378
ACT
TATGGGATGCAGCAGGGAAT
23976
SpyCas9-
41
0






SpRY




 


379
GAAGCTT
tgtcTTCTCCAGCTCCAGGGGGA
23977
BlatCas9
41
0



T







 


380
GAAGC
tgtcTTCTCCAGCTCCAGGGGGA
23978
BlatCas9
41
0


 


381
GAAGCT
TCTTCTCCAGCTCCAGGGGGA
23979
cCas9-v16
41
0


 


382
GAAGCT
TCTTCTCCAGCTCCAGGGGGA
23980
cCas9-v21
41
0


 


383
AGAAG
GTCTTCTCCAGCTCCAGGGGG
23981
SauCas9KKH
42
0


 


384
AG
TCTTCTCCAGCTCCAGGGGG
23982
SpyCas9-NG
42
0


 


385
AG
TCTTCTCCAGCTCCAGGGGG
23983
SpyCas9-
42
0






xCas




 


386
AG
TCTTCTCCAGCTCCAGGGGG
23984
SpyCas9-
42
0






xCas-NG




 


387
AGA
TCTTCTCCAGCTCCAGGGGG
23985
SpyCas9-
42
0






SpG




 


388
AGA
TCTTCTCCAGCTCCAGGGGG
23986
SpyCas9-
42
0






SpRY




 


389
TAC
CTATGGGATGCAGCAGGGAA
23987
SpyCas9-
42
0






SpRY




 


390
AGAAGC
GTCTTCTCCAGCTCCAGGGGG
23988
cCas9-v17
42
0


 


391
AGAAGC
GTCTTCTCCAGCTCCAGGGGG
23989
cCas9-v42
42
0


 


392
AGAAGCT
gctgTCTTCTCCAGCTCCAGGGGG
23990
NmeCas9
42
0



T







 


393
AGAA
TCTTCTCCAGCTCCAGGGGG
23991
SpyCas9-
42
0






3var-NRRH




 


394
AGAA
TCTTCTCCAGCTCCAGGGGG
23992
SpyCas9-
42
0






VQR




 


395
TACT
CTATGGGATGCAGCAGGGAA
23993
SpyCas9-
42
0






3var-NRCH




 


396
GAGAA
gcTGTCTTCTCCAGCTCCAGGGG
23994
SauCas9
43
0


 


397
GAGAA
TGTCTTCTCCAGCTCCAGGGG
23995
SauCas9KKH
43
0


 


398
GAG
GTCTTCTCCAGCTCCAGGGG
23996
ScaCas9
43
0


 


399
GAG
GTCTTCTCCAGCTCCAGGGG
23997
ScaCas9-
43
0






HiFi-Sc++




 


400
GAG
GTCTTCTCCAGCTCCAGGGG
23998
ScaCas9-
43
0






Sc++




 


401
GAG
GTCTTCTCCAGCTCCAGGGG
23999
SpyCas9-
43
0






SpRY




 


402
ATA
CCTATGGGATGCAGCAGGGA
24000
SpyCas9-
43
0






SpRY




 


403
GAGAAG
TGTCTTCTCCAGCTCCAGGGG
24001
cCas9-v17
43
0


 


404
GAGAAG
TGTCTTCTCCAGCTCCAGGGG
24002
cCas9-v42
43
0


 


405
GAGA
GTCTTCTCCAGCTCCAGGGG
24003
SpyCas9-
43
0






3var-NRRH




 


406
GGAGA
CTGTCTTCTCCAGCTCCAGGG
24004
SauCas9KKH
44
0


 


407
GGAG
CTGTCTTCTCCAGCTCCAGGG
24005
SauriCas9-
44
0






KKH




 


408
GGAG
TGTCTTCTCCAGCTCCAGGG
24006
SpyCas9-
44
0






VQR




 


409
GG
TGTCTTCTCCAGCTCCAGGG
24007
SpyCas9-NG
44
0


 


410
GG
TGTCTTCTCCAGCTCCAGGG
24008
SpyCas9-
44
0






xCas




 


411
GG
TGTCTTCTCCAGCTCCAGGG
24009
SpyCas9-
44
0






xCas-NG




 


412
GGA
TGTCTTCTCCAGCTCCAGGG
24010
SpyCas9-
44
0






SpG




 


413
GGA
TGTCTTCTCCAGCTCCAGGG
24011
SpyCas9-
44
0






SpRY




 


414
AAT
GCCTATGGGATGCAGCAGGG
24012
SpyCas9-
44
0






SpRY




 


415
AATACTG
atggCCTATGGGATGCAGCAGGG
24013
BlatCas9
44
0



A







 


416
AATAC
atggCCTATGGGATGCAGCAGGG
24014
BlatCas9
44
0


 


417
GGAGAA
CTGTCTTCTCCAGCTCCAGGG
24015
cCas9-v17
44
0


 


418
GGAGAA
CTGTCTTCTCCAGCTCCAGGG
24016
cCas9-v42
44
0


 


419
AATA
GCCTATGGGATGCAGCAGGG
24017
SpyCas9-
44
0






3var-NRTH




 


420
GGGAG
tgGCTGTCTTCTCCAGCTCCAGG
24018
SauCas9
45
0


 


421
GGGAG
GCTGTCTTCTCCAGCTCCAGG
24019
SauCas9KKH
45
0


 


422
GGG
CTGTCTTCTCCAGCTCCAGG
24020
ScaCas9
45
0


 


423
GGG
CTGTCTTCTCCAGCTCCAGG
24021
ScaCas9-
45
0






HiFi-Sc++




 


424
GGG
CTGTCTTCTCCAGCTCCAGG
24022
ScaCas9-
45
0






Sc++




 


425
GGG
CTGTCTTCTCCAGCTCCAGG
24023
SpyCas9
45
0


 


426
GGG
CTGTCTTCTCCAGCTCCAGG
24024
SpyCas9-
45
0






HF1




 


427
GGG
CTGTCTTCTCCAGCTCCAGG
24025
SpyCas9-
45
0






SpG




 


428
GGG
CTGTCTTCTCCAGCTCCAGG
24026
SpyCas9-
45
0






SpRY




 


429
GG
CTGTCTTCTCCAGCTCCAGG
24027
SpyCas9-NG
45
0


 


430
GG
CTGTCTTCTCCAGCTCCAGG
24028
SpyCas9-
45
0






xCas




 


431
GG
CTGTCTTCTCCAGCTCCAGG
24029
SpyCas9-
45
0






xCas-NG




 


432
GAA
GGCCTATGGGATGCAGCAGG
24030
SpyCas9-
45
0






SpRY




 


433
GAA
GGCCTATGGGATGCAGCAGG
24031
SpyCas9-
45
0






xCas




 


434
GGGAGA
GCTGTCTTCTCCAGCTCCAGG
24032
cCas9-v17
45
0


 


435
GGGAGA
GCTGTCTTCTCCAGCTCCAGG
24033
cCas9-v42
45
0


 


436
GAATACT
ATGGCCTATGGGATGCAGCAGG
24034
CdiCas9
45
0


 


437
GAAT
GGCCTATGGGATGCAGCAGG
24035
SpyCas9-
45
0






3var-NRRH




 


438
GAAT
tgGCCTATGGGATGCAGCAGG
24036
iSpyMacCas9
45
0


 


439
GGGA
CTGTCTTCTCCAGCTCCAGG
24037
SpyCas9-
45
0






3var-NRRH




 


440
GGGGA
atGGCTGTCTTCTCCAGCTCCAG
24038
SauCas9
46
0


 


441
GGGGA
GGCTGTCTTCTCCAGCTCCAG
24039
SauCas9KKH
46
0


 


442
GGGG
GGCTGTCTTCTCCAGCTCCAG
24040
SauriCas9
46
0


 


443
GGGG
GGCTGTCTTCTCCAGCTCCAG
24041
SauriCas9-
46
0






KKH




 


444
GGG
GCTGTCTTCTCCAGCTCCAG
24042
ScaCas9
46
0


 


445
GGG
GCTGTCTTCTCCAGCTCCAG
24043
ScaCas9-
46
0






HiFi-Sc++




 


446
GGG
GCTGTCTTCTCCAGCTCCAG
24044
ScaCas9-
46
0






Sc++




 


447
GGG
GCTGTCTTCTCCAGCTCCAG
24045
SpyCas9
46
0


 


448
GGG
GCTGTCTTCTCCAGCTCCAG
24046
SpyCas9-
46
0






HF1




 


449
GGG
GCTGTCTTCTCCAGCTCCAG
24047
SpyCas9-
46
0






SpG




 


450
GGG
GCTGTCTTCTCCAGCTCCAG
24048
SpyCas9-
46
0






SpRY




 


451
GG
GCTGTCTTCTCCAGCTCCAG
24049
SpyCas9-NG
46
0


 


452
GG
GCTGTCTTCTCCAGCTCCAG
24050
SpyCas9-
46
0






xCas




 


453
GG
GCTGTCTTCTCCAGCTCCAG
24051
SpyCas9-
46
0






xCas-NG




 


454
GG
TGGCCTATGGGATGCAGCAG
24052
SpyCas9-NG
46
0


 


455
GG
TGGCCTATGGGATGCAGCAG
24053
SpyCas9-
46
0






xCas




 


456
GG
TGGCCTATGGGATGCAGCAG
24054
SpyCas9-
46
0






xCas-NG




 


457
GGA
TGGCCTATGGGATGCAGCAG
24055
SpyCas9-
46
0






SpG




 


458
GGA
TGGCCTATGGGATGCAGCAG
24056
SpyCas9-
46
0






SpRY




 


459
GGGGAG
GGCTGTCTTCTCCAGCTCCAG
24057
cCas9-v17
46
0


 


460
GGGGAG
GGCTGTCTTCTCCAGCTCCAG
24058
cCas9-v42
46
0


 


461
GGAATAC
AATGGCCTATGGGATGCAGCAG
24059
CdiCas9
46
0


 


462
GGAA
TGGCCTATGGGATGCAGCAG
24060
SpyCas9-
46
0






3var-NRRH




 


463
GGAA
TGGCCTATGGGATGCAGCAG
24061
SpyCas9-
46
0






VQR




 


464
GGGGG
gaTGGCTGTCTTCTCCAGCTCCA
24062
SauCas9
47
0


 


465
GGGGG
TGGCTGTCTTCTCCAGCTCCA
24063
SauCas9KKH
47
0


 


466
GGGAA
caAATGGCCTATGGGATGCAGCA
24064
SauCas9
47
0


 


467
GGGAA
AATGGCCTATGGGATGCAGCA
24065
SauCas9KKH
47
0


 


468
GGGAAT
caAATGGCCTATGGGATGCAGCA
24066
SauCas9
47
0


 


469
GGGAAT
AATGGCCTATGGGATGCAGCA
24067
SauCas9KKH
47
0


 


470
GGGAAT
AATGGCCTATGGGATGCAGCA
24068
cCas9-v17
47
0


 


471
GGGAAT
AATGGCCTATGGGATGCAGCA
24069
cCas9-v42
47
0


 


472
GGGG
TGGCTGTCTTCTCCAGCTCCA
24070
SauriCas9
47
0


 


473
GGGG
TGGCTGTCTTCTCCAGCTCCA
24071
SauriCas9-
47
0






KKH




 


474
GGG
GGCTGTCTTCTCCAGCTCCA
24072
ScaCas9
47
0


 


475
GGG
GGCTGTCTTCTCCAGCTCCA
24073
ScaCas9-
47
0






HiFi-Sc++




 


476
GGG
GGCTGTCTTCTCCAGCTCCA
24074
ScaCas9-
47
0






Sc++




 


477
GGG
GGCTGTCTTCTCCAGCTCCA
24075
SpyCas9
47
0


 


478
GGG
GGCTGTCTTCTCCAGCTCCA
24076
SpyCas9-
47
0






HF1




 


479
GGG
GGCTGTCTTCTCCAGCTCCA
24077
SpyCas9-
47
0






SpG




 


480
GGG
GGCTGTCTTCTCCAGCTCCA
24078
SpyCas9-
47
0






SpRY




 


481
GGG
ATGGCCTATGGGATGCAGCA
24079
ScaCas9
47
0


 


482
GGG
ATGGCCTATGGGATGCAGCA
24080
ScaCas9-
47
0






HiFi-Sc++




 


483
GGG
ATGGCCTATGGGATGCAGCA
24081
ScaCas9-
47
0






Sc++




 


484
GGG
ATGGCCTATGGGATGCAGCA
24082
SpyCas9
47
0


 


485
GGG
ATGGCCTATGGGATGCAGCA
24083
SpyCas9-
47
0






HF1




 


486
GGG
ATGGCCTATGGGATGCAGCA
24084
SpyCas9-
47
0






SpG




 


487
GGG
ATGGCCTATGGGATGCAGCA
24085
SpyCas9-
47
0






SpRY




 


488
GG
GGCTGTCTTCTCCAGCTCCA
24086
SpyCas9-NG
47
0


 


489
GG
GGCTGTCTTCTCCAGCTCCA
24087
SpyCas9-
47
0






xCas




 


490
GG
GGCTGTCTTCTCCAGCTCCA
24088
SpyCas9-
47
0






xCas-NG




 


491
GG
ATGGCCTATGGGATGCAGCA
24089
SpyCas9-NG
47
0


 


492
GG
ATGGCCTATGGGATGCAGCA
24090
SpyCas9-
47
0






xCas




 


493
GG
ATGGCCTATGGGATGCAGCA
24091
SpyCas9-
47
0






xCas-NG




 


494
GGGGGA
TGGCTGTCTTCTCCAGCTCCA
24092
cCas9-v17
47
0


 


495
GGGGGA
TGGCTGTCTTCTCCAGCTCCA
24093
cCas9-v42
47
0


 


496
GGGAATA
caAATGGCCTATGGGATGCAGCA
24094
CjeCas9
47
0



C







 


497
GGGA
ATGGCCTATGGGATGCAGCA
24095
SpyCas9-
47
0






3var-NRRH




 


498
AGGGG
ggATGGCTGTCTTCTCCAGCTCC
24096
SauCas9
48
0


 


499
AGGGG
ATGGCTGTCTTCTCCAGCTCC
24097
SauCas9KKH
48
0


 


500
AGGGA
acAAATGGCCTATGGGATGCAGC
24098
SauCas9
48
0


 


501
AGGGA
AAATGGCCTATGGGATGCAGC
24099
SauCas9KKH
48
0


 


502
AGGG
ATGGCTGTCTTCTCCAGCTCC
24100
SauriCas9
48
0


 


503
AGGG
ATGGCTGTCTTCTCCAGCTCC
24101
SauriCas9-
48
0






KKH




 


504
AGGG
AAATGGCCTATGGGATGCAGC
24102
SauriCas9
48
0


 


505
AGGG
AAATGGCCTATGGGATGCAGC
24103
SauriCas9-
48
0






KKH




 


506
AGG
TGGCTGTCTTCTCCAGCTCC
24104
ScaCas9
48
0


 


507
AGG
TGGCTGTCTTCTCCAGCTCC
24105
ScaCas9-
48
0






HiFi-Sc++




 


508
AGG
TGGCTGTCTTCTCCAGCTCC
24106
ScaCas9-
48
0






Sc++




 


509
AGG
TGGCTGTCTTCTCCAGCTCC
24107
SpyCas9
48
0


 


510
AGG
TGGCTGTCTTCTCCAGCTCC
24108
SpyCas9-
48
0






HF1




 


511
AGG
TGGCTGTCTTCTCCAGCTCC
24109
SpyCas9-
48
0






SpG




 


512
AGG
TGGCTGTCTTCTCCAGCTCC
24110
SpyCas9-
48
0






SpRY




 


513
AGG
AATGGCCTATGGGATGCAGC
24111
ScaCas9
48
0


 


514
AGG
AATGGCCTATGGGATGCAGC
24112
ScaCas9-
48
0






HiFi-Sc++




 


515
AGG
AATGGCCTATGGGATGCAGC
24113
ScaCas9-
48
0






Sc++




 


516
AGG
AATGGCCTATGGGATGCAGC
24114
SpyCas9
48
0


 


517
AGG
AATGGCCTATGGGATGCAGC
24115
SpyCas9-
48
0






HF1




 


518
AGG
AATGGCCTATGGGATGCAGC
24116
SpyCas9-
48
0






SpG




 


519
AGG
AATGGCCTATGGGATGCAGC
24117
SpyCas9-
48
0






SpRY




 


520
AG
TGGCTGTCTTCTCCAGCTCC
24118
SpyCas9-NG
48
0


 


521
AG
TGGCTGTCTTCTCCAGCTCC
24119
SpyCas9-
48
0






xCas




 


522
AG
TGGCTGTCTTCTCCAGCTCC
24120
SpyCas9-
48
0






xCas-NG




 


523
AG
AATGGCCTATGGGATGCAGC
24121
SpyCas9-NG
48
0


 


524
AG
AATGGCCTATGGGATGCAGC
24122
SpyCas9-
48
0






xCas




 


525
AG
AATGGCCTATGGGATGCAGC
24123
SpyCas9-
48
0






xCas-NG




 


526
AGGGAAT
AATGGCCTATGGGATGCAGC
24124
St1Cas9
48
0


 


527
AGGGGG
ATGGCTGTCTTCTCCAGCTCC
24125
cCas9-v17
48
0


 


528
AGGGGG
ATGGCTGTCTTCTCCAGCTCC
24126
cCas9-v42
48
0


 


529
AGGGAA
AAATGGCCTATGGGATGCAGC
24127
cCas9-v17
48
0


 


530
AGGGAA
AAATGGCCTATGGGATGCAGC
24128
cCas9-v42
48
0


 


531
CAGGG
tgGATGGCTGTCTTCTCCAGCTC
24129
SauCas9
49
0


 


532
CAGGG
GATGGCTGTCTTCTCCAGCTC
24130
SauCas9KKH
49
0


 


533
CAGGG
caCAAATGGCCTATGGGATGCAG
24131
SauCas9
49
0


 


534
CAGGG
CAAATGGCCTATGGGATGCAG
24132
SauCas9KKH
49
0


 


535
CAGG
GATGGCTGTCTTCTCCAGCTC
24133
SauriCas9
49
0


 


536
CAGG
GATGGCTGTCTTCTCCAGCTC
24134
SauriCas9-
49
0






KKH




 


537
CAGG
CAAATGGCCTATGGGATGCAG
24135
SauriCas9
49
0


 


538
CAGG
CAAATGGCCTATGGGATGCAG
24136
SauriCas9-
49
0






KKH




 


539
CAG
ATGGCTGTCTTCTCCAGCTC
24137
ScaCas9
49
0


 


540
CAG
ATGGCTGTCTTCTCCAGCTC
24138
ScaCas9-
49
0






HiFi-Sc++




 


541
CAG
ATGGCTGTCTTCTCCAGCTC
24139
ScaCas9-
49
0






Sc++




 


542
CAG
ATGGCTGTCTTCTCCAGCTC
24140
SpyCas9-
49
0






SpRY




 


543
CAG
AAATGGCCTATGGGATGCAG
24141
ScaCas9
49
0


 


544
CAG
AAATGGCCTATGGGATGCAG
24142
ScaCas9-
49
0






HiFi-Sc++




 


545
CAG
AAATGGCCTATGGGATGCAG
24143
ScaCas9-
49
0






Sc++




 


546
CAG
AAATGGCCTATGGGATGCAG
24144
SpyCas9-
49
0






SpRY




 


547
CAGGGG
GATGGCTGTCTTCTCCAGCTC
24145
cCas9-v17
49
0


 


548
CAGGGG
GATGGCTGTCTTCTCCAGCTC
24146
cCas9-v42
49
0


 


549
CAGGGA
CAAATGGCCTATGGGATGCAG
24147
cCas9-v17
49
0


 


550
CAGGGA
CAAATGGCCTATGGGATGCAG
24148
cCas9-v42
49
0


 


551
CCAGG
GGATGGCTGTCTTCTCCAGCT
24149
SauCas9KKH
50
0


 


552
GCAGG
ACAAATGGCCTATGGGATGCA
24150
SauCas9KKH
50
0


 


553
CCAG
GGATGGCTGTCTTCTCCAGCT
24151
SauriCas9-
50
0






KKH




 


554
GCAG
ACAAATGGCCTATGGGATGCA
24152
SauriCas9-
50
0






KKH




 


555
CCA
GATGGCTGTCTTCTCCAGCT
24153
SpyCas9-
50
0






SpRY




 


556
GCA
CAAATGGCCTATGGGATGCA
24154
SpyCas9-
50
0






SpRY




 


557
CCAGGG
GGATGGCTGTCTTCTCCAGCT
24155
cCas9-v17
50
0


 


558
CCAGGG
GGATGGCTGTCTTCTCCAGCT
24156
cCas9-v42
50
0


 


559
GCAGGG
ACAAATGGCCTATGGGATGCA
24157
cCas9-v17
50
0


 


560
GCAGGG
ACAAATGGCCTATGGGATGCA
24158
cCas9-v42
50
0


 


561
TCCAG
TGGATGGCTGTCTTCTCCAGC
24159
SauCas9KKH
51
0


 


562
AGCAG
CACAAATGGCCTATGGGATGC
24160
SauCas9KKH
51
0


 


563
AG
ACAAATGGCCTATGGGATGC
24161
SpyCas9-NG
51
0


 


564
AG
ACAAATGGCCTATGGGATGC
24162
SpyCas9-
51
0






xCas




 


565
AG
ACAAATGGCCTATGGGATGC
24163
SpyCas9-
51
0






xCas-NG




 


566
AGC
ACAAATGGCCTATGGGATGC
24164
SpyCas9-
51
0






SpG




 


567
AGC
ACAAATGGCCTATGGGATGC
24165
SpyCas9-
51
0






SpRY




 


568
TCC
GGATGGCTGTCTTCTCCAGC
24166
SpyCas9-
51
0






SpRY




 


569
TCCAGG
TGGATGGCTGTCTTCTCCAGC
24167
cCas9-v17
51
0


 


570
TCCAGG
TGGATGGCTGTCTTCTCCAGC
24168
cCas9-v42
51
0


 


571
AGCAGG
CACAAATGGCCTATGGGATGC
24169
cCas9-v17
51
0


 


572
AGCAGG
CACAAATGGCCTATGGGATGC
24170
cCas9-v42
51
0


 


573
AGCA
ACAAATGGCCTATGGGATGC
24171
SpyCas9-
51
0






3var-NRCH




 


574
CAG
CACAAATGGCCTATGGGATG
24172
ScaCas9
52
0


 


575
CAG
CACAAATGGCCTATGGGATG
24173
ScaCas9-
52
0






HiFi-Sc++




 


576
CAG
CACAAATGGCCTATGGGATG
24174
ScaCas9-
52
0






Sc++




 


577
CAG
CACAAATGGCCTATGGGATG
24175
SpyCas9-
52
0






SpRY




 


578
CTC
TGGATGGCTGTCTTCTCCAG
24176
SpyCas9-
52
0






SpRY




 


579
CAGC
CACAAATGGCCTATGGGATG
24177
SpyCas9-
52
0






3var-NRRH




 


580
GCAG
GGCACAAATGGCCTATGGGAT
24178
SauriCas9-
53
0






KKH




 


581
GCT
TTGGATGGCTGTCTTCTCCA
24179
SpyCas9-
53
0






SpRY




 


582
GCA
GCACAAATGGCCTATGGGAT
24180
SpyCas9-
53
0






SpRY




 


583
GCTCCAG
atttTGGATGGCTGTCTTCTCCA
24181
BlatCas9
53
0



G







 


584
GCAGCAG
ggggCACAAATGGCCTATGGGAT
24182
BlatCas9
53
0



G







 


585
GCTCC
atttTGGATGGCTGTCTTCTCCA
24183
BlatCas9
53
0


 


586
GCAGC
ggggCACAAATGGCCTATGGGAT
24184
BlatCas9
53
0


 


587
AGCTCC
taATTTTGGATGGCTGTCTTCTCC
24185
Nme2Cas9
54
0


 


588
TGCAG
GGGCACAAATGGCCTATGGGA
24186
SauCas9KKH
54
0


 


589
AG
TTTGGATGGCTGTCTTCTCC
24187
SpyCas9-NG
54
0


 


590
AG
TTTGGATGGCTGTCTTCTCC
24188
SpyCas9-
54
0






xCas




 


591
AG
TTTGGATGGCTGTCTTCTCC
24189
SpyCas9-
54
0






xCas-NG




 


592
TG
GGCACAAATGGCCTATGGGA
24190
SpyCas9-NG
54
0


 


593
TG
GGCACAAATGGCCTATGGGA
24191
SpyCas9-
54
0






xCas




 


594
TG
GGCACAAATGGCCTATGGGA
24192
SpyCas9-
54
0






xCas-NG




 


595
AGC
TTTGGATGGCTGTCTTCTCC
24193
SpyCas9-
54
0






SpG




 


596
AGC
TTTGGATGGCTGTCTTCTCC
24194
SpyCas9-
54
0






SpRY




 


597
TGC
GGCACAAATGGCCTATGGGA
24195
SpyCas9-
54
0






SpG




 


598
TGC
GGCACAAATGGCCTATGGGA
24196
SpyCas9-
54
0






SpRY




 


599
AGCTCCA
aattTTGGATGGCTGTCTTCTCC
24197
BlatCas9
54
0



G







 


600
AGCTC
aattTTGGATGGCTGTCTTCTCC
24198
BlatCas9
54
0


 


601
TGCAGC
GGGCACAAATGGCCTATGGGA
24199
cCas9-v17
54
0


 


602
TGCAGC
GGGCACAAATGGCCTATGGGA
24200
cCas9-v42
54
0


 


603
AGCT
TTTGGATGGCTGTCTTCTCC
24201
SpyCas9-
54
0






3var-NRCH




 


604
TGCA
GGCACAAATGGCCTATGGGA
24202
SpyCas9-
54
0






3var-NRCH




 


605
CAG
TTTTGGATGGCTGTCTTCTC
24203
ScaCas9
55
0


 


606
CAG
TTTTGGATGGCTGTCTTCTC
24204
ScaCas9-
55
0






HiFi-Sc++




 


607
CAG
TTTTGGATGGCTGTCTTCTC
24205
ScaCas9-
55
0






Sc++




 


608
CAG
TTTTGGATGGCTGTCTTCTC
24206
SpyCas9-
55
0






SpRY




 


609
ATG
GGGCACAAATGGCCTATGGG
24207
ScaCas9
55
0


 


610
ATG
GGGCACAAATGGCCTATGGG
24208
ScaCas9-
55
0






HiFi-Sc++




 


611
ATG
GGGCACAAATGGCCTATGGG
24209
ScaCas9-
55
0






Sc++




 


612
ATG
GGGCACAAATGGCCTATGGG
24210
SpyCas9-
55
0






SpRY




 


613
CAGCTCC
AATTTTGGATGGCTGTCTTCTC
24211
CdiCas9
55
0


 


614
CAGC
TTTTGGATGGCTGTCTTCTC
24212
SpyCas9-
55
0






3var-NRRH




 


615
CCAG
AATTTTGGATGGCTGTCTTCT
24213
SauriCas9-
56
0






KKH




 


616
GAT
GGGGCACAAATGGCCTATGG
24214
SpyCas9-
56
0






SpRY




 


617
GAT
GGGGCACAAATGGCCTATGG
24215
SpyCas9-
56
0






xCas




 


618
CCA
ATTTTGGATGGCTGTCTTCT
24216
SpyCas9-
56
0






SpRY




 


619
CCAGC
gtaaTTTTGGATGGCTGTCTTCT
24217
BlatCas9
56
0


 


620
GATGC
gaagGGGCACAAATGGCCTATGG
24218
BlatCas9
56
0


 


621
CCAGCT
AATTTTGGATGGCTGTCTTCT
24219
cCas9-v16
56
0


 


622
CCAGCT
AATTTTGGATGGCTGTCTTCT
24220
cCas9-v21
56
0


 


623
TCCAG
TAATTTTGGATGGCTGTCTTC
24221
SauCas9KKH
57
0


 


624
GG
AGGGGCACAAATGGCCTATG
24222
SpyCas9-NG
57
0


 


625
GG
AGGGGCACAAATGGCCTATG
24223
SpyCas9-
57
0






xCas




 


626
GG
AGGGGCACAAATGGCCTATG
24224
SpyCas9-
57
0






xCas-NG




 


627
GGA
AGGGGCACAAATGGCCTATG
24225
SpyCas9-
57
0






SpG




 


628
GGA
AGGGGCACAAATGGCCTATG
24226
SpyCas9-
57
0






SpRY




 


629
TCC
AATTTTGGATGGCTGTCTTC
24227
SpyCas9-
57
0






SpRY




 


630
TCCAGC
TAATTTTGGATGGCTGTCTTC
24228
cCas9-v17
57
0


 


631
TCCAGC
TAATTTTGGATGGCTGTCTTC
24229
cCas9-v42
57
0


 


632
GGAT
AGGGGCACAAATGGCCTATG
24230
SpyCas9-
57
0






3var-NRRH




 


633
GGAT
AGGGGCACAAATGGCCTATG
24231
SpyCas9-
57
0






VQR




 


634
GGG
AAGGGGCACAAATGGCCTAT
24232
ScaCas9
58
0


 


635
GGG
AAGGGGCACAAATGGCCTAT
24233
ScaCas9-
58
0






HiFi-Sc++




 


636
GGG
AAGGGGCACAAATGGCCTAT
24234
ScaCas9-
58
0






Sc++




 


637
GGG
AAGGGGCACAAATGGCCTAT
24235
SpyCas9
58
0


 


638
GGG
AAGGGGCACAAATGGCCTAT
24236
SpyCas9-
58
0






HF1




 


639
GGG
AAGGGGCACAAATGGCCTAT
24237
SpyCas9-
58
0






SpG




 


640
GGG
AAGGGGCACAAATGGCCTAT
24238
SpyCas9-
58
0






SpRY




 


641
GG
AAGGGGCACAAATGGCCTAT
24239
SpyCas9-NG
58
0


 


642
GG
AAGGGGCACAAATGGCCTAT
24240
SpyCas9-
58
0






xCas




 


643
GG
AAGGGGCACAAATGGCCTAT
24241
SpyCas9-
58
0






xCas-NG




 


644
CTC
TAATTTTGGATGGCTGTCTT
24242
SpyCas9-
58
0






SpRY




 


645
GGGATGC
GAAGGGGCACAAATGGCCTAT
24243
cCas9-v16
58
0


 


646
GGGATGC
GAAGGGGCACAAATGGCCTAT
24244
cCas9-v21
58
0


 


647
GGGA
AAGGGGCACAAATGGCCTAT
24245
SpyCas9-
58
0






3var-NRRH




 


648
TGGGA
agAGAAGGGGCACAAATGGCCTA
24246
SauCas9
59
0


 


649
TGGGA
AGAAGGGGCACAAATGGCCTA
24247
SauCas9KKH
59
0


 


650
TGGGAT
agAGAAGGGGCACAAATGGCCTA
24248
SauCas9
59
0


 


651
TGGGAT
AGAAGGGGCACAAATGGCCTA
24249
SauCas9KKH
59
0


 


652
TGGGAT
AGAAGGGGCACAAATGGCCTA
24250
cCas9-v17
59
0


 


653
TGGGAT
AGAAGGGGCACAAATGGCCTA
24251
cCas9-v42
59
0


 


654
TGGG
AGAAGGGGCACAAATGGCCTA
24252
SauriCas9
59
0


 


655
TGGG
AGAAGGGGCACAAATGGCCTA
24253
SauriCas9-
59
0






KKH




 


656
TGG
GAAGGGGCACAAATGGCCTA
24254
ScaCas9
59
0


 


657
TGG
GAAGGGGCACAAATGGCCTA
24255
ScaCas9-
59
0






HiFi-Sc++




 


658
TGG
GAAGGGGCACAAATGGCCTA
24256
ScaCas9-
59
0






Sc++




 


659
TGG
GAAGGGGCACAAATGGCCTA
24257
SpyCas9
59
0


 


660
TGG
GAAGGGGCACAAATGGCCTA
24258
SpyCas9-
59
0






HF1




 


661
TGG
GAAGGGGCACAAATGGCCTA
24259
SpyCas9-
59
0






SpG




 


662
TGG
GAAGGGGCACAAATGGCCTA
24260
SpyCas9-
59
0






SpRY




 


663
TG
GAAGGGGCACAAATGGCCTA
24261
SpyCas9-NG
59
0


 


664
TG
GAAGGGGCACAAATGGCCTA
24262
SpyCas9-
59
0






xCas




 


665
TG
GAAGGGGCACAAATGGCCTA
24263
SpyCas9-
59
0






xCas-NG




 


666
TCT
GTAATTTTGGATGGCTGTCT
24264
SpyCas9-
59
0






SpRY




 


667
TCTCC
agtgTAATTTTGGATGGCTGTCT
24265
BlatCas9
59
0


 


668
TTCTCC
acAGTGTAATTTTGGATGGCTGTC
24266
Nme2Cas9
60
0


 


669
ATGGG
gaGAGAAGGGGCACAAATGGCCT
24267
SauCas9
60
0


 


670
ATGGG
GAGAAGGGGCACAAATGGCCT
24268
SauCas9KKH
60
0


 


671
ATGG
GAGAAGGGGCACAAATGGCCT
24269
SauriCas9
60
0


 


672
ATGG
GAGAAGGGGCACAAATGGCCT
24270
SauriCas9-
60
0






KKH




 


673
ATG
AGAAGGGGCACAAATGGCCT
24271
ScaCas9
60
0


 


674
ATG
AGAAGGGGCACAAATGGCCT
24272
ScaCas9-
60
0






HiFi-Sc++




 


675
ATG
AGAAGGGGCACAAATGGCCT
24273
ScaCas9-
60
0






Sc++




 


676
ATG
AGAAGGGGCACAAATGGCCT
24274
SpyCas9-
60
0






SpRY




 


677
TTC
TGTAATTTTGGATGGCTGTC
24275
SpyCas9-
60
0






SpRY




 


678
TTCTCCAG
cagtGTAATTTTGGATGGCTGTC
24276
BlatCas9
60
0


 


679
TTCTC
cagtGTAATTTTGGATGGCTGTC
24277
BlatCas9
60
0


 


680
ATGGGA
GAGAAGGGGCACAAATGGCCT
24278
cCas9-v17
60
0


 


681
ATGGGA
GAGAAGGGGCACAAATGGCCT
24279
cCas9-v42
60
0


 


682
TATGG
AGAGAAGGGGCACAAATGGCC
24280
SauCas9KKH
61
0


 


683
TAT
GAGAAGGGGCACAAATGGCC
24281
SpyCas9-
61
0






SpRY




 


684
CTT
GTGTAATTTTGGATGGCTGT
24282
SpyCas9-
61
0






SpRY




 


685
TCT
AGTGTAATTTTGGATGGCTG
24283
SpyCas9-
62
0






SpRY




 


686
CTA
AGAGAAGGGGCACAAATGGC
24284
SpyCas9-
62
0






SpRY




 


687
TCTTC
gacaGTGTAATTTTGGATGGCTG
24285
BlatCas9
62
0


 


688
GTC
CAGTGTAATTTTGGATGGCT
24286
SpyCas9-
63
0






SpRY




 


689
CCT
GAGAGAAGGGGCACAAATGG
24287
SpyCas9-
63
0






SpRY




 


690
TG
ACAGTGTAATTTTGGATGGC
24288
SpyCas9-NG
64
0


 


691
TG
ACAGTGTAATTTTGGATGGC
24289
SpyCas9-
64
0






xCas




 


692
TG
ACAGTGTAATTTTGGATGGC
24290
SpyCas9-
64
0






xCas-NG




 


693
TGT
ACAGTGTAATTTTGGATGGC
24291
SpyCas9-
64
0






SpG




 


694
TGT
ACAGTGTAATTTTGGATGGC
24292
SpyCas9-
64
0






SpRY




 


695
GCC
TGAGAGAAGGGGCACAAATG
24293
SpyCas9-
64
0






SpRY




 


696
TGTC
ACAGTGTAATTTTGGATGGC
24294
SpyCas9-
64
0






3var-NRTH




 


697
CTG
GACAGTGTAATTTTGGATGG
24295
ScaCas9
65
0


 


698
CTG
GACAGTGTAATTTTGGATGG
24296
ScaCas9-
65
0






HiFi-Sc++




 


699
CTG
GACAGTGTAATTTTGGATGG
24297
ScaCas9-
65
0






Sc++




 


700
CTG
GACAGTGTAATTTTGGATGG
24298
SpyCas9-
65
0






SpRY




 


701
GG
ATGAGAGAAGGGGCACAAAT
24299
SpyCas9-NG
65
0


 


702
GG
ATGAGAGAAGGGGCACAAAT
24300
SpyCas9-
65
0






xCas




 


703
GG
ATGAGAGAAGGGGCACAAAT
24301
SpyCas9-
65
0






xCas-NG




 


704
GGC
ATGAGAGAAGGGGCACAAAT
24302
SpyCas9-
65
0






SpG




 


705
GGC
ATGAGAGAAGGGGCACAAAT
24303
SpyCas9-
65
0






SpRY




 


706
CTGTC
cgtgACAGTGTAATTTTGGATGG
24304
BlatCas9
65
0


 


707
CTGTCTT
GTGACAGTGTAATTTTGGATGG
24305
CdiCas9
65
0


 


708
GGCC
ATGAGAGAAGGGGCACAAAT
24306
SpyCas9-
65
0






3var-NRCH




 


709
TGG
GATGAGAGAAGGGGCACAAA
24307
ScaCas9
66
0


 


710
TGG
GATGAGAGAAGGGGCACAAA
24308
ScaCas9-
66
0






HiFi-Sc++




 


711
TGG
GATGAGAGAAGGGGCACAAA
24309
ScaCas9-
66
0






Sc++




 


712
TGG
GATGAGAGAAGGGGCACAAA
24310
SpyCas9
66
0


 


713
TGG
GATGAGAGAAGGGGCACAAA
24311
SpyCas9-
66
0






HF1




 


714
TGG
GATGAGAGAAGGGGCACAAA
24312
SpyCas9-
66
0






SpG




 


715
TGG
GATGAGAGAAGGGGCACAAA
24313
SpyCas9-
66
0






SpRY




 


716
TG
GATGAGAGAAGGGGCACAAA
24314
SpyCas9-NG
66
0


 


717
TG
GATGAGAGAAGGGGCACAAA
24315
SpyCas9-
66
0






xCas




 


718
TG
GATGAGAGAAGGGGCACAAA
24316
SpyCas9-
66
0






xCas-NG




 


719
GCT
TGACAGTGTAATTTTGGATG
24317
SpyCas9-
66
0






SpRY




 


720
TGGCCTAT
tgagATGAGAGAAGGGGCACAAA
24318
BlatCas9
66
0


 


721
TGGCC
tgagATGAGAGAAGGGGCACAAA
24319
BlatCas9
66
0


 


722
TGGC
GATGAGAGAAGGGGCACAAA
24320
SpyCas9-
66
0






3var-NRRH




 


723
ATGGCC
ggTGAGATGAGAGAAGGGGCAC
24321
Nme2Cas9
67
0




AA






 


724
ATGG
GAGATGAGAGAAGGGGCACAA
24322
SauriCas9
67
0


 


725
ATGG
GAGATGAGAGAAGGGGCACAA
24323
SauriCas9-
67
0






KKH




 


726
ATG
AGATGAGAGAAGGGGCACAA
24324
ScaCas9
67
0


 


727
ATG
AGATGAGAGAAGGGGCACAA
24325
ScaCas9-
67
0






HiFi-Sc++




 


728
ATG
AGATGAGAGAAGGGGCACAA
24326
ScaCas9-
67
0






Sc++




 


729
ATG
AGATGAGAGAAGGGGCACAA
24327
SpyCas9-
67
0






SpRY




 


730
GG
GTGACAGTGTAATTTTGGAT
24328
SpyCas9-NG
67
0


 


731
GG
GTGACAGTGTAATTTTGGAT
24329
SpyCas9-
67
0






xCas




 


732
GG
GTGACAGTGTAATTTTGGAT
24330
SpyCas9-
67
0






xCas-NG




 


733
GGC
GTGACAGTGTAATTTTGGAT
24331
SpyCas9-
67
0






SpG




 


734
GGC
GTGACAGTGTAATTTTGGAT
24332
SpyCas9-
67
0






SpRY




 


735
ATGGCCT
gtgaGATGAGAGAAGGGGCACAA
24333
BlatCas9
67
0



A







 


736
ATGGC
gtgaGATGAGAGAAGGGGCACAA
24334
BlatCas9
67
0


 


737
GGCTGTCT
ctccGTGACAGTGTAATTTTGGAT
24335
NmeCas9
67
0


 


738
GGCT
GTGACAGTGTAATTTTGGAT
24336
SpyCas9-
67
0






3var-NRCH




 


739
AATGG
TGAGATGAGAGAAGGGGCACA
24337
SauCas9KKH
68
0


 


740
TGG
CGTGACAGTGTAATTTTGGA
24338
ScaCas9
68
0


 


741
TGG
CGTGACAGTGTAATTTTGGA
24339
ScaCas9-
68
0






HiFi-Sc++




 


742
TGG
CGTGACAGTGTAATTTTGGA
24340
ScaCas9-
68
0






Sc++




 


743
TGG
CGTGACAGTGTAATTTTGGA
24341
SpyCas9
68
0


 


744
TGG
CGTGACAGTGTAATTTTGGA
24342
SpyCas9-
68
0






HF1




 


745
TGG
CGTGACAGTGTAATTTTGGA
24343
SpyCas9-
68
0






SpG




 


746
TGG
CGTGACAGTGTAATTTTGGA
24344
SpyCas9-
68
0






SpRY




 


747
TG
CGTGACAGTGTAATTTTGGA
24345
SpyCas9-NG
68
0


 


748
TG
CGTGACAGTGTAATTTTGGA
24346
SpyCas9-
68
0






xCas




 


749
TG
CGTGACAGTGTAATTTTGGA
24347
SpyCas9-
68
0






xCas-NG




 


750
AAT
GAGATGAGAGAAGGGGCACA
24348
SpyCas9-
68
0






SpRY




 


751
TGGC
CGTGACAGTGTAATTTTGGA
24349
SpyCas9-
68
0






3var-NRRH




 


752
ATGG
TCCGTGACAGTGTAATTTTGG
24350
SauriCas9
69
0


 


753
ATGG
TCCGTGACAGTGTAATTTTGG
24351
SauriCas9-
69
0






KKH




 


754
ATG
CCGTGACAGTGTAATTTTGG
24352
ScaCas9
69
0


 


755
ATG
CCGTGACAGTGTAATTTTGG
24353
ScaCas9-
69
0






HiFi-Sc++




 


756
ATG
CCGTGACAGTGTAATTTTGG
24354
ScaCas9-
69
0






Sc++




 


757
ATG
CCGTGACAGTGTAATTTTGG
24355
SpyCas9-
69
0






SpRY




 


758
AAA
TGAGATGAGAGAAGGGGCAC
24356
SpyCas9-
69
0






SpRY




 


759
ATGGCTG
actcCGTGACAGTGTAATTTTGG
24357
BlatCas9
69
0



T







 


760
ATGGC
actcCGTGACAGTGTAATTTTGG
24358
BlatCas9
69
0


 


761
ATGGCT
TCCGTGACAGTGTAATTTTGG
24359
cCas9-v16
69
0


 


762
ATGGCT
TCCGTGACAGTGTAATTTTGG
24360
cCas9-v21
69
0


 


763
AAAT
TGAGATGAGAGAAGGGGCAC
24361
SpyCas9-
69
0






3var-NRRH




 


764
AAAT
gtGAGATGAGAGAAGGGGCAC
24362
iSpyMacCas9
69
0


 


765
GATGG
CTCCGTGACAGTGTAATTTTG
24363
SauCas9KKH
70
0


 


766
GAT
TCCGTGACAGTGTAATTTTG
24364
SpyCas9-
70
0






SpRY




 


767
GAT
TCCGTGACAGTGTAATTTTG
24365
SpyCas9-
70
0






xCas




 


768
CAA
GTGAGATGAGAGAAGGGGCA
24366
SpyCas9-
70
0






SpRY




 


769
CAAA
GTGAGATGAGAGAAGGGGCA
24367
SpyCas9-
70
0






3var-NRRH




 


770
CAAA
ggTGAGATGAGAGAAGGGGCA
24368
iSpyMacCas9
70
0


 


771
ACAAA
GGGTGAGATGAGAGAAGGGGC
24369
SauCas9KKH
71
0


 


772
ACAAAT
GGGTGAGATGAGAGAAGGGGC
24370
SauCas9KKH
71
0


 


773
ACAAAT
GGGTGAGATGAGAGAAGGGGC
24371
cCas9-v17
71
0


 


774
ACAAAT
GGGTGAGATGAGAGAAGGGGC
24372
cCas9-v42
71
0


 


775
GG
CTCCGTGACAGTGTAATTTT
24373
SpyCas9-NG
71
0


 


776
GG
CTCCGTGACAGTGTAATTTT
24374
SpyCas9-
71
0






xCas




 


777
GG
CTCCGTGACAGTGTAATTTT
24375
SpyCas9-
71
0






xCas-NG




 


778
GGA
CTCCGTGACAGTGTAATTTT
24376
SpyCas9-
71
0






SpG




 


779
GGA
CTCCGTGACAGTGTAATTTT
24377
SpyCas9-
71
0






SpRY




 


780
ACA
GGTGAGATGAGAGAAGGGGC
24378
SpyCas9-
71
0






SpRY




 


781
GGAT
CTCCGTGACAGTGTAATTTT
24379
SpyCas9-
71
0






3var-NRRH




 


782
GGAT
CTCCGTGACAGTGTAATTTT
24380
SpyCas9-
71
0






VQR




 


783
CACAA
GGGGTGAGATGAGAGAAGGGG
24381
SauCas9KKH
72
0


 


784
TGG
ACTCCGTGACAGTGTAATTT
24382
ScaCas9
72
0


 


785
TGG
ACTCCGTGACAGTGTAATTT
24383
ScaCas9-
72
0






HiFi-Sc++




 


786
TGG
ACTCCGTGACAGTGTAATTT
24384
ScaCas9-
72
0






Sc++




 


787
TGG
ACTCCGTGACAGTGTAATTT
24385
SpyCas9
72
0


 


788
TGG
ACTCCGTGACAGTGTAATTT
24386
SpyCas9-
72
0






HF1




 


789
TGG
ACTCCGTGACAGTGTAATTT
24387
SpyCas9-
72
0






SpG




 


790
TGG
ACTCCGTGACAGTGTAATTT
24388
SpyCas9-
72
0






SpRY




 


791
TG
ACTCCGTGACAGTGTAATTT
24389
SpyCas9-NG
72
0


 


792
TG
ACTCCGTGACAGTGTAATTT
24390
SpyCas9-
72
0






xCas




 


793
TG
ACTCCGTGACAGTGTAATTT
24391
SpyCas9-
72
0






xCas-NG




 


794
CAC
GGGTGAGATGAGAGAAGGGG
24392
SpyCas9-
72
0






SpRY




 


795
CACAAA
GGGGTGAGATGAGAGAAGGGG
24393
cCas9-v17
72
0


 


796
CACAAA
GGGGTGAGATGAGAGAAGGGG
24394
cCas9-v42
72
0


 


797
TGGA
ACTCCGTGACAGTGTAATTT
24395
SpyCas9-
72
0






3var-NRRH




 


798
CACA
GGGTGAGATGAGAGAAGGGG
24396
SpyCas9-
72
0






3var-NRCH




 


799
TTGGA
tgGAACTCCGTGACAGTGTAATT
24397
SauCas9
73
0


 


800
TTGGA
GAACTCCGTGACAGTGTAATT
24398
SauCas9KKH
73
0


 


801
TTGGAT
tgGAACTCCGTGACAGTGTAATT
24399
SauCas9
73
0


 


802
TTGGAT
GAACTCCGTGACAGTGTAATT
24400
SauCas9KKH
73
0


 


803
TTGGAT
GAACTCCGTGACAGTGTAATT
24401
cCas9-v17
73
0


 


804
TTGGAT
GAACTCCGTGACAGTGTAATT
24402
cCas9-v42
73
0


 


805
TTGG
GAACTCCGTGACAGTGTAATT
24403
SauriCas9
73
0


 


806
TTGG
GAACTCCGTGACAGTGTAATT
24404
SauriCas9-
73
0






KKH




 


807
TTG
AACTCCGTGACAGTGTAATT
24405
ScaCas9
73
0


 


808
TTG
AACTCCGTGACAGTGTAATT
24406
ScaCas9-
73
0






HiFi-Sc++




 


809
TTG
AACTCCGTGACAGTGTAATT
24407
ScaCas9-
73
0






Sc++




 


810
TTG
AACTCCGTGACAGTGTAATT
24408
SpyCas9-
73
0






SpRY




 


811
GCA
GGGGTGAGATGAGAGAAGGG
24409
SpyCas9-
73
0






SpRY




 


812
GCACAA
GGGGTGAGATGAGAGAAGGG
24410
St1Cas9-
73
0






CNRZ1066




 


813
TTTGG
GGAACTCCGTGACAGTGTAAT
24411
SauCas9KKH
74
0


 


814
GG
CGGGGTGAGATGAGAGAAGG
24412
SpyCas9-NG
74
0


 


815
GG
CGGGGTGAGATGAGAGAAGG
24413
SpyCas9-
74
0






xCas




 


816
GG
CGGGGTGAGATGAGAGAAGG
24414
SpyCas9-
74
0






xCas-NG




 


817
GGC
CGGGGTGAGATGAGAGAAGG
24415
SpyCas9-
74
0






SpG




 


818
GGC
CGGGGTGAGATGAGAGAAGG
24416
SpyCas9-
74
0






SpRY




 


819
TTT
GAACTCCGTGACAGTGTAAT
24417
SpyCas9-
74
0






SpRY




 


820
GGCACAA
aatcGGGGTGAGATGAGAGAAGG
24418
BlatCas9
74
0



A







 


821
GGCACAA
aatcGGGGTGAGATGAGAGAAGG
24419
BlatCas9
74
0



A







 


822
GGCACAA
aaTCGGGGTGAGATGAGAGAAGG
24420
GeoCas9
74
0



A







 


823
GGCAC
aatcGGGGTGAGATGAGAGAAGG
24421
BlatCas9
74
0


 


824
GGCA
CGGGGTGAGATGAGAGAAGG
24422
SpyCas9-
74
0






3var-NRCH




 


825
GGG
TCGGGGTGAGATGAGAGAAG
24423
ScaCas9
75
0


 


826
GGG
TCGGGGTGAGATGAGAGAAG
24424
ScaCas9-
75
0






HiFi-Sc++




 


827
GGG
TCGGGGTGAGATGAGAGAAG
24425
ScaCas9-
75
0






Sc++




 


828
GGG
TCGGGGTGAGATGAGAGAAG
24426
SpyCas9
75
0


 


829
GGG
TCGGGGTGAGATGAGAGAAG
24427
SpyCas9-
75
0






HF1




 


830
GGG
TCGGGGTGAGATGAGAGAAG
24428
SpyCas9-
75
0






SpG




 


831
GGG
TCGGGGTGAGATGAGAGAAG
24429
SpyCas9-
75
0






SpRY




 


832
GG
TCGGGGTGAGATGAGAGAAG
24430
SpyCas9-NG
75
0


 


833
GG
TCGGGGTGAGATGAGAGAAG
24431
SpyCas9-
75
0






xCas




 


834
GG
TCGGGGTGAGATGAGAGAAG
24432
SpyCas9-
75
0






xCas-NG




 


835
TTT
GGAACTCCGTGACAGTGTAA
24433
SpyCas9-
75
0






SpRY




 


836
GGGC
TCGGGGTGAGATGAGAGAAG
24434
SpyCas9-
75
0






3var-NRRH




 


837
GGGG
AATCGGGGTGAGATGAGAGAA
24435
SauriCas9
76
0


 


838
GGGG
AATCGGGGTGAGATGAGAGAA
24436
SauriCas9-
76
0






KKH




 


839
GGG
ATCGGGGTGAGATGAGAGAA
24437
ScaCas9
76
0


 


840
GGG
ATCGGGGTGAGATGAGAGAA
24438
ScaCas9-
76
0






HiFi-Sc++




 


841
GGG
ATCGGGGTGAGATGAGAGAA
24439
ScaCas9-
76
0






Sc++




 


842
GGG
ATCGGGGTGAGATGAGAGAA
24440
SpyCas9
76
0


 


843
GGG
ATCGGGGTGAGATGAGAGAA
24441
SpyCas9-
76
0






HF1




 


844
GGG
ATCGGGGTGAGATGAGAGAA
24442
SpyCas9-
76
0






SpG




 


845
GGG
ATCGGGGTGAGATGAGAGAA
24443
SpyCas9-
76
0






SpRY




 


846
GG
ATCGGGGTGAGATGAGAGAA
24444
SpyCas9-NG
76
0


 


847
GG
ATCGGGGTGAGATGAGAGAA
24445
SpyCas9-
76
0






xCas




 


848
GG
ATCGGGGTGAGATGAGAGAA
24446
SpyCas9-
76
0






xCas-NG




 


849
ATT
TGGAACTCCGTGACAGTGTA
24447
SpyCas9-
76
0






SpRY




 


850
GGGGC
ggaaTCGGGGTGAGATGAGAGAA
24448
BlatCas9
76
0


 


851
AGGGG
agGAATCGGGGTGAGATGAGAG
24449
SauCas9
77
0




A






 


852
AGGGG
GAATCGGGGTGAGATGAGAGA
24450
SauCas9KKH
77
0


 


853
AGGG
GAATCGGGGTGAGATGAGAGA
24451
SauriCas9
77
0


 


854
AGGG
GAATCGGGGTGAGATGAGAGA
24452
SauriCas9-
77
0






KKH




 


855
AGG
AATCGGGGTGAGATGAGAGA
24453
ScaCas9
77
0


 


856
AGG
AATCGGGGTGAGATGAGAGA
24454
ScaCas9-
77
0






HiFi-Sc++




 


857
AGG
AATCGGGGTGAGATGAGAGA
24455
ScaCas9-
77
0






Sc++




 


858
AGG
AATCGGGGTGAGATGAGAGA
24456
SpyCas9
77
0


 


859
AGG
AATCGGGGTGAGATGAGAGA
24457
SpyCas9-
77
0






HF1




 


860
AGG
AATCGGGGTGAGATGAGAGA
24458
SpyCas9-
77
0






SpG




 


861
AGG
AATCGGGGTGAGATGAGAGA
24459
SpyCas9-
77
0






SpRY




 


862
AG
AATCGGGGTGAGATGAGAGA
24460
SpyCas9-NG
77
0


 


863
AG
AATCGGGGTGAGATGAGAGA
24461
SpyCas9-
77
0






xCas




 


864
AG
AATCGGGGTGAGATGAGAGA
24462
SpyCas9-
77
0






xCas-NG




 


865
AAT
CTGGAACTCCGTGACAGTGT
24463
SpyCas9-
77
0






SpRY




 


866
AGGGGC
GAATCGGGGTGAGATGAGAGA
24464
cCas9-v17
77
0


 


867
AGGGGC
GAATCGGGGTGAGATGAGAGA
24465
cCas9-v42
77
0


 


868
AGGGGCA
agGAATCGGGGTGAGATGAGAG
24466
CjeCas9
77
0



C
A






 


869
AATT
CTGGAACTCCGTGACAGTGT
24467
SpyCas9-
77
0






3var-NRTH




 


870
AAGGG
aaGGAATCGGGGTGAGATGAGAG
24468
SauCas9
78
0


 


871
AAGGG
GGAATCGGGGTGAGATGAGAG
24469
SauCas9KKH
78
0


 


872
AAGG
GGAATCGGGGTGAGATGAGAG
24470
SauriCas9
78
0


 


873
AAGG
GGAATCGGGGTGAGATGAGAG
24471
SauriCas9-
78
0






KKH




 


874
AAG
GAATCGGGGTGAGATGAGAG
24472
ScaCas9
78
0


 


875
AAG
GAATCGGGGTGAGATGAGAG
24473
ScaCas9-
78
0






HiFi-Sc++




 


876
AAG
GAATCGGGGTGAGATGAGAG
24474
ScaCas9-
78
0






Sc++




 


877
AAG
GAATCGGGGTGAGATGAGAG
24475
SpyCas9-
78
0






SpRY




 


878
TAA
GCTGGAACTCCGTGACAGTG
24476
SpyCas9-
78
0






SpRY




 


879
AAGGGG
GGAATCGGGGTGAGATGAGAG
24477
cCas9-v17
78
0


 


880
AAGGGG
GGAATCGGGGTGAGATGAGAG
24478
cCas9-v42
78
0


 


881
TAATTTT
GGGCTGGAACTCCGTGACAGTG
24479
CdiCas9
78
0


 


882
TAAT
GCTGGAACTCCGTGACAGTG
24480
SpyCas9-
78
0






3var-NRRH




 


883
TAAT
ggCTGGAACTCCGTGACAGTG
24481
iSpyMacCas9
78
0


 


884
GAAGG
AGGAATCGGGGTGAGATGAGA
24482
SauCas9KKH
79
0


 


885
GAAG
AGGAATCGGGGTGAGATGAGA
24483
SauriCas9-
79
0






KKH




 


886
GAAG
GGAATCGGGGTGAGATGAGA
24484
SpyCas9-
79
0






QQR1




 


887
GAAG
agGAATCGGGGTGAGATGAGA
24485
iSpyMacCas9
79
0


 


888
GAA
GGAATCGGGGTGAGATGAGA
24486
SpyCas9-
79
0






SpRY




 


889
GAA
GGAATCGGGGTGAGATGAGA
24487
SpyCas9-
79
0






xCas




 


890
GTA
GGCTGGAACTCCGTGACAGT
24488
SpyCas9-
79
0






SpRY




 


891
GTAATT
GGGCTGGAACTCCGTGACAGT
24489
cCas9-v16
79
0


 


892
GTAATT
GGGCTGGAACTCCGTGACAGT
24490
cCas9-v21
79
0


 


893
GAAGGG
AGGAATCGGGGTGAGATGAGA
24491
cCas9-v17
79
0


 


894
GAAGGG
AGGAATCGGGGTGAGATGAGA
24492
cCas9-v42
79
0


 


895
GTAATTT
GGGGCTGGAACTCCGTGACAGT
24493
CdiCas9
79
0


 


896
TGTAATT
agaGGGGCTGGAACTCCGTGACA
24494
PpnCas9
80
0




G






 


897
TGTAA
GGGGCTGGAACTCCGTGACAG
24495
SauCas9KKH
80
0


 


898
AGAAG
AAGGAATCGGGGTGAGATGAG
24496
SauCas9KKH
80
0


 


899
TGTAAT
GGGGCTGGAACTCCGTGACAG
24497
SauCas9KKH
80
0


 


900
TG
GGGCTGGAACTCCGTGACAG
24498
SpyCas9-NG
80
0


 


901
TG
GGGCTGGAACTCCGTGACAG
24499
SpyCas9-
80
0






xCas




 


902
TG
GGGCTGGAACTCCGTGACAG
24500
SpyCas9-
80
0






xCas-NG




 


903
AG
AGGAATCGGGGTGAGATGAG
24501
SpyCas9-NG
80
0


 


904
AG
AGGAATCGGGGTGAGATGAG
24502
SpyCas9-
80
0






xCas




 


905
AG
AGGAATCGGGGTGAGATGAG
24503
SpyCas9-
80
0






xCas-NG




 


906
TGT
GGGCTGGAACTCCGTGACAG
24504
SpyCas9-
80
0






SpG




 


907
TGT
GGGCTGGAACTCCGTGACAG
24505
SpyCas9-
80
0






SpRY




 


908
AGA
AGGAATCGGGGTGAGATGAG
24506
SpyCas9-
80
0






SpG




 


909
AGA
AGGAATCGGGGTGAGATGAG
24507
SpyCas9-
80
0






SpRY




 


910
AGAAGG
AAGGAATCGGGGTGAGATGAG
24508
cCas9-v17
80
0


 


911
AGAAGG
AAGGAATCGGGGTGAGATGAG
24509
cCas9-v42
80
0


 


912
AGAA
AGGAATCGGGGTGAGATGAG
24510
SpyCas9-
80
0






3var-NRRH




 


913
AGAA
AGGAATCGGGGTGAGATGAG
24511
SpyCas9-
80
0






VQR




 


914
TGTA
GGGCTGGAACTCCGTGACAG
24512
SpyCas9-
80
0






3var-NRTH




 


915
GAGAA
taGAAGGAATCGGGGTGAGATGA
24513
SauCas9
81
0


 


916
GAGAA
GAAGGAATCGGGGTGAGATGA
24514
SauCas9KKH
81
0


 


917
GTG
GGGGCTGGAACTCCGTGACA
24515
ScaCas9
81
0


 


918
GTG
GGGGCTGGAACTCCGTGACA
24516
ScaCas9-
81
0






HiFi-Sc++




 


919
GTG
GGGGCTGGAACTCCGTGACA
24517
ScaCas9-
81
0






Sc++




 


920
GTG
GGGGCTGGAACTCCGTGACA
24518
SpyCas9-
81
0






SpRY




 


921
GAG
AAGGAATCGGGGTGAGATGA
24519
ScaCas9
81
0


 


922
GAG
AAGGAATCGGGGTGAGATGA
24520
ScaCas9-
81
0






HiFi-Sc++




 


923
GAG
AAGGAATCGGGGTGAGATGA
24521
ScaCas9-
81
0






Sc++




 


924
GAG
AAGGAATCGGGGTGAGATGA
24522
SpyCas9-
81
0






SpRY




 


925
GAGAAG
GAAGGAATCGGGGTGAGATGA
24523
cCas9-v17
81
0


 


926
GAGAAG
GAAGGAATCGGGGTGAGATGA
24524
cCas9-v42
81
0


 


927
GTGTAAT
GAGGGGCTGGAACTCCGTGACA
24525
CdiCas9
81
0


 


928
GAGA
AAGGAATCGGGGTGAGATGA
24526
SpyCas9-
81
0






3var-NRRH




 


929
AGAGA
AGAAGGAATCGGGGTGAGATG
24527
SauCas9KKH
82
0


 


930
AGAG
AGAAGGAATCGGGGTGAGATG
24528
SauriCas9-
82
0






KKH




 


931
AGAG
GAAGGAATCGGGGTGAGATG
24529
SpyCas9-
82
0






VQR




 


932
AG
AGGGGCTGGAACTCCGTGAC
24530
SpyCas9-NG
82
0


 


933
AG
AGGGGCTGGAACTCCGTGAC
24531
SpyCas9-
82
0






xCas




 


934
AG
AGGGGCTGGAACTCCGTGAC
24532
SpyCas9-
82
0






xCas-NG




 


935
AG
GAAGGAATCGGGGTGAGATG
24533
SpyCas9-NG
82
0


 


936
AG
GAAGGAATCGGGGTGAGATG
24534
SpyCas9-
82
0






xCas




 


937
AG
GAAGGAATCGGGGTGAGATG
24535
SpyCas9-
82
0






xCas-NG




 


938
AGT
AGGGGCTGGAACTCCGTGAC
24536
SpyCas9-
82
0






SpG




 


939
AGT
AGGGGCTGGAACTCCGTGAC
24537
SpyCas9-
82
0






SpRY




 


940
AGA
GAAGGAATCGGGGTGAGATG
24538
SpyCas9-
82
0






SpG




 


941
AGA
GAAGGAATCGGGGTGAGATG
24539
SpyCas9-
82
0






SpRY




 


942
AGAGAA
AGAAGGAATCGGGGTGAGATG
24540
cCas9-v17
82
0


 


943
AGAGAA
AGAAGGAATCGGGGTGAGATG
24541
cCas9-v42
82
0


 


944
GAGAG
tgTAGAAGGAATCGGGGTGAGAT
24542
SauCas9
83
0


 


945
GAGAG
TAGAAGGAATCGGGGTGAGAT
24543
SauCas9KKH
83
0


 


946
CAG
GAGGGGCTGGAACTCCGTGA
24544
ScaCas9
83
0


 


947
CAG
GAGGGGCTGGAACTCCGTGA
24545
ScaCas9-
83
0






HiFi-Sc++




 


948
CAG
GAGGGGCTGGAACTCCGTGA
24546
ScaCas9-
83
0






Sc++




 


949
CAG
GAGGGGCTGGAACTCCGTGA
24547
SpyCas9-
83
0






SpRY




 


950
GAG
AGAAGGAATCGGGGTGAGAT
24548
ScaCas9
83
0


 


951
GAG
AGAAGGAATCGGGGTGAGAT
24549
ScaCas9-
83
0






HiFi-Sc++




 


952
GAG
AGAAGGAATCGGGGTGAGAT
24550
ScaCas9-
83
0






Sc++




 


953
GAG
AGAAGGAATCGGGGTGAGAT
24551
SpyCas9-
83
0






SpRY




 


954
GAGAGA
TAGAAGGAATCGGGGTGAGAT
24552
cCas9-v17
83
0


 


955
GAGAGA
TAGAAGGAATCGGGGTGAGAT
24553
cCas9-v42
83
0


 


956
CAGT
GAGGGGCTGGAACTCCGTGA
24554
SpyCas9-
83
0






3var-NRRH




 


957
GAGA
AGAAGGAATCGGGGTGAGAT
24555
SpyCas9-
83
0






3var-NRRH




 


958
TGAGA
GTAGAAGGAATCGGGGTGAGA
24556
SauCas9KKH
84
0


 


959
ACAG
TAGAGGGGCTGGAACTCCGTG
24557
SauriCas9-
84
0






KKH




 


960
TGAG
GTAGAAGGAATCGGGGTGAGA
24558
SauriCas9-
84
0






KKH




 


961
TGAG
TAGAAGGAATCGGGGTGAGA
24559
SpyCas9-
84
0






VQR




 


962
TG
TAGAAGGAATCGGGGTGAGA
24560
SpyCas9-NG
84
0


 


963
TG
TAGAAGGAATCGGGGTGAGA
24561
SpyCas9-
84
0






xCas




 


964
TG
TAGAAGGAATCGGGGTGAGA
24562
SpyCas9-
84
0






xCas-NG




 


965
TGA
TAGAAGGAATCGGGGTGAGA
24563
SpyCas9-
84
0






SpG




 


966
TGA
TAGAAGGAATCGGGGTGAGA
24564
SpyCas9-
84
0






SpRY




 


967
ACA
AGAGGGGCTGGAACTCCGTG
24565
SpyCas9-
84
0






SpRY




 


968
ACAGTG
TAGAGGGGCTGGAACTCCGTG
24566
cCas9-v16
84
0


 


969
ACAGTG
TAGAGGGGCTGGAACTCCGTG
24567
cCas9-v21
84
0


 


970
TGAGAG
GTAGAAGGAATCGGGGTGAGA
24568
cCas9-v17
84
0


 


971
TGAGAG
GTAGAAGGAATCGGGGTGAGA
24569
cCas9-v42
84
0


 


972
ATGAG
gaTGTAGAAGGAATCGGGGTGAG
24570
SauCas9
85
0


 


973
ATGAG
TGTAGAAGGAATCGGGGTGAG
24571
SauCas9KKH
85
0


 


974
GACAG
ATAGAGGGGCTGGAACTCCGT
24572
SauCas9KKH
85
0


 


975
GACAGT
ATAGAGGGGCTGGAACTCCGT
24573
SauCas9KKH
85
0


 


976
GACAGT
ATAGAGGGGCTGGAACTCCGT
24574
cCas9-v17
85
0


 


977
GACAGT
ATAGAGGGGCTGGAACTCCGT
24575
cCas9-v42
85
0


 


978
ATG
GTAGAAGGAATCGGGGTGAG
24576
ScaCas9
85
0


 


979
ATG
GTAGAAGGAATCGGGGTGAG
24577
ScaCas9-
85
0






HiFi-Sc++




 


980
ATG
GTAGAAGGAATCGGGGTGAG
24578
ScaCas9-
85
0






Sc++




 


981
ATG
GTAGAAGGAATCGGGGTGAG
24579
SpyCas9-
85
0






SpRY




 


982
GAC
TAGAGGGGCTGGAACTCCGT
24580
SpyCas9-
85
0






SpRY




 


983
ATGAGA
TGTAGAAGGAATCGGGGTGAG
24581
cCas9-v17
85
0


 


984
ATGAGA
TGTAGAAGGAATCGGGGTGAG
24582
cCas9-v42
85
0


 


985
GACA
TAGAGGGGCTGGAACTCCGT
24583
SpyCas9-
85
0






3var-NRCH




 


986
GATGA
ATGTAGAAGGAATCGGGGTGA
24584
SauCas9KKH
86
0


 


987
TG
ATAGAGGGGCTGGAACTCCG
24585
SpyCas9-NG
86
0


 


988
TG
ATAGAGGGGCTGGAACTCCG
24586
SpyCas9-
86
0






xCas




 


989
TG
ATAGAGGGGCTGGAACTCCG
24587
SpyCas9-
86
0






xCas-NG




 


990
TGA
ATAGAGGGGCTGGAACTCCG
24588
SpyCas9-
86
0






SpG




 


991
TGA
ATAGAGGGGCTGGAACTCCG
24589
SpyCas9-
86
0






SpRY




 


992
GAT
TGTAGAAGGAATCGGGGTGA
24590
SpyCas9-
86
0






SpRY




 


993
GAT
TGTAGAAGGAATCGGGGTGA
24591
SpyCas9-
86
0






xCas




 


994
TGAC
ATAGAGGGGCTGGAACTCCG
24592
SpyCas9-
86
0






3var-NRRH




 


995
TGAC
ATAGAGGGGCTGGAACTCCG
24593
SpyCas9-
86
0






VQR




 


996
GTG
AATAGAGGGGCTGGAACTCC
24594
ScaCas9
87
0


 


997
GTG
AATAGAGGGGCTGGAACTCC
24595
ScaCas9-
87
0






HiFi-Sc++




 


998
GTG
AATAGAGGGGCTGGAACTCC
24596
ScaCas9-
87
0






Sc++




 


999
GTG
AATAGAGGGGCTGGAACTCC
24597
SpyCas9-
87
0






SpRY




 


1000
AG
ATGTAGAAGGAATCGGGGTG
24598
SpyCas9-NG
87
0


 


1001
AG
ATGTAGAAGGAATCGGGGTG
24599
SpyCas9-
87
0






xCas




 


1002
AG
ATGTAGAAGGAATCGGGGTG
24600
SpyCas9-
87
0






xCas-NG




 


1003
AGA
ATGTAGAAGGAATCGGGGTG
24601
SpyCas9-
87
0






SpG




 


1004
AGA
ATGTAGAAGGAATCGGGGTG
24602
SpyCas9-
87
0






SpRY




 


1005
GTGACAG
cgtaATAGAGGGGCTGGAACTCC
24603
BlatCas9
87
0



T







 


1006
GTGAC
cgtaATAGAGGGGCTGGAACTCC
24604
BlatCas9
87
0


 


1007
AGAT
ATGTAGAAGGAATCGGGGTG
24605
SpyCas9-
87
0






3var-NRRH




 


1008
AGAT
ATGTAGAAGGAATCGGGGTG
24606
SpyCas9-
87
0






VQR




 


1009
CGTGA
GTAATAGAGGGGCTGGAACTC
24607
SauCas9KKH
88
0


 


1010
GAG
GATGTAGAAGGAATCGGGGT
24608
ScaCas9
88
0


 


1011
GAG
GATGTAGAAGGAATCGGGGT
24609
ScaCas9-
88
0






HiFi-Sc++




 


1012
GAG
GATGTAGAAGGAATCGGGGT
24610
ScaCas9-
88
0






Sc++




 


1013
GAG
GATGTAGAAGGAATCGGGGT
24611
SpyCas9-
88
0






SpRY




 


1014
CG
TAATAGAGGGGCTGGAACTC
24612
SpyCas9-NG
88
0


 


1015
CG
TAATAGAGGGGCTGGAACTC
24613
SpyCas9-
88
0






xCas




 


1016
CG
TAATAGAGGGGCTGGAACTC
24614
SpyCas9-
88
0






xCas-NG




 


1017
CGT
TAATAGAGGGGCTGGAACTC
24615
SpyCas9-
88
0






SpG




 


1018
CGT
TAATAGAGGGGCTGGAACTC
24616
SpyCas9-
88
0






SpRY




 


1019
GAGATGA
TGATGTAGAAGGAATCGGGGT
24617
cCas9-v16
88
0


 


1020
GAGATGA
TGATGTAGAAGGAATCGGGGT
24618
cCas9-v21
88
0


 


1021
GAGA
GATGTAGAAGGAATCGGGGT
24619
SpyCas9-
88
0






3var-NRRH




 


1022
TGAGA
GTGATGTAGAAGGAATCGGGG
24620
SauCas9KKH
89
0


 


1023
TGAGAT
GTGATGTAGAAGGAATCGGGG
24621
SauCas9KKH
89
0


 


1024
TGAGAT
GTGATGTAGAAGGAATCGGGG
24622
cCas9-v17
89
0


 


1025
TGAGAT
GTGATGTAGAAGGAATCGGGG
24623
cCas9-v42
89
0


 


1026
TGAG
GTGATGTAGAAGGAATCGGGG
24624
SauriCas9-
89
0






KKH




 


1027
TGAG
TGATGTAGAAGGAATCGGGG
24625
SpyCas9-
89
0






VQR




 


1028
CCG
GTAATAGAGGGGCTGGAACT
24626
ScaCas9
89
0


 


1029
CCG
GTAATAGAGGGGCTGGAACT
24627
ScaCas9-
89
0






HiFi-Sc++




 


1030
CCG
GTAATAGAGGGGCTGGAACT
24628
ScaCas9-
89
0






Sc++




 


1031
CCG
GTAATAGAGGGGCTGGAACT
24629
SpyCas9-
89
0






SpRY




 


1032
TG
TGATGTAGAAGGAATCGGGG
24630
SpyCas9-NG
89
0


 


1033
TG
TGATGTAGAAGGAATCGGGG
24631
SpyCas9-
89
0






xCas




 


1034
TG
TGATGTAGAAGGAATCGGGG
24632
SpyCas9-
89
0






xCas-NG




 


1035
TGA
TGATGTAGAAGGAATCGGGG
24633
SpyCas9-
89
0






SpG




 


1036
TGA
TGATGTAGAAGGAATCGGGG
24634
SpyCas9-
89
0






SpRY




 


1037
CCGTGAC
ccacGTAATAGAGGGGCTGGAACT
24635
NmeCas9
89
0



A







 


1038
GTGAG
gcTGTGATGTAGAAGGAATCGGG
24636
SauCas9
90
0


 


1039
GTGAG
TGTGATGTAGAAGGAATCGGG
24637
SauCas9KKH
90
0


 


1040
GTG
GTGATGTAGAAGGAATCGGG
24638
ScaCas9
90
0


 


1041
GTG
GTGATGTAGAAGGAATCGGG
24639
ScaCas9-
90
0






HiFi-Sc++




 


1042
GTG
GTGATGTAGAAGGAATCGGG
24640
ScaCas9-
90
0






Sc++




 


1043
GTG
GTGATGTAGAAGGAATCGGG
24641
SpyCas9-
90
0






SpRY




 


1044
TCC
CGTAATAGAGGGGCTGGAAC
24642
SpyCas9-
90
0






SpRY




 


1045
TCCGTG
ACGTAATAGAGGGGCTGGAAC
24643
cCas9-v16
90
0


 


1046
TCCGTG
ACGTAATAGAGGGGCTGGAAC
24644
cCas9-v21
90
0


 


1047
GTGAGA
TGTGATGTAGAAGGAATCGGG
24645
cCas9-v17
90
0


 


1048
GTGAGA
TGTGATGTAGAAGGAATCGGG
24646
cCas9-v42
90
0


 


1049
GGTGA
CTGTGATGTAGAAGGAATCGG
24647
SauCas9KKH
91
0


 


1050
GG
TGTGATGTAGAAGGAATCGG
24648
SpyCas9-NG
91
0


 


1051
GG
TGTGATGTAGAAGGAATCGG
24649
SpyCas9-
91
0






xCas




 


1052
GG
TGTGATGTAGAAGGAATCGG
24650
SpyCas9-
91
0






xCas-NG




 


1053
GGT
TGTGATGTAGAAGGAATCGG
24651
SpyCas9-
91
0






SpG




 


1054
GGT
TGTGATGTAGAAGGAATCGG
24652
SpyCas9-
91
0






SpRY




 


1055
CTC
ACGTAATAGAGGGGCTGGAA
24653
SpyCas9-
91
0






SpRY




 


1056
GGG
CTGTGATGTAGAAGGAATCG
24654
ScaCas9
92
0


 


1057
GGG
CTGTGATGTAGAAGGAATCG
24655
ScaCas9-
92
0






HiFi-Sc++




 


1058
GGG
CTGTGATGTAGAAGGAATCG
24656
ScaCas9-
92
0






Sc++




 


1059
GGG
CTGTGATGTAGAAGGAATCG
24657
SpyCas9
92
0


 


1060
GGG
CTGTGATGTAGAAGGAATCG
24658
SpyCas9-
92
0






HF1




 


1061
GGG
CTGTGATGTAGAAGGAATCG
24659
SpyCas9-
92
0






SpG




 


1062
GGG
CTGTGATGTAGAAGGAATCG
24660
SpyCas9-
92
0






SpRY




 


1063
GG
CTGTGATGTAGAAGGAATCG
24661
SpyCas9-NG
92
0


 


1064
GG
CTGTGATGTAGAAGGAATCG
24662
SpyCas9-
92
0






xCas




 


1065
GG
CTGTGATGTAGAAGGAATCG
24663
SpyCas9-
92
0






xCas-NG




 


1066
ACT
CACGTAATAGAGGGGCTGGA
24664
SpyCas9-
92
0






SpRY




 


1067
ACTCCGT
tgccACGTAATAGAGGGGCTGGA
24665
BlatCas9
92
0



G







 


1068
ACTCC
tgccACGTAATAGAGGGGCTGGA
24666
BlatCas9
92
0


 


1069
GGGT
CTGTGATGTAGAAGGAATCG
24667
SpyCas9-
92
0






3var-NRRH




 


1070
AACTCC
tcTGCCACGTAATAGAGGGGCTG
24668
Nme2Cas9
93
0




G






 


1071
GGGG
GGCTGTGATGTAGAAGGAATC
24669
SauriCas9
93
0


 


1072
GGGG
GGCTGTGATGTAGAAGGAATC
24670
SauriCas9-
93
0






KKH




 


1073
GGG
GCTGTGATGTAGAAGGAATC
24671
ScaCas9
93
0


 


1074
GGG
GCTGTGATGTAGAAGGAATC
24672
ScaCas9-
93
0






HiFi-Sc++




 


1075
GGG
GCTGTGATGTAGAAGGAATC
24673
ScaCas9-
93
0






Sc++




 


1076
GGG
GCTGTGATGTAGAAGGAATC
24674
SpyCas9
93
0


 


1077
GGG
GCTGTGATGTAGAAGGAATC
24675
SpyCas9-
93
0






HF1




 


1078
GGG
GCTGTGATGTAGAAGGAATC
24676
SpyCas9-
93
0






SpG




 


1079
GGG
GCTGTGATGTAGAAGGAATC
24677
SpyCas9-
93
0






SpRY




 


1080
GG
GCTGTGATGTAGAAGGAATC
24678
SpyCas9-NG
93
0


 


1081
GG
GCTGTGATGTAGAAGGAATC
24679
SpyCas9-
93
0






xCas




 


1082
GG
GCTGTGATGTAGAAGGAATC
24680
SpyCas9-
93
0






xCas-NG




 


1083
AAC
CCACGTAATAGAGGGGCTGG
24681
SpyCas9-
93
0






SpRY




 


1084
AACTCCG
ctgcCACGTAATAGAGGGGCTGG
24682
BlatCas9
93
0



T







 


1085
AACTC
ctgcCACGTAATAGAGGGGCTGG
24683
BlatCas9
93
0


 


1086
GGGGTG
GGCTGTGATGTAGAAGGAATC
24684
cCas9-v16
93
0


 


1087
GGGGTG
GGCTGTGATGTAGAAGGAATC
24685
cCas9-v21
93
0


 


1088
AACT
CCACGTAATAGAGGGGCTGG
24686
SpyCas9-
93
0






3var-NRCH




 


1089
CGGGG
ttGGGCTGTGATGTAGAAGGAAT
24687
SauCas9
94
0


 


1090
CGGGG
GGGCTGTGATGTAGAAGGAAT
24688
SauCas9KKH
94
0


 


1091
CGGGGT
ttGGGCTGTGATGTAGAAGGAAT
24689
SauCas9
94
0


 


1092
CGGGGT
GGGCTGTGATGTAGAAGGAAT
24690
SauCas9KKH
94
0


 


1093
CGGGGT
GGGCTGTGATGTAGAAGGAAT
24691
cCas9-v17
94
0


 


1094
CGGGGT
GGGCTGTGATGTAGAAGGAAT
24692
cCas9-v42
94
0


 


1095
CGGG
GGGCTGTGATGTAGAAGGAAT
24693
SauriCas9
94
0


 


1096
CGGG
GGGCTGTGATGTAGAAGGAAT
24694
SauriCas9-
94
0






KKH




 


1097
CGG
GGCTGTGATGTAGAAGGAAT
24695
ScaCas9
94
0


 


1098
CGG
GGCTGTGATGTAGAAGGAAT
24696
ScaCas9-
94
0






HiFi-Sc++




 


1099
CGG
GGCTGTGATGTAGAAGGAAT
24697
ScaCas9-
94
0






Sc++




 


1100
CGG
GGCTGTGATGTAGAAGGAAT
24698
SpyCas9
94
0


 


1101
CGG
GGCTGTGATGTAGAAGGAAT
24699
SpyCas9-
94
0






HF1




 


1102
CGG
GGCTGTGATGTAGAAGGAAT
24700
SpyCas9-
94
0






SpG




 


1103
CGG
GGCTGTGATGTAGAAGGAAT
24701
SpyCas9-
94
0






SpRY




 


1104
CG
GGCTGTGATGTAGAAGGAAT
24702
SpyCas9-NG
94
0


 


1105
CG
GGCTGTGATGTAGAAGGAAT
24703
SpyCas9-
94
0






xCas




 


1106
CG
GGCTGTGATGTAGAAGGAAT
24704
SpyCas9-
94
0






xCas-NG




 


1107
GAA
GCCACGTAATAGAGGGGCTG
24705
SpyCas9-
94
0






SpRY




 


1108
GAA
GCCACGTAATAGAGGGGCTG
24706
SpyCas9-
94
0






xCas




 


1109
GAACTCC
CTGCCACGTAATAGAGGGGCTG
24707
CdiCas9
94
0


 


1110
GAAC
GCCACGTAATAGAGGGGCTG
24708
SpyCas9-
94
0






3var-NRRH




 


1111
GAAC
tgCCACGTAATAGAGGGGCTG
24709
iSpyMacCas9
94
0


 


1112
TCGGG
ttTGGGCTGTGATGTAGAAGGAA
24710
SauCas9
95
0


 


1113
TCGGG
TGGGCTGTGATGTAGAAGGAA
24711
SauCas9KKH
95
0


 


1114
TCGG
TGGGCTGTGATGTAGAAGGAA
24712
SauriCas9
95
0


 


1115
TCGG
TGGGCTGTGATGTAGAAGGAA
24713
SauriCas9-
95
0






KKH




 


1116
TCG
GGGCTGTGATGTAGAAGGAA
24714
ScaCas9
95
0


 


1117
TCG
GGGCTGTGATGTAGAAGGAA
24715
ScaCas9-
95
0






HiFi-Sc++




 


1118
TCG
GGGCTGTGATGTAGAAGGAA
24716
ScaCas9-
95
0






Sc++




 


1119
TCG
GGGCTGTGATGTAGAAGGAA
24717
SpyCas9-
95
0






SpRY




 


1120
GG
TGCCACGTAATAGAGGGGCT
24718
SpyCas9-NG
95
0


 


1121
GG
TGCCACGTAATAGAGGGGCT
24719
SpyCas9-
95
0






xCas




 


1122
GG
TGCCACGTAATAGAGGGGCT
24720
SpyCas9-
95
0






xCas-NG




 


1123
GGA
TGCCACGTAATAGAGGGGCT
24721
SpyCas9-
95
0






SpG




 


1124
GGA
TGCCACGTAATAGAGGGGCT
24722
SpyCas9-
95
0






SpRY




 


1125
GGAAC
ctctGCCACGTAATAGAGGGGCT
24723
BlatCas9
95
0


 


1126
GGAACT
CTGCCACGTAATAGAGGGGCT
24724
cCas9-v16
95
0


 


1127
GGAACT
CTGCCACGTAATAGAGGGGCT
24725
cCas9-v21
95
0


 


1128
TCGGGG
TGGGCTGTGATGTAGAAGGAA
24726
cCas9-v17
95
0


 


1129
TCGGGG
TGGGCTGTGATGTAGAAGGAA
24727
cCas9-v42
95
0


 


1130
GGAACTC
TCTGCCACGTAATAGAGGGGCT
24728
CdiCas9
95
0


 


1131
GGAA
TGCCACGTAATAGAGGGGCT
24729
SpyCas9-
95
0






3var-NRRH




 


1132
GGAA
TGCCACGTAATAGAGGGGCT
24730
SpyCas9-
95
0






VQR




 


1133
TGGAA
tcTCTGCCACGTAATAGAGGGGC
24731
SauCas9
96
0


 


1134
TGGAA
TCTGCCACGTAATAGAGGGGC
24732
SauCas9KKH
96
0


 


1135
ATCGG
TTGGGCTGTGATGTAGAAGGA
24733
SauCas9KKH
96
0


 


1136
TGG
CTGCCACGTAATAGAGGGGC
24734
ScaCas9
96
0


 


1137
TGG
CTGCCACGTAATAGAGGGGC
24735
ScaCas9-
96
0






HiFi-Sc++




 


1138
TGG
CTGCCACGTAATAGAGGGGC
24736
ScaCas9-
96
0






Sc++




 


1139
TGG
CTGCCACGTAATAGAGGGGC
24737
SpyCas9
96
0


 


1140
TGG
CTGCCACGTAATAGAGGGGC
24738
SpyCas9-
96
0






HF1




 


1141
TGG
CTGCCACGTAATAGAGGGGC
24739
SpyCas9-
96
0






SpG




 


1142
TGG
CTGCCACGTAATAGAGGGGC
24740
SpyCas9-
96
0






SpRY




 


1143
TG
CTGCCACGTAATAGAGGGGC
24741
SpyCas9-NG
96
0


 


1144
TG
CTGCCACGTAATAGAGGGGC
24742
SpyCas9-
96
0






xCas




 


1145
TG
CTGCCACGTAATAGAGGGGC
24743
SpyCas9-
96
0






xCas-NG




 


1146
ATC
TGGGCTGTGATGTAGAAGGA
24744
SpyCas9-
96
0






SpRY




 


1147
TGGAAC
TCTGCCACGTAATAGAGGGGC
24745
cCas9-v17
96
0


 


1148
TGGAAC
TCTGCCACGTAATAGAGGGGC
24746
cCas9-v42
96
0


 


1149
ATCGGG
TTGGGCTGTGATGTAGAAGGA
24747
cCas9-v17
96
0


 


1150
ATCGGG
TTGGGCTGTGATGTAGAAGGA
24748
cCas9-v42
96
0


 


1151
TGGAACT
CTCTGCCACGTAATAGAGGGGC
24749
CdiCas9
96
0


 


1152
TGGA
CTGCCACGTAATAGAGGGGC
24750
SpyCas9-
96
0






3var-NRRH




 


1153
CTGGA
ctCTCTGCCACGTAATAGAGGGG
24751
SauCas9
97
0


 


1154
CTGGA
CTCTGCCACGTAATAGAGGGG
24752
SauCas9KKH
97
0


 


1155
CTGG
CTCTGCCACGTAATAGAGGGG
24753
SauriCas9
97
0


 


1156
CTGG
CTCTGCCACGTAATAGAGGGG
24754
SauriCas9-
97
0






KKH




 


1157
CTG
TCTGCCACGTAATAGAGGGG
24755
ScaCas9
97
0


 


1158
CTG
TCTGCCACGTAATAGAGGGG
24756
ScaCas9-
97
0






HiFi-Sc++




 


1159
CTG
TCTGCCACGTAATAGAGGGG
24757
ScaCas9-
97
0






Sc++




 


1160
CTG
TCTGCCACGTAATAGAGGGG
24758
SpyCas9-
97
0






SpRY




 


1161
AAT
TTGGGCTGTGATGTAGAAGG
24759
SpyCas9-
97
0






SpRY




 


1162
CTGGAA
CTCTGCCACGTAATAGAGGGG
24760
cCas9-v17
97
0


 


1163
CTGGAA
CTCTGCCACGTAATAGAGGGG
24761
cCas9-v42
97
0


 


1164
AATC
TTGGGCTGTGATGTAGAAGG
24762
SpyCas9-
97
0






3var-NRTH




 


1165
GCTGG
TCTCTGCCACGTAATAGAGGG
24763
SauCas9KKH
98
0


 


1166
GAA
TTTGGGCTGTGATGTAGAAG
24764
SpyCas9-
98
0






SpRY




 


1167
GAA
TTTGGGCTGTGATGTAGAAG
24765
SpyCas9-
98
0






xCas




 


1168
GCT
CTCTGCCACGTAATAGAGGG
24766
SpyCas9-
98
0






SpRY




 


1169
GAATCGG
gcatTTGGGCTGTGATGTAGAAG
24767
BlatCas9
98
0



G







 


1170
GAATC
gcatTTGGGCTGTGATGTAGAAG
24768
BlatCas9
98
0


 


1171
GAAT
TTTGGGCTGTGATGTAGAAG
24769
SpyCas9-
98
0






3var-NRRH




 


1172
GAAT
atTTGGGCTGTGATGTAGAAG
24770
iSpyMacCas9
98
0


 


1173
GG
TCTCTGCCACGTAATAGAGG
24771
SpyCas9-NG
99
0


 


1174
GG
TCTCTGCCACGTAATAGAGG
24772
SpyCas9-
99
0






xCas




 


1175
GG
TCTCTGCCACGTAATAGAGG
24773
SpyCas9-
99
0






xCas-NG




 


1176
GG
ATTTGGGCTGTGATGTAGAA
24774
SpyCas9-NG
99
0


 


1177
GG
ATTTGGGCTGTGATGTAGAA
24775
SpyCas9-
99
0






xCas




 


1178
GG
ATTTGGGCTGTGATGTAGAA
24776
SpyCas9-
99
0






xCas-NG




 


1179
GGC
TCTCTGCCACGTAATAGAGG
24777
SpyCas9-
99
0






SpG




 


1180
GGC
TCTCTGCCACGTAATAGAGG
24778
SpyCas9-
99
0






SpRY




 


1181
GGA
ATTTGGGCTGTGATGTAGAA
24779
SpyCas9-
99
0






SpG




 


1182
GGA
ATTTGGGCTGTGATGTAGAA
24780
SpyCas9-
99
0






SpRY




 


1183
GGAA
ATTTGGGCTGTGATGTAGAA
24781
SpyCas9-
99
0






3var-NRRH




 


1184
GGAA
ATTTGGGCTGTGATGTAGAA
24782
SpyCas9-
99
0






VQR




 


1185
GGCT
TCTCTGCCACGTAATAGAGG
24783
SpyCas9-
99
0






3var-NRCH




 


1186
AGGAA
caGCATTTGGGCTGTGATGTAGA
24784
SauCas9
100
0


 


1187
AGGAA
GCATTTGGGCTGTGATGTAGA
24785
SauCas9KKH
100
0


 


1188
AGGAAT
caGCATTTGGGCTGTGATGTAGA
24786
SauCas9
100
0


 


1189
AGGAAT
GCATTTGGGCTGTGATGTAGA
24787
SauCas9KKH
100
0


 


1190
AGGAAT
GCATTTGGGCTGTGATGTAGA
24788
cCas9-v17
100
0


 


1191
AGGAAT
GCATTTGGGCTGTGATGTAGA
24789
cCas9-v42
100
0


 


1192
GGG
CTCTCTGCCACGTAATAGAG
24790
ScaCas9
100
0


 


1193
GGG
CTCTCTGCCACGTAATAGAG
24791
ScaCas9-
100
0






HiFi-Sc++




 


1194
GGG
CTCTCTGCCACGTAATAGAG
24792
ScaCas9-
100
0






Sc++




 


1195
GGG
CTCTCTGCCACGTAATAGAG
24793
SpyCas9
100
0


 


1196
GGG
CTCTCTGCCACGTAATAGAG
24794
SpyCas9-
100
0






HF1




 


1197
GGG
CTCTCTGCCACGTAATAGAG
24795
SpyCas9-
100
0






SpG




 


1198
GGG
CTCTCTGCCACGTAATAGAG
24796
SpyCas9-
100
0






SpRY




 


1199
AGG
CATTTGGGCTGTGATGTAGA
24797
ScaCas9
100
0


 


1200
AGG
CATTTGGGCTGTGATGTAGA
24798
ScaCas9-
100
0






HiFi-Sc++




 


1201
AGG
CATTTGGGCTGTGATGTAGA
24799
ScaCas9-
100
0






Sc++




 


1202
AGG
CATTTGGGCTGTGATGTAGA
24800
SpyCas9
100
0


 


1203
AGG
CATTTGGGCTGTGATGTAGA
24801
SpyCas9-
100
0






HF1




 


1204
AGG
CATTTGGGCTGTGATGTAGA
24802
SpyCas9-
100
0






SpG




 


1205
AGG
CATTTGGGCTGTGATGTAGA
24803
SpyCas9-
100
0






SpRY




 


1206
GG
CTCTCTGCCACGTAATAGAG
24804
SpyCas9-NG
100
0


 


1207
GG
CTCTCTGCCACGTAATAGAG
24805
SpyCas9-
100
0






xCas




 


1208
GG
CTCTCTGCCACGTAATAGAG
24806
SpyCas9-
100
0






xCas-NG




 


1209
AG
CATTTGGGCTGTGATGTAGA
24807
SpyCas9-NG
100
0


 


1210
AG
CATTTGGGCTGTGATGTAGA
24808
SpyCas9-
100
0






xCas




 


1211
AG
CATTTGGGCTGTGATGTAGA
24809
SpyCas9-
100
0






xCas-NG




 


1212
AGGAATC
AGCATTTGGGCTGTGATGTAGA
24810
CdiCas9
100
0


 


1213
GGGC
CTCTCTGCCACGTAATAGAG
24811
SpyCas9-
100
0






3var-NRRH




 


1214
AGGA
CATTTGGGCTGTGATGTAGA
24812
SpyCas9-
100
0






3var-NRRH









In the exemplary template sequences provided herein, capital letters indicate “core nucleotides” while lower case letters indicate “flanking nucleotides.” Herein, when an RNA sequence (e.g., a template RNA sequence) is said to comprise a particular sequence (e.g., a sequence of Tables 1A, 1B, 1C, or 1D or a portion thereof) that comprises thymine (T), it is of course understood that the RNA sequence may (and frequently does) comprise uracil (U) in place of T. For instance, the RNA sequence may comprise U at every position shown as T in the sequence in Tables 1A, 1B, 1C, or 1D. More specifically, the present disclosure provides an RNA sequence according to every gRNA spacer sequence shown in Tables 1A, 1B, 1C, or 1D, wherein the RNA sequence has a U in place of each T in the sequence in Tables 1A, 1B, 1C, or 1D.

In some embodiments of the systems and methods herein, the heterologous object sequence comprises the core nucleotides of an RT template sequence from Table 3A, Table 3B, Table 3C, or Table 3D. In some embodiments, the heterologous object sequence additionally comprises one or more (e.g., 2, 3, 4, 5, 10, 20, 30, 40, or all) consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the RT template sequence. In some embodiments, the heterologous object sequence comprises the core nucleotides of the RT template sequence of Table 3A, Table 3B, Table 3C, or Table 3D that corresponds to the gRNA spacer sequence. In the context of the sequence tables, a first component “corresponds to” a second component when both components have the same ID number in the referenced table. For example, for a gRNA spacer of ID #1, the corresponding RT template would be the RT template also having ID #1 in a table referencing the same mutation. In some embodiments, the heterologous object sequence additionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the RT template sequence.
In some embodiments, the primer binding site (PBS) sequence has a sequence comprising the core nucleotides of a PBS sequence from the same row of Table 3A, Table 3B, Table 3C, or Table 3D as the RT template sequence. In some embodiments, the PBS sequence additionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or all) consecutive nucleotides starting with the 5′ end of the flanking nucleotides of the primer region.







TABLE 3A







Exmplary RT sequence (heterologous object sequence) and PBS sequence pairs


Table 3A provides exemplified PBS sequences and heterologous object sequences (reverse


transcription template regions) of a template RNA for correcting the pathogenic R408W mutation


in PAH. The gRNA spacers from Table 1A were filtered, e.g., filtered by occurrence within 15 nt


of the desired editing location and use of a Tier 1 Cas enzyme. PBS sequences and heterologous


object sequences (reverse transcription template regions) were designed relative to the nick


site directed by the cognate gRNA from Table 1A, as described in this application. For


exemplification, these regions were designed to be 8-17 nt (priming) and 1-50 nt extended


beyond the location of the edit (RT). Without wishing to be limited by example, given


 variability of length, sequences are provided that use the maximum length parameters and


comprise all templates of shorter length within the given parameters. Sequences are shown with


uppercase letters indicating core sequence and lowercase letters indicating flanking sequence


that may be truncated within the described length parameters.













SEQ

SEQ




ID
PBS
ID


ID
RT Template Sequence
NO
Sequence
NO














1
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCG
24813
AGGTATTGtg
25003





gcagcaa



 


2
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCG
24814
GCCCTTCTca
25004





gttcgct



 


6
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCG
24815
GCCCTTCTca
25005





gttcgct



 


7
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGA
24816
GGTATTGTgg
25006





cagcaaa



 


10
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGG
24817
CCCTTCTCag
25007





ttcgcta



 


13
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGG
24818
CCCTTCTCag
25008





ttcgcta



 


14
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGG
24819
CCCTTCTCag
25009





ttcgcta



 


17
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGG
24820
CCCTTCTCag
25010





ttcgcta



 


18
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAG
24821
GTATTGTGgc
25011





agcaaag



 


21
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGG
24822
CCCTTCTCag
25012





ttcgcta



 


25
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAG
24823
GTATTGTGgc
25013





agcaaag



 


26
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAG
24824
GTATTGTGgc
25014





agcaaag



 


29
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGG
24825
TATTGTGGca
25015





gcaaagt



 


30
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGC
24826
CCTTCTCAgtt
25016





cgctac



 


31
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGC
24827
CCTTCTCAgtt
25017





cgctac



 


34
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGG
24828
TATTGTGGca
25018





gcaaagt



 


35
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGG
24829
TATTGTGGca
25019





gcaaagt



 


38
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGG
24830
TATTGTGGca
25020





gcaaagt



 


41
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGC
24831
CCTTCTCAgtt
25021





cgctac



 


42
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGC
24832
CCTTCTCAgtt
25022





cgctac



 


43
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGG
24833
TATTGTGGca
25023





gcaaagt



 


46
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGG
24834
TATTGTGGca
25024





gcaaagt



 


47
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGG
24835
TATTGTGGca
25025





gcaaagt



 


50
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGT
24836
ATTGTGGCag
25026





caaagtt



 


51
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCC
24837
CTTCTCAGttc
25027





gctacg



 


52
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCC
24838
CTTCTCAGttc
25028





gctacg



 


55
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGT
24839
ATTGTGGCag
25029





caaagtt



 


56
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGT
24840
ATTGTGGCag
25030





caaagtt



 


57
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCC
24841
CTTCTCAGttc
25031





gctacg



 


62
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGT
24842
ATTGTGGCag
25032





caaagtt



 


63
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGT
24843
ATTGTGGCag
25033





caaagtt



 


64
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCC
24844
CTTCTCAGttc
25034





gctacg



 


65
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGT
24845
ATTGTGGCag
25035





caaagtt



 


66
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCC
24846
TTCTCAGTtc
25036





gctacga



 


67
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTA
24847
TTGTGGCAgc
25037





aaagttc



 


68
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCC
24848
TTCTCAGTtc
25038





gctacga



 


69
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTA
24849
TTGTGGCAgc
25039





aaagttc



 


72
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCT
24850
TCTCAGTTcg
25040





ctacgac



 


73
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCT
24851
TCTCAGTTcg
25041





ctacgac



 


74
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTAT
24852
TGTGGCAGc
25042





aaagttcc



 


77
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTT
24853
CTCAGTTCgc
25043





tacgacc



 


81
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTT
24854
CTCAGTTCgc
25044





tacgacc



 


82
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATT
24855
GTGGCAGCa
25045





aagttcct



 


86
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTC
24856
TCAGTTCGct
25046





acgaccc



 


87
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTC
24857
TCAGTTCGct
25047





acgaccc



 


90
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTC
24858
TCAGTTCGct
25048





acgaccc



 


91
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTC
24859
TCAGTTCGct
25049





acgaccc



 


94
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24860
TGGCAGCAa
25050





agttccta



 


95
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTC
24861
TCAGTTCGct
25051





acgaccc



 


96
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTC
24862
TCAGTTCGct
25052





acgaccc



 


99
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCT
24863
CAGTTCGCta
25053





cgaccca



 


100
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCT
24864
CAGTTCGCta
25054





cgaccca



 


101
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCT
24865
CAGTTCGCta
25055





cgaccca



 


104
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCT
24866
CAGTTCGCta
25056





cgaccca



 


105
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCT
24867
CAGTTCGCta
25057





cgaccca



 


108
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCT
24868
CAGTTCGCta
25058





cgaccca



 


109
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCT
24869
CAGTTCGCta
25059





cgaccca



 


112
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24870
GGCAGCAAa
25060



T

gttcctaa



 


113
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCT
24871
CAGTTCGCta
25061





cgaccca



 


114
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCT
24872
CAGTTCGCta
25062





cgaccca



 


115
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24873
GGCAGCAAa
25063



T

gttcctaa



 


116
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCT
24874
CAGTTCGCta
25064





cgaccca



 


117
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24875
GGCAGCAAa
25065



T

gttcctaa



 


119
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24876
GCAGCAAAg
25066



TG

ttcctaag



 


120
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24877
AGTTCGCTac
25067





gacccat



 


121
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24878
AGTTCGCTac
25068





gacccat



 


122
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24879
AGTTCGCTac
25069





gacccat



 


123
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24880
AGTTCGCTac
25070





gacccat



 


126
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24881
AGTTCGCTac
25071





gacccat



 


127
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24882
AGTTCGCTac
25072





gacccat



 


128
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24883
GCAGCAAAg
25073



TG

ttcctaag



 


129
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24884
GCAGCAAAg
25074



TG

ttcctaag



 


130
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24885
GCAGCAAAg
25075



TG

ttcctaag



 


134
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24886
GTTCGCTAcg
25076



A

acccata



 


135
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24887
GTTCGCTAcg
25077



A

acccata



 


138
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24888
GTTCGCTAcg
25078



A

acccata



 


140
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24889
CAGCAAAGtt
25079



TGG

cctaaga



 


146
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24890
TTCGCTACga
25080



AG

cccatac



 


147
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24891
TTCGCTACga
25081



AG

cccatac



 


151
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24892
TTCGCTACga
25082



AG

cccatac



 


152
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24893
AGCAAAGTtc
25083



TGGC

ctaagac



 


158
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24894
TCGCTACGac
25084



AGT

ccataca



 


159
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24895
TCGCTACGac
25085



AGT

ccataca



 


160
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24896
GCAAAGTTc
25086



TGGCA

ctaagacc



 


161
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24897
GCAAAGTTc
25087



TGGCA

ctaagacc



 


166
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24898
TCGCTACGac
25088



AGT

ccataca



 


167
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24899
TCGCTACGac
25089



AGT

ccataca



 


168
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24900
GCAAAGTTc
25090



TGGCA

ctaagacc



 


174
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24901
CGCTACGAc
25091



AGTT

ccatacac



 


175
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24902
CGCTACGAc
25092



AGTT

ccatacac



 


177
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24903
CGCTACGAc
25093



AGTT

ccatacac



 


181
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24904
CGCTACGAc
25094



AGTT

ccatacac



 


182
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24905
CAAAGTTCct
25095



TGGCAG

aagacca



 


187
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24906
GCTACGACc
25096



AGTTC

catacacc



 


188
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24907
GCTACGACc
25097



AGTTC

catacacc



 


191
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24908
GCTACGACc
25098



AGTTC

catacacc



 


192
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24909
GCTACGACc
25099



AGTTC

catacacc



 


193
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24910
AAAGTTCCta
25100



TGGCAGC

agaccaa



 


194
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24911
AAAGTTCCta
25101



TGGCAGC

agaccaa



 


195
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24912
AAAGTTCCta
25102



TGGCAGC

agaccaa



 


198
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24913
CTACGACCca
25103



AGTTCG

tacaccc



 


199
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24914
AAGTTCCTaa
25104



TGGCAGCA

gaccaaa



 


203
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24915
AAGTTCCTaa
25105



TGGCAGCA

gaccaaa



 


204
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24916
CTACGACCca
25106



AGTTCG

tacaccc



 


208
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24917
AGTTCCTAag
25107



TGGCAGCAA

accaaaa



 


209
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24918
AGTTCCTAag
25108



TGGCAGCAA

accaaaa



 


210
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24919
TACGACCCat
25109



AGTTCGC

acaccca



 


211
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24920
AGTTCCTAag
25110



TGGCAGCAA

accaaaa



 


214
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24921
GTTCCTAAga
25111



TGGCAGCAAA

ccaaaac



 


215
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24922
ACGACCCAta
25112



AGTTCGCT

cacccaa



 


217
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24923
GTTCCTAAga
25113



TGGCAGCAAA

ccaaaac



 


218
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24924
GTTCCTAAga
25114



TGGCAGCAAA

ccaaaac



 


221
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24925
CGACCCATac
25115



AGTTCGCTA

acccaaa



 


224
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24926
TTCCTAAGac
25116



TGGCAGCAAAG

caaaacc



 


228
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24927
CGACCCATac
25117



AGTTCGCTA

acccaaa



 


230
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24928
TTCCTAAGac
25118



TGGCAGCAAAG

caaaacc



 


231
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24929
CGACCCATac
25119



AGTTCGCTA

acccaaa



 


232
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24930
CGACCCATac
25120



AGTTCGCTA

acccaaa



 


238
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24931
GACCCATAc
25121



AGTTCGCTAC

acccaaag



 


239
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24932
GACCCATAc
25122



AGTTCGCTAC

acccaaag



 


242
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24933
GACCCATAc
25123



AGTTCGCTAC

acccaaag



 


243
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24934
GACCCATAc
25124



AGTTCGCTAC

acccaaag



 


246
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24935
TCCTAAGAcc
25125



TGGCAGCAAAGT

aaaacca



 


247
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24936
TCCTAAGAcc
25126



TGGCAGCAAAGT

aaaacca



 


251
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24937
ACCCATACac
25127



AGTTCGCTACG

ccaaagg



 


252
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24938
ACCCATACac
25128



AGTTCGCTACG

ccaaagg



 


256
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24939
ACCCATACac
25129



AGTTCGCTACG

ccaaagg



 


257
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24940
CCTAAGACc
25130



TGGCAGCAAAGTT

aaaaccac



 


258
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24941
CCTAAGACc
25131



TGGCAGCAAAGTT

aaaaccac



 


264
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24942
CCCATACAcc
25132



AGTTCGCTACGA

caaagga



 


265
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24943
CCCATACAcc
25133



AGTTCGCTACGA

caaagga



 


266
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24944
CTAAGACCa
25134



TGGCAGCAAAGTTC

aaaccaca



 


268
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24945
CCATACACcc
25135



AGTTCGCTACGAC

aaaggat



 


269
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24946
CCATACACcc
25136



AGTTCGCTACGAC

aaaggat



 


270
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24947
TAAGACCAa
25137



TGGCAGCAAAGTTCC

aaccacag



 


271
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24948
CCATACACcc
25138



AGTTCGCTACGAC

aaaggat



 


272
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24949
CCATACACcc
25139



AGTTCGCTACGAC

aaaggat



 


274
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24950
CCATACACcc
25140



AGTTCGCTACGAC

aaaggat



 


275
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24951
CATACACCca
25141



AGTTCGCTACGACC

aaggatt



 


276
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24952
CATACACCca
25142



AGTTCGCTACGACC

aaggatt



 


280
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24953
CATACACCca
25143



AGTTCGCTACGACC

aaggatt



 


281
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24954
AAGACCAAa
25144



TGGCAGCAAAGTTCCT

accacagg



 


286
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24955
ATACACCCaa
25145



AGTTCGCTACGACCC

aggattg



 


287
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24956
ATACACCCaa
25146



AGTTCGCTACGACCC

aggattg



 


288
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24957
AGACCAAAa
25147



TGGCAGCAAAGTTCCTA

ccacaggc



 


293
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24958
GACCAAAAC
25148



TGGCAGCAAAGTTCCTAA

cacaggct



 


297
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24959
GACCAAAAc
25149



TGGCAGCAAAGTTCCTAA

cacaggct



 


298
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24960
TACACCCAaa
25150



AGTTCGCTACGACCCA

ggattga



 


299
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24961
GACCAAAAC
25151



TGGCAGCAAAGTTCCTAA

cacaggct



 


300
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24962
GACCAAAAC
25152



TGGCAGCAAAGTTCCTAA

cacaggct



 


306
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24963
ACCAAAACc
25153



TGGCAGCAAAGTTCCTAAG

acaggctt



 


307
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24964
ACCAAAACc
25154



TGGCAGCAAAGTTCCTAAG

acaggctt



 


310
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24965
ACCAAAACc
25155



TGGCAGCAAAGTTCCTAAG

acaggctt



 


311
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24966
ACCAAAACc
25156



TGGCAGCAAAGTTCCTAAG

acaggctt



 


314
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24967
ACCAAAACc
25157



TGGCAGCAAAGTTCCTAAG

acaggctt



 


315
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24968
ACACCCAAa
25158



AGTTCGCTACGACCCAT

ggattgag



 


318
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24969
ACCAAAACc
25159



TGGCAGCAAAGTTCCTAAG

acaggctt



 


322
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24970
ACACCCAAa
25160



AGTTCGCTACGACCCAT

ggattgag



 


328
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24971
CCAAAACCa
25161



TGGCAGCAAAGTTCCTAAGA

caggcttg



 


329
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24972
CCAAAACCa
25162



TGGCAGCAAAGTTCCTAAGA

caggcttg



 


330
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24973
CCAAAACCa
25163



TGGCAGCAAAGTTCCTAAGA

caggcttg



 


331
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24974
CCAAAACCa
25164



TGGCAGCAAAGTTCCTAAGA

caggcttg



 


334
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24975
CACCCAAAg
25165



AGTTCGCTACGACCCATA

gattgagg



 


335
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24976
CACCCAAAg
25166



AGTTCGCTACGACCCATA

gattgagg



 


338
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24977
CACCCAAAg
25167



AGTTCGCTACGACCCATA

gattgagg



 


341
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24978
CCAAAACCa
25168



TGGCAGCAAAGTTCCTAAGA

caggcttg



 


342
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24979
CCAAAACCa
25169



TGGCAGCAAAGTTCCTAAGA

caggcttg



 


343
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24980
CACCCAAAg
25170



AGTTCGCTACGACCCATA

gattgagg



 


346
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24981
CACCCAAAg
25171



AGTTCGCTACGACCCATA

gattgagg



 


347
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24982
CACCCAAAg
25172



AGTTCGCTACGACCCATA

gattgagg



 


351
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24983
CAAAACCAc
25173



TGGCAGCAAAGTTCCTAAGAC

aggcttga



 


352
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24984
ACCCAAAGg
25174



AGTTCGCTACGACCCATAC

attgaggt



 


353
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24985
ACCCAAAGg
25175



AGTTCGCTACGACCCATAC

attgaggt



 


354
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24986
CAAAACCAc
25176



TGGCAGCAAAGTTCCTAAGAC

aggcttga



 


357
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24987
ACCCAAAGg
25177



AGTTCGCTACGACCCATAC

attgaggt



 


358
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24988
ACCCAAAGg
25178



AGTTCGCTACGACCCATAC

attgaggt



 


361
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24989
ACCCAAAGg
25179



AGTTCGCTACGACCCATAC

attgaggt



 


362
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24990
ACCCAAAGg
25180



AGTTCGCTACGACCCATAC

attgaggt



 


365
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24991
CAAAACCAc
25181



TGGCAGCAAAGTTCCTAAGAC

aggcttga



 


366
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24992
CAAAACCAc
25182



TGGCAGCAAAGTTCCTAAGAC

aggcttga



 


369
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24993
CCCAAAGGat
25183



AGTTCGCTACGACCCATACA

tgaggtc



 


370
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24994
CCCAAAGGat
25184



AGTTCGCTACGACCCATACA

tgaggtc



 


371
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24995
CCCAAAGGat
25185



AGTTCGCTACGACCCATACA

tgaggtc



 


372
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24996
CCCAAAGGat
25186



AGTTCGCTACGACCCATACA

tgaggtc



 


375
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
24997
AAAACCACa
25187



TGGCAGCAAAGTTCCTAAGACC

ggcttgag



 


376
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24998
CCCAAAGGat
25188



AGTTCGCTACGACCCATACA

tgaggtc



 


377
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
24999
CCCAAAGGat
25189



AGTTCGCTACGACCCATACA

tgaggtc



 


380
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
25000
CCCAAAGGat
25190



AGTTCGCTACGACCCATACA

tgaggtc



 


381
tcactcaagcctgtggttttggtcttaggaactttgctgccacaataccTCGGCCCTTCTC
25001
CCCAAAGGat
25191



AGTTCGCTACGACCCATACA

tgaggtc



 


382
caagacctcaatcctttgggtgtatgggtcgtagcgaactgagaagggcCGAGGTATTG
25002
AAAACCACa
25192



TGGCAGCAAAGTTCCTAAGACC

ggcttgag
















TABLE 3B







Exemplary RT sequence (heterologous object sequence) and PBS sequence pairs


Table 3B provides exemplified PBS sequences and heterologous object sequences


(reverse transcription template regions) of a template RNA for correcting


the pathogenic R261Q mutation in PAH. The gRNA spacers from Table 1B were


filtered, e.g., filtered by occurrence within 15 nt of the desired editing


location and use of a Tier 1 Cas enzyme. PBS sequences and heterologous


object sequences (reverse transcription template regions) were designed


relative to the nick site directed by the cognate gRNA from Table 1B,


as described in this application. For exemplification, these regions were


designed to be 8-17 nt (priming) and 1-50 nt extended beyond the location


of the edit (RT). Without wishing to be limited by example, given


variability of length, sequences are provided that use the maximum


length parameters and comprise all templates of shorter length within


the given parameters. Sequences are shown with uppercase letters


indicating core sequence and lowercase letters indicating flanking


sequence that may be truncated within the described length parameters.













SEQ

SEQ




ID

ID


ID
RT Template Sequence
NO
PBS Sequence
NO





  1
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTC
25193
GGAAGGCCa
25370





ggccaccc



 


  2
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTC
25194
GGAAGGCCa
25371





ggccaccc



 


  3
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTC
25195
GGAAGGCCa
25372





ggccaccc



 


  4
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGA
25196
GTCTTCCActg
25373





cacaca



 


  5
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGA
25197
GTCTTCCActg
25374





cacaca



 


  8
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGA
25198
GTCTTCCActg
25375





cacaca



 


 10
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCG
25199
GAAGGCCAg
25376





gccaccca



 


 13
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAG
25200
TCTTCCACtgc
25377





acacag



 


 14
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGG
25201
AAGGCCAGg
25378





ccacccaa



 


 17
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAG
25202
TCTTCCACtgc
25379





acacag



 


 21
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGG
25203
AAGGCCAGg
25380





ccacccaa



 


 23
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAG
25204
TCTTCCACtgc
25381





acacag



 


 29
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGT
25205
CTTCCACTgc
25382





acacagt



 


 30
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGT
25206
CTTCCACTgc
25383





acacagt



 


 33
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGA
25207
AGGCCAGGc
25384





cacccaag



 


 34
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGA
25208
AGGCCAGGc
25385





cacccaag



 


 37
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGT
25209
CTTCCACTgc
25386





acacagt



 


 38
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGT
25210
CTTCCACTgc
25387





acacagt



 


 41
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGT
25211
CTTCCACTgc
25388





acacagt



 


 42
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGT
25212
CTTCCACTgc
25389





acacagt



 


 45
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGA
25213
AGGCCAGGc
25390





cacccaag



 


 51
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTC
25214
TTCCACTGca
25391





cacagta



 


 52
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTC
25215
TTCCACTGca
25392





cacagta



 


 53
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTC
25216
TTCCACTGca
25393





cacagta



 


 54
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTC
25217
TTCCACTGca
25394





cacagta



 


 55
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAA
25218
GGCCAGGCca
25395





cccaaga



 


 60
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTC
25219
TTCCACTGca
25396





cacagta



 


 61
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTC
25220
TTCCACTGca
25397





cacagta



 


 62
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAA
25221
GGCCAGGCca
25398





cccaaga



 


 65
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAG
25222
GCCAGGCCac
25399





ccaagaa



 


 66
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCT
25223
TCCACTGCac
25400





acagtac



 


 67
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAG
25224
GCCAGGCCac
25401





ccaagaa



 


 70
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCT
25225
TCCACTGCac
25402





acagtac



 


 71
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAG
25226
GCCAGGCCac
25403





ccaagaa



 


 73
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGG
25227
CCAGGCCAcc
25404





caagaaa



 


 74
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTT
25228
CCACTGCAca
25405





cagtaca



 


 75
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGG
25229
CCAGGCCAcc
25406





caagaaa



 


 78
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTC
25230
CACTGCACac
25407





agtacat



 


 79
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGC
25231
CAGGCCACcc
25408





aagaaat



 


 81
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCC
25232
ACTGCACAca
25409





gtacatc



 


 85
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCC
25233
ACTGCACAca
25410





gtacatc



 


 86
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25234
AGGCCACCca
25411





agaaatc



 


 87
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25235
AGGCCACCca
25412





agaaatc



 


 88
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25236
AGGCCACCca
25413





agaaatc



 


 91
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25237
GGCCACCCaa
25414



A

gaaatcc



 


 94
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25238
CTGCACACag
25415





tacatca



 


 95
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25239
CTGCACACag
25416





tacatca



 


 96
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25240
GGCCACCCaa
25417



A

gaaatcc



 


 97
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25241
CTGCACACag
25418





tacatca



 


 98
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25242
GGCCACCCaa
25419



A

gaaatcc



 


 99
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25243
GGCCACCCaa
25420



A

gaaatcc



 


100
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25244
CTGCACACag
25421





tacatca



 


101
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25245
GGCCACCCaa
25422



A

gaaatcc



 


106
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25246
TGCACACAgt
25423



C

acatcag



 


107
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25247
TGCACACAgt
25424



C

acatcag



 


110
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25248
TGCACACAgt
25425



C

acatcag



 


112
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25249
GCCACCCAag
25426



AG

aaatccc



 


115
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25250
GCACACAGta
25427



CT

catcaga



 


116
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25251
GCACACAGta
25428



CT

catcaga



 


119
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25252
CCACCCAAga
25429



AGG

aatcccg



 


123
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25253
GCACACAGta
25430



CT

catcaga



 


125
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25254
CCACCCAAga
25431



AGG

aatcccg



 


132
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25255
CACACAGTac
25432



CTG

atcagac



 


133
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25256
CACACAGTac
25433



CTG

atcagac



 


136
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25257
CACACAGTac
25434



CTG

atcagac



 


137
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25258
CACACAGTac
25435



CTG

atcagac



 


140
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25259
CACACAGTac
25436



CTG

atcagac



 


143
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25260
CACCCAAGaa
25437



AGGC

atcccga



 


144
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25261
CACCCAAGaa
25438



AGGC

atcccga



 


147
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25262
CACCCAAGaa
25439



AGGC

atcccga



 


148
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25263
CACACAGTac
25440



CTG

atcagac



 


151
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25264
CACCCAAGaa
25441



AGGC

atcccga



 


154
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25265
CACCCAAGaa
25442



AGGC

atcccga



 


160
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25266
ACCCAAGAaa
25443



AGGCC

tcccgag



 


161
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25267
ACACAGTAca
25444



CTGC

tcagaca



 


162
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25268
ACACAGTAca
25445



CTGC

tcagaca



 


163
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25269
ACACAGTAca
25446



CTGC

tcagaca



 


164
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25270
ACACAGTAca
25447



CTGC

tcagaca



 


165
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25271
ACCCAAGAaa
25448



AGGCC

tcccgag



 


166
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25272
ACCCAAGAaa
25449



AGGCC

tcccgag



 


169
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25273
ACACAGTAca
25450



CTGC

tcagaca



 


170
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25274
ACACAGTAca
25451



CTGC

tcagaca



 


173
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25275
ACCCAAGAaa
25452



AGGCC

tcccgag



 


174
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25276
ACCCAAGAaa
25453



AGGCC

tcccgag



 


175
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25277
ACCCAAGAaa
25454



AGGCC

tcccgag



 


176
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25278
ACCCAAGAaa
25455



AGGCC

tcccgag



 


179
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25279
CACAGTACat
25456



CTGCA

cagacat



 


180
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25280
CCCAAGAAat
25457



AGGCCA

cccgaga



 


181
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25281
CACAGTACat
25458



CTGCA

cagacat



 


185
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25282
CACAGTACat
25459



CTGCA

cagacat



 


186
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25283
CCCAAGAAat
25460



AGGCCA

cccgaga



 


189
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25284
ACAGTACAtc
25461



CTGCAC

agacatg



 


190
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25285
ACAGTACAtc
25462



CTGCAC

agacatg



 


191
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25286
CCAAGAAAtc
25463



AGGCCAC

ccgagag



 


193
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25287
CAGTACATca
25464



CTGCACA

gacatgg



 


194
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25288
CAAGAAATcc
25465



AGGCCACC

cgagagg



 


198
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25289
CAAGAAATcc
25466



AGGCCACC

cgagagg



 


199
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25290
CAGTACATca
25467



CTGCACA

gacatgg



 


203
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25291
AGTACATCag
25468



CTGCACAC

acatgga



 


204
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25292
AGTACATCag
25469



CTGCACAC

acatgga



 


209
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25293
AAGAAATCcc
25470



AGGCCACCC

gagagga



 


210
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25294
AAGAAATCcc
25471



AGGCCACCC

gagagga



 


213
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25295
AAGAAATCcc
25472



AGGCCACCC

gagagga



 


214
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25296
AGTACATCag
25473



CTGCACAC

acatgga



 


217
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25297
AAGAAATCcc
25474



AGGCCACCC

gagagga



 


221
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25298
AGTACATCag
25475



CTGCACAC

acatgga



 


222
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25299
AAGAAATCcc
25476



AGGCCACCC

gagagga



 


223
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25300
AAGAAATCcc
25477



AGGCCACCC

gagagga



 


226
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25301
AGAAATCCcg
25478



AGGCCACCCA

agaggaa



 


227
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25302
AGAAATCCcg
25479



AGGCCACCCA

agaggaa



 


228
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25303
AGAAATCCcg
25480



AGGCCACCCA

agaggaa



 


231
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25304
GTACATCAga
25481



CTGCACACA

catggat



 


232
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25305
GTACATCAga
25482



CTGCACACA

catggat



 


235
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25306
AGAAATCCcg
25483



AGGCCACCCA

agaggaa



 


236
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25307
AGAAATCCcg
25484



AGGCCACCCA

agaggaa



 


237
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25308
AGAAATCCcg
25485



AGGCCACCCA

agaggaa



 


238
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25309
AGAAATCCcg
25486



AGGCCACCCA

agaggaa



 


239
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25310
GAAATCCCga
25487



AGGCCACCCAA

gaggaaa



 


240
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25311
TACATCAGac
25488



CTGCACACAG

atggatc



 


243
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25312
GAAATCCCga
25489



AGGCCACCCAA

gaggaaa



 


247
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25313
TACATCAGac
25490



CTGCACACAG

atggatc



 


249
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25314
GAAATCCCga
25491



AGGCCACCCAA

gaggaaa



 


250
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25315
TACATCAGac
25492



CTGCACACAG

atggatc



 


251
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25316
TACATCAGac
25493



CTGCACACAG

atggatc



 


254
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25317
ACATCAGAca
25494



CTGCACACAGT

tggatcc



 


255
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25318
ACATCAGAca
25495



CTGCACACAGT

tggatcc



 


258
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25319
AAATCCCGag
25496



AGGCCACCCAAG

aggaaag



 


259
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25320
AAATCCCGag
25497



AGGCCACCCAAG

aggaaag



 


262
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25321
AAATCCCGag
25498



AGGCCACCCAAG

aggaaag



 


263
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25322
AAATCCCGag
25499



AGGCCACCCAAG

aggaaag



 


267
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25323
AATCCCGAga
25500



AGGCCACCCAAGA

ggaaagc



 


268
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25324
AATCCCGAga
25501



AGGCCACCCAAGA

ggaaagc



 


271
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25325
AATCCCGAga
25502



AGGCCACCCAAGA

ggaaagc



 


272
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25326
AATCCCGAga
25503



AGGCCACCCAAGA

ggaaagc



 


275
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25327
AATCCCGAga
25504



AGGCCACCCAAGA

ggaaagc



 


276
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25328
CATCAGACat
25505



CTGCACACAGTA

ggatcca



 


279
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25329
AATCCCGAga
25506



AGGCCACCCAAGA

ggaaagc



 


283
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25330
CATCAGACat
25507



CTGCACACAGTA

ggatcca



 


286
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25331
ATCCCGAGag
25508



AGGCCACCCAAGAA

gaaagca



 


287
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25332
ATCCCGAGag
25509



AGGCCACCCAAGAA

gaaagca



 


288
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25333
ATCCCGAGag
25510



AGGCCACCCAAGAA

gaaagca



 


289
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25334
ATCCCGAGag
25511



AGGCCACCCAAGAA

gaaagca



 


292
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25335
ATCCCGAGag
25512



AGGCCACCCAAGAA

gaaagca



 


293
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25336
ATCCCGAGag
25513



AGGCCACCCAAGAA

gaaagca



 


296
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25337
ATCAGACAtg
25514



CTGCACACAGTAC

gatccaa



 


297
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25338
ATCAGACAtg
25515



CTGCACACAGTAC

gatccaa



 


300
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25339
ATCCCGAGag
25516



AGGCCACCCAAGAA

gaaagca



 


301
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25340
ATCCCGAGag
25517



AGGCCACCCAAGAA

gaaagca



 


302
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25341
TCCCGAGAgg
25518



AGGCCACCCAAGAAA

aaagcag



 


303
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25342
TCAGACATgg
25519



CTGCACACAGTACA

atccaag



 


304
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25343
TCCCGAGAgg
25520



AGGCCACCCAAGAAA

aaagcag



 


305
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25344
CAGACATGga
25521



CTGCACACAGTACAT

tccaagc



 


306
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25345
CCCGAGAGg
25522



AGGCCACCCAAGAAAT

aaagcagg



 


308
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25346
AGACATGGat
25523



CTGCACACAGTACATC

ccaagcc



 


309
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25347
CCGAGAGGa
25524



AGGCCACCCAAGAAATC

aagcaggc



 


310
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25348
GACATGGAtc
25525



CTGCACACAGTACATCA

caagccc



 


314
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25349
GACATGGAtc
25526



CTGCACACAGTACATCA

caagccc



 


315
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25350
CGAGAGGAa
25527



AGGCCACCCAAGAAATCC

agcaggcc



 


316
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25351
GACATGGAtc
25528



CTGCACACAGTACATCA

caagccc



 


317
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25352
GACATGGAtc
25529



CTGCACACAGTACATCA

caagccc



 


321
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25353
ACATGGATcc
25530



CTGCACACAGTACATCAG

aagccca



 


322
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25354
ACATGGATcc
25531



CTGCACACAGTACATCAG

aagccca



 


323
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25355
GAGAGGAAa
25532



AGGCCACCCAAGAAATCCC

gcaggcca



 


324
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25356
GAGAGGAAa
25533



AGGCCACCCAAGAAATCCC

gcaggcca



 


325
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25357
GAGAGGAAa
25534



AGGCCACCCAAGAAATCCC

gcaggcca



 


327
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25358
CATGGATCca
25535



CTGCACACAGTACATCAGA

agcccat



 


329
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25359
AGAGGAAAg
25536



AGGCCACCCAAGAAATCCCG

caggccag



 


332
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25360
ATGGATCCaa
25537



CTGCACACAGTACATCAGAC

gcccatg



 


335
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25361
GAGGAAAGc
25538



AGGCCACCCAAGAAATCCCGA

aggccagc



 


339
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25362
ATGGATCCaa
25539



CTGCACACAGTACATCAGAC

gcccatg



 


341
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25363
GAGGAAAGc
25540



AGGCCACCCAAGAAATCCCGA

aggccagc



 


350
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25364
TGGATCCAag
25541



CTGCACACAGTACATCAGACA

cccatgt



 


351
tgtggctggcctgctttcctctcgggatttcttgggtggcctggccttcCGAGTCTTCCA
25365
TGGATCCAag
25542



CTGCACACAGTACATCAGACA

cccatgt



 


354
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25366
AGGAAAGCa
25543



AGGCCACCCAAGAAATCCCGAG

ggccagcc



 


355
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25367
AGGAAAGCa
25544



AGGCCACCCAAGAAATCCCGAG

ggccagcc



 


358
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25368
AGGAAAGCa
25545



AGGCCACCCAAGAAATCCCGAG

ggccagcc



 


359
tatacatgggcttggatccatgtctgatgtactgtgtgcagtggaagacTCGGAAGGCC
25369
AGGAAAGCa
25546



AGGCCACCCAAGAAATCCCGAG

ggccagcc
















TABLE 3C







Exemplary RT sequence (heterologous object sequence) and PBS sequence pairs


Table 3C provides exemplified PBS sequences and heterologous object sequences


(reverse transcription template regions) of a template RNA for correcting


the pathogenic R243Q mutation in PAH. The gRNA spacers from Table 1C were


filtered, e.g., filtered by occurrence within 15 nt of the desired editing


location and use of a Tier 1 Cas enzyme. PBS sequences and heterologous


object sequences (reverse transcription template regions) were designed


relative to the nick site directed by the cognate gRNA from Table 1C, as


described in this application. For exemplification, these regions were


designed to be 8-17 nt (priming) and 1-50 nt extended beyond the location


of the edit (RT). Without wishing to be limited by example, given variability


of length, sequences are provided that use the maximum length parameters and


comprise all templates of shorter length within the given parameters.


Sequences are shown with uppercase letters indicating core sequence and


lowercase letters indicating flanking sequence that may be truncated within


the described length parameters.













SEQ

SEQ




ID

ID


ID
RT Template Sequence
NO
PBS Sequence
NO





  3
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTC
25547
GGAGGCGGa
25732





aaccagtg



 


  4
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTC
25548
GGAGGCGGa
25733





aaccagtg



 


  5
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGA
25549
CCTGTGGCtg
25734





gcctgct



 


  6
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGA
25550
CCTGTGGCtg
25735





gcctgct



 


  9
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGA
25551
CCTGTGGCtg
25736





gcctgct



 


 10
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGA
25552
CCTGTGGCtg
25737





gcctgct



 


 11
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCG
25553
GAGGCGGAa
25738





accagtgc



 


 12
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGA
25554
CCTGTGGCtg
25739





gcctgct



 


 13
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGA
25555
CCTGTGGCtg
25740





gcctgct



 


 14
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGAC
25556
CTGTGGCTg
25741





gcctgctt



 


 15
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGAC
25557
CTGTGGCTg
25742





gcctgctt



 


 17
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGAC
25558
CTGTGGCTg
25743





gcctgctt



 


 21
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGAC
25559
CTGTGGCTg
25744





gcctgctt



 


 22
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGG
25560
AGGCGGAAa
25745





ccagtgca



 


 25
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACC
25561
TGTGGCTGg
25746





cctgcttt



 


 26
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACC
25562
TGTGGCTGg
25747





cctgcttt



 


 29
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACC
25563
TGTGGCTGg
25748





cctgcttt



 


 30
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACC
25564
TGTGGCTGg
25749





cctgcttt



 


 33
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACC
25565
TGTGGCTGg
25750





cctgcttt



 


 34
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACC
25566
TGTGGCTGg
25751





cctgcttt



 


 37
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGA
25567
GGCGGAAAc
25752





cagtgcaa



 


 42
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCT
25568
GTGGCTGGc
25753





ctgctttc



 


 43
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCT
25569
GTGGCTGGc
25754





ctgctttc



 


 44
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCT
25570
GTGGCTGGc
25755





ctgctttc



 


 45
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCT
25571
GTGGCTGGc
25756





ctgctttc



 


 48
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCT
25572
GTGGCTGGc
25757





ctgctttc



 


 49
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCT
25573
GTGGCTGGc
25758





ctgctttc



 


 50
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAG
25574
GCGGAAACC
25759





agtgcaag



 


 51
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAG
25575
GCGGAAACC
25760





agtgcaag



 


 52
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAG
25576
GCGGAAACC
25761





agtgcaag



 


 57
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGG
25577
CGGAAACCa
25762





gtgcaagc



 


 58
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTG
25578
TGGCTGGCct
25763





gctttcc



 


 59
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTG
25579
TGGCTGGCct
25764





gctttcc



 


 60
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGG
25580
CGGAAACCa
25765





gtgcaagc



 


 61
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGG
25581
CGGAAACCa
25766





gtgcaagc



 


 62
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGG
25582
CGGAAACCa
25767





gtgcaagc



 


 64
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25583
GGAAACCAg
25768





tgcaagct



 


 65
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGT
25584
GGCTGGCCt
25769





gctttcct



 


 69
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGT
25585
GGCTGGCCt
25770





gctttcct



 


 70
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25586
GGAAACCAg
25771





tgcaagct



 


 76
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTG
25587
GCTGGCCTg
25772





ctttcctc



 


 77
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTG
25588
GCTGGCCTg
25773





ctttcctc



 


 80
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTG
25589
GCTGGCCTg
25774





ctttcctc



 


 81
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTG
25590
GCTGGCCTg
25775





ctttcctc



 


 84
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25591
GAAACCAGt
25776



G

gcaagctg



 


 86
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGG
25592
CTGGCCTGct
25777





ttcctct



 


 87
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGG
25593
CTGGCCTGct
25778





ttcctct



 


 90
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGG
25594
CTGGCCTGct
25779





ttcctct



 


 91
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGG
25595
CTGGCCTGct
25780





ttcctct



 


 92
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25596
AAACCAGTg
25781



GG

caagctgg



 


 93
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25597
AAACCAGTg
25782



GG

caagctgg



 


 96
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25598
AACCAGTGc
25783



GGA

aagctggg



 


 97
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGC
25599
TGGCCTGCtt
25784





tcctctc



 


 98
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGC
25600
TGGCCTGCtt
25785





tcctctc



 


 99
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGC
25601
TGGCCTGCtt
25786





tcctctc



 


102
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGC
25602
TGGCCTGCtt
25787





tcctctc



 


103
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGC
25603
TGGCCTGCtt
25788





tcctctc



 


104
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25604
AACCAGTGc
25789



GGA

aagctggg



 


105
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25605
AACCAGTGc
25790



GGA

aagctggg



 


106
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25606
AACCAGTGc
25791



GGA

aagctggg



 


107
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25607
AACCAGTGc
25792



GGA

aagctggg



 


108
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCT
25608
GGCCTGCTtt
25793





cctctcg



 


109
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25609
ACCAGTGCa
25794



GGAA

agctggga



 


112
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCT
25610
GGCCTGCTtt
25795





cctctcg



 


114
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25611
ACCAGTGCa
25796



GGAA

agctggga



 


121
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25612
CCAGTGCAa
25797



GGAAA

gctgggat



 


122
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25613
CCAGTGCAa
25798



GGAAA

gctgggat



 


123
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTG
25614
GCCTGCTTtc
25799





ctctcgg



 


124
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25615
CCAGTGCAa
25800



GGAAA

gctgggat



 


126
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25616
CAGTGCAAg
25801



GGAAAC

ctgggatg



 


127
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25617
CCTGCTTTcc
25802





tctcggg



 


128
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25618
CAGTGCAAg
25803



GGAAAC

ctgggatg



 


129
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25619
CCTGCTTTcc
25804





tctcggg



 


130
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25620
CCTGCTTTcc
25805





tctcggg



 


131
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25621
CAGTGCAAg
25806



GGAAAC

ctgggatg



 


132
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25622
CTGCTTTCct
25807



C

ctcggga



 


136
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25623
CTGCTTTCct
25808



C

ctcggga



 


137
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25624
AGTGCAAGc
25809



GGAAACC

tgggatga



 


141
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25625
TGCTTTCCtct
25810



CC

cgggat



 


142
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25626
TGCTTTCCtct
25811



CC

cgggat



 


143
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25627
GTGCAAGCt
25812



GGAAACCA

gggatgaa



 


144
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25628
TGCTTTCCtct
25813



CC

cgggat



 


145
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25629
GTGCAAGCt
25814



GGAAACCA

gggatgaa



 


148
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25630
GCTTTCCTct
25815



CCT

cgggatt



 


149
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25631
TGCAAGCTg
25816



GGAAACCAG

ggatgaaa



 


150
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25632
GCTTTCCTct
25817



CCT

cgggatt



 


151
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25633
GCTTTCCTct
25818



CCT

cgggatt



 


152
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25634
TGCAAGCTg
25819



GGAAACCAG

ggatgaaa



 


153
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25635
GCTTTCCTct
25820



CCT

cgggatt



 


154
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25636
TGCAAGCTg
25821



GGAAACCAG

ggatgaaa



 


155
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25637
CTTTCCTCtc
25822



CCTG

gggattt



 


156
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25638
GCAAGCTGg
25823



GGAAACCAGT

gatgaaaa



 


160
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25639
GCAAGCTGg
25824



GGAAACCAGT

gatgaaaa



 


161
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25640
CTTTCCTCtc
25825



CCTG

gggattt



 


167
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25641
CAAGCTGGg
25826



GGAAACCAGTG

atgaaaag



 


168
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25642
CAAGCTGGg
25827



GGAAACCAGTG

atgaaaag



 


171
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25643
CAAGCTGGg
25828



GGAAACCAGTG

atgaaaag



 


172
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25644
TTTCCTCTcg
25829



CCTGC

ggatttc



 


175
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25645
CAAGCTGGg
25830



GGAAACCAGTG

atgaaaag



 


179
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25646
TTTCCTCTcg
25831



CCTGC

ggatttc



 


183
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25647
AAGCTGGGa
25832



GGAAACCAGTGC

tgaaaaga



 


184
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25648
AAGCTGGGa
25833



GGAAACCAGTGC

tgaaaaga



 


187
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25649
TTCCTCTCgg
25834



CCTGCT

gatttct



 


188
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25650
TTCCTCTCgg
25835



CCTGCT

gatttct



 


191
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25651
TTCCTCTCgg
25836



CCTGCT

gatttct



 


194
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25652
AAGCTGGGa
25837



GGAAACCAGTGC

tgaaaaga



 


195
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25653
AAGCTGGGa
25838



GGAAACCAGTGC

tgaaaaga



 


196
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25654
TTCCTCTCgg
25839



CCTGCT

gatttct



 


199
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25655
TTCCTCTCgg
25840



CCTGCT

gatttct



 


203
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25656
TCCTCTCGg
25841



CCTGCTT

gatttctt



 


204
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25657
AGCTGGGAt
25842



GGAAACCAGTGCA

gaaaagaa



 


205
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25658
AGCTGGGAt
25843



GGAAACCAGTGCA

gaaaagaa



 


206
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25659
AGCTGGGAt
25844



GGAAACCAGTGCA

gaaaagaa



 


207
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25660
TCCTCTCGg
25845



CCTGCTT

gatttctt



 


208
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25661
TCCTCTCGg
25846



CCTGCTT

gatttctt



 


211
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25662
TCCTCTCGg
25847



CCTGCTT

gatttctt



 


212
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25663
TCCTCTCGg
25848



CCTGCTT

gatttctt



 


213
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25664
AGCTGGGAt
25849



GGAAACCAGTGCA

gaaaagaa



 


214
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25665
TCCTCTCGg
25850



CCTGCTT

gatttctt



 


215
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25666
TCCTCTCGg
25851



CCTGCTT

gatttctt



 


217
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25667
CCTCTCGGg
25852



CCTGCTTT

atttcttg



 


218
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25668
CCTCTCGGg
25853



CCTGCTTT

atttcttg



 


219
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25669
GCTGGGATg
25854



GGAAACCAGTGCAA

aaaagaag



 


220
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25670
CCTCTCGGg
25855



CCTGCTTT

atttcttg



 


224
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25671
CTCTCGGGat
25856



CCTGCTTTC

ttcttgg



 


225
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25672
CTCTCGGGat
25857



CCTGCTTTC

ttcttgg



 


229
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25673
CTCTCGGGat
25858



CCTGCTTTC

ttcttgg



 


230
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25674
CTGGGATGa
25859



GGAAACCAGTGCAAG

aaagaaga



 


236
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25675
TCTCGGGAtt
25860



CCTGCTTTCC

tcttggg



 


237
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25676
TCTCGGGAtt
25861



CCTGCTTTCC

tcttggg



 


238
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25677
TGGGATGAa
25862



GGAAACCAGTGCAAGC

aagaagaa



 


242
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25678
TGGGATGAa
25863



GGAAACCAGTGCAAGC

aagaagaa



 


243
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25679
TGGGATGAa
25864



GGAAACCAGTGCAAGC

aagaagaa



 


244
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25680
TGGGATGAa
25865



GGAAACCAGTGCAAGC

aagaagaa



 


249
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25681
CTCGGGATtt
25866



CCTGCTTTCCT

cttgggt



 


254
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25682
GGGATGAAa
25867



GGAAACCAGTGCAAGCT

agaagaaa



 


255
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25683
GGGATGAAa
25868



GGAAACCAGTGCAAGCT

agaagaaa



 


256
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25684
CTCGGGATtt
25869



CCTGCTTTCCT

cttgggt



 


257
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25685
CTCGGGATtt
25870



CCTGCTTTCCT

cttgggt



 


258
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25686
CTCGGGATtt
25871



CCTGCTTTCCT

cttgggt



 


260
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25687
TCGGGATTtc
25872



CCTGCTTTCCTC

ttgggtg



 


261
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25688
TCGGGATTtc
25873



CCTGCTTTCCTC

ttgggtg



 


263
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25689
GGATGAAAa
25874



GGAAACCAGTGCAAGCTG

gaagaaag



 


264
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25690
GGATGAAAa
25875



GGAAACCAGTGCAAGCTG

gaagaaag



 


269
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25691
CGGGATTTct
25876



CCTGCTTTCCTCT

tgggtgg



 


270
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25692
CGGGATTTct
25877



CCTGCTTTCCTCT

tgggtgg



 


274
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25693
CGGGATTTct
25878



CCTGCTTTCCTCT

tgggtgg



 


275
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25694
GATGAAAAg
25879



GGAAACCAGTGCAAGCTGG

aagaaaga



 


281
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25695
GGGATTTCtt
25880



CCTGCTTTCCTCTC

gggtggc



 


282
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25696
GGGATTTCtt
25881



CCTGCTTTCCTCTC

gggtggc



 


285
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25697
GGGATTTCtt
25882



CCTGCTTTCCTCTC

gggtggc



 


286
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25698
GGGATTTCtt
25883



CCTGCTTTCCTCTC

gggtggc



 


289
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25699
GGGATTTCtt
25884



CCTGCTTTCCTCTC

gggtggc



 


290
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25700
GGGATTTCtt
25885



CCTGCTTTCCTCTC

gggtggc



 


293
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25701
ATGAAAAGa
25886



GGAAACCAGTGCAAGCTGGG

agaaagaa



 


297
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25702
ATGAAAAGa
25887



GGAAACCAGTGCAAGCTGGG

agaaagaa



 


303
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25703
GGATTTCTtg
25888



CCTGCTTTCCTCTCG

ggtggcc



 


304
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25704
GGATTTCTtg
25889



CCTGCTTTCCTCTCG

ggtggcc



 


305
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25705
GGATTTCTtg
25890



CCTGCTTTCCTCTCG

ggtggcc



 


306
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25706
GGATTTCTtg
25891



CCTGCTTTCCTCTCG

ggtggcc



 


309
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25707
GGATTTCTtg
25892



CCTGCTTTCCTCTCG

ggtggcc



 


310
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25708
GGATTTCTtg
25893



CCTGCTTTCCTCTCG

ggtggcc



 


313
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25709
TGAAAAGAa
25894



GGAAACCAGTGCAAGCTGGGA

gaaagaaa



 


314
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25710
TGAAAAGAa
25895



GGAAACCAGTGCAAGCTGGGA

gaaagaaa



 


315
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25711
GGATTTCTtg
25896



CCTGCTTTCCTCTCG

ggtggcc



 


319
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25712
GATTTCTTgg
25897



CCTGCTTTCCTCTCGG

gtggcct



 


320
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25713
GATTTCTTgg
25898



CCTGCTTTCCTCTCGG

gtggcct



 


322
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25714
GAAAAGAA
25899



GGAAACCAGTGCAAGCTGGGAT

gaaagaaaa



 


323
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25715
GATTTCTTgg
25900



CCTGCTTTCCTCTCGG

gtggcct



 


327
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25716
GATTTCTTgg
25901



CCTGCTTTCCTCTCGG

gtggcct



 


328
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25717
GAAAAGAA
25902



GGAAACCAGTGCAAGCTGGGAT

gaaagaaaa



 


329
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25718
GATTTCTTgg
25903



CCTGCTTTCCTCTCGG

gtggcct



 


330
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25719
GAAAAGAA
25904



GGAAACCAGTGCAAGCTGGGAT

gaaagaaaa



 


331
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25720
GAAAAGAA
25905



GGAAACCAGTGCAAGCTGGGAT

gaaagaaaa



 


336
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25721
ATTTCTTGgg
25906



CCTGCTTTCCTCTCGGG

tggcctg



 


337
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25722
ATTTCTTGgg
25907



CCTGCTTTCCTCTCGGG

tggcctg



 


338
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25723
AAAAGAAG
25908



GGAAACCAGTGCAAGCTGGGATG

aaagaaaac



 


341
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25724
ATTTCTTGgg
25909



CCTGCTTTCCTCTCGGG

tggcctg



 


342
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25725
ATTTCTTGgg
25910



CCTGCTTTCCTCTCGGG

tggcctg



 


343
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25726
AAAAGAAG
25911



GGAAACCAGTGCAAGCTGGGATG

aaagaaaac



 


350
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25727
TTTCTTGGgt
25912



CCTGCTTTCCTCTCGGGA

ggcctgg



 


351
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25728
TTTCTTGGgt
25913



CCTGCTTTCCTCTCGGGA

ggcctgg



 


353
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25729
TTTCTTGGgt
25914



CCTGCTTTCCTCTCGGGA

ggcctgg



 


357
ttgagttttctttcttcttttcatcccagcttgcactggtttccgcctcCGACCTGTGGCTGG
25730
TTTCTTGGgt
25915



CCTGCTTTCCTCTCGGGA

ggcctgg



 


358
aggccaggccacccaagaaatcccgagaggaaagcaggccagccacaggTCGGAGGC
25731
AAAGAAGA
25916



GGAAACCAGTGCAAGCTGGGATGA

aagaaaact
















TABLE 3D







Exemplary RT sequence (heterologous object sequence) and PBS sequence pairs


Table 3D provides exemplified PBS sequences and heterologous object sequences


(reverse transcription template regions) of a template RNA for correcting


the pathogenic IVS10-11G > A mutation in PAH. The gRNA spacers from Table 1D


were filtered, e.g., filtered by occurrence within 15 nt of the desired


editing location and use of a Tier 1 Cas enzyme. PBS sequences and


heterologous object sequences (reverse transcription template regions) were


designed relative to the nick site directed by the cognate gRNA from


Table 1D, as described in this application. For exemplification, these


regions were designed to be 8-17 nt (priming) and 1-50 nt extended beyond the


location of the edit (RT). Without wishing to be limited by example, given


variability of length, sequences are provided that use the maximum length


parameters and comprise all templates of shorter length within the given


parameters. Sequences are shown with uppercase letters indicating core


sequence and lowercase letters indicating flanking sequence that may be


truncated within the described length parameters.













SEQ

SEQ




ID

ID


ID
RT Template Sequence
NO
PBS Sequence
NO





  1
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCC
25917
AAGTGAAAa
26066





gttattat



 


  2
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGG
25918
GGCCTACAgt
26067





actgctt



 


  3
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCA
25919
AGTGAAAAg
26068





ttattatc



 


  6
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGG
25920
GGCCTACAgt
26069





actgctt



 


 10
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCA
25921
AGTGAAAAg
26070





ttattatc



 


 12
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGG
25922
GGCCTACAgt
26071





actgctt



 


 16
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAA
25923
GTGAAAAGtt
26072





attatca



 


 17
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAA
25924
GTGAAAAGtt
26073





attatca



 


 20
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAA
25925
GTGAAAAGtt
26074





attatca



 


 23
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGG
25926
GCCTACAGta
26075





ctgctta



 


 24
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGG
25927
GCCTACAGta
26076





ctgctta



 


 25
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAA
25928
GTGAAAAGtt
26077





attatca



 


 28
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAA
25929
GTGAAAAGtt
26078





attatca



 


 31
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAG
25930
TGAAAAGTta
26079





ttatcac



 


 32
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAG
25931
TGAAAAGTta
26080





ttatcac



 


 33
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAG
25932
TGAAAAGTta
26081





ttatcac



 


 34
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGG
25933
CCTACAGTac
26082





tgcttat



 


 39
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAG
25934
TGAAAAGTta
26083





ttatcac



 


 40
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAG
25935
TGAAAAGTta
26084





ttatcac



 


 43
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAG
25936
TGAAAAGTta
26085





ttatcac



 


 44
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAG
25937
TGAAAAGTta
26086





ttatcac



 


 47
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGG
25938
CCTACAGTac
26087





tgcttat



 


 48
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAG
25939
TGAAAAGTta
26088





ttatcac



 


 49
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAG
25940
TGAAAAGTta
26089





ttatcac



 


 52
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGT
25941
GAAAAGTTat
26090





tatcact



 


 53
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGT
25942
GAAAAGTTat
26091





tatcact



 


 54
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGC
25943
CTACAGTAct
26092





gcttatc



 


 55
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGC
25944
CTACAGTAct
26093





gcttatc



 


 58
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGT
25945
GAAAAGTTat
26094





tatcact



 


 59
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGT
25946
GAAAAGTTat
26095





tatcact



 


 62
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGT
25947
GAAAAGTTat
26096





tatcact



 


 63
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGT
25948
GAAAAGTTat
26097





tatcact



 


 64
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGC
25949
CTACAGTAct
26098





gcttatc



 


 67
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCC
25950
TACAGTACtg
26099





cttatca



 


 68
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTG
25951
AAAAGTTAtt
26100





atcactg



 


 69
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTG
25952
AAAAGTTAtt
26101





atcactg



 


 70
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCC
25953
TACAGTACtg
26102





cttatca



 


 71
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTG
25954
AAAAGTTAtt
26103





atcactg



 


 74
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGA
25955
AAAGTTATta
26104





tcactgt



 


 75
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCT
25956
ACAGTACTg
26105





cttatcag



 


 76
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGA
25957
AAAGTTATta
26106





tcactgt



 


 77
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTA
25958
CAGTACTGct
26107





tatcaga



 


 78
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAA
25959
AAGTTATTat
26108





cactgtt



 


 79
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTAC
25960
AGTACTGCtt
26109





atcagag



 


 83
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTAC
25961
AGTACTGCtt
26110





atcagag



 


 84
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
25962
AGTTATTAtc
26111





actgtta



 


 85
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTAC
25963
AGTACTGCtt
26112





atcagag



 


 86
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTAC
25964
AGTACTGCtt
26113





atcagag



 


 87
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTAC
25965
AGTACTGCtt
26114





atcagag



 


 90
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25966
GTACTGCTtat
26115





cagaga



 


 93
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25967
GTACTGCTtat
26116





cagaga



 


 94
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25968
GTACTGCTtat
26117





cagaga



 


 97
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25969
GTACTGCTtat
26118





cagaga



 


 98
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25970
GTACTGCTtat
26119





cagaga



 


101
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
25971
GTTATTATca
26120



A

ctgttaa



 


102
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25972
GTACTGCTtat
26121





cagaga



 


103
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25973
GTACTGCTtat
26122





cagaga



 


106
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25974
TACTGCTTat
26123



G

cagagaa



 


107
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25975
TACTGCTTat
26124



G

cagagaa



 


108
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25976
TACTGCTTat
26125



G

cagagaa



 


111
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25977
TACTGCTTat
26126



G

cagagaa



 


112
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25978
TACTGCTTat
26127



G

cagagaa



 


113
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
25979
TTATTATCact
26128



AG

gttaaa



 


114
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
25980
TTATTATCact
26129



AG

gttaaa



 


115
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25981
TACTGCTTat
26130



G

cagagaa



 


116
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
25982
TTATTATCact
26131



AG

gttaaa



 


119
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25983
ACTGCTTAtc
26132



GT

agagaag



 


120
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25984
ACTGCTTAtc
26133



GT

agagaag



 


121
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25985
ACTGCTTAtc
26134



GT

agagaag



 


122
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
25986
TATTATCAct
26135



AGT

gttaaat



 


125
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25987
CTGCTTATca
26136



GTA

gagaagc



 


126
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25988
CTGCTTATca
26137



GTA

gagaagc



 


130
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25989
CTGCTTATca
26138



GTA

gagaagc



 


131
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
25990
ATTATCACtg
26139



AGTT

ttaaatc



 


132
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
25991
ATTATCACtg
26140



AGTT

ttaaatc



 


136
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25992
TGCTTATCag
26141



GTAC

agaagcc



 


137
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25993
TGCTTATCag
26142



GTAC

agaagcc



 


138
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
25994
TTATCACTgtt
26143



AGTTA

aaatca



 


139
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25995
GCTTATCAga
26144



GTACT

gaagcca



 


140
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
25996
TATCACTGtta
26145



AGTTAT

aatcag



 


141
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
25997
CTTATCAGag
26146



GTACTG

aagccaa



 


142
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
25998
ATCACTGTta
26147



AGTTATT

aatcagg



 


145
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
25999
TCACTGTTaa
26148



AGTTATTA

atcagga



 


146
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26000
TTATCAGAga
26149



GTACTGC

agccaaa



 


150
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26001
TATCAGAGa
26150



GTACTGCT

agccaaag



 


154
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26002
TATCAGAGa
26151



GTACTGCT

agccaaag



 


155
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26003
CACTGTTAaa
26152



AGTTATTAT

tcaggat



 


156
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26004
TATCAGAGa
26153



GTACTGCT

agccaaag



 


157
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26005
CACTGTTAaa
26154



AGTTATTAT

tcaggat



 


158
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26006
TATCAGAGa
26155



GTACTGCT

agccaaag



 


159
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26007
CACTGTTAaa
26156



AGTTATTAT

tcaggat



 


164
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26008
ACTGTTAAat
26157



AGTTATTATC

caggatc



 


167
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26009
ATCAGAGAa
26158



GTACTGCTT

gccaaagc



 


168
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26010
ATCAGAGAa
26159



GTACTGCTT

gccaaagc



 


169
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26011
ACTGTTAAat
26160



AGTTATTATC

caggatc



 


173
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26012
TCAGAGAAg
26161



GTACTGCTTA

ccaaagct



 


174
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26013
CTGTTAAAtc
26162



AGTTATTATCA

aggatca



 


175
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26014
TCAGAGAAg
26163



GTACTGCTTA

ccaaagct



 


179
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26015
CAGAGAAGc
26164



GTACTGCTTAT

caaagctt



 


180
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26016
CAGAGAAGc
26165



GTACTGCTTAT

caaagctt



 


183
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26017
CAGAGAAGc
26166



GTACTGCTTAT

caaagctt



 


187
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26018
CAGAGAAGc
26167



GTACTGCTTAT

caaagctt



 


188
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26019
TGTTAAATca
26168



AGTTATTATCAC

ggatcag



 


191
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26020
GTTAAATCag
26169



AGTTATTATCACT

gatcagt



 


192
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26021
GTTAAATCag
26170



AGTTATTATCACT

gatcagt



 


195
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26022
AGAGAAGCc
26171



GTACTGCTTATC

aaagcttc



 


196
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26023
AGAGAAGCc
26172



GTACTGCTTATC

aaagcttc



 


197
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26024
GTTAAATCag
26173



AGTTATTATCACT

gatcagt



 


201
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26025
GAGAAGCCa
26174



GTACTGCTTATCA

aagcttct



 


204
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26026
TTAAATCAg
26175



AGTTATTATCACTG

gatcagta



 


207
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26027
GAGAAGCCa
26176



GTACTGCTTATCA

aagcttct



 


209
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26028
TTAAATCAg
26177



AGTTATTATCACTG

gatcagta



 


210
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26029
GAGAAGCCa
26178



GTACTGCTTATCA

aagcttct



 


211
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26030
GAGAAGCCa
26179



GTACTGCTTATCA

aagcttct



 


215
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26031
AGAAGCCAa
26180



GTACTGCTTATCAG

agcttctc



 


218
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26032
TAAATCAGg
26181



AGTTATTATCACTGT

atcagtat



 


219
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26033
TAAATCAGg
26182



AGTTATTATCACTGT

atcagtat



 


220
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26034
AGAAGCCAa
26183



GTACTGCTTATCAG

agcttctc



 


223
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26035
GAAGCCAAa
26184



GTACTGCTTATCAGA

gcttctcc



 


224
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26036
AAATCAGGat
26185



AGTTATTATCACTGTT

cagtatt



 


225
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26037
AAATCAGGat
26186



AGTTATTATCACTGTT

cagtatt



 


226
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26038
AAATCAGGat
26187



AGTTATTATCACTGTT

cagtatt



 


229
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26039
GAAGCCAAa
26188



GTACTGCTTATCAGA

gcttctcc



 


232
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26040
AAATCAGGat
26189



AGTTATTATCACTGTT

cagtatt



 


236
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26041
AATCAGGAtc
26190



AGTTATTATCACTGTTA

agtattc



 


237
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26042
AATCAGGAtc
26191



AGTTATTATCACTGTTA

agtattc



 


238
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26043
AAGCCAAAg
26192



GTACTGCTTATCAGAG

cttctccc



 


242
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26044
AAGCCAAAg
26193



GTACTGCTTATCAGAG

cttctccc



 


247
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26045
ATCAGGATc
26194



AGTTATTATCACTGTTAA

agtattcc



 


250
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26046
AGCCAAAGC
26195



GTACTGCTTATCAGAGA

ttctcccc



 


251
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26047
AGCCAAAGc
26196



GTACTGCTTATCAGAGA

ttctcccc



 


252
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26048
ATCAGGATc
26197



AGTTATTATCACTGTTAA

agtattcc



 


255
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26049
GCCAAAGCtt
26198



GTACTGCTTATCAGAGAA

ctccccc



 


256
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26050
TCAGGATCa
26199



AGTTATTATCACTGTTAAA

gtattccc



 


257
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26051
GCCAAAGCtt
26200



GTACTGCTTATCAGAGAA

ctccccc



 


258
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26052
TCAGGATCa
26201



AGTTATTATCACTGTTAAA

gtattccc



 


261
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26053
TCAGGATCa
26202



AGTTATTATCACTGTTAAA

gtattccc



 


262
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26054
GCCAAAGCtt
26203



GTACTGCTTATCAGAGAA

ctccccc



 


264
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26055
CAGGATCAgt
26204



AGTTATTATCACTGTTAAAT

attccct



 


265
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26056
CCAAAGCTtc
26205



GTACTGCTTATCAGAGAAG

tccccct



 


269
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26057
CAAAGCTTct
26206



GTACTGCTTATCAGAGAAGC

ccccctg



 


270
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26058
AGGATCAGta
26207



AGTTATTATCACTGTTAAATC

ttccctg



 


271
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26059
AGGATCAGta
26208



AGTTATTATCACTGTTAAATC

ttccctg



 


272
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26060
AGGATCAGta
26209



AGTTATTATCACTGTTAAATC

ttccctg



 


273
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26061
GGATCAGTat
26210



AGTTATTATCACTGTTAAATCA

tccctgc



 


274
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26062
AAAGCTTCtc
26211



GTACTGCTTATCAGAGAAGCC

cccctgg



 


275
gctccagggggagaagctttggcttctctgataagcagtactgtaggccCCAAGTGAAA
26063
GGATCAGTat
26212



AGTTATTATCACTGTTAAATCA

tccctgc



 


276
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26064
AAAGCTTCtc
26213



GTACTGCTTATCAGAGAAGCC

cccctgg



 


277
gcagcagggaatactgatcctgatttaacagtgataataacttttcactTGGGGCCTACA
26065
AAAGCTTCtc
26214



GTACTGCTTATCAGAGAAGCC

cccctgg









Capital letters indicate “core nucleotides” while lower case letters indicate “flanking nucleotides.” Herein, when an RNA sequence (e.g., a template RNA sequence) is said to comprise a particular sequence (e.g., a sequence of Table 3A, Table 3B, Table 3C, or Table 3D or a portion thereof) that comprises thymine (T), it is of course understood that the RNA sequence may (and frequently does) comprise uracil (U) in place of T. For instance, the RNA sequence may comprise U at every position shown as T in the sequence in Table 3A, Table 3B, Table 3C, or Table 3D. More specifically, the present disclosure provides an RNA sequence according to every heterologous object sequence and PBS sequence shown in Table 3A, Table 3B, Table 3C, or Table 3D, wherein the RNA sequence has a U in place of each T in the sequence of Table 3A, Table 3B, Table 3C, or Table 3D.
In some embodiments of the systems and methods herein, the template RNA comprises a gRNA scaffold (e.g., that binds a gene modifying polypeptide, e.g., a Cas polypeptide) that comprises a sequence of a gRNA scaffold of Table 12. In some embodiments, the gRNA scaffold comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a gRNA scaffold of Table 12. In some embodiments, the gRNA scaffold comprises a sequence of a scaffold region of Table 12 that corresponds to the RT template sequence, the spacer sequence, or both, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.
In some embodiments of the systems and methods herein, the system further comprises a second strand-targeting gRNA that directs a nick to the second strand of the human PAH gene. In some embodiments, the second strand-targeting gRNA comprises a left gRNA spacer sequence or a right gRNA spacer sequence from Table 2A, Table 2B, Table 2C, or Table 2D. In some embodiments, the gRNA spacer additionally comprises one or more (e.g., 2, 3, or all) consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the left gRNA spacer sequence or right gRNA spacer sequence. In some embodiments, the second strand-targeting gRNA comprises a sequence comprising the core nucleotides of a second nick gRNA sequence from Table 4A, Table 4B, Table 4C, or Table 4D, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the second nick gRNA sequence additionally comprises one or more consecutive nucleotides starting with the 3′ end of the flanking nucleotides of the second nick gRNA sequence. In some embodiments, the second nick gRNA comprises a gRNA scaffold sequence that is orthogonal to the Cas domain of the gene modifying polypeptide. In some embodiments, the second nick gRNA comprises a gRNA scaffold sequence of Table 12.







TABLE 2A







Exemplary left gRNA spacer and right gRNA spacer pairs


Table 2A provides exemplified second-nick gRNA species for optional use for correcting the


pathogenic R408W mutation in PAH. The gRNA spacers from Table 1A were filtered, e.g., filtered


by occurrence within 15 nt of the desired editing location and use of a Tier 1 Cas enzyme.


Second-nick gRNAs were generated by searching the opposite strand of DNA in the regions −40 to


−140 (″left″) and +40 to +140 (″right″), relative to the first nick site defined by the first


gRNA, for the PAM utilized by the corresponding Cas variant. One exemplary spacer is shown for


each side of the target nick site.















SEQ


SEQ





ID


ID
Right


ID
Left gRNA spacer
NO
Left PAM
Right gRNA spacer
NO
PAM
















1
CTCGTAAGGTGTAAATTA
26215
TAC
AGTTCTCAGTGGCATTTTA
26595
TTC



CT


C




 


2
GGCTGATTCCATTAACAG
26216
AG
TTCCTAAAAAAGAAGTAA
26596
TG



TA


AA




 


6
CCATTAACAGTAAGTAAT
26217
ACA
CCTAAAAAAGAAGTAAAA
26597
CCA



TT


TG




 


7
TCGTAAGGTGTAAATTAC
26218
ACT
GTTCTCAGTGGCATTTTAC
26598
TCT



TT


T




 


10
tttTGGCTGATTCCATTAAC
26219
AGTAAT
gaaGACCCTGCTCTAGGGA
26599
CCGTGT



AGTA

T
GGTGT

T


 


13
CAGTAAGTAATTTACACC
26220
ACG
AAAAAAGAAGTAAAATG
26600
CTG



TT


CCA




 


14
TAAGTAATTTACACCTTA
26221
AGG
TTGAAGACCCTGCTCTAG
26601
AGG



CG


GG




 


17
CATTAACAGTAAGTAATT
26222
CAC
CTAAAAAAGAAGTAAAAT
26602
CAC



TA


GC




 


18
GTAAGGTGTAAATTACTT
26223
TG
TGGCATTTTACTTCTTTTT
26603
AG



AC


T




 


21
AGTAAGTAATTTACACCT
26224
CG
AAAAAGAAGTAAAATGCC
26604
TG



TA


AC




 


25
CGTAAGGTGTAAATTACT
26225
CTG
TTCTCAGTGGCATTTTACT
26605
CTT



TA


T




 


26
gtggCCTCGTAAGGTGTAA
26226
CTTACT
aagaGAGTTCTCAGTGGCAT
26606
ACTTC



ATTA

GT
TTT




 


29
gtAAATTACTTACTGTTAA
26227
TCAGCC
ttTTACTTCTTTTTTAGGAA
26607
GACACC



TGGAA


CACG




 


30
AGTAAGTAATTTACACCT
26228
GAGG
CTTAAGACTACCTTTCTCC
26608
ATGG



TAC


AA




 


31
CAGTAAGTAATTTACACC
26229
CGAG
AAAAAAGAAGTAAAATG
26609
TGAG



TTA


CCAC




 


34
CGTAAGGTGTAAATTACT
26230
CTG
GTGGCATTTTACTTCTTTT
26610
TAG



TA


T




 


35
TGTAAATTACTTACTGTT
26231
TGG
TGGCATTTTACTTCTTTTT
26611
AGG



AA


T




 


38
GTAAGGTGTAAATTACTT
26232
TGT
TCTCAGTGGCATTTTACTT
26612
TTT



AC


C




 


41
CAGTAAGTAATTTACACC
26233
ACG
AAAAAAGAAGTAAAATG
26613
CTG



TT


CCA




 


42
ATTAACAGTAAGTAATTT
26234
ACC
TAAAAAAGAAGTAAAATG
26614
ACT



AC


CC




 


43
GTAAGGTGTAAATTACTT
26235
TG
TGGCATTTTACTTCTTTTT
26615
AG



AC


T




 


46
gtggCCTCGTAAGGTGTAA
26236
CTTACT
aagaGAGTTCTCAGTGGCAT
26616
ACTTC



ATTA

GT
TTT




 


47
gtggCCTCGTAAGGTGTAA
26237
CTTACT
aagaGAGTTCTCAGTGGCAT
26617
ACTTC



ATTA

GT
TTT




 


50
gtAAATTACTTACTGTTAA
26238
TCAGCC
ttTTACTTCTTTTTTAGGAA
26618
GACACC



TGGAA


CACG




 


51
AACAGTAAGTAATTTACA
26239
TACGA
TAAAAAAGAAGTAAAATG
26619
CTGAG



CCT


CCA




 


52
AACAGTAAGTAATTTACA
26240
TACGA
TAAAAAAGAAGTAAAATG
26620
CTGAG



CCT


CCA




 


55
GGTGTAAATTACTTACTG
26241
ATGG
AGTGGCATTTTACTTCTTT
26621
TAGG



TTA


TT




 


56
GGTGTAAATTACTTACTG
26242
ATGG
CAGTGGCATTTTACTTCTT
26622
TTAG



TTA


TT




 


57
CAGTAAGTAATTTACACC
26243
CGAG
AAAAAAGAAGTAAAATG
26623
TGAG



TTA


CCAC




 


62
CGTAAGGTGTAAATTACT
26244
CTG
GTGGCATTTTACTTCTTTT
26624
TAG



TA


T




 


63
TAAGGTGTAAATTACTTA
26245
GTT
CTCAGTGGCATTTTACTTC
26625
TTT



CT


T




 


64
TTAACAGTAAGTAATTTA
26246
CCT
AAAAAAGAAGTAAAATG
26626
CTG



CA


CCA




 


65
gtggCCTCGTAAGGTGTAA
26247
CTTACT
agtgGCATTTTACTTCTTTTT
26627
GGAAC



ATTA

GT
TA




 


66
AACAGTAAGTAATTTACA
26248
TACGA
AAAAAAGAAGTAAAATG
26628
TGAGA



CCT


CCAC




 


67
GTAAGGTGTAAATTACTT
26249
GTTAAT
TCAGTGGCATTTTACTTCT
26629
TTTAG



ACT


TT




 


68
TAACAGTAAGTAATTTAC
26250
CTT
AAAAAGAAGTAAAATGCC
26630
TGA



AC


AC




 


69
AAGGTGTAAATTACTTAC
26251
TTA
TCAGTGGCATTTTACTTCT
26631
TTT



TG


T




 


72
AACAGTAAGTAATTTACA
26252
TACGA
AAAAAGAAGTAAAATGCC
26632
GAGAA



CCT


ACT




 


73
AACAGTAAGTAATTTACA
26253
TTA
AAAAGAAGTAAAATGCCA
26633
GAG



CC


CT




 


74
AGGTGTAAATTACTTACT
26254
TAA
CAGTGGCATTTTACTTCTT
26634
TTT



GT


T




 


77
AGTAAGTAATTTACACCT
26255
CG
AAAGAAGTAAAATGCCAC
26635
AG



TA


TG




 


81
ACAGTAAGTAATTTACAC
26256
TAC
AAAGAAGTAAAATGCCAC
26636
AGA



CT


TG




 


82
GGTGTAAATTACTTACTG
26257
AAT
AGTGGCATTTTACTTCTTT
26637
TTA



TT


T




 


86
CAGTAAGTAATTTACACC
26258
ACG
AAAAGAAGTAAAATGCCA
26638
GAG



TT


CT




 


87
TAAGTAATTTACACCTTA
26259
AGG
TAAGACTACCTTTCTCCA
26639
TGG



CG


AA




 


90
CAGTAAGTAATTTACACC
26260
ACG
AAGAAGTAAAATGCCACT
26640
GAA



TT


GA




 


91
AGTAAGTAATTTACACCT
26261
CG
AAAGAAGTAAAATGCCAC
26641
AG



TA


TG




 


94
GTGTAAATTACTTACTGT
26262
ATG
GTGGCATTTTACTTCTTTT
26642
TAG



TA


T




 


95
acagTAAGTAATTTACACCT
26263
GAGGC
aaaaAAGAAGTAAAATGCC
26643
AGAAC



TAC


ACTG




 


96
acagTAAGTAATTTACACCT
26264
GAGGC
aaaaAAGAAGTAAAATGCC
26644
AGAAC



TAC


ACTG




 


99
aaCAGTAAGTAATTTACAC
26265
GAGGCC
tcCGTGTTCCTAAAAAAGA
26645
AATGCC



CTTAC


AGTAA




 


100
AGTAAGTAATTTACACCT
26266
GAGG
CTTAAGACTACCTTTCTCC
26646
ATGG



TAC


AA




 


101
CAGTAAGTAATTTACACC
26267
CGAG
AAAAAAGAAGTAAAATG
26647
TGAG



TTA


CCAC




 


104
GTAAGTAATTTACACCTT
26268
GAG
AAAAGAAGTAAAATGCCA
26648
GAG



AC


CT




 


105
TAAGTAATTTACACCTTA
26269
AGG
TAAGACTACCTTTCTCCA
26649
TGG



CG


AA




 


108
AGTAAGTAATTTACACCT
26270
CGA
AGAAGTAAAATGCCACTG
26650
AAC



TA


AG




 


109
AGTAAGTAATTTACACCT
26271
CG
AAAGAAGTAAAATGCCAC
26651
AG



TA


TG




 


112
TGTAAATTACTTACTGTT
26272
TGG
TGGCATTTTACTTCTTTTT
26652
AGG



AA


T




 


113
acagTAAGTAATTTACACCT
26273
GAGGC
aaaaGAAGTAAAATGCCAC
26653
AACTC



TAC


TGAG




 


114
acagTAAGTAATTTACACCT
26274
GAGGC
aaaaGAAGTAAAATGCCAC
26654
AACTC



TAC


TGAG




 


115
gtgtAAATTACTTACTGTTA
26275
GAATC
agtgGCATTTTACTTCTTTTT
26655
GGAAC



ATG


TA




 


116
acagTAAGTAATTTACACCT
26276
GAGGC
aaaaGAAGTAAAATGCCAC
26656
AACTC



TAC


TGAG




 


117
gtgtAAATTACTTACTGTTA
26277
GAATC
agtgGCATTTTACTTCTTTTT
26657
GGAAC



ATG


TA




 


119
gtAAATTACTTACTGTTAA
26278
TCAGCC
ttTTACTTCTTTTTTAGGAA
26658
GACACC



TGGAA


CACG




 


120
taACAGTAAGTAATTTACA
26279
ACGAG
taAAAAAGAAGTAAAATG
26659
GAGAA



CCTT


CCACT




 


121
CAGTAAGTAATTTACACC
26280
CGAGG
AAAAAGAAGTAAAATGCC
26660
GAGAA



TTA


ACT




 


122
AGTAAGTAATTTACACCT
26281
GAGG
CTTAAGACTACCTTTCTCC
26661
ATGG



TAC


AA




 


123
AGTAAGTAATTTACACCT
26282
GAGG
AAAAAAGAAGTAAAATG
26662
TGAG



TAC


CCAC




 


126
GTAAGTAATTTACACCTT
26283
GAG
AAAAGAAGTAAAATGCCA
26663
GAG



AC


CT




 


127
GTAAGTAATTTACACCTT
26284
GAG
GAAGTAAAATGCCACTGA
26664
ACT



AC


GA




 


128
GTAAATTACTTACTGTTA
26285
GGA
GGCATTTTACTTCTTTTTT
26665
GGA



AT


A




 


129
gtgtAAATTACTTACTGTTA
26286
GAATC
agtgGCATTTTACTTCTTTTT
26666
GGAAC



ATG


TA




 


130
gtgtAAATTACTTACTGTTA
26287
GAATC
agtgGCATTTTACTTCTTTTT
26667
GGAAC



ATG


TA




 


134
CAGTAAGTAATTTACACC
26288
CGAGG
TAAAATGCCACTGAGAAC
26668
CTTAA



TTA


TCT




 


135
AGTAAGTAATTTACACCT
26289
GAGG
AAATGCCACTGAGAACTC
26669
TAAG



TAC


TCT




 


138
TAAGTAATTTACACCTTA
26290
AGG
AAGTAAAATGCCACTGAG
26670
CTC



CG


AA




 


140
TAAATTACTTACTGTTAA
26291
GAA
GCATTTTACTTCTTTTTTA
26671
GAA



TG


G




 


146
CAGTAAGTAATTTACACC
26292
CGAGG
TAAAATGCCACTGAGAAC
26672
CTTAA



TTA


TCT




 


147
AAGTAATTTACACCTTAC
26293
GG
AAAGAAGTAAAATGCCAC
26673
AG



GA


TG




 


151
AAGTAATTTACACCTTAC
26294
GGC
AGTAAAATGCCACTGAGA
26674
TCT



GA


AC




 


152
AAATTACTTACTGTTAAT
26295
AAT
CATTTTACTTCTTTTTTAG
26675
AAC



GG


G




 


158
taACAGTAAGTAATTTACA
26296
ACGAG
taAAAAAGAAGTAAAATG
26676
GAGAA



CCTT


CCACT




 


159
CAGTAAGTAATTTACACC
26297
CGAGG
TAAAATGCCACTGAGAAC
26677
CTTAA



TTA


TCT




 


160
AAATTACTTACTGTTAAT
26298
ATCAG
ATTTTACTTCTTTTTTAGG
26678
CACGG



GGA


AA




 


161
AAATTACTTACTGTTAAT
26299
ATCAG
ATTTTACTTCTTTTTTAGG
26679
CACGG



GGA


AA




 


166
TAAGTAATTTACACCTTA
26300
AGG
ATGCCACTGAGAACTCTC
26680
AAG



CG


TT




 


167
AGTAATTTACACCTTACG
26301
GCC
GTAAAATGCCACTGAGAA
26681
CTC



AG


CT




 


168
AATTACTTACTGTTAATG
26302
ATC
ATTTTACTTCTTTTTTAGG
26682
ACA



GA


A




 


174
AATTTACACCTTACGAGG
26303
CTCGG
TAAAATGCCACTGAGAAC
26683
CTTAA



CCA


TCT




 


175
ATTTACACCTTACGAGGC
26304
TCGG
AAATGCCACTGAGAACTC
26684
TAAG



CAC


TCT




 


177
AAGTAATTTACACCTTAC
26305
GG
TGCCACTGAGAACTCTCT
26685
AG



GA


TA




 


181
GTAATTTACACCTTACGA
26306
CCA
TAAAATGCCACTGAGAAC
26686
TCT



GG


TC




 


182
ATTACTTACTGTTAATGG
26307
TCA
TTTTACTTCTTTTTTAGGA
26687
CAC



AA


A




 


187
taACAGTAAGTAATTTACA
26308
ACGAG
taAAAAAGAAGTAAAATG
26688
GAGAA



CCTT


CCACT




 


188
AATTTACACCTTACGAGG
26309
CTCGG
TAAAATGCCACTGAGAAC
26689
CTTAA



CCA


TCT




 


191
TTTACACCTTACGAGGCC
26310
TCG
ATGCCACTGAGAACTCTC
26690
AAG



AC


TT




 


192
TAATTTACACCTTACGAG
26311
CAC
AAAATGCCACTGAGAACT
26691
CTT



GC


CT




 


193
TTACTTACTGTTAATGGA
26312
CAG
TTTACTTCTTTTTTAGGAA
26692
ACG



AT


C




 


194
aaatTACTTACTGTTAATGG
26313
CAGCC
atttTACTTCTTTTTTAGGAA
26693
CGGAC



AAT


CA




 


195
aaatTACTTACTGTTAATGG
26314
CAGCC
atttTACTTCTTTTTTAGGAA
26694
CGGAC



AAT


CA




 


198
AATTTACACCTTACGAGG
26315
CTCGG
AAAATGCCACTGAGAACT
26695
TTAAG



CCA


CTC




 


199
TACTTACTGTTAATGGAA
26316
AG
TTACTTCTTTTTTAGGAAC
26696
CG



TC


A




 


203
TACTTACTGTTAATGGAA
26317
AGC
TTACTTCTTTTTTAGGAAC
26697
CGG



TC


A




 


204
AATTTACACCTTACGAGG
26318
ACT
AAATGCCACTGAGAACTC
26698
TTA



CC


TC




 


208
TTACTTACTGTTAATGGA
26319
CAG
TTACTTCTTTTTTAGGAAC
26699
CGG



AT


A




 


209
ACTTACTGTTAATGGAAT
26320
GCC
TACTTCTTTTTTAGGAACA
26700
GGA



CA


C




 


210
ATTTACACCTTACGAGGC
26321
CTC
AATGCCACTGAGAACTCT
26701
TAA



CA


CT




 


211
aaatTACTTACTGTTAATGG
26322
CAGCC
atttTACTTCTTTTTTAGGAA
26702
CGGAC



AAT


CA




 


214
gtAAATTACTTACTGTTAA
26323
TCAGCC
ttTTACTTCTTTTTTAGGAA
26703
GACACC



TGGAA


CACG




 


215
TTTACACCTTACGAGGCC
26324
TCG
ATGCCACTGAGAACTCTC
26704
AAG



AC


TT




 


217
CTTACTGTTAATGGAATC
26325
CCA
ACTTCTTTTTTAGGAACAC
26705
GAC



AG


G




 


218
cttaCTGTTAATGGAATCAG
26326
AAATCT
tttaCTTCTTTTTTAGGAACA
26706
GACAC



CCA

TA
CG




 


221
TTACACCTTACGAGGCCA
26327
CG
TGCCACTGAGAACTCTCT
26707
AG



CT


TA




 


224
TACTTACTGTTAATGGAA
26328
AG
TACTTCTTTTTTAGGAACA
26708
GG



TC


C




 


228
TTACACCTTACGAGGCCA
26329
CGG
TGCCACTGAGAACTCTCT
26709
AGA



CT


TA




 


230
TTACTGTTAATGGAATCA
26330
CAA
CTTCTTTTTTAGGAACACG
26710
ACA



GC


G




 


231
tttaCACCTTACGAGGCCAC
26331
GTTTC
aaaaTGCCACTGAGAACTCT
26711
AAGAC



TCG


CTT




 


232
tttaCACCTTACGAGGCCAC
26332
GTTTC
aaaaTGCCACTGAGAACTCT
26712
AAGAC



TCG


CTT




 


238
taACAGTAAGTAATTTACA
26333
ACGAG
taAAAAAGAAGTAAAATG
26713
GAGAA



CCTT


CCACT




 


239
AATTTACACCTTACGAGG
26334
CTCGGT
AAATGCCACTGAGAACTC
26714
TAAGA



CCA


TCT




 


242
TTACACCTTACGAGGCCA
26335
CGG
ATGCCACTGAGAACTCTC
26715
AAG



CT


TT




 


243
TACACCTTACGAGGCCAC
26336
GGT
GCCACTGAGAACTCTCTT
26716
GAC



TC


AA




 


246
TTACTTACTGTTAATGGA
26337
CAG
TTACTTCTTTTTTAGGAAC
26717
CGG



AT


A




 


247
TACTGTTAATGGAATCAG
26338
AAA
TTCTTTTTTAGGAACACGG
26718
CAC



CC


A




 


251
AATTTACACCTTACGAGG
26339
CTCGGT
AAATGCCACTGAGAACTC
26719
TAAGA



CCA


TCT




 


252
TACACCTTACGAGGCCAC
26340
GG
TGCCACTGAGAACTCTCT
26720
AG



TC


TA




 


256
ACACCTTACGAGGCCACT
26341
GTT
CCACTGAGAACTCTCTTA
26721
ACT



CG


AG




 


257
ACTGTTAATGGAATCAGC
26342
AAA
TCTTTTTTAGGAACACGG
26722
ACC



CA


AC




 


258
cttaCTGTTAATGGAATCAG
26343
AAATC
acttCTTTTTTAGGAACACG
26723
ACCTC



CCA


GAC




 


264
TTACACCTTACGAGGCCA
26344
CGG
ATGCCACTGAGAACTCTC
26724
AAG



CT


TT




 


265
CACCTTACGAGGCCACTC
26345
TTT
CACTGAGAACTCTCTTAA
26725
CTA



GG


GA




 


266
CTGTTAATGGAATCAGCC
26364
AAT
CTTTTTTAGGAACACGGA
26726
CCT



AA


CA




 


268
CTTACGAGGCCACTCGGT
26347
TCAG
AAATGCCACTGAGAACTC
26727
TAAG



TTC


TCT




 


269
ACCTTACGAGGCCACTCG
26348
TTC
ACTGAGAACTCTCTTAAG
26728
TAC



GT


AC




 


270
TGTTAATGGAATCAGCCA
26349
ATC
TTTTTTAGGAACACGGAC
26729
CTC



AA


AC




 


271
tacaCCTTACGAGGCCACTC
26350
TTCTCA
tgccACTGAGAACTCTCTTA
26730
CTACCT



GGT

GT
AGA

TT


 


272
tacaCCTTACGAGGCCACTC
26351
TTCTCA
tgccACTGAGAACTCTCTTA
26731
CTACCT



GGT

GT
AGA

TT


 


274
tacaCCTTACGAGGCCACTC
26352
TTCTCA
tgccACTGAGAACTCTCTTA
26732
CTACCT



GGT

GT
AGA

TT


 


275
CCTTACGAGGCCACTCGG
26353
CTCAG
ACTCTCTTAAGACTACCTT
26733
TCCAA



TTT


TC




 


276
ACGAGGCCACTCGGTTTC
26354
AG
TGCCACTGAGAACTCTCT
26734
AG



TC


TA




 


280
CCTTACGAGGCCACTCGG
26355
TCT
CTGAGAACTCTCTTAAGA
26735
ACC



TT


CT




 


281
GTTAATGGAATCAGCCAA
26356
TCT
TTTTTAGGAACACGGACA
26736
TCC



AA


CC




 


286
TACGAGGCCACTCGGTTT
26357
CAG
ATGCCACTGAGAACTCTC
26737
AAG



CT


TT




 


287
CTTACGAGGCCACTCGGT
26358
CTC
TGAGAACTCTCTTAAGAC
26738
CCT



TT


TA




 


288
TTAATGGAATCAGCCAAA
26359
CTT
TTTTAGGAACACGGACAC
26739
CCC



AT


CT




 


293
TGGAATCAGCCAAAATCT
26360
AG
AGGAACACGGACACCTCC
26740
AG



TA


CT




 


297
TAATGGAATCAGCCAAAA
26361
TTA
TTTAGGAACACGGACACC
26741
CCT



TC


TC




 


298
TTACGAGGCCACTCGGTT
26362
TCA
GAGAACTCTCTTAAGACT
26742
CTT



TC


AC




 


299
gttaATGGAATCAGCCAAA
26363
TAAGC
ttctTTTTTAGGAACACGGA
26743
CTCCCT



ATCT


CAC

AG


 


300
gttaATGGAATCAGCCAAA
26364
TAAGC
ttctTTTTTAGGAACACGGA
26744
CTCCCT



ATCT


CAC

AG


 


306
gaATCAGCCAAAATCTTAA
26365
CTGGG
ttTTAGGAACACGGACACC
26745
TAGAG



GCTG


TCCC




 


307
TTAATGGAATCAGCCAAA
26366
TTAAG
TTTAGGAACACGGACACC
26746
CTAGA



ATC


TCC




 


310
ATGGAATCAGCCAAAATC
26367
AAG
TAGGAACACGGACACCTC
26747
TAG



TT


CC




 


311
CAGCCAAAATCTTAAGCT
26368
TGG
CACGGACACCTCCCTAGA
26748
AGG



GC


GC




 


314
AATGGAATCAGCCAAAAT
26369
TAA
TTAGGAACACGGACACCT
26749
CTA



CT


CC




 


315
ACGAGGCCACTCGGTTTC
26370
AG
TGCCACTGAGAACTCTCT
26750
AG



TC


TA




 


318
TGGAATCAGCCAAAATCT
26371
AG
AGGAACACGGACACCTCC
26751
AG



TA


CT




 


322
TACGAGGCCACTCGGTTT
26372
CAG
AGAACTCTCTTAAGACTA
26752
TTT



CT


CC




 


328
gaATCAGCCAAAATCTTAA
26373
CTGGG
ttTTAGGAACACGGACACC
26753
TAGAG



GCTG


TCCC




 


329
TTAATGGAATCAGCCAAA
26374
TTAAG
TTAGGAACACGGACACCT
26754
TAGAG



ATC


CCC




 


330
ATCAGCCAAAATCTTAAG
26375
CTGG
AACACGGACACCTCCCTA
26755
CAGG



CTG


GAG




 


331
TAATGGAATCAGCCAAAA
26376
TAAG
TAGGAACACGGACACCTC
26756
AGAG



TCT


CCT




 


334
TACGAGGCCACTCGGTTT
26377
CAG
TTAAGACTACCTTTCTCCA
26757
ATG



CT


A




 


335
TTACACCTTACGAGGCCA
26378
CGG
TAAGACTACCTTTCTCCA
26758
TGG



CT


AA




 


338
ACGAGGCCACTCGGTTTC
26379
AGT
GAACTCTCTTAAGACTAC
26759
TTC



TC


CT




 


341
ATGGAATCAGCCAAAATC
26380
AAG
TAGGAACACGGACACCTC
26760
TAG



TT


CC




 


342
ATGGAATCAGCCAAAATC
26381
AAG
TAGGAACACGGACACCTC
26761
TAG



TT


CC




 


343
ACGAGGCCACTCGGTTTC
26382
AG
TGCCACTGAGAACTCTCT
26762
AG



TC


TA




 


346
tacgAGGCCACTCGGTTTCT
26383
TAATCG
tgagAACTCTCTTAAGACTA
26763
TTCTC



CAG

AA
CCT




 


347
tacgAGGCCACTCGGTTTCT
26384
TAATCG
tgagAACTCTCTTAAGACTA
26764
TTCTC



CAG

AA
CCT




 


351
TTAATGGAATCAGCCAAA
26385
TTAAG
TTAGGAACACGGACACCT
26765
TAGAG



ATC


CCC




 


352
ATTTACACCTTACGAGGC
26386
TCGG
CTTAAGACTACCTTTCTCC
26766
ATGG



CAC


AA




 


353
CTTACGAGGCCACTCGGT
26387
TCAG
CTTAAGACTACCTTTCTCC
26767
ATGG



TTC


AA




 


354
TAATGGAATCAGCCAAAA
26388
TAAG
TAGGAACACGGACACCTC
26768
AGAG



TCT


CCT




 


357
TACGAGGCCACTCGGTTT
26389
CAG
TTAAGACTACCTTTCTCCA
26769
ATG



CT


A




 


358
TTACACCTTACGAGGCCA
26390
CGG
TAAGACTACCTTTCTCCA
26770
TGG



CT


AA




 


361
CGAGGCCACTCGGTTTCT
26391
GTA
AACTCTCTTAAGACTACC
26771
TCT



CA


TT




 


362
ACGAGGCCACTCGGTTTC
26392
AG
TAAGACTACCTTTCTCCA
26772
TG



TC


AA




 


365
TGGAATCAGCCAAAATCT
26393
AGC
AGGAACACGGACACCTCC
26773
AGA



TA


CT




 


366
GTGTAAATTACTTACTGT
26394
ATGGAA
AGTGGCATTTTACTTCTTT
26774
TTAGGA



TA

T
T

A


 


369
gaGGCCACTCGGTTTCTCA
26395
TCGAA
taAAAAAGAAGTAAAATG
26775
GAGAA



GTAA


CCACT




 


370
TACGAGGCCACTCGGTTT
26396
AGTAAT
ACTCTCTTAAGACTACCTT
26776
TCCAA



CTC


TC




 


371
gaGGCCACTCGGTTTCTCA
26397
TCGAA
taAAAAAGAAGTAAAATG
26777
GAGAA



GTAA


CCACT




 


372
TACGAGGCCACTCGGTTT
26398
AGTAAT
ACTCTCTTAAGACTACCTT
26778
TCCAA



CTC


TC




 


375
AATCAGCCAAAATCTTAA
26399
GCTGG
GGAACACGGACACCTCCC
26779
AGCAG



GCT


TAG




 


376
ATTTACACCTTACGAGGC
26400
TCGG
CTTAAGACTACCTTTCTCC
26780
ATGG



CAC


AA




 


377
CTTACGAGGCCACTCGGT
26401
TCAG
CTTAAGACTACCTTTCTCC
26781
ATGG



TTC


AA




 


380
TACGAGGCCACTCGGTTT
26402
CAG
TTAAGACTACCTTTCTCCA
26782
ATG



CT


A




 


381
GAGGCCACTCGGTTTCTC
26403
TAA
ACTCTCTTAAGACTACCTT
26783
CTC



AG


T




 


382
GGAATCAGCCAAAATCTT
26404
GCT
GGAACACGGACACCTCCC
26784
GAG



AA


TA
















TABLE 2B







Exemplary left gRNA spacer and right gRNA spacer pairs


Table 2B provides exemplified second-nick gRNA species for optional use for correcting the


pathogenic R261Q mutation in PAH. The gRNA spacers from Table 1B were filtered, e.g., filtered


by occurrence within 15 nt of the desired editing location and use of a Tier 1 Cas enzyme.


Second-nick gRNAs were generated by searching the opposite strand of DNA in the regions −40 to


−140 (″left″) and +40 to +140 (″right″), relative to the first nick site defined by the first


gRNA, for the PAM utilized by the corresponding Cas variant. One exemplary spacer is shown for


each side of the target nick site.















SEQ


SEQ





ID


ID
Right


ID
Left gRNA spacer
NO
Left PAM
Right gRNA spacer
NO
PAM
















1
gcAGCAGGAAAAGATGGC
26975
TGTGCC
aaGAAGAAAGAAAACTCA
27329
ATCACC



GCTCAT


AAGCTC




 


2
AGGAAAAGATGGCGCTCA
26976
GTG
AAAAGAAGAAAGAAAAC
27330
AAG



TT


TCA




 


3
agcaGGAAAAGATGGCGCT
26977
GTGCCT
gatgAAAAGAAGAAAGAAA
27331
AAAGC



CATT

GG
ACTC




 


4
AGCTACCAGTTGCCAGGC
26978
ATGAG
CTGACTCAGTGGTGATGA
27332
TTGAGT



ACA


GCT




 


5
GCTACCAGTTGCCAGGCA
26979
TGAG
TGACTCAGTGGTGATGAG
27333
TGAG



CAA


CTT




 


8
GTTGCCAGGCACAATGAG
26980
CCA
GGTGATGAGCTTTGAGTT
27334
CTT



CG


TT




 


10
GGAAAAGATGGCGCTCAT
26981
TGC
AAAGAAGAAAGAAAACT
27335
AGC



TG


CAA




 


13
AGCTACCAGTTGCCAGGC
26982
ATGAG
CTGACTCAGTGGTGATGA
27336
TTGAGT



ACA


GCT




 


14
GGAAAAGATGGCGCTCAT
26983
TG
AAAGAAGAAAGAAAACT
27337
AG



TG


CAA




 


17
CAGTTGCCAGGCACAATG
26984
CG
CTCAGTGGTGATGAGCTT
27338
AG



AG


TG




 


21
GAAAAGATGGCGCTCATT
26985
GCC
AAGAAGAAAGAAAACTC
27339
GCT



GT


AAA




 


23
TTGCCAGGCACAATGAGC
26986
CAT
GTGATGAGCTTTGAGTTTT
27340
TTT



GC


C




 


29
ccAGCTACCAGTTGCCAGG
26987
ATGAG
ctCTGACTCAGTGGTGATG
27341
TTGAG



CACA


AGCT




 


30
AGCTACCAGTTGCCAGGC
26988
ATGAG
CTGACTCAGTGGTGATGA
27342
TTGAGT



ACA


GCT




 


33
AAAGATGGCGCTCATTGT
26989
CTG
AAAAGAAGAAAGAAAAC
27343
AAG



GC


TCA




 


34
AAAAGATGGCGCTCATTG
26990
CCT
AGAAGAAAGAAAACTCA
27344
CTC



TG


AAG




 


37
CCAGTTGCCAGGCACAAT
26991
GCG
ACTCAGTGGTGATGAGCT
27345
GAG



GA


TT




 


38
TCCTCCAGCTACCAGTTG
26992
AGG
TCCTAGTGCCTCTGACTCA
27346
TGG



CC


G




 


41
TGCCAGGCACAATGAGCG
26993
ATC
TGATGAGCTTTGAGTTTTC
27347
TTC



CC


T




 


42
CAGTTGCCAGGCACAATG
26994
CG
CTCAGTGGTGATGAGCTT
27348
AG



AG


TG




 


45
ggaaAAGATGGCGCTCATT
26995
CTGGC
aaagAAGAAAGAAAACTCA
27349
TCATC



GTGC


AAGC




 


51
ccAGCTACCAGTTGCCAGG
26996
ATGAG
ctCTGACTCAGTGGTGATG
27350
TTGAG



CACA


AGCT




 


52
AGCTACCAGTTGCCAGGC
26997
ATGAG
AGTTTTCTTTCTTCTTTTC
27351
CCCAG



ACA


AT




 


53
TGTCCTCCAGCTACCAGTT
26998
CAGG
TTCTTTTCATCCCAGCTTG
27352
CTGG



GC


CA




 


54
GCTACCAGTTGCCAGGCA
26999
TGAG
TGACTCAGTGGTGATGAG
27353
TGAG



CAA


CTT




 


55
AAAAGATGGCGCTCATTG
27000
CTGG
TGAAAAGAAGAAAGAAA
27354
AAAG



TGC


ACTC




 


60
CCAGTTGCCAGGCACAAT
27001
GCG
ACTCAGTGGTGATGAGCT
27355
GAG



GA


TT




 


61
GCCAGGCACAATGAGCGC
27002
TCT
GATGAGCTTTGAGTTTTCT
27356
TCT



CA


T




 


62
AAAGATGGCGCTCATTGT
27003
CTG
GAAGAAAGAAAACTCAA
27357
TCA



GC


AGC




 


65
AAGATGGCGCTCATTGTG
27004
GGCAA
GAAAACTCAAAGCTCATC
27358
ACTGA



CCT


ACC




 


66
ATGAGCGCCATCTTTTCCT
27005
TGCAA
AGTTTTCTTTCTTCTTTTC
27359
CCCAG



GC


AT




 


67
AAGATGGCGCTCATTGTG
27006
GGCAA
GAAAACTCAAAGCTCATC
27360
ACTGA



CCT


ACC




 


70
CCAGGCACAATGAGCGCC
27007
CTT
ATGAGCTTTGAGTTTTCTT
27361
CTT



AT


T




 


71
AAGATGGCGCTCATTGTG
27008
TGG
AAGAAAGAAAACTCAAA
27362
CAT



CC


GCT




 


73
AAGATGGCGCTCATTGTG
27009
GGCAA
GAAAACTCAAAGCTCATC
27363
ACTGA



CCT


ACC




 


74
CAGGCACAATGAGCGCCA
27010
TTT
TGAGCTTTGAGTTTTCTTT
27364
TTC



TC


C




 


75
AGATGGCGCTCATTGTGC
27011
GGC
AGAAAGAAAACTCAAAG
27365
ATC



CT


CTC




 


78
AGGCACAATGAGCGCCAT
27012
TTT
GAGCTTTGAGTTTTCTTTC
27366
TCT



CT


T




 


79
GATGGCGCTCATTGTGCC
27013
GCA
GAAAGAAAACTCAAAGCT
27367
TCA



TG


CA




 


81
CAATGAGCGCCATCTTTT
27014
TG
TTCTTTCTTCTTTTCATCC
27368
AG



CC


C




 


85
GGCACAATGAGCGCCATC
27015
TTC
AGCTTTGAGTTTTCTTTCT
27369
CTT



TT


T




 


86
ATGGCGCTCATTGTGCCT
27016
CAA
AAAGAAAACTCAAAGCTC
27370
CAC



GG


AT




 


87
aaagATGGCGCTCATTGTGC
27017
GCAACT
gaagAAAGAAAACTCAAAG
27371
TCACC



CTG

GG
CTCA




 


88
aaagATGGCGCTCATTGTGC
27018
GCAACT
gaagAAAGAAAACTCAAAG
27372
TCACC



CTG

GG
CTCA




 


91
gcAGCAGGAAAAGATGGC
27019
TGTGCC
aaGAAGAAAGAAAACTCA
27373
ATCACC



GCTCAT


AAGCTC




 


94
ACAATGAGCGCCATCTTT
27020
CTG
TTTCTTTCTTCTTTTCATCC
27374
CAG



TC







 


95
GCACAATGAGCGCCATCT
27021
TCC
GCTTTGAGTTTTCTTTCTT
27375
TTT



TT


C




 


96
TGGCGCTCATTGTGCCTG
27022
AAC
AAGAAAACTCAAAGCTCA
27376
ACC



GC


TC




 


97
aggcACAATGAGCGCCATC
27023
CCTGC
tgagCTTTGAGTTTTCTTTCT
27377
TTTTC



TTTT


TC




 


98
aaagATGGCGCTCATTGTGC
27024
GCAACT
agaaAGAAAACTCAAAGCT
27378
ACCAC



CTG

GG
CATC




 


99
aaagATGGCGCTCATTGTGC
27025
GCAACT
agaaAGAAAACTCAAAGCT
27379
ACCAC



CTG

GG
CATC




 


100
aggcACAATGAGCGCCATC
27026
CCTGC
tgagCTTTGAGTTTTCTTTCT
27380
TTTTC



TTTT


TC




 


101
aaagATGGCGCTCATTGTGC
27027
GCAACT
agaaAGAAAACTCAAAGCT
27381
ACCAC



CTG

GG
CATC




 


106
ATGAGCGCCATCTTTTCCT
27028
TGCAA
AGTTTTCTTTCTTCTTTTC
27382
CCCAG



GC


AT




 


107
GAGCGCCATCTTTTCCTGC
27029
CAAG
GTTTTCTTTCTTCTTTTCAT
27383
CCAG



TG


C




 


110
CACAATGAGCGCCATCTT
27030
CCT
CTTTGAGTTTTCTTTCTTC
27384
TTT



TT


T




 


112
GGCGCTCATTGTGCCTGG
27031
ACT
AGAAAACTCAAAGCTCAT
27385
CCA



CA


CA




 


115
ATGAGCGCCATCTTTTCCT
27032
TGCAA
AGTTTTCTTTCTTCTTTTC
27386
CCCAG



GC


AT




 


116
CAATGAGCGCCATCTTTT
27033
TG
TTCTTTCTTCTTTTCATCC
27387
AG



CC


C




 


119
CGCTCATTGTGCCTGGCA
27034
TG
AACTCAAAGCTCATCACC
27388
TG



AC


AC




 


123
ACAATGAGCGCCATCTTT
27035
CTG
TTTGAGTTTTCTTTCTTCT
27389
TTC



TC


T




 


125
GCGCTCATTGTGCCTGGC
27036
CTG
GAAAACTCAAAGCTCATC
27390
CAC



AA


AC




 


132
tgAGCGCCATCTTTTCCTG
27037
AAGAA
ctCTGACTCAGTGGTGATG
27391
TTGAGT



CTGC


AGCT




 


133
ATGAGCGCCATCTTTTCCT
27038
TGCAA
AGTTTTCTTTCTTCTTTTC
27392
CCCAG



GC


AT




 


136
ACAATGAGCGCCATCTTT
27039
CTG
TTTCTTTCTTCTTTTCATCC
27393
CAG



TC







 


137
CTTTTCCTGCTGCAAGAAT
27040
AGG
CTTTTCATCCCAGCTTGCA
27394
TGG



G


C




 


140
CAATGAGCGCCATCTTTT
27041
TGC
TTGAGTTTTCTTTCTTCTT
27395
TCA



CC


T




 


143
CGCTCATTGTGCCTGGCA
27042
TGG
AAACTCAAAGCTCATCAC
27396
CTG



AC


CA




 


144
CGCTCATTGTGCCTGGCA
27043
TGG
GCTCATCACCACTGAGTC
27397
AGG



AC


AG




 


147
CGCTCATTGTGCCTGGCA
27044
TGG
AAAACTCAAAGCTCATCA
27398
ACT



AC


CC




 


148
CAATGAGCGCCATCTTTT
27045
TG
TTCTTTCTTCTTTTCATCC
27399
AG



CC


C




 


151
CGCTCATTGTGCCTGGCA
27046
TG
AACTCAAAGCTCATCACC
27400
TG



AC


AC




 


154
gcgcTCATTGTGCCTGGCAA
27047
GTAGCT
aaaaCTCAAAGCTCATCACC
27401
GAGTCA



CTG

GG
ACT

GA


 


160
gcAGCAGGAAAAGATGGC
27048
TGTGCC
aaGAAGAAAGAAAACTCA
27402
ATCACC



GCTCAT


AAGCTC




 


161
tgAGCGCCATCTTTTCCTG
27049
AAGAA
ctCTGACTCAGTGGTGATG
27403
TTGAGT



CTGC


AGCT




 


162
ATGAGCGCCATCTTTTCCT
27050
TGCAA
AGTTTTCTTTCTTCTTTTC
27404
CCCAG



GC


AT




 


163
ATCTTTTCCTGCTGCAAGA
27051
GAGG
TTCTTTTCATCCCAGCTTG
27405
CTGG



AT


CA




 


164
GAGCGCCATCTTTTCCTGC
27052
CAAG
GTTTTCTTTCTTCTTTTCAT
27406
CCAG



TG


C




 


165
GGCGCTCATTGTGCCTGG
27053
CTGG
AAGCTCATCACCACTGAG
27407
GAGG



CAA


TCA




 


166
GCTCATTGTGCCTGGCAA
27054
GTAG
AAACTCAAAGCTCATCAC
27408
TGAG



CTG


CAC




 


169
ATGAGCGCCATCTTTTCCT
27055
CTG
TTTCTTTCTTCTTTTCATCC
27409
CAG



G







 


170
AATGAGCGCCATCTTTTC
27056
GCT
TGAGTTTTCTTTCTTCTTT
27410
CAT



CT


T




 


173
CGCTCATTGTGCCTGGCA
27057
TGG
AAACTCAAAGCTCATCAC
27411
CTG



AC


CA




 


174
GCTCATTGTGCCTGGCAA
27058
GGT
AAACTCAAAGCTCATCAC
27412
CTG



CT


CA




 


175
gcgcTCATTGTGCCTGGCAA
27059
GTAGCT
aaaaCTCAAAGCTCATCACC
27413
GAGTCA



CTG

GG
ACT

GA


 


176
gcgcTCATTGTGCCTGGCAA
27060
GTAGCT
aaaaCTCAAAGCTCATCACC
27414
GAGTCA



CTG

GG
ACT

GA


 


179
ATGAGCGCCATCTTTTCCT
27061
TGCAA
AGTTTTCTTTCTTCTTTTC
27415
CCCAG



GC


AT




 


180
CGCTCATTGTGCCTGGCA
27062
GGTAG
AAAACTCAAAGCTCATCA
27416
CTGAGT



ACT


CCA




 


181
TGAGCGCCATCTTTTCCTG
27063
TG
TTCTTTCTTCTTTTCATCC
27417
AG



C


C




 


185
ATGAGCGCCATCTTTTCCT
27064
CTG
GAGTTTTCTTTCTTCTTTT
27418
ATC



G


C




 


186
CTCATTGTGCCTGGCAAC
27065
GTA
AACTCAAAGCTCATCACC
27419
TGA



TG


AC




 


189
ATGAGCGCCATCTTTTCCT
27066
CTG
TTTCTTTCTTCTTTTCATCC
27420
CAG



G







 


190
TGAGCGCCATCTTTTCCTG
27067
TGC
AGTTTTCTTTCTTCTTTTC
27421
TCC



C


A




 


191
TCATTGTGCCTGGCAACT
27068
TAG
ACTCAAAGCTCATCACCA
27422
GAG



GG


CT




 


193
GAGCGCCATCTTTTCCTGC
27069
CAAG
GTTTTCTTTCTTCTTTTCAT
27423
CCAG



TG


C




 


194
CATTGTGCCTGGCAACTG
27070
AG
CTCAAAGCTCATCACCAC
27424
AG



GT


TG




 


198
CATTGTGCCTGGCAACTG
27071
AGC
CTCAAAGCTCATCACCAC
27425
AGT



GT


TG




 


199
GAGCGCCATCTTTTCCTGC
27072
GCA
GTTTTCTTTCTTCTTTTCAT
27426
CCC



T







 


203
GAGCGCCATCTTTTCCTGC
27073
CAAGA
AGTTTTCTTTCTTCTTTTC
27427
CCCAG



TG


AT




 


204
GAGCGCCATCTTTTCCTGC
27074
CAAGA
AGTTTTCTTTCTTCTTTTC
27428
CCCAG



TG


AT




 


209
TTGTGCCTGGCAACTGGT
27075
CTG
ACTCAAAGCTCATCACCA
27429
GAG



AG


CT




 


210
TGTGCCTGGCAACTGGTA
27076
TGG
GCTCATCACCACTGAGTC
27430
AGG



GC


AG




 


213
ATTGTGCCTGGCAACTGG
27077
GCT
TCAAAGCTCATCACCACT
27431
GTC



TA


GA




 


214
TGAGCGCCATCTTTTCCTG
27078
TG
TTCTTTCTTCTTTTCATCC
27432
AG



C


C




 


217
CATTGTGCCTGGCAACTG
27079
AG
CTCAAAGCTCATCACCAC
27433
AG



GT


TG




 


221
AGCGCCATCTTTTCCTGCT
27080
CAA
TTTTCTTTCTTCTTTTCATC
27434
CCA



G







 


222
gcgcTCATTGTGCCTGGCAA
27081
GTAGCT
aaaaCTCAAAGCTCATCACC
27435
GAGTCA



CTG

GG
ACT

GA


 


223
gcgcTCATTGTGCCTGGCAA
27082
GTAGCT
aaaaCTCAAAGCTCATCACC
27436
GAGTCA



CTG

GG
ACT

GA


 


226
gcAGCAGGAAAAGATGGC
27083
TGTGCC
aaGAAGAAAGAAAACTCA
27437
ATCACC



GCTCAT


AAGCTC




 


227
ATTGTGCCTGGCAACTGG
27084
CTGG
AAGCTCATCACCACTGAG
27438
GAGG



TAG


TCA




 


228
ATTGTGCCTGGCAACTGG
27085
CTGG
TCAAAGCTCATCACCACT
27439
TCAG



TAG


GAG




 


231
GCGCCATCTTTTCCTGCTG
27086
AAG
TTTCTTTCTTCTTTTCATCC
27440
CAG



C







 


232
GCGCCATCTTTTCCTGCTG
27087
AAG
TTTCTTTCTTCTTTTCATCC
27441
CAG



C







 


235
TTGTGCCTGGCAACTGGT
27088
CTG
AAAGCTCATCACCACTGA
27442
CAG



AG


GT




 


236
TTGTGCCTGGCAACTGGT
27089
CTG
CAAAGCTCATCACCACTG
27443
TCA



AG


AG




 


237
gcgcTCATTGTGCCTGGCAA
27090
GTAGCT
aaaaCTCAAAGCTCATCACC
27444
GAGTCA



CTG

GG
ACT

GA


 


238
gcgcTCATTGTGCCTGGCAA
27091
GTAGCT
aaaaCTCAAAGCTCATCACC
27445
GAGTCA



CTG

GG
ACT

GA


 


239
TTGTGCCTGGCAACTGGT
27092
TGGAG
CAAAGCTCATCACCACTG
27446
CAGAG



AGC


AGT




 


240
CGCCATCTTTTCCTGCTGC
27093
AG
TTCTTTCTTCTTTTCATCC
27447
AG



A


C




 


243
TGTGCCTGGCAACTGGTA
27094
TG
AAGCTCATCACCACTGAG
27448
AG



GC


TC




 


247
CGCCATCTTTTCCTGCTGC
27095
AGA
TTCTTTCTTCTTTTCATCC
27449
AGC



A


C




 


249
TGTGCCTGGCAACTGGTA
27096
TGG
AAAGCTCATCACCACTGA
27450
CAG



GC


GT




 


250
cacaATGAGCGCCATCTTTT
27097
GCTGC
gagtTTTCTTTCTTCTTTTCA
27451
CCAGCT



CCT


TC

TG


 


251
cacaATGAGCGCCATCTTTT
27098
GCTGC
gagtTTTCTTTCTTCTTTTCA
27452
CCAGCT



CCT


TC

TG


 


254
GCGCCATCTTTTCCTGCTG
27099
AAG
TTTCTTTCTTCTTTTCATCC
27453
CAG



C







 


255
GCCATCTTTTCCTGCTGCA
27100
GAA
TCTTTCTTCTTTTCATCCC
27454
GCT



A


A




 


258
TGTGCCTGGCAACTGGTA
27101
TGG
AGCTCATCACCACTGAGT
27455
GAG



GC


CA




 


259
TGTGCCTGGCAACTGGTA
27102
TGG
GCTCATCACCACTGAGTC
27456
AGG



GC


AG




 


262
GTGCCTGGCAACTGGTAG
27103
GGA
AAGCTCATCACCACTGAG
27457
AGA



CT


TC




 


263
GTGCCTGGCAACTGGTAG
27104
GG
AAGCTCATCACCACTGAG
27458
AG



CT


TC




 


267
GTGCCTGGCAACTGGTAG
27105
GAGG
AAGCTCATCACCACTGAG
27459
GAGG



CTG


TCA




 


268
GTGCCTGGCAACTGGTAG
27106
GAGG
AAGCTCATCACCACTGAG
27460
GAGG



CTG


TCA




 


271
TGCCTGGCAACTGGTAGC
27107
GAG
AGCTCATCACCACTGAGT
27461
GAG



TG


CA




 


272
GCCTGGCAACTGGTAGCT
27108
AGG
GCTCATCACCACTGAGTC
27462
AGG



GG


AG




 


275
TGCCTGGCAACTGGTAGC
27109
GAG
AGCTCATCACCACTGAGT
27463
GAG



TG


CA




 


276
CGCCATCTTTTCCTGCTGC
27110
AG
TTCTTTCTTCTTTTCATCC
27464
AG



A


C




 


279
GTGCCTGGCAACTGGTAG
27111
GG
AAGCTCATCACCACTGAG
27465
AG



CT


TC




 


283
CCATCTTTTCCTGCTGCAA
27112
AAT
CTTTCTTCTTTTCATCCCA
27466
CTT



G


G




 


286
ttGTGCCTGGCAACTGGTA
27113
GAGGA
ctCAAAGCTCATCACCACT
27467
CAGAG



GCTG


GAGT




 


287
GTGCCTGGCAACTGGTAG
27114
GAGGA
AAAGCTCATCACCACTGA
27468
AGAGG



CTG


GTC




 


288
ttGTGCCTGGCAACTGGTA
27115
GAGGA
ctCAAAGCTCATCACCACT
27469
CAGAG



GCTG


GAGT




 


289
GTGCCTGGCAACTGGTAG
27116
GAGGA
AAAGCTCATCACCACTGA
27470
AGAGG



CTG


GTC




 


292
GTGCCTGGCAACTGGTAG
27117
GAGG
AAGCTCATCACCACTGAG
27471
GAGG



CTG


TCA




 


293
GTGCCTGGCAACTGGTAG
27118
GAGG
AAGCTCATCACCACTGAG
27472
GAGG



CTG


TCA




 


296
CATCTTTTCCTGCTGCAAG
27119
ATG
TTTCTTCTTTTCATCCCAG
27473
TTG



A


C




 


297
CATCTTTTCCTGCTGCAAG
27120
ATG
TTTCTTCTTTTCATCCCAG
27474
TTG



A


C




 


300
GCCTGGCAACTGGTAGCT
27121
AGG
GCTCATCACCACTGAGTC
27475
AGG



GG


AG




 


301
GCCTGGCAACTGGTAGCT
27122
AGG
GCTCATCACCACTGAGTC
27476
AGG



GG


AG




 


302
CCTGGCAACTGGTAGCTG
27123
GACAGT
CATCACCACTGAGTCAGA
27477
ACTAG



GAG


GGC




 


303
ATCTTTTCCTGCTGCAAGA
27124
TGA
TTCTTCTTTTCATCCCAGC
27478
TGC



A


T




 


304
CCTGGCAACTGGTAGCTG
27125
GGA
CTCATCACCACTGAGTCA
27479
GGC



GA


GA




 


305
TCTTTTCCTGCTGCAAGAA
27126
GAG
TCTTCTTTTCATCCCAGCT
27480
GCA



T


T




 


306
CTGGCAACTGGTAGCTGG
27127
GAC
TCATCACCACTGAGTCAG
2781
GCA



AG


AG




 


308
CTTTTCCTGCTGCAAGAAT
27128
AGG
CTTCTTTTCATCCCAGCTT
27482
CAC



G


G




 


309
TGGCAACTGGTAGCTGGA
27129
ACA
CATCACCACTGAGTCAGA
27483
CAC



GG


GG




 


310
TTTTCCTGCTGCAAGAAT
27130
GG
CTTTTCATCCCAGCTTGCA
27484
TG



GA


C




 


314
TTTTCCTGCTGCAAGAAT
27131
GGT
TTCTTTTCATCCCAGCTTG
27485
ACT



GA


C




 


315
GGCAACTGGTAGCTGGAG
27132
CAG
ATCACCACTGAGTCAGAG
27486
ACT



GA


GC




 


316
tcctGCTGCAAGAATGAGGT
27133
GGTTCA
tcttTCTTCTTTTCATCCCAG
27487
TGCACT



TTG

TT
CT

GG


 


317
tcctGCTGCAAGAATGAGGT
27134
GGTTCA
tcttTCTTCTTTTCATCCCAG
27488
TGCACT



TTG

TT
CT

GG


 


321
CTTTTCCTGCTGCAAGAAT
27135
AGG
TCTTTTCATCCCAGCTTGC
27489
CTG



G


A




 


322
TTTCCTGCTGCAAGAATG
27136
GTT
TCTTTTCATCCCAGCTTGC
27490
CTG



AG


A




 


323
GCAACTGGTAGCTGGAGG
27137
AGT
TCACCACTGAGTCAGAGG
27491
CTA



AC


CA




 


324
ctggCAACTGGTAGCTGGA
27138
AGTAC
caccACTGAGTCAGAGGCA
27492
GAGAC



GGAC


CTAG




 


325
ctggCAACTGGTAGCTGGA
27139
AGTAC
caccACTGAGTCAGAGGCA
27493
GAGAC



GGAC


CTAG




 


327
TTCCTGCTGCAAGAATGA
27140
TTT
CTTTTCATCCCAGCTTGCA
27494
TGG



GG


C




 


329
CAACTGGTAGCTGGAGGA
27141
GTA
CACCACTGAGTCAGAGGC
27495
TAG



CA


AC




 


332
CCTGCTGCAAGAATGAGG
27142
TG
TTTTCATCCCAGCTTGCAC
27496
GG



TT


T




 


335
GCAACTGGTAGCTGGAGG
27143
AG
ACCACTGAGTCAGAGGCA
27497
AG



AC


CT




 


339
TCCTGCTGCAAGAATGAG
27144
TTG
TTTTCATCCCAGCTTGCAC
27498
GGT



GT


T




 


341
AACTGGTAGCTGGAGGAC
27145
TAC
ACCACTGAGTCAGAGGCA
27499
AGG



AG


CT




 


350
CCTGCTGCAAGAATGAGG
27146
TGG
CTTTTCATCCCAGCTTGCA
27500
TGG



TT


C




 


351
CCTGCTGCAAGAATGAGG
27147
TGG
TTTCATCCCAGCTTGCACT
27501
GTT



TT


G




 


354
GTAGCTGGAGGACAGTAC
27148
ACG
ACCACTGAGTCAGAGGCA
27502
AGG



TC


CT




 


355
TAGCTGGAGGACAGTACT
27149
CGG
ACCACTGAGTCAGAGGCA
27503
AGG



CA


CT




 


358
ACTGGTAGCTGGAGGACA
27150
ACT
CCACTGAGTCAGAGGCAC
27504
GGA



GT


TA




 


359
GCAACTGGTAGCTGGAGG
27151
AG
CCACTGAGTCAGAGGCAC
27505
GG



AC


TA
















TABLE 2C







Exemplary left gRNA spacer and right gRNA spacer pairs


Table 2C provides exemplified second-nick gRNA species for optional use for correcting the


pathogenic R243Q mutation in PAH. The gRNA spacers from Table 1C were filtered, e.g., filtered


by occurrence within 15 nt of the desired editing location and use of a Tier 1 Cas enzyme.


Second-nick gRNAs were generated by searching the opposite strand of DNA in the regions −40 to


−140 (″left″) and +40 to +140 (″right″), relative to the first nick site defined by the first


gRNA, for the PAM utilized by the corresponding Cas variant. One exemplary spacer is shown for


each side of the target nick site.















SEQ


SEQ





ID


ID
Right


ID
Left gRNA spacer
NO
Left PAM
Right gRNA spacer
NO
PAM
















3
CACGGTTCGGGGGTATAC
27683
GGG
GAGACCTTTAGGTAGTGG
28053
TAG



AT


AG




 


4
ACGGTTCGGGGGTATACA
27684
GGC
ACCTTTAGGTAGTGGAGT
28054
TAC



TG


AG




 


5
ACTGCACACAGTACATCA
27685
ATGG
TGTGTACTACTCCACTAC
28055
AAGG



GAC


CTA




 


6
CCCATGTATACCCCCGAA
27686
TGAG
ATGTGTACTACTCCACTA
28056
AAAG



CCG


CCT




 


9
ATCCAAGCCCATGTATAC
27687
CCG
GTGTACTACTCCACTACC
28057
AAG



CC


TA




 


10
TGGATCCAAGCCCATGTA
27688
CCC
CAGTTATGTGTACTACTC
28058
CTA



TA


CA




 


11
CGGTTCGGGGGTATACAT
27689
GCT
CCTTTAGGTAGTGGAGTA
28059
ACA



GG


GT




 


12
acatGGATCCAAGCCCATGT
27690
CCCCCG
gggcAGTTATGTGTACTACT
28060
CTACCT



ATA

AA
CCA

AA


 


13
acatGGATCCAAGCCCATGT
27691
CCCCCG
gggcAGTTATGTGTACTACT
28061
CTACCT



ATA

AA
CCA

AA


 


14
GGATCCAAGCCCATGTAT
27692
CCCGA
GTTATGTGTACTACTCCA
28062
CCTAA



ACC


CTA




 


15
CCCATGTATACCCCCGAA
27693
TGAG
ATGTGTACTACTCCACTA
28063
AAAG



CCG


CCT




 


17
TCCAAGCCCATGTATACC
27694
CG
TGTACTACTCCACTACCT
28064
AG



CC


AA




 


21
GGATCCAAGCCCATGTAT
27695
CCC
AGTTATGTGTACTACTCC
28065
TAC



AC


AC




 


22
GGTTCGGGGGTATACATG
27696
CTT
CTTTAGGTAGTGGAGTAG
28066
CAC



GG


TA




 


25
tgGATCCAAGCCCATGTAT
27697
CCGAA
gcCTAGCGTCAAAGCCTAT
28067
CTGGG



ACCC


GTCC




 


26
GGATCCAAGCCCATGTAT
27698
CCCGA
GTTATGTGTACTACTCCA
28068
CCTAA



ACC


CTA




 


29
ATCCAAGCCCATGTATAC
27699
CCG
GTGTACTACTCCACTACC
28069
AAG



CC


TA




 


30
TGCACACAGTACATCAGA
27700
TGG
TGTACTACTCCACTACCT
28070
AGG



CA


AA




 


33
GATCCAAGCCCATGTATA
27701
CCC
GTTATGTGTACTACTCCA
28071
ACC



CC


CT




 


34
TCCAAGCCCATGTATACC
27702
CG
TGTACTACTCCACTACCT
28072
AG



CC


AA




 


37
GTTCGGGGGTATACATGG
27703
TTG
TTTAGGTAGTGGAGTAGT
28073
ACA



GC


AC




 


42
tgGATCCAAGCCCATGTAT
27704
CCGAA
gcCTAGCGTCAAAGCCTAT
28074
CTGGG



ACCC


GTCC




 


43
GATCCAAGCCCATGTATA
27705
CCGAA
GTTATGTGTACTACTCCA
28075
CCTAA



CCC


CTA




 


44
ACTGCACACAGTACATCA
27706
ATGG
TGTGTACTACTCCACTAC
28076
AAGG



GAC


CTA




 


45
CCCATGTATACCCCCGAA
27707
TGAG
ATGTGTACTACTCCACTA
28077
AAAG



CCG


CCT




 


48
ATCCAAGCCCATGTATAC
27708
CCG
GTGTACTACTCCACTACC
28078
AAG



CC


TA




 


49
ATCCAAGCCCATGTATAC
27709
CCG
TTATGTGTACTACTCCACT
28079
CCT



CC


A




 


50
TTCGGGGGTATACATGGG
27710
TGG
TTAGGTAGTGGAGTAGTA
28080
CAT



CT


CA




 


51
ggttCGGGGGTATACATGG
27711
GGATC
ctttAGGTAGTGGAGTAGTA
28081
ATAAC



GCTT


CAC




 


52
ggttCGGGGGTATACATGG
27712
GGATC
ctttAGGTAGTGGAGTAGTA
28082
ATAAC



GCTT


CAC




 


57
cgGTTCGGGGGTATACATG
27713
GGATCC
ttAGGTAGTGGAGTAGTAC
28083
ACTGCC



GGCTT


ACATA




 


58
GATCCAAGCCCATGTATA
27714
CCGAA
TTATGTGTACTACTCCACT
28084
CTAAA



CCC


AC




 


59
TCCAAGCCCATGTATACC
27715
CGA
TATGTGTACTACTCCACT
28085
CTA



CC


AC




 


60
TCGGGGGTATACATGGGC
27716
GGA
TAGGTAGTGGAGTAGTAC
28086
ATA



TT


AC




 


61
ggttCGGGGGTATACATGG
27717
GGATC
ctttAGGTAGTGGAGTAGTA
28087
ATAAC



GCTT


CAC




 


62
ggttCGGGGGTATACATGG
27718
GGATC
ctttAGGTAGTGGAGTAGTA
28088
ATAAC



GCTT


CAC




 


64
GGTTCGGGGGTATACATG
27719
TTGGAT
TTTAGGTAGTGGAGTAGT
28089
CATAA



GGC


ACA




 


65
TCCAAGCCCATGTATACC
27720
CG
TGTACTACTCCACTACCT
28090
AG



CC


AA




 


69
CCAAGCCCATGTATACCC
27721
GAA
ATGTGTACTACTCCACTA
28091
TAA



CC


CC




 


70
CGGGGGTATACATGGGCT
27722
GAT
AGGTAGTGGAGTAGTACA
28092
TAA



TG


CA




 


76
AGCCCATGTATACCCCCG
27723
CCG
GTGTACTACTCCACTACC
28093
AAG



AA


TA




 


77
TGCACACAGTACATCAGA
27724
TGG
TGTACTACTCCACTACCT
28094
AGG



CA


AA




 


80
CAAGCCCATGTATACCCC
27725
AAC
TGTGTACTACTCCACTAC
28095
AAA



CG


CT




 


81
TCCAAGCCCATGTATACC
27726
CG
TGTACTACTCCACTACCT
28096
AG



CC


AA




 


84
GGGGGTATACATGGGCTT
27727
ATC
GGTAGTGGAGTAGTACAC
28097
AAC



GG


AT




 


86
ACTGCACACAGTACATCA
27728
ATGG
TGTGTACTACTCCACTAC
28098
AAGG



GAC


CTA




 


87
CCCATGTATACCCCCGAA
27729
TGAG
TGTGTACTACTCCACTAC
28099
AAGG



CCG


CTA




 


90
AGCCCATGTATACCCCCG
27730
CCG
GTGTACTACTCCACTACC
28100
AAG



AA


TA




 


91
AAGCCCATGTATACCCCC
27731
ACC
GTGTACTACTCCACTACC
28101
AAG



GA


TA




 


92
GGGGTATACATGGGCTTG
27732
TCC
GTAGTGGAGTAGTACACA
28102
ACT



GA


TA




 


93
gttcGGGGGTATACATGGG
27733
GATCCA
taggTAGTGGAGTAGTACA
28103
ACTGC



CTTG

TG
CATA




 


96
cgGTTCGGGGGTATACATG
27734
GGATCC
taGGTAGTGGAGTAGTACA
28104
CTGCCC



GGCTT


CATAA




 


97
aacCGTGAGTACTGTCCTC
27735
ACCAGT
ctaGCGTCAAAGCCTATGT
28105
GGCAGT



CAGCT

T
CCCTG

T


 


98
AGCCCATGTATACCCCCG
27736
CGTGA
ATGTGTACTACTCCACTA
28106
AAAGGT



AAC


CCT




 


99
AGCCCATGTATACCCCCG
27737
CGTGA
ATGTGTACTACTCCACTA
28107
AAAGGT



AAC


CCT




 


102
CCCATGTATACCCCCGAA
27738
TGAG
TGTGTACTACTCCACTAC
28108
AAGG



CCG


CTA




 


103
AGCCCATGTATACCCCCG
27739
CCG
TGTACTACTCCACTACCT
28109
AGG



AA


AA




 


104
GGGTATACATGGGCTTGG
27740
CCA
TAGTGGAGTAGTACACAT
28110
CTG



AT


AA




 


105
ggggTATACATGGGCTTGG
27741
ATGTCT
aggtAGTGGAGTAGTACAC
28111
CTGCCC



ATCC

GA
ATAA

AG


 


106
ggggTATACATGGGCTTGG
27742
ATGTCT
aggtAGTGGAGTAGTACAC
28112
CTGCCC



ATCC

GA
ATAA

AG


 


107
ggggTATACATGGGCTTGG
27743
ATGTCT
aggtAGTGGAGTAGTACAC
28113
CTGCCC



ATCC

GA
ATAA

AG


 


108
AGCCCATGTATACCCCCG
27744
CGTGA
ATGTGTACTACTCCACTA
28114
AAAGGT



AAC


CCT




 


109
TATACATGGGCTTGGATC
27745
TG
AGTGGAGTAGTACACATA
28115
TG



CA


AC




 


112
GCCCATGTATACCCCCGA
27746
CGT
GTACTACTCCACTACCTA
28116
GGT



AC


AA




 


114
GGTATACATGGGCTTGGA
27747
CAT
AGTGGAGTAGTACACATA
28117
TGC



TC


AC




 


121
GTATACATGGGCTTGGAT
27748
ATG
TAGTGGAGTAGTACACAT
28118
CTG



CC


AA




 


122
GTATACATGGGCTTGGAT
27749
ATG
GTGGAGTAGTACACATAA
28119
GCC



CC


CT




 


123
CCCATGTATACCCCCGAA
27750
GTG
TACTACTCCACTACCTAA
28120
GTC



CC


AG




 


124
ggggTATACATGGGCTTGG
27751
ATGTC
ggtaGTGGAGTAGTACACA
28121
TGCCC



ATCC


TAAC




 


126
cgGTTCGGGGGTATACATG
27752
GGATCC
taGGTAGTGGAGTAGTACA
28122
CTGCCC



GGCTT


CATAA




 


127
CCATGTATACCCCCGAAC
27753
TGA
ACTACTCCACTACCTAAA
28123
TCT



CG


GG




 


128
TATACATGGGCTTGGATC
27754
TGT
TGGAGTAGTACACATAAC
28124
CCC



CA


TG




 


129
cccaTGTATACCCCCGAAC
27755
AGTAC
tgtaCTACTCCACTACCTAA
28125
TCTCCT



CGTG


AGG

AG


 


130
cccaTGTATACCCCCGAAC
27756
AGTAC
tgtaCTACTCCACTACCTAA
28126
TCTCCT



CGTG


AGG

AG


 


131
ggggTATACATGGGCTTGG
27757
ATGTC
ggtaGTGGAGTAGTACACA
28127
TGCCC



ATCC


TAAC




 


132
ATGTATACCCCCGAACCG
27758
AG
GTACTACTCCACTACCTA
28128
GG



TG


AA




 


136
CATGTATACCCCCGAACC
27759
GAG
CTACTCCACTACCTAAAG
28129
CTC



GT


GT




 


137
ATACATGGGCTTGGATCC
27760
GTC
GGAGTAGTACACATAACT
28130
CCA



AT


GC




 


141
CATGTATACCCCCGAACC
27761
GAG
TCCACTACCTAAAGGTCT
28131
TAG



GT


CC




 


142
ATGTATACCCCCGAACCG
27762
AGT
TACTCCACTACCTAAAGG
28132
TCC



TG


TC




 


143
TACATGGGCTTGGATCCA
27763
TCT
GAGTAGTACACATAACTG
28133
CAG



TG


CC




 


144
cccaTGTATACCCCCGAAC
27764
AGTAC
tgtaCTACTCCACTACCTAA
28134
TCTCCT



CGTG


AGG

AG


 


145
ggggTATACATGGGCTTGG
27765
ATGTC
ggagTAGTACACATAACTG
28135
GGGAC



ATCC


CCCA




 


148
atGTATACCCCCGAACCGT
27766
CTGTCC
taCTCCACTACCTAAAGGT
28136
AGTGCC



GAGTA


CTCCT




 


149
cgGTTCGGGGGTATACATG
27767
GGATCC
taGGTAGTGGAGTAGTACA
28137
CTGCCC



GGCTT


CATAA




 


150
CCCATGTATACCCCCGAA
27768
TGAG
ACTCCACTACCTAAAGGT
28138
CTAG



CCG


CTC




 


151
TGTATACCCCCGAACCGT
27769
GTA
ACTCCACTACCTAAAGGT
28139
CCT



GA


CT




 


152
ACATGGGCTTGGATCCAT
27770
CTG
AGTAGTACACATAACTGC
28140
AGG



GT


CC




 


153
cccaTGTATACCCCCGAAC
27771
AGTAC
actcCACTACCTAAAGGTCT
28141
AGTGC



CGTG


CCT




 


154
catgGGCTTGGATCCATGTC
27772
TGTACT
ggagTAGTACACATAACTG
28142
GGGAC



TGA

GT
CCCA




 


155
GCCCATGTATACCCCCGA
27773
GTGAGT
TACTCCACTACCTAAAGG
28143
CCTAGT



ACC


TCT




 


156
CATGGGCTTGGATCCATG
27774
TG
GTAGTACACATAACTGCC
28144
GG



TC


CA




 


160
CATGGGCTTGGATCCATG
27775
TGA
GTAGTACACATAACTGCC
28145
GGG



TC


CA




 


161
GTATACCCCCGAACCGTG
27776
TAC
CTCCACTACCTAAAGGTC
28146
CTA



AG


TC




 


167
TGGGCTTGGATCCATGTC
27777
ATG
GTAGTACACATAACTGCC
28147
GGG



TG


CA




 


168
TTCGGGGGTATACATGGG
27778
TGG
GTAGTACACATAACTGCC
28148
GGG



CT


CA




 


171
ATGGGCTTGGATCCATGT
27779
GAT
TAGTACACATAACTGCCC
28149
GGA



CT


AG




 


172
TACCCCCGAACCGTGAGT
27780
TG
CCACTACCTAAAGGTCTC
28150
AG



AC


CT




 


175
CATGGGCTTGGATCCATG
27781
TG
TAGTACACATAACTGCCC
28151
GG



TC


AG




 


179
TATACCCCCGAACCGTGA
27782
ACT
TCCACTACCTAAAGGTCT
28152
TAG



GT


CC




 


183
ATGTCTGATGTACTGTGT
27783
GTGG
GAGTAGTACACATAACTG
28153
AGGG



GCA


CCC




 


184
TCCATGTCTGATGTACTG
27784
GCAG
GAGTAGTACACATAACTG
28154
AGGG



TGT


CCC




 


187
ATACCCCCGAACCGTGAG
27785
CTG
CACTACCTAAAGGTCTCC
28155
GTG



TA


TA




 


188
TCCTCCAGCTACCAGTTG
27786
AGG
TGTACTACTCCACTACCT
28156
AGG



CC


AA




 


191
ATACCCCCGAACCGTGAG
27787
CTG
CCACTACCTAAAGGTCTC
28157
AGT



TA


CT




 


194
TGGGCTTGGATCCATGTC
27788
ATG
GTAGTACACATAACTGCC
28158
GGG



TG


CA




 


195
TGGGCTTGGATCCATGTC
27789
ATG
AGTACACATAACTGCCCA
28159
GAC



TG


GG




 


196
TACCCCCGAACCGTGAGT
27790
TG
CCACTACCTAAAGGTCTC
28160
AG



AC


CT




 


199
tgtaTACCCCCGAACCGTGA
27791
CTGTC
actcCACTACCTAAAGGTCT
28161
AGTGC



GTA


CCT




 


203
atGTATACCCCCGAACCGT
27792
CTGTCC
taCTCCACTACCTAAAGGT
28162
AGTGCC



GAGTA


CTCCT




 


204
aacTGGTAGCTGGAGGACA
27793
CACGGT

28163




GTACT

T





 


205
ATACATGGGCTTGGATCC
27794
TCTGAT
GTACACATAACTGCCCAG
28164
CATAG



ATG


GGA




 


206
ATACATGGGCTTGGATCC
27795
TCTGAT
GTACACATAACTGCCCAG
28165
CATAG



ATG


GGA




 


207
TGTCCTCCAGCTACCAGT
27796
CAGG
TGTGTACTACTCCACTAC
28166
AAGG



TGC


CTA




 


208
CCGAACCGTGAGTACTGT
27797
CCAG
ACTCCACTACCTAAAGGT
28167
CTAG



CCT


CTC




 


211
ATACCCCCGAACCGTGAG
27798
CTG
CACTACCTAAAGGTCTCC
28168
GTG



TA


TA




 


212
TACCCCCGAACCGTGAGT
27799
TGT
CACTACCTAAAGGTCTCC
28169
GTG



AC


TA




 


213
GGGCTTGGATCCATGTCT
27800
TGT
GTACACATAACTGCCCAG
28170
ACA



GA


GG




 


214
gtatACCCCCGAACCGTGAG
27801
TGTCC
ctccACTACCTAAAGGTCTC
28171
GTGCC



TAC


CTA




 


215
gtatACCCCCGAACCGTGAG
27802
TGTCC
ctccACTACCTAAAGGTCTC
28172
GTGCC



TAC


CTA




 


217
CCCGAACCGTGAGTACTG
27803
TCCAG
ACCTAAAGGTCTCCTAGT
28173
TCTGA



TCC


GCC




 


218
CCGAACCGTGAGTACTGT
27804
CCAG
ACTCCACTACCTAAAGGT
28174
CTAG



CCT


CTC




 


219
GGCTTGGATCCATGTCTG
27805
GTA
TACACATAACTGCCCAGG
28175
CAT



AT


GA




 


220
ACCCCCGAACCGTGAGTA
27806
GTC
ACTACCTAAAGGTCTCCT
28176
TGC



CT


AG




 


224
CCCGAACCGTGAGTACTG
27807
TCCAG
ACCTAAAGGTCTCCTAGT
28177
TCTGA



TCC


GCC




 


225
TACCCCCGAACCGTGAGT
27808
TG
ACTACCTAAAGGTCTCCT
28178
TG



AC


AG




 


229
CCCCCGAACCGTGAGTAC
27809
TCC
CTACCTAAAGGTCTCCTA
28179
GCC



TG


GT




 


230
GCTTGGATCCATGTCTGA
27810
TAC
ACACATAACTGCCCAGGG
28180
ATA



TG


AC




 


236
GAACCGTGAGTACTGTCC
27811
CAG
CACTACCTAAAGGTCTCC
28181
GTG



TC


TA




 


237
CCCCGAACCGTGAGTACT
27812
CCT
TACCTAAAGGTCTCCTAG
28182
CCT



GT


TG




 


238
TGGATCCATGTCTGATGT
27813
TG
ACATAACTGCCCAGGGAC
28183
AG



AC


AT




 


242
CTTGGATCCATGTCTGAT
27814
ACT
CACATAACTGCCCAGGGA
28184
TAG



GT


CA




 


243
catgGGCTTGGATCCATGTC
27815
TGTACT
gtacACATAACTGCCCAGG
28185
TAGGCT



TGA

GT
GACA

TT


 


244
catgGGCTTGGATCCATGTC
27816
TGTACT
gtacACATAACTGCCCAGG
28186
TAGGCT



TGA

GT
GACA

TT


 


249
CCGAACCGTGAGTACTGT
27817
CCAG
ACTCCACTACCTAAAGGT
28187
CTAG



CCT


CTC




 


254
TTGGATCCATGTCTGATG
27818
CTG
ACATAACTGCCCAGGGAC
28188
AGG



TA


AT




 


255
TTGGATCCATGTCTGATG
27819
CTG
ACATAACTGCCCAGGGAC
28189
AGG



TA


AT




 


256
CCCGAACCGTGAGTACTG
27820
CTC
ACCTAAAGGTCTCCTAGT
28190
CTC



TC


GC




 


257
taccCCCGAACCGTGAGTA
27821
CCTCC
ccacTACCTAAAGGTCTCCT
28191
GCCTC



CTGT


AGT




 


258
taccCCCGAACCGTGAGTA
27822
CCTCC
ccacTACCTAAAGGTCTCCT
28192
GCCTC



CTGT


AGT




 


260
CCCGAACCGTGAGTACTG
27823
TCCAG
ACCTAAAGGTCTCCTAGT
28193
TCTGA



TCC


GCC




 


261
CCGAACCGTGAGTACTGT
27824
TCC
CCTAAAGGTCTCCTAGTG
28194
TCT



CC


CC




 


263
TGGATCCATGTCTGATGT
27825
TGT
CATAACTGCCCAGGGACA
28195
GGC



AC


TA




 


264
ttggATCCATGTCTGATGTA
27826
TGTGCA
gtacACATAACTGCCCAGG
28196
TAGGCT



CTG

GT
GACA

TT


 


269
CCCGAACCGTGAGTACTG
27827
TCCAG
ACCTAAAGGTCTCCTAGT
28197
TCTGA



TCC


GCC




 


270
AACCGTGAGTACTGTCCT
27828
AG
TAAAGGTCTCCTAGTGCC
28198
TG



CC


TC




 


274
CGAACCGTGAGTACTGTC
27829
CCA
CTAAAGGTCTCCTAGTGC
28199
CTG



CT


CT




 


275
GGATCCATGTCTGATGTA
27830
GTG
ATAACTGCCCAGGGACAT
28200
GCT



CT


AG




 


281
aaGCCCATGTATACCCCCG
27831
GTGAGT
ctAGTGCCTCTGACTCAGT
28201
ATGAG



AACC


GGTG




 


282
CCCGAACCGTGAGTACTG
27832
TCCAG
ACCTAAAGGTCTCCTAGT
28202
TCTGA



TCC


GCC




 


285
GAACCGTGAGTACTGTCC
27833
CAG
CTAAAGGTCTCCTAGTGC
28203
CTG



TC


CT




 


286
TCCTCCAGCTACCAGTTG
27834
AGG
TCCTAGTGCCTCTGACTC
28204
TGG



CC


AG




 


289
GAACCGTGAGTACTGTCC
27835
CAG
TAAAGGTCTCCTAGTGCC
28205
TGA



TC


TC




 


290
AACCGTGAGTACTGTCCT
27836
AG
TAAAGGTCTCCTAGTGCC
28206
TG



CC


TC




 


293
GATCCATGTCTGATGTAC
27837
TG
CATAACTGCCCAGGGACA
28207
GG



TG


TA




 


297
GATCCATGTCTGATGTAC
27838
TGT
TAACTGCCCAGGGACATA
28208
CTT



TG


GG




 


303
aaGCCCATGTATACCCCCG
27839
GTGAGT
ctAGTGCCTCTGACTCAGT
28209
ATGAG



AACC


GGTG




 


304
CCCGAACCGTGAGTACTG
27840
TCCAG
AAGGTCTCCTAGTGCCTC
28210
CTCAG



TCC


TGA




 


305
TGTCCTCCAGCTACCAGT
27841
CAGG
TCTCCTAGTGCCTCTGACT
28211
GTGG



TGC


CA




 


306
CCGAACCGTGAGTACTGT
27842
CCAG
AGGTCTCCTAGTGCCTCT
28212
TCAG



CCT


GAC




 


309
GAACCGTGAGTACTGTCC
27843
CAG
CTAAAGGTCTCCTAGTGC
28213
CTG



TC


CT




 


310
AACCGTGAGTACTGTCCT
27844
AGC
AAAGGTCTCCTAGTGCCT
28214
GAC



CC


CT




 


313
ATCCATGTCTGATGTACT
27845
GTG
ACTGCCCAGGGACATAGG
28215
TTG



GT


CT




 


314
ATCCATGTCTGATGTACT
27846
GTG
AACTGCCCAGGGACATAG
28216
TTT



GT


GC




 


315
AGCGCCATCTTTTCCTGCT
27847
CAAGAA

28217




G

T





 


319
CGTGAGTACTGTCCTCCA
27848
ACCAGT
AAGGTCTCCTAGTGCCTC
28218
CTCAG



GCT


TGA




 


320
CCGAACCGTGAGTACTGT
27849
CCAG
AGGTCTCCTAGTGCCTCT
28219
TCAG



CCT


GAC




 


322
TCCATGTCTGATGTACTG
27850
GCAG
ACACATAACTGCCCAGGG
28220
TAGG



TGT


ACA




 


323
AACCGTGAGTACTGTCCT
27851
AG
TAAAGGTCTCCTAGTGCC
28221
TG



CC


TC




 


327
ACCGTGAGTACTGTCCTC
27852
GCT
AAGGTCTCCTAGTGCCTC
28222
ACT



CA


TG




 


328
TCCATGTCTGATGTACTG
27853
TGC
ACTGCCCAGGGACATAGG
28223
TTG



TG


CT




 


329
AGCGCCATCTTTTCCTGCT
27854
CAAGAA

28224




G

T





 


330
ttggATCCATGTCTGATGTA
27855
TGTGCA
ataaCTGCCCAGGGACATA
28225
TTGAC



CTG

GT
GGCT




 


331
ttggATCCATGTCTGATGTA
27856
TGTGCA
ataaCTGCCCAGGGACATA
28226
TTGAC



CTG

GT
GGCT




 


336
aaGCCCATGTATACCCCCG
27857
GTGAGT
ctAGTGCCTCTGACTCAGT
28227
ATGAG



AACC


GGTG




 


337
CGTGAGTACTGTCCTCCA
27858
ACCAGT
AAGGTCTCCTAGTGCCTC
28228
CTCAG



GCT


TGA




 


338
ATCCATGTCTGATGTACT
27859
TGCAGT
TAACTGCCCAGGGACATA
28229
TTTGA



GTG


GGC




 


341
GAACCGTGAGTACTGTCC
27860
CAG
GTCTCCTAGTGCCTCTGA
28230
CAG



TC


CT




 


342
CCGTGAGTACTGTCCTCC
27861
CTA
AGGTCTCCTAGTGCCTCT
28231
CTC



AG


GA




 


343
CCATGTCTGATGTACTGT
27862
GCA
CTGCCCAGGGACATAGGC
28232
TGA



GT


TT




 


350
CGTGAGTACTGTCCTCCA
27863
ACCAGT
AAGGTCTCCTAGTGCCTC
28233
CTCAG



GCT


TGA




 


351
GTGAGTACTGTCCTCCAG
27864
CCAG
AGGTCTCCTAGTGCCTCT
28234
TCAG



CTA


GAC




 


353
AACCGTGAGTACTGTCCT
27865
AG
TCTCCTAGTGCCTCTGACT
28235
AG



CC


C




 


357
CGTGAGTACTGTCCTCCA
27866
TAC
GGTCTCCTAGTGCCTCTG
28236
TCA



GC


AC




 


358
CATGTCTGATGTACTGTG
27867
CAG
TGCCCAGGGACATAGGCT
28237
GAC



TG


TT
















TABLE 2D







Exemplary left gRNA spacer and right gRNA spacer pairs


Table 2D provides exemplified second-nick gRNA species for optional use for correcting the


pathogenic IVS10-11G > A mutation in PAH. The gRNA spacers from Table 1D were filtered, e.g.,


filtered by occurrence within 15 nt of the desired editing location and use of a Tier 1 Cas


enzyme. Second-nick gRNAs were generated by searching the opposite strand of DNA in the regions


−40 to −140 (″left″) and +40 to +140 (″right″), relative to the first nick site defined by the


first gRNA, for the PAM utilized by the corresponding Cas variant. One exemplary spacer is


shown for each side of the target nick site.















SEQ


SEQ





ID


ID
Right


ID
Left gRNA spacer
NO
Left PAM
Right gRNA spacer
NO
PAM
















1
TCTCTGCCACGTAATAGA
28423
GGC
TCACCCCGATTCCTTCTAC
28721
TCA



GG


A




 


2
GGAGTTCCAGCCCCTCTA
28424
CGTGG
GCATTTGGGCTGTGATGTA
28722
AGGAAT



TTA


GA




 


3
TCTCTGCCACGTAATAGA
28425
GG
CCCGATTCCTTCTACATCA
28723
AG



GG


C




 


6
GAGTTCCAGCCCCTCTAT
28426
CG
ATTTGGGCTGTGATGTAGA
28724
GG



TA


A




 


10
CTCTGCCACGTAATAGAG
28427
GCT
CACCCCGATTCCTTCTACA
28725
CAC



GG


T




 


12
GGAGTTCCAGCCCCTCTA
28428
ACG
TTTGGGCTGTGATGTAGAA
28726
GAA



TT


G




 


16
TCTGCCACGTAATAGAGG
28429
CTG
CCCCGATTCCTTCTACATC
28727
CAG



GG


A




 


17
CTGCCACGTAATAGAGG
28430
TGG
CAAATTACTTTGCACATAC
28728
AGG



GGC


T




 


20
TCTGCCACGTAATAGAGG
28431
CTG
ACCCCGATTCCTTCTACAT
28729
ACA



GG


C




 


23
GGAGTTCCAGCCCCTCTA
28432
ACG
GGGCTGTGATGTAGAAGG
28730
TCG



TT


AA




 


24
GAGTTCCAGCCCCTCTAT
28433
CGT
TTGGGCTGTGATGTAGAA
28731
AAT



TA


GG




 


25
CTGCCACGTAATAGAGG
28434
TG
CCCGATTCCTTCTACATCA
28732
AG



GGC


C




 


28
ctctGCCACGTAATAGAGG
28435
GGAAC
ctcaCCCCGATTCCTTCTACA
28733
ACAGC



GGCT


TC




 


31
tcTGCCACGTAATAGAGG
28436
AACTCC
ctCACCCCGATTCCTTCTAC
28734
CAGCCC



GGCTGG


ATCA




 


32
CTCTGCCACGTAATAGAG
28437
CTGG
GCCAAATTACTTTGCACAT
28735
TAGG



GGG


AC




 


33
CTCTGCCACGTAATAGAG
28438
CTGG
CACCCCGATTCCTTCTACA
28736
ACAG



GGG


TC




 


34
GAGTTCCAGCCCCTCTAT
28439
GTGG
TGGGCTGTGATGTAGAAG
28737
TCGG



TAC


GAA




 


39
CTGCCACGTAATAGAGG
28440
TGG
CCCCGATTCCTTCTACATC
28738
CAG



GGC


A




 


40
CTGCCACGTAATAGAGG
28441
TGG
CAAATTACTTTGCACATAC
28739
AGG



GGC


T




 


43
CTGCCACGTAATAGAGG
28442
TGG
CCCCGATTCCTTCTACATC
28740
CAG



GGC


A




 


44
CTGCCACGTAATAGAGG
28443
TG
CCCGATTCCTTCTACATCA
28741
AG



GGC


C




 


47
AGTTCCAGCCCCTCTATT
28444
GTG
TGGGCTGTGATGTAGAAG
28742
ATC



AC


GA




 


48
ctctGCCACGTAATAGAGG
28445
GGAAC
tcacCCCGATTCCTTCTACAT
28743
CAGCCC



GGCT


CA

AA


 


49
ctctGCCACGTAATAGAGG
28446
GGAAC
tcacCCCGATTCCTTCTACAT
28744
CAGCCC



GGCT


CA

AA


 


52
tcTCTGCCACGTAATAGAG
28447
TGGAA
ttCTACATCACAGCCCAAAT
28745
GTGAG



GGGC


GCT




 


53
TCTGCCACGTAATAGAGG
28448
TGGAA
CCGATTCCTTCTACATCAC
28746
CCCAA



GGC


AG




 


54
GTTCCAGCCCCTCTATTA
28449
GGCAG
TGGGCTGTGATGTAGAAG
28747
TCGGG



CGT


GAA




 


55
GTTCCAGCCCCTCTATTA
28450
GGCAG
TGGGCTGTGATGTAGAAG
28748
TCGGG



CGT


GAA




 


58
CTCTGCCACGTAATAGAG
28451
CTGG
GCCAAATTACTTTGCACAT
28749
TAGG



GGG


AC




 


59
CTCTGCCACGTAATAGAG
28452
CTGG
CACCCCGATTCCTTCTACA
28750
ACAG



GGG


TC




 


62
CTGCCACGTAATAGAGG
28453
TGG
CCCCGATTCCTTCTACATC
28751
CAG



GGC


A




 


63
TGCCACGTAATAGAGGG
28454
GGA
CCCGATTCCTTCTACATCA
28752
AGC



GCT


C




 


64
GTTCCAGCCCCTCTATTA
28455
TGG
GGGCTGTGATGTAGAAGG
28753
TCG



CG


AA




 


67
GTTCCAGCCCCTCTATTA
28456
GGCAG
GGGCTGTGATGTAGAAGG
28754
CGGGG



CGT


AAT




 


68
TCTGCCACGTAATAGAGG
28457
TGGAA
CCGATTCCTTCTACATCAC
28755
CCCAA



GGC


AG




 


69
CTCTGCCACGTAATAGAG
28458
CTGG
CACCCCGATTCCTTCTACA
28756
ACAG



GGG


TC




 


70
TTCCAGCCCCTCTATTAC
28459
GGC
GGCTGTGATGTAGAAGGA
28757
CGG



GT


AT




 


71
GCCACGTAATAGAGGGG
28460
GAA
CCGATTCCTTCTACATCAC
28758
GCC



CTG


A




 


74
TCTGCCACGTAATAGAGG
28461
TGGAA
CCGATTCCTTCTACATCAC
28759
CCCAA



GGC


AG




 


75
TCCAGCCCCTCTATTACG
28462
GCA
GCTGTGATGTAGAAGGAA
28760
GGG



TG


TC




 


76
CCACGTAATAGAGGGGC
28463
AAC
CGATTCCTTCTACATCACA
28761
CCC



TGG


G




 


77
CCAGCCCCTCTATTACGT
28464
CAG
CTGTGATGTAGAAGGAAT
28762
GGG



GG


CG




 


78
CACGTAATAGAGGGGCT
28465
ACT
GATTCCTTCTACATCACAG
28763
CCA



GGA


C




 


79
CAGCCCCTCTATTACGTG
28466
AG
TGTGATGTAGAAGGAATC
28764
GG



GC


GG




 


83
CAGCCCCTCTATTACGTG
28467
AGA
TGTGATGTAGAAGGAATC
28765
GGT



GC


GG




 


84
ACGTAATAGAGGGGCTG
28468
CTC
ATTCCTTCTACATCACAGC
28766
CAA



GAA


C




 


85
cggaGTTCCAGCCCCTCTA
28469
GTGGC
gcatTTGGGCTGTGATGTAG
28767
GAATCG



TTAC


AAG

GG


 


86
cggaGTTCCAGCCCCTCTA
28470
GTGGC
gcatTTGGGCTGTGATGTAG
28768
GAATCG



TTAC


AAG

GG


 


87
cggaGTTCCAGCCCCTCTA
28471
GTGGC
gcatTTGGGCTGTGATGTAG
28769
GAATCG



TTAC


AAG

GG


 


90
taTTACGTGGCAGAGAGTT
28472
GATGCC
ggTGAGATGAGAGAAGGG
28770
ATGGCC



TTAAT


GCACAA




 


93
AGCCCCTCTATTACGTGG
28473
GAG
GTGATGTAGAAGGAATCG
28771
GTG



CA


GG




 


94
GTTCCAGCCCCTCTATTA
28474
TGG
CTGTGATGTAGAAGGAAT
28772
GGG



CG


CG




 


97
AGCCCCTCTATTACGTGG
28475
GAG
GTGATGTAGAAGGAATCG
28773
GTG



CA


GG




 


98
GCCCCTCTATTACGTGGC
28476
AG
TGTGATGTAGAAGGAATC
28774
GG



AG


GG




 


101
CGTAATAGAGGGGCTGG
28477
TCC
TTCCTTCTACATCACAGCC
28775
AAA



AAC


C




 


102
cggaGTTCCAGCCCCTCTA
28478
GTGGCA
gcatTTGGGCTGTGATGTAG
28776
GAATCG



TTAC

GA
AAG

GG


 


103
cggaGTTCCAGCCCCTCTA
28479
GTGGCA
gcatTTGGGCTGTGATGTAG
28777
GAATCG



TTAC

GA
AAG

GG


 


106
taTTACGTGGCAGAGAGTT
28480
GATGCC
ggTGAGATGAGAGAAGGG
28778
ATGGCC



TTAAT


GCACAA




 


107
GAGTTCCAGCCCCTCTAT
28481
GTGG
GGCTGTGATGTAGAAGGA
28779
GGGG



TAC


ATC




 


108
AGCCCCTCTATTACGTGG
28482
AGAG
GTGATGTAGAAGGAATCG
28780
TGAG



CAG


GGG




 


111
CCCCTCTATTACGTGGCA
28483
GAG
GTGATGTAGAAGGAATCG
28781
GTG



GA


GG




 


112
GCCCCTCTATTACGTGGC
28484
AGA
TGATGTAGAAGGAATCGG
28782
TGA



AG


GG




 


113
GTAATAGAGGGGCTGGA
28485
CCG
TCCTTCTACATCACAGCCC
28783
AAT



ACT


A




 


114
cgtaATAGAGGGGCTGGAA
28486
GTGAC
gattCCTTCTACATCACAGCC
28784
AATGCT



CTCC


CA

GT


 


115
cggaGTTCCAGCCCCTCTA
28487
GTGGCA
gcatTTGGGCTGTGATGTAG
28785
GAATCG



TTAC

GA
AAG

GG


 


116
cgtaATAGAGGGGCTGGAA
28488
GTGAC
gattCCTTCTACATCACAGCC
28786
AATGCT



CTCC


CA

GT


 


119
CAGCCCCTCTATTACGTG
28489
GAGAGT
GTGATGTAGAAGGAATCG
28787
TGAGAT



GCA


GGG




 


120
AGCCCCTCTATTACGTGG
28490
AGAG
GTGATGTAGAAGGAATCG
28788
TGAG



CAG


GGG




 


121
CCCCTCTATTACGTGGCA
28491
GAG
GATGTAGAAGGAATCGGG
28789
GAG



GA


GT




 


122
TAATAGAGGGGCTGGAA
28492
CGT
CCTTCTACATCACAGCCCA
28790
ATG



CTC


A




 


125
CTCTATTACGTGGCAGAG
28493
TTTAA
ATGTAGAAGGAATCGGGG
28791
GATGA



AGT


TGA




 


126
CCCTCTATTACGTGGCAG
28494
AG
ATGTAGAAGGAATCGGGG
28792
AG



AG


TG




 


130
CCCTCTATTACGTGGCAG
28495
AGT
ATGTAGAAGGAATCGGGG
28793
AGA



AG


TG




 


131
AATAGAGGGGCTGGAAC
28496
GTG
CTTCTACATCACAGCCCAA
28794
TGC



TCC


A




 


132
cgtaATAGAGGGGCTGGAA
28497
GTGAC
gattCCTTCTACATCACAGCC
28795
AATGCT



CTCC


CA

GT


 


136
CCCCTCTATTACGTGGCA
28498
GAG
GTAGAAGGAATCGGGGTG
28796
ATG



GA


AG




 


137
CCTCTATTACGTGGCAGA
28499
GTT
TGTAGAAGGAATCGGGGT
28797
GAT



GA


GA




 


138
ATAGAGGGGCTGGAACT
28500
TGA
TTCTACATCACAGCCCAAA
28798
GCT



CCG


T




 


139
CTCTATTACGTGGCAGAG
28501
TTT
GTAGAAGGAATCGGGGTG
28799
ATG



AG


AG




 


140
TAGAGGGGCTGGAACTC
28502
GAC
TCTACATCACAGCCCAAAT
28800
CTG



CGT


G




 


141
TCTATTACGTGGCAGAGA
28503
TTT
TAGAAGGAATCGGGGTGA
28801
TGA



GT


GA




 


142
AGAGGGGCTGGAACTCC
28504
ACA
CTACATCACAGCCCAAAT
28802
TGT



GTG


GC




 


145
GAGGGGCTGGAACTCCG
28505
CAG
TACATCACAGCCCAAATG
28803
GTG



TGA


CT




 


146
CTATTACGTGGCAGAGAG
28506
TTA
AGAAGGAATCGGGGTGAG
28804
GAG



TT


AT




 


150
TACGTGGCAGAGAGTTTT
28507
TG
GAAGGAATCGGGGTGAGA
28805
AG



AA


TG




 


154
TATTACGTGGCAGAGAGT
28508
TAA
GAAGGAATCGGGGTGAGA
28806
AGA



TT


TG




 


155
AGGGGCTGGAACTCCGT
28509
AGT
ACATCACAGCCCAAATGC
28807
TGA



GAC


TG




 


156
attaCGTGGCAGAGAGTTTT
28510
GATGCC
ggaaTCGGGGTGAGATGAG
28808
GGGGC



AAT

AA
AGAA




 


157
cgtaATAGAGGGGCTGGAA
28511
GTGAC
tctaCATCACAGCCCAAATG
28809
TGAGCC



CTCC


CTG

AA


 


158
attaCGTGGCAGAGAGTTTT
28512
GATGCC
ggaaTCGGGGTGAGATGAG
28810
GGGGC



AAT

AA
AGAA




 


159
cgtaATAGAGGGGCTGGAA
28513
GTGAC
tctaCATCACAGCCCAAATG
28811
TGAGCC



CTCC


CTG

AA


 


164
GGGGCTGGAACTCCGTG
28514
TGTAA
ATCACAGCCCAAATGCTG
28812
GCCAA



ACAG


TGA




 


167
TTACGTGGCAGAGAGTTT
28515
ATG
AAGGAATCGGGGTGAGAT
28813
GAG



TA


GA




 


168
ATTACGTGGCAGAGAGTT
28516
AAT
AAGGAATCGGGGTGAGAT
28814
GAG



TT


GA




 


169
GGGGCTGGAACTCCGTG
28517
GTG
CATCACAGCCCAAATGCT
28815
GAG



ACA


GT




 


173
GGCAGAGAGTTTTAATGA
28518
CAAG
AGGAATCGGGGTGAGATG
28816
GAAG



TGC


AGA




 


174
GGGCTGGAACTCCGTGAC
28519
TGT
ATCACAGCCCAAATGCTG
28817
AGC



AG


TG




 


175
TTACGTGGCAGAGAGTTT
28520
ATG
AGGAATCGGGGTGAGATG
28818
AGA



TA


AG




 


179
TATTACGTGGCAGAGAGT
28521
AATGA
AGGAATCGGGGTGAGATG
28819
GAAGG



TTT


AGA




 


180
TATTACGTGGCAGAGAGT
28522
AATGA
AGGAATCGGGGTGAGATG
28820
GAAGG



TTT


AGA




 


183
TACGTGGCAGAGAGTTTT
28523
TG
AGGAATCGGGGTGAGATG
28821
AG



AA


AG




 


187
TACGTGGCAGAGAGTTTT
28524
TGA
GGAATCGGGGTGAGATGA
28822
GAA



AA


GA




 


188
GGCTGGAACTCCGTGACA
28525
GTA
TCACAGCCCAAATGCTGT
28823
GCC



GT


GA




 


191
GGGGCTGGAACTCCGTG
28526
TGTAA
TCACAGCCCAAATGCTGT
28824
CCAAA



ACAG


GAG




 


192
GGGGCTGGAACTCCGTG
28527
TGTAA
TCACAGCCCAAATGCTGT
28825
CCAAA



ACAG


GAG




 


195
CGTGGCAGAGAGTTTTAA
28528
ATG
GAATCGGGGTGAGATGAG
28826
AAG



TG


AG




 


196
ACGTGGCAGAGAGTTTTA
28529
GAT
GAATCGGGGTGAGATGAG
28827
AAG



AT


AG




 


197
GCTGGAACTCCGTGACAG
28530
TAA
CACAGCCCAAATGCTGTG
28828
CCA



TG


AG




 


201
GGCAGAGAGTTTTAATGA
28531
CAAG
GAATCGGGGTGAGATGAG
28829
AGGG



TGC


AGA




 


204
GGGCTGGAACTCCGTGAC
28532
TG
ATCACAGCCCAAATGCTG
28830
AG



AG


TG




 


207
CGTGGCAGAGAGTTTTAA
28533
ATG
AATCGGGGTGAGATGAGA
28831
AGG



TG


GA




 


209
CTGGAACTCCGTGACAGT
28534
AAT
ACAGCCCAAATGCTGTGA
28832
CAA



GT


GC




 


210
ttacGTGGCAGAGAGTTTTA
28535
ATGCCA
ggaaTCGGGGTGAGATGAG
28833
GGGGC



ATG

AG
AGAA




 


211
ttacGTGGCAGAGAGTTTTA
28536
ATGCCA
ggaaTCGGGGTGAGATGAG
28834
GGGGC



ATG

AG
AGAA




 


215
GTGGCAGAGAGTTTTAAT
28537
GCCAA
GAATCGGGGTGAGATGAG
28835
AGGGG



GAT


AGA




 


218
AACTCCGTGACAGTGTAA
28538
TTG
CATCACAGCCCAAATGCT
28836
GAG



TT


GT




 


219
TGGAACTCCGTGACAGTG
28539
ATT
CAGCCCAAATGCTGTGAG
28837
AAA



TA


CC




 


220
GTGGCAGAGAGTTTTAAT
28540
TGC
ATCGGGGTGAGATGAGAG
28838
GGG



GA


AA




 


223
GTGGCAGAGAGTTTTAAT
28541
GCCAA
GAATCGGGGTGAGATGAG
28839
AGGGG



GAT


AGA




 


224
GGAACTCCGTGACAGTGT
28542
TTTGG
TCACAGCCCAAATGCTGT
28840
CCAAAT



AAT


GAG




 


225
GGAACTCCGTGACAGTGT
28543
TTTGG
TCACAGCCCAAATGCTGT
28841
CCAAAT



AAT


GAG




 


226
ACTCCGTGACAGTGTAAT
28544
TG
ATCACAGCCCAAATGCTG
28842
AG



TT


TG




 


229
TGGCAGAGAGTTTTAATG
28545
GCC
TCGGGGTGAGATGAGAGA
28843
GGG



AT


AG




 


232
GGAACTCCGTGACAGTGT
28546
TTT
AGCCCAAATGCTGTGAGC
28844
AAT



AA


CA




 


236
AACTCCGTGACAGTGTAA
28547
TTG
ATGCTGTGAGCCAAATTA
28845
TTG



TT


CT




 


237
GAACTCCGTGACAGTGTA
28548
TTT
GCCCAAATGCTGTGAGCC
28846
ATT



AT


AA




 


238
GTGGCAGAGAGTTTTAAT
28549
TG
CGGGGTGAGATGAGAGAA
28847
GG



GA


GG




 


242
GGCAGAGAGTTTTAATGA
28550
CCA
CGGGGTGAGATGAGAGAA
28848
GGC



TG


GG




 


247
GAACTCCGTGACAGTGTA
28551
TTGG
TACATCACAGCCCAAATG
28849
TGAG



ATT


CTG




 


250
CAGAGAGTTTTAATGATG
28552
AAG
TCGGGGTGAGATGAGAGA
28850
GGG



CC


AG




 


251
GCAGAGAGTTTTAATGAT
28553
CAA
GGGGTGAGATGAGAGAAG
28851
GCA



GC


GG




 


252
AACTCCGTGACAGTGTAA
28554
TTG
CCCAAATGCTGTGAGCCA
28852
TTA



TT


AA




 


255
GCAGAGAGTTTTAATGAT
28555
AAGGA
GGGGTGAGATGAGAGAAG
28853
CACAA



GCC


GGG




 


256
GAACTCCGTGACAGTGTA
28556
TTGGA
TCACAGCCCAAATGCTGT
28854
CCAAAT



ATT


GAG




 


257
GCAGAGAGTTTTAATGAT
28557
AAGGA
GGGGTGAGATGAGAGAAG
28855
CACAA



GCC


GGG




 


258
GAACTCCGTGACAGTGTA
28558
TTGGA
TCACAGCCCAAATGCTGT
28856
CCAAAT



ATT


GAG




 


261
ACTCCGTGACAGTGTAAT
28559
TGG
CCAAATGCTGTGAGCCAA
28857
TAC



TT


AT




 


262
CAGAGAGTTTTAATGATG
28560
AAG
GGGTGAGATGAGAGAAGG
28858
CAC



CC


GG




 


264
CTCCGTGACAGTGTAATT
28561
GGA
CAAATGCTGTGAGCCAAA
28859
ACT



TT


TT




 


265
AGAGAGTTTTAATGATGC
28562
AGG
GGTGAGATGAGAGAAGGG
28860
ACA



CA


GC




 


269
GAGAGTTTTAATGATGCC
28563
GGA
GTGAGATGAGAGAAGGGG
28861
CAA



AA


CA




 


270
TCCGTGACAGTGTAATTT
28564
GAT
AAATGCTGTGAGCCAAAT
28862
CTT



TG


TA




 


271
actcCGTGACAGTGTAATTT
28565
ATGGC
ccaaATGCTGTGAGCCAAAT
28863
TTTGC



TGG


TAC




 


272
actcCGTGACAGTGTAATTT
28566
ATGGC
ccaaATGCTGTGAGCCAAAT
28864
TTTGC



TGG


TAC




 


273
CTCCGTGACAGTGTAATT
28567
GATGG
TCACAGCCCAAATGCTGT
28865
CCAAAT



TTG


GAG




 


274
AGAGTTTTAATGATGCCA
28568
GAG
TGAGATGAGAGAAGGGGC
28866
AAA



AG


AC




 


275
CCGTGACAGTGTAATTTT
28569
ATG
AATGCTGTGAGCCAAATT
28867
TTT



GG


AC




 


276
ttacGTGGCAGAGAGTTTTA
28570
ATGCCA
gtgaGATGAGAGAAGGGGC
28868
ATGGCC



ATG

AG
ACAA

TA


 


277
ttacGTGGCAGAGAGTTTTA
28571
ATGCCA
gtgaGATGAGAGAAGGGGC
28869
ATGGCC



ATG

AG
ACAA

TA









Capital letters indicate “core nucleotides” while lower case letters indicate “flanking nucleotides.” Herein, when an RNA sequence (e.g., a gRNA to produce a second nick) is said to comprise a particular sequence (e.g., a sequence of Table 2A, Table 2B, Table 2C, or Table 2D or a portion thereof) that comprises thymine (T), it is of course understood that the RNA sequence may (and frequently does) comprise uracil (U) in place of T. For instance, the RNA sequence may comprise U at every position shown as T in the sequence in Table 2A, Table 2B, Table 2C, or Table 2D. More specifically, the present disclosure provides an RNA sequence according to every gRNA spacer sequence shown in Table 2A, Table 2B, Table 2C, or Table 2D, wherein the RNA sequence has a U in place of each T in the sequence in Table 2A, Table 2B, Table 2C, or Table 2D.
In some embodiments, the systems and methods provided herein may comprise a template sequence listed in Table 4A, Table 4B, Table 4C, or Table 4D. Table 4A, Table 4B, Table 4C, or Table 4D provides exemplary template RNA sequences (column 4) and optional second-nick gRNA spacer sequences (column 5) designed to be paired with a gene modifying polypeptide to correct a mutation in the PAH gene. The templates in Table 4A, Table 4B, Table 4C, or Table 4D are meant to exemplify the total sequence of: (1) gRNA spacer (e.g., for targeting for first strand nick), (2) gRNA scaffold, (3) heterologous object sequence, and (4) PBS sequence (e.g., for initiating TPRT at first strand nick).







TABLE 4A







Exemplary template RNA sequences and second nick gRNA spacer sequences


Table 4A provides design of RNA components of gene modifying systems for correcting the pathogenic R408W, mutation in PAH. The gRNA


spacers from Table 1A were filtered, e.g., filtered by occurrence within 15 nt of the desired editing location and use of a Tier 1 Cas


enzyme. For each gRNA ID, this table details the sequence of a complete template RNA, optional second-nick gRNA, and Cas variant for use in


a Cas-RT fusion gene modifying polypeptide. For exemplification, PBS sequences and post-edit homology regions (after the location of the


edit) are set to 12 nt and 30 nt, respectively. Additionally, a second-nick gRNA is selected with preference for a distance near 100 nt from


the first nick and a first preference for a design resulting in a PAM-in system, as described elsewhere in this application.

















SEQ

SEQ



Cas


ID

ID


ID
species
strand
Template RNA
NO
second-nick gRNA
NO
















1
SpyCas9-
−
TTGCTGCCACAATACCTTGGGTTTTAGAGCTAGAAATAGC
29019
CTCGTAAGGTGTAAATTACTGTTTTAGAG
29209



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCgtatgggtcgtagcgaactgagaagggcCGAG

GTCCGTTATCAACTTGAAAAAGTGGCACC






GTATTGtggc

GAGTCGGTGC



 


2
SpyCas9-
+
AGCGAACTGAGAAGGGCCAAGTTTTAGAGCTAGAAATAG
29020
TTCCTAAAAAAGAAGTAAAAGTTTTAGAG
29210



NG

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCtcttaggaactttgctgccacaataccTCGGC

GTCCGTTATCAACTTGAAAAAGTGGCACC






CCTTCTcagt

GAGTCGGTGC



 


6
SpyCas9-
+
AGCGAACTGAGAAGGGCCAAGTTTTAGAGCTAGAAATAG
29021
CCTAAAAAAGAAGTAAAATGGTTTTAGAG
29211



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCtcttaggaactttgctgccacaataccTCGGC

GTCCGTTATCAACTTGAAAAAGTGGCACC






CCTTCTcagt

GAGTCGGTGC



 


7
SpyCas9-
−
TTTGCTGCCACAATACCTTGGTTTTAGAGCTAGAAATAGC
29022
TCGTAAGGTGTAAATTACTTGTTTTAGAG
29212



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCtatgggtcgtagcgaactgagaagggcCGAGG

GTCCGTTATCAACTTGAAAAAGTGGCACC






TATTGTggca

GAGTCGGTGC



 


10
PpnCas9
+
gtcGTAGCGAACTGAGAAGGGCCAGTTGTAGCTCCCTTTTT
29023
gaaGACCCTGCTCTAGGGAGGTGTGTTGTA
29213





CATTTCGCGAAAGCGAAATGAAAAACGTTGTTACAATAA

GCTCCCTTTTTCATTTCGCGAAAGCGAAA






GAGATGAATTTCTCGCAAAGCTCTGCCTCTTGAAATTTCG

TGAAAAACGTTGTTACAATAAGAGATGAA






GTTTCAAGAGGCATCcttaggaactttgctgccacaataccTCGGCCCTT

TTTCTCGCAAAGCTCTGCCTCTTGAAATTT






CTCagtt

CGGTTTCAAGAGGCATC



 


13
ScaCas9-
+
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAG
29024
AAAAAAGAAGTAAAATGCCAGTTTTAGA
29214



Sc++

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

GCTAGAAATAGCAAGTTAAAATAAGGCT






GTGGCACCGAGTCGGTGCcttaggaactttgctgccacaataccTCGGCC

AGTCCGTTATCAACTTGAAAAAGTGGCAC






CTTCTCagtt

CGAGTCGGTGC



 


14
SpyCas9
+
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAG
29025
TTGAAGACCCTGCTCTAGGGGTTTTAGAG
29215





CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCcttaggaactttgctgccacaataccTCGGCC

GTCCGTTATCAACTTGAAAAAGTGGCACC






CTTCTCagtt

GAGTCGGTGC



 


17
SpyCas9-
+
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAG
29026
CTAAAAAAGAAGTAAAATGCGTTTTAGAG
29216



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCcttaggaactttgctgccacaataccTCGGCC

GTCCGTTATCAACTTGAAAAAGTGGCACC






CTTCTCagtt

GAGTCGGTGC



 


18
SpyCas9-
−
CTTTGCTGCCACAATACCTTGTTTTAGAGCTAGAAATAGC
29027
GTAAGGTGTAAATTACTTACGTTTTAGAG
29217



NG

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCatgggtcgtagcgaactgagaagggcCGAGG

GTCCGTTATCAACTTGAAAAAGTGGCACC






TATTGTGgcag

GAGTCGGTGC



 


21
SpyCas9-
+
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAG
29028
AAAAAGAAGTAAAATGCCACGTTTTAGA
29218



NG

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

GCTAGAAATAGCAAGTTAAAATAAGGCT






GTGGCACCGAGTCGGTGCcttaggaactttgctgccacaataccTCGGCC

AGTCCGTTATCAACTTGAAAAAGTGGCAC






CTTCTCagtt

CGAGTCGGTGC



 


25
SpyCas9-
−
CTTTGCTGCCACAATACCTTGTTTTAGAGCTAGAAATAGC
29029
CGTAAGGTGTAAATTACTTAGTTTTAGAG
29219



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCatgggtcgtagcgaactgagaagggcCGAGG

GTCCGTTATCAACTTGAAAAAGTGGCACC






TATTGTGgcag

GAGTCGGTGC



 


26
BlatCas9
−
gaacTTTGCTGCCACAATACCTTGCTATAGTTCCTTACTGAA
29030
gtggCCTCGTAAGGTGTAAATTAGCTATAGT
29220





AGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCGT

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATAT

AGGGCAACAGACCCGAGGCGTTGGGGAT






TCAAAATAATGACAGACGAGCACCTTGGAGCATTTATCT

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






CCGAGGTGCTatgggtcgtagcgaactgagaagggcCGAGGTATTGTGg

ATATTCAAAATAATGACAGACGAGCACCT






cag

TGGAGCATTTATCTCCGAGGTGCT



 


29
Nme2Cas9
−
agGAACTTTGCTGCCACAATACCTGTTGTAGCTCCCTTTCT
29031
gtAAATTACTTACTGTTAATGGAAGTTGTA
29221





CATTTCGGAAACGAAATGAGAACCGTTGCTACAATAAGG

GCTCCCTTTCTCATTTCGGAAACGAAATG






CCGTCTGAAAAGATGTGCCGCAACGCTCTGCCCCTTAAA

AGAACCGTTGCTACAATAAGGCCGTCTGA






GCTTCTGCTTTAAGGGGCATCGTTTAtgggtcgtagcgaactgagaag

AAAGATGTGCCGCAACGCTCTGCCCCTTA






ggcCGAGGTATTGTGGcagc

AAGCTTCTGCTTTAAGGGGCATCGTTTA



 


30
SauriCas9
+
CGTAGCGAACTGAGAAGGGCCGTTTTAGTACTCTGGAAA
29032
CTTAAGACTACCTTTCTCCAAGTTTTAGTA
29222





CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

CTCTGGAAACAGAATCTACTAAAACAAGG






CGTCAACTTGTTGGCGAGAttaggaactttgctgccacaataccTCGGCC

CAAAATGCCGTGTTTATCTCGTCAACTTGT






CTTCTCAgttc

TGGCGAGA



 


31
SauriCas9-
+
CGTAGCGAACTGAGAAGGGCCGTTTTAGTACTCTGGAAA
29033
AAAAAAGAAGTAAAATGCCACGTTTTAGT
29223



KKH

CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

ACTCTGGAAACAGAATCTACTAAAACAAG






CGTCAACTTGTTGGCGAGAttaggaactttgctgccacaataccTCGGCC

GCAAAATGCCGTGTTTATCTCGTCAACTT






CTTCTCAgttc

GTTGGCGAGA



 


34
ScaCas9-
−
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGC
29034
CGTAAGGTGTAAATTACTTAGTTTTAGAG
29224



Sc++

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCtgggtcgtagcgaactgagaagggcCGAGGT

GTCCGTTATCAACTTGAAAAAGTGGCACC






ATTGTGGcagc

GAGTCGGTGC



 


35
SpyCas9
−
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGC
29035
TGTAAATTACTTACTGTTAAGTTTTAGAGC
29225





AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

TAGAAATAGCAAGTTAAAATAAGGCTAGT






TGGCACCGAGTCGGTGCtgggtcgtagcgaactgagaagggcCGAGGT

CCGTTATCAACTTGAAAAAGTGGCACCGA






ATTGTGGcagc

GTCGGTGC



 


38
SpyCas9-
−
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGC
29036
GTAAGGTGTAAATTACTTACGTTTTAGAG
29226



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCtgggtcgtagcgaactgagaagggcCGAGGT

GTCCGTTATCAACTTGAAAAAGTGGCACC






ATTGTGGcagc

GAGTCGGTGC



 


41
ScaCas9-
+
GTAGCGAACTGAGAAGGGCCGTTTTAGAGCTAGAAATAG
29037
AAAAAAGAAGTAAAATGCCAGTTTTAGA
29227



Sc++

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

GCTAGAAATAGCAAGTTAAAATAAGGCT






GTGGCACCGAGTCGGTGCttaggaactttgctgccacaataccTCGGCC

AGTCCGTTATCAACTTGAAAAAGTGGCAC






CTTCTCAgttc

CGAGTCGGTGC



 


42
SpyCas9-
+
GTAGCGAACTGAGAAGGGCCGTTTTAGAGCTAGAAATAG
29038
TAAAAAAGAAGTAAAATGCCGTTTTAGAG
29228



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCttaggaactttgctgccacaataccTCGGCC

GTCCGTTATCAACTTGAAAAAGTGGCACC






CTTCTCAgttc

GAGTCGGTGC



 


43
SpyCas9-
−
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGC
29039
GTAAGGTGTAAATTACTTACGTTTTAGAG
29229



NG

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCtgggtcgtagcgaactgagaagggcCGAGGT

GTCCGTTATCAACTTGAAAAAGTGGCACC






ATTGTGGcagc

GAGTCGGTGC



 


46
BlatCas9
−
ggaaCTTTGCTGCCACAATACCTGCTATAGTTCCTTACTGA
29040
gtggCCTCGTAAGGTGTAAATTAGCTATAGT
29230





AAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCG

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATA

AGGGCAACAGACCCGAGGCGTTGGGGAT






TTCAAAATAATGACAGACGAGCACCTTGGAGCATTTATC

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






TCCGAGGTGCTtgggtcgtagcgaactgagaagggcCGAGGTATTGTG

ATATTCAAAATAATGACAGACGAGCACCT






Gcagc

TGGAGCATTTATCTCCGAGGTGCT



 


47
BlatCas9
−
ggaaCTTTGCTGCCACAATACCTGCTATAGTTCCTTACTGA
29041
gtggCCTCGTAAGGTGTAAATTAGCTATAGT
29231





AAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCG

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATA

AGGGCAACAGACCCGAGGCGTTGGGGAT






TTCAAAATAATGACAGACGAGCACCTTGGAGCATTTATC

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






TCCGAGGTGCTtgggtcgtagcgaactgagaagggcCGAGGTATTGTG

ATATTCAAAATAATGACAGACGAGCACCT






Gcagc

TGGAGCATTTATCTCCGAGGTGCT



 


50
Nme2Cas9
−
taGGAACTTTGCTGCCACAATACCGTTGTAGCTCCCTTTCT
29042
gtAAATTACTTACTGTTAATGGAAGTTGTA
29232





CATTTCGGAAACGAAATGAGAACCGTTGCTACAATAAGG

GCTCCCTTTCTCATTTCGGAAACGAAATG






CCGTCTGAAAAGATGTGCCGCAACGCTCTGCCCCTTAAA

AGAACCGTTGCTACAATAAGGCCGTCTGA






GCTTCTGCTTTAAGGGGCATCGTTTAgggtcgtagcgaactgagaag

AAAGATGTGCCGCAACGCTCTGCCCCTTA






ggcCGAGGTATTGTGGCagca

AAGCTTCTGCTTTAAGGGGCATCGTTTA



 


51
SauCas9KKH
+
TCGTAGCGAACTGAGAAGGGCGTTTTAGTACTCTGGAAA
29043
TAAAAAAGAAGTAAAATGCCAGTTTTAGT
29233





CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

ACTCTGGAAACAGAATCTACTAAAACAAG






CGTCAACTTGTTGGCGAGAtaggaactttgctgccacaataccTCGGCC

GCAAAATGCCGTGTTTATCTCGTCAACTT






CTTCTCAGttcg

GTTGGCGAGA



 


52
SauCas9KKH
+
TCGTAGCGAACTGAGAAGGGCGTTTTAGTACTCTGGAAA
29044
TAAAAAAGAAGTAAAATGCCAGTTTTAGT
29234





CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

ACTCTGGAAACAGAATCTACTAAAACAAG






CGTCAACTTGTTGGCGAGAtaggaactttgctgccacaataccTCGGCC

GCAAAATGCCGTGTTTATCTCGTCAACTT






CTTCTCAGttcg

GTTGGCGAGA



 


55
SauriCas9
−
GAACTTTGCTGCCACAATACCGTTTTAGTACTCTGGAAAC
29045
GGTGTAAATTACTTACTGTTAGTTTTAGTA
29235





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

CTCTGGAAACAGAATCTACTAAAACAAGG






GTCAACTTGTTGGCGAGAgggtcgtagcgaactgagaagggcCGAGG

CAAAATGCCGTGTTTATCTCGTCAACTTGT






TATTGTGGCagca

TGGCGAGA



 


56
SauriCas9-
−
GAACTTTGCTGCCACAATACCGTTTTAGTACTCTGGAAAC
29046
GGTGTAAATTACTTACTGTTAGTTTTAGTA
29236



KKH

AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

CTCTGGAAACAGAATCTACTAAAACAAGG






GTCAACTTGTTGGCGAGAgggtcgtagcgaactgagaagggcCGAGG

CAAAATGCCGTGTTTATCTCGTCAACTTGT






TATTGTGGCagca

TGGCGAGA



 


57
SauriCas9-
+
TCGTAGCGAACTGAGAAGGGCGTTTTAGTACTCTGGAAA
29047
AAAAAAGAAGTAAAATGCCACGTTTTAGT
29237



KKH

CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

ACTCTGGAAACAGAATCTACTAAAACAAG






CGTCAACTTGTTGGCGAGAtaggaactttgctgccacaataccTCGGCC

GCAAAATGCCGTGTTTATCTCGTCAACTT






CTTCTCAGttcg

GTTGGCGAGA



 


62
ScaCas9-
−
AACTTTGCTGCCACAATACCGTTTTAGAGCTAGAAATAGC
29048
CGTAAGGTGTAAATTACTTAGTTTTAGAG
29238



Sc++

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCgggtcgtagcgaactgagaagggcCGAGGT

GTCCGTTATCAACTTGAAAAAGTGGCACC






ATTGTGGCagca

GAGTCGGTGC



 


63
SpyCas9-
−
AACTTTGCTGCCACAATACCGTTTTAGAGCTAGAAATAGC
29049
TAAGGTGTAAATTACTTACTGTTTTAGAG
29239



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCgggtcgtagcgaactgagaagggcCGAGGT

GTCCGTTATCAACTTGAAAAAGTGGCACC






ATTGTGGCagca

GAGTCGGTGC



 


64
SpyCas9-
+
CGTAGCGAACTGAGAAGGGCGTTTTAGAGCTAGAAATAG
29050
AAAAAAGAAGTAAAATGCCAGTTTTAGA
29240



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

GCTAGAAATAGCAAGTTAAAATAAGGCT






GTGGCACCGAGTCGGTGCtaggaactttgctgccacaataccTCGGCCC

AGTCCGTTATCAACTTGAAAAAGTGGCAC






TTCTCAGttcg

CGAGTCGGTGC



 


65
BlatCas9
−
aggaACTTTGCTGCCACAATACCGCTATAGTTCCTTACTGA
29051
gtggCCTCGTAAGGTGTAAATTAGCTATAGT
29241





AAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCG

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATA

AGGGCAACAGACCCGAGGCGTTGGGGAT






TTCAAAATAATGACAGACGAGCACCTTGGAGCATTTATC

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






TCCGAGGTGCTgggtcgtagcgaactgagaagggcCGAGGTATTGTGG

ATATTCAAAATAATGACAGACGAGCACCT






Cagca

TGGAGCATTTATCTCCGAGGTGCT



 


66
SauCas9KKH
+
GTCGTAGCGAACTGAGAAGGGGTTTTAGTACTCTGGAAA
29052
AAAAAAGAAGTAAAATGCCACGTTTTAGT
29242





CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

ACTCTGGAAACAGAATCTACTAAAACAAG






CGTCAACTTGTTGGCGAGAaggaactttgctgccacaataccTCGGCC

GCAAAATGCCGTGTTTATCTCGTCAACTT






CTTCTCAGTtcgc

GTTGGCGAGA



 


67
SauCas9KKH
−
GGAACTTTGCTGCCACAATACGTTTTAGTACTCTGGAAAC
29053
GTAAGGTGTAAATTACTTACTGTTTTAGT
29243





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

ACTCTGGAAACAGAATCTACTAAAACAAG






GTCAACTTGTTGGCGAGAggtcgtagcgaactgagaagggcCGAGGT

GCAAAATGCCGTGTTTATCTCGTCAACTT






ATTGTGGCAgcaa

GTTGGCGAGA



 


68
SpyCas9-
+
TCGTAGCGAACTGAGAAGGGGTTTTAGAGCTAGAAATAG
29054
AAAAAGAAGTAAAATGCCACGTTTTAGA
29244



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

GCTAGAAATAGCAAGTTAAAATAAGGCT






GTGGCACCGAGTCGGTGCaggaactttgctgccacaataccTCGGCCC

AGTCCGTTATCAACTTGAAAAAGTGGCAC






TTCTCAGTtcgc

CGAGTCGGTGC



 


69
SpyCas9-
−
GAACTTTGCTGCCACAATACGTTTTAGAGCTAGAAATAG
29055
AAGGTGTAAATTACTTACTGGTTTTAGAG
29245



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCggtcgtagcgaactgagaagggcCGAGGT

GTCCGTTATCAACTTGAAAAAGTGGCACC






ATTGTGGCAgcaa

GAGTCGGTGC



 


72
SauCas9KKH
+
GGTCGTAGCGAACTGAGAAGGGTTTTAGTACTCTGGAAA
29056
AAAAAGAAGTAAAATGCCACTGTTTTAGT
29246





CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

ACTCTGGAAACAGAATCTACTAAAACAAG






CGTCAACTTGTTGGCGAGAggaactttgctgccacaataccTCGGCCC

GCAAAATGCCGTGTTTATCTCGTCAACTT






TTCTCAGTTcgct

GTTGGCGAGA



 


73
SpyCas9-
+
GTCGTAGCGAACTGAGAAGGGTTTTAGAGCTAGAAATAG
29057
AAAAGAAGTAAAATGCCACTGTTTTAGAG
29247



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCggaactttgctgccacaataccTCGGCCCT

GTCCGTTATCAACTTGAAAAAGTGGCACC






TCTCAGTTcgct

GAGTCGGTGC



 


74
SpyCas9-
−
GGAACTTTGCTGCCACAATAGTTTTAGAGCTAGAAATAG
29058
AGGTGTAAATTACTTACTGTGTTTTAGAG
29248



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCgtcgtagcgaactgagaagggcCGAGGTA

GTCCGTTATCAACTTGAAAAAGTGGCACC






TTGTGGCAGcaaa

GAGTCGGTGC



 


77
SpyCas9-
+
GGTCGTAGCGAACTGAGAAGGTTTTAGAGCTAGAAATAG
29059
AAAGAAGTAAAATGCCACTGGTTTTAGAG
29249



NG

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCgaactttgctgccacaataccTCGGCCCTT

GTCCGTTATCAACTTGAAAAAGTGGCACC






CTCAGTTCgcta

GAGTCGGTGC



 


81
SpyCas9-
+
GGTCGTAGCGAACTGAGAAGGTTTTAGAGCTAGAAATAG
29060
AAAGAAGTAAAATGCCACTGGTTTTAGAG
29250



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCgaactttgctgccacaataccTCGGCCCTT

GTCCGTTATCAACTTGAAAAAGTGGCACC






CTCAGTTCgcta

GAGTCGGTGC



 


82
SpyCas9-
−
AGGAACTTTGCTGCCACAATGTTTTAGAGCTAGAAATAG
29061
GGTGTAAATTACTTACTGTTGTTTTAGAGC
29251



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

TAGAAATAGCAAGTTAAAATAAGGCTAGT






GTGGCACCGAGTCGGTGCtcgtagcgaactgagaagggcCGAGGTAT

CCGTTATCAACTTGAAAAAGTGGCACCGA






TGTGGCAGCaaag

GTCGGTGC



 


86
ScaCas9-
+
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAG
29062
AAAAGAAGTAAAATGCCACTGTTTTAGAG
29252



Sc++

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCaactttgctgccacaataccTCGGCCCTTC

GTCCGTTATCAACTTGAAAAAGTGGCACC






TCAGTTCGctac

GAGTCGGTGC



 


87
SpyCas9
+
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAG
29063
TAAGACTACCTTTCTCCAAAGTTTTAGAG
29253





CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCaactttgctgccacaataccTCGGCCCTTC

GTCCGTTATCAACTTGAAAAAGTGGCACC






TCAGTTCGctac

GAGTCGGTGC



 


90
SpyCas9-
+
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAG
29064
AAGAAGTAAAATGCCACTGAGTTTTAGAG
29254



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCaactttgctgccacaataccTCGGCCCTTC

GTCCGTTATCAACTTGAAAAAGTGGCACC






TCAGTTCGctac

GAGTCGGTGC



 


91
SpyCas9-
+
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAG
29065
AAAGAAGTAAAATGCCACTGGTTTTAGAG
29255



NG

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCaactttgctgccacaataccTCGGCCCTTC

GTCCGTTATCAACTTGAAAAAGTGGCACC






TCAGTTCGctac

GAGTCGGTGC



 


94
SpyCas9-
−
TAGGAACTTTGCTGCCACAAGTTTTAGAGCTAGAAATAG
29066
GTGTAAATTACTTACTGTTAGTTTTAGAGC
29256



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

TAGAAATAGCAAGTTAAAATAAGGCTAGT






GTGGCACCGAGTCGGTGCcgtagcgaactgagaagggcCGAGGTAT

CCGTTATCAACTTGAAAAAGTGGCACCGA






TGTGGCAGCAaagt

GTCGGTGC



 


95
BlatCas9
+
tatgGGTCGTAGCGAACTGAGAAGCTATAGTTCCTTACTGA
29067
aaaaAAGAAGTAAAATGCCACTGGCTATAG
29257





AAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCG

TTCCTTACTGAAAGGTAAGTTGCTATAGT






TTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATA

AAGGGCAACAGACCCGAGGCGTTGGGGA






TTCAAAATAATGACAGACGAGCACCTTGGAGCATTTATC

TCGCCTAGCCCGTGTTTACGGGCTCTCCCC






TCCGAGGTGCTaactttgctgccacaataccTCGGCCCTTCTCAGTTC

ATATTCAAAATAATGACAGACGAGCACCT






Gctac

TGGAGCATTTATCTCCGAGGTGCT



 


96
BlatCas9
+
tatgGGTCGTAGCGAACTGAGAAGCTATAGTTCCTTACTGA
29068
aaaaAAGAAGTAAAATGCCACTGGCTATAG
29258





AAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCG

TTCCTTACTGAAAGGTAAGTTGCTATAGT






TTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATA

AAGGGCAACAGACCCGAGGCGTTGGGGA






TTCAAAATAATGACAGACGAGCACCTTGGAGCATTTATC

TCGCCTAGCCCGTGTTTACGGGCTCTCCCC






TCCGAGGTGCTaactttgctgccacaataccTCGGCCCTTCTCAGTTC

ATATTCAAAATAATGACAGACGAGCACCT






Gctac

TGGAGCATTTATCTCCGAGGTGCT



 


99
Nme2Cas9
+
tgTATGGGTCGTAGCGAACTGAGAGTTGTAGCTCCCTTTCT
29069
tcCGTGTTCCTAAAAAAGAAGTAAGTTGTA
29259





CATTTCGGAAACGAAATGAGAACCGTTGCTACAATAAGG

GCTCCCTTTCTCATTTCGGAAACGAAATG






CCGTCTGAAAAGATGTGCCGCAACGCTCTGCCCCTTAAA

AGAACCGTTGCTACAATAAGGCCGTCTGA






GCTTCTGCTTTAAGGGGCATCGTTTAactttgctgccacaataccTCG

AAAGATGTGCCGCAACGCTCTGCCCCTTA






GCCCTTCTCAGTTCGCtacg

AAGCTTCTGCTTTAAGGGGCATCGTTTA



 


100
SauriCas9
+
ATGGGTCGTAGCGAACTGAGAGTTTTAGTACTCTGGAAA
29070
CTTAAGACTACCTTTCTCCAAGTTTTAGTA
29260





CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

CTCTGGAAACAGAATCTACTAAAACAAGG






CGTCAACTTGTTGGCGAGAactttgctgccacaataccTCGGCCCTT

CAAAATGCCGTGTTTATCTCGTCAACTTGT






CTCAGTTCGCtacg

TGGCGAGA



 


101
SauriCas9-
+
ATGGGTCGTAGCGAACTGAGAGTTTTAGTACTCTGGAAA
29071
AAAAAAGAAGTAAAATGCCACGTTTTAGT
29261



KKH

CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

ACTCTGGAAACAGAATCTACTAAAACAAG






CGTCAACTTGTTGGCGAGAactttgctgccacaataccTCGGCCCTT

GCAAAATGCCGTGTTTATCTCGTCAACTT






CTCAGTTCGCtacg

GTTGGCGAGA



 


104
ScaCas9-
+
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAG
29072
AAAAGAAGTAAAATGCCACTGTTTTAGAG
29262



Sc++

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCactttgctgccacaataccTCGGCCCTTC

GTCCGTTATCAACTTGAAAAAGTGGCACC






TCAGTTCGCtacg

GAGTCGGTGC



 


105
SpyCas9
+
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAG
29073
TAAGACTACCTTTCTCCAAAGTTTTAGAG
29263





CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCactttgctgccacaataccTCGGCCCTTC

GTCCGTTATCAACTTGAAAAAGTGGCACC






TCAGTTCGCtacg

GAGTCGGTGC



 


108
SpyCas9-
+
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAG
29074
AGAAGTAAAATGCCACTGAGGTTTTAGAG
29264



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCactttgctgccacaataccTCGGCCCTTC

GTCCGTTATCAACTTGAAAAAGTGGCACC






TCAGTTCGCtacg

GAGTCGGTGC



 


109
SpyCas9-
+
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAG
29075
AAAGAAGTAAAATGCCACTGGTTTTAGAG
29265



NG

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCactttgctgccacaataccTCGGCCCTTC

GTCCGTTATCAACTTGAAAAAGTGGCACC






TCAGTTCGCtacg

GAGTCGGTGC



 


112
SpyCas9-
−
TTAGGAACTTTGCTGCCACAGTTTTAGAGCTAGAAATAGC
29076
TGTAAATTACTTACTGTTAAGTTTTAGAGC
29266



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

TAGAAATAGCAAGTTAAAATAAGGCTAGT






TGGCACCGAGTCGGTGCgtagcgaactgagaagggcCGAGGTATTG

CCGTTATCAACTTGAAAAAGTGGCACCGA






TGGCAGCAAagtt

GTCGGTGC



 


113
BlatCas9
+
gtatGGGTCGTAGCGAACTGAGAGCTATAGTTCCTTACTGA
29077
aaaaGAAGTAAAATGCCACTGAGGCTATAG
29267





AAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCG

TTCCTTACTGAAAGGTAAGTTGCTATAGT






TTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATA

AAGGGCAACAGACCCGAGGCGTTGGGGA






TTCAAAATAATGACAGACGAGCACCTTGGAGCATTTATC

TCGCCTAGCCCGTGTTTACGGGCTCTCCCC






TCCGAGGTGCTactttgctgccacaataccTCGGCCCTTCTCAGTTCG

ATATTCAAAATAATGACAGACGAGCACCT






Ctacg

TGGAGCATTTATCTCCGAGGTGCT



 


114
BlatCas9
+
gtatGGGTCGTAGCGAACTGAGAGCTATAGTTCCTTACTGA
29078
aaaaGAAGTAAAATGCCACTGAGGCTATAG
29268





AAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCG

TTCCTTACTGAAAGGTAAGTTGCTATAGT






TTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATA

AAGGGCAACAGACCCGAGGCGTTGGGGA






TTCAAAATAATGACAGACGAGCACCTTGGAGCATTTATC

TCGCCTAGCCCGTGTTTACGGGCTCTCCCC






TCCGAGGTGCTactttgctgccacaataccTCGGCCCTTCTCAGTTCG

ATATTCAAAATAATGACAGACGAGCACCT






Ctacg

TGGAGCATTTATCTCCGAGGTGCT



 


115
BlatCas9
−
gtctTAGGAACTTTGCTGCCACAGCTATAGTTCCTTACTGAA
29079
gtgtAAATTACTTACTGTTAATGGCTATAGT
29269





AGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCGT

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATAT

AGGGCAACAGACCCGAGGCGTTGGGGAT






TCAAAATAATGACAGACGAGCACCTTGGAGCATTTATCT

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






CCGAGGTGCTgtagcgaactgagaagggcCGAGGTATTGTGGCAGC

ATATTCAAAATAATGACAGACGAGCACCT






AAagtt

TGGAGCATTTATCTCCGAGGTGCT



 


116
BlatCas9
+
gtatGGGTCGTAGCGAACTGAGAGCTATAGTTCCTTACTGA
29080
aaaaGAAGTAAAATGCCACTGAGGCTATAG
29270





AAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCG

TTCCTTACTGAAAGGTAAGTTGCTATAGT






TTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATA

AAGGGCAACAGACCCGAGGCGTTGGGGA






TTCAAAATAATGACAGACGAGCACCTTGGAGCATTTATC

TCGCCTAGCCCGTGTTTACGGGCTCTCCCC






TCCGAGGTGCTactttgctgccacaataccTCGGCCCTTCTCAGTTCG

ATATTCAAAATAATGACAGACGAGCACCT






Ctacg

TGGAGCATTTATCTCCGAGGTGCT



 


117
BlatCas9
−
gtctTAGGAACTTTGCTGCCACAGCTATAGTTCCTTACTGAA
29081
gtgtAAATTACTTACTGTTAATGGCTATAGT
29271





AGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCGT

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATAT

AGGGCAACAGACCCGAGGCGTTGGGGAT






TCAAAATAATGACAGACGAGCACCTTGGAGCATTTATCT

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






CCGAGGTGCTgtagcgaactgagaagggcCGAGGTATTGTGGCAGC

ATATTCAAAATAATGACAGACGAGCACCT






AAagtt

TGGAGCATTTATCTCCGAGGTGCT



 


119
Nme2Cas9
−
tgGTCTTAGGAACTTTGCTGCCACGTTGTAGCTCCCTTTCT
29082
gtAAATTACTTACTGTTAATGGAAGTTGTA
29272





CATTTCGGAAACGAAATGAGAACCGTTGCTACAATAAGG

GCTCCCTTTCTCATTTCGGAAACGAAATG






CCGTCTGAAAAGATGTGCCGCAACGCTCTGCCCCTTAAA

AGAACCGTTGCTACAATAAGGCCGTCTGA






GCTTCTGCTTTAAGGGGCATCGTTTAtagcgaactgagaagggcCG

AAAGATGTGCCGCAACGCTCTGCCCCTTA






AGGTATTGTGGCAGCAAAgttc

AAGCTTCTGCTTTAAGGGGCATCGTTTA



 


120
SauCas9
+
tgTATGGGTCGTAGCGAACTGAGGTTTTAGTACTCTGGAA
29083
taAAAAAGAAGTAAAATGCCACTGTTTTAG
29273





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATC

TACTCTGGAAACAGAATCTACTAAAACAA






TCGTCAACTTGTTGGCGAGActttgctgccacaataccTCGGCCCTT

GGCAAAATGCCGTGTTTATCTCGTCAACT






CTCAGTTCGCTacga

TGTTGGCGAGA



 


121
SauCas9KKH
+
TATGGGTCGTAGCGAACTGAGGTTTTAGTACTCTGGAAA
29084
AAAAAGAAGTAAAATGCCACTGTTTTAGT
29274





CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

ACTCTGGAAACAGAATCTACTAAAACAAG






CGTCAACTTGTTGGCGAGActttgctgccacaataccTCGGCCCTTC

GCAAAATGCCGTGTTTATCTCGTCAACTT






TCAGTTCGCTacga

GTTGGCGAGA



 


122
SauriCas9
+
TATGGGTCGTAGCGAACTGAGGTTTTAGTACTCTGGAAA
29085
CTTAAGACTACCTTTCTCCAAGTTTTAGTA
29275





CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

CTCTGGAAACAGAATCTACTAAAACAAGG






CGTCAACTTGTTGGCGAGActttgctgccacaataccTCGGCCCTTC

CAAAATGCCGTGTTTATCTCGTCAACTTGT






TCAGTTCGCTacga

TGGCGAGA



 


123
SauriCas9-
+
TATGGGTCGTAGCGAACTGAGGTTTTAGTACTCTGGAAA
29086
AAAAAAGAAGTAAAATGCCACGTTTTAGT
29276



KKH

CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

ACTCTGGAAACAGAATCTACTAAAACAAG






CGTCAACTTGTTGGCGAGActttgctgccacaataccTCGGCCCTTC

GCAAAATGCCGTGTTTATCTCGTCAACTT






TCAGTTCGCTacga

GTTGGCGAGA



 


126
ScaCas9-
+
ATGGGTCGTAGCGAACTGAGGTTTTAGAGCTAGAAATAG
29087
AAAAGAAGTAAAATGCCACTGTTTTAGAG
29277



Sc++

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCctttgctgccacaataccTCGGCCCTTCT

GTCCGTTATCAACTTGAAAAAGTGGCACC






CAGTTCGCTacga

GAGTCGGTGC



 


127
SpyCas9-
+
ATGGGTCGTAGCGAACTGAGGTTTTAGAGCTAGAAATAG
29088
GAAGTAAAATGCCACTGAGAGTTTTAGAG
29278



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCctttgctgccacaataccTCGGCCCTTCT

GTCCGTTATCAACTTGAAAAAGTGGCACC






CAGTTCGCTacga

GAGTCGGTGC



 


128
SpyCas9-
−
CTTAGGAACTTTGCTGCCACGTTTTAGAGCTAGAAATAGC
29089
GTAAATTACTTACTGTTAATGTTTTAGAGC
29279



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

TAGAAATAGCAAGTTAAAATAAGGCTAGT






TGGCACCGAGTCGGTGCtagcgaactgagaagggcCGAGGTATTGT

CCGTTATCAACTTGAAAAAGTGGCACCGA






GGCAGCAAAgttc

GTCGGTGC



 


129
BlatCas9
−
ggtcTTAGGAACTTTGCTGCCACGCTATAGTTCCTTACTGA
29090
gtgtAAATTACTTACTGTTAATGGCTATAGT
29280





AAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCG

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATA

AGGGCAACAGACCCGAGGCGTTGGGGAT






TTCAAAATAATGACAGACGAGCACCTTGGAGCATTTATC

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






TCCGAGGTGCTtagcgaactgagaagggcCGAGGTATTGTGGCAGC

ATATTCAAAATAATGACAGACGAGCACCT






AAAgttc

TGGAGCATTTATCTCCGAGGTGCT



 


130
BlatCas9
−
ggtcTTAGGAACTTTGCTGCCACGCTATAGTTCCTTACTGA
29091
gtgtAAATTACTTACTGTTAATGGCTATAGT
29281





AAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCG

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATA

AGGGCAACAGACCCGAGGCGTTGGGGAT






TTCAAAATAATGACAGACGAGCACCTTGGAGCATTTATC

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






TCCGAGGTGCTtagcgaactgagaagggcCGAGGTATTGTGGCAGC

ATATTCAAAATAATGACAGACGAGCACCT






AAAgttc

TGGAGCATTTATCTCCGAGGTGCT



 


134
SauCas9KKH
+
GTATGGGTCGTAGCGAACTGAGTTTTAGTACTCTGGAAA
29092
TAAAATGCCACTGAGAACTCTGTTTTAGT
29282





CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

ACTCTGGAAACAGAATCTACTAAAACAAG






CGTCAACTTGTTGGCGAGAtttgctgccacaataccTCGGCCCTTCT

GCAAAATGCCGTGTTTATCTCGTCAACTT






CAGTTCGCTAcgac

GTTGGCGAGA



 


135
SauriCas9-
+
GTATGGGTCGTAGCGAACTGAGTTTTAGTACTCTGGAAA
29093
AAATGCCACTGAGAACTCTCTGTTTTAGT
29283



KKH

CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

ACTCTGGAAACAGAATCTACTAAAACAAG






CGTCAACTTGTTGGCGAGAtttgctgccacaataccTCGGCCCTTCT

GCAAAATGCCGTGTTTATCTCGTCAACTT






CAGTTCGCTAcgac

GTTGGCGAGA



 


138
SpyCas9-
+
TATGGGTCGTAGCGAACTGAGTTTTAGAGCTAGAAATAG
29094
AAGTAAAATGCCACTGAGAAGTTTTAGAG
29284



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCtttgctgccacaataccTCGGCCCTTCTC

GTCCGTTATCAACTTGAAAAAGTGGCACC






AGTTCGCTAcgac

GAGTCGGTGC



 


140
SpyCas9-
−
TCTTAGGAACTTTGCTGCCAGTTTTAGAGCTAGAAATAGC
29095
TAAATTACTTACTGTTAATGGTTTTAGAGC
29285



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

TAGAAATAGCAAGTTAAAATAAGGCTAGT






TGGCACCGAGTCGGTGCagcgaactgagaagggcCGAGGTATTGT

CCGTTATCAACTTGAAAAAGTGGCACCGA






GGCAGCAAAGttcc

GTCGGTGC



 


146
SauCas9KKH
+
TGTATGGGTCGTAGCGAACTGGTTTTAGTACTCTGGAAAC
29096
TAAAATGCCACTGAGAACTCTGTTTTAGT
29286





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

ACTCTGGAAACAGAATCTACTAAAACAAG






GTCAACTTGTTGGCGAGAttgctgccacaataccTCGGCCCTTCTC

GCAAAATGCCGTGTTTATCTCGTCAACTT






AGTTCGCTACgacc

GTTGGCGAGA



 


147
SpyCas9-
+
GTATGGGTCGTAGCGAACTGGTTTTAGAGCTAGAAATAG
29097
AAAGAAGTAAAATGCCACTGGTTTTAGAG
29287



NG

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCttgctgccacaataccTCGGCCCTTCTC

GTCCGTTATCAACTTGAAAAAGTGGCACC






AGTTCGCTACgacc

GAGTCGGTGC



 


151
SpyCas9-
+
GTATGGGTCGTAGCGAACTGGTTTTAGAGCTAGAAATAG
29098
AGTAAAATGCCACTGAGAACGTTTTAGAG
29288



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCttgctgccacaataccTCGGCCCTTCTC

GTCCGTTATCAACTTGAAAAAGTGGCACC






AGTTCGCTACgacc

GAGTCGGTGC



 


152
SpyCa
−
GTCTTAGGAACTTTGCTGCCGTTTTAGAGCTAGAAATAGC
29099
AAATTACTTACTGTTAATGGGTTTTAGAG
29289



s9-

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA




SpRY

TGGCACCGAGTCGGTGCgcgaactgagaagggcCGAGGTATTGTG

GTCCGTTATCAACTTGAAAAAGTGGCACC






GCAGCAAAGTtcct

GAGTCGGTGC



 


158
SauCas9
+
ggGTGTATGGGTCGTAGCGAACTGTTTTAGTACTCTGGAA
29100
taAAAAAGAAGTAAAATGCCACTGTTTTAG
29290





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATC

TACTCTGGAAACAGAATCTACTAAAACAA






TCGTCAACTTGTTGGCGAGAtgctgccacaataccTCGGCCCTTCT

GGCAAAATGCCGTGTTTATCTCGTCAACT






CAGTTCGCTACGaccc

TGTTGGCGAGA



 


159
SauCas9KKH
+
GTGTATGGGTCGTAGCGAACTGTTTTAGTACTCTGGAAAC
29101
TAAAATGCCACTGAGAACTCTGTTTTAGT
29291





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

ACTCTGGAAACAGAATCTACTAAAACAAG






GTCAACTTGTTGGCGAGAtgctgccacaataccTCGGCCCTTCTCA

GCAAAATGCCGTGTTTATCTCGTCAACTT






GTTCGCTACGaccc

GTTGGCGAGA



 


160
SauCas9KKH
−
TGGTCTTAGGAACTTTGCTGCGTTTTAGTACTCTGGAAAC
29102
AAATTACTTACTGTTAATGGAGTTTTAGT
29292





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

ACTCTGGAAACAGAATCTACTAAAACAAG






GTCAACTTGTTGGCGAGAcgaactgagaagggcCGAGGTATTGT

GCAAAATGCCGTGTTTATCTCGTCAACTT






GGCAGCAAAGTTccta

GTTGGCGAGA



 


161
SauCas9KKH
−
TGGTCTTAGGAACTTTGCTGCGTTTTAGTACTCTGGAAAC
29103
AAATTACTTACTGTTAATGGAGTTTTAGT
29293





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

ACTCTGGAAACAGAATCTACTAAAACAAG






GTCAACTTGTTGGCGAGAcgaactgagaagggcCGAGGTATTGT

GCAAAATGCCGTGTTTATCTCGTCAACTT






GGCAGCAAAGTTccta

GTTGGCGAGA



 


166
ScaCas9-
+
TGTATGGGTCGTAGCGAACTGTTTTAGAGCTAGAAATAG
29104
ATGCCACTGAGAACTCTCTTGTTTTAGAG
29294



Sc++

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCtgctgccacaataccTCGGCCCTTCTC

GTCCGTTATCAACTTGAAAAAGTGGCACC






AGTTCGCTACGaccc

GAGTCGGTGC



 


167
SpyCas9-
+
TGTATGGGTCGTAGCGAACTGTTTTAGAGCTAGAAATAG
29105
GTAAAATGCCACTGAGAACTGTTTTAGAG
29295



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCtgctgccacaataccTCGGCCCTTCTC

GTCCGTTATCAACTTGAAAAAGTGGCACC






AGTTCGCTACGaccc

GAGTCGGTGC



 


168
SpyCas9-
−
GGTCTTAGGAACTTTGCTGCGTTTTAGAGCTAGAAATAGC
29106
AATTACTTACTGTTAATGGAGTTTTAGAG
29296



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCcgaactgagaagggcCGAGGTATTGTG

GTCCGTTATCAACTTGAAAAAGTGGCACC






GCAGCAAAGTTccta

GAGTCGGTGC



 


174
SauCas9KKH
+
GGTGTATGGGTCGTAGCGAACGTTTTAGTACTCTGGAAA
29107
TAAAATGCCACTGAGAACTCTGTTTTAGT
29297





CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

ACTCTGGAAACAGAATCTACTAAAACAAG






CGTCAACTTGTTGGCGAGAgctgccacaataccTCGGCCCTTCTC

GCAAAATGCCGTGTTTATCTCGTCAACTT






AGTTCGCTACGAccca

GTTGGCGAGA



 


175
SauriCas9-
+
GGTGTATGGGTCGTAGCGAACGTTTTAGTACTCTGGAAA
29108
AAATGCCACTGAGAACTCTCTGTTTTAGT
29298



KKH

CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

ACTCTGGAAACAGAATCTACTAAAACAAG






CGTCAACTTGTTGGCGAGAgctgccacaataccTCGGCCCTTCTC

GCAAAATGCCGTGTTTATCTCGTCAACTT






AGTTCGCTACGAccca

GTTGGCGAGA



 


177
SpyCas9-
+
GTGTATGGGTCGTAGCGAACGTTTTAGAGCTAGAAATAG
29109
TGCCACTGAGAACTCTCTTAGTTTTAGAG
29299



NG

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCgctgccacaataccTCGGCCCTTCTCA

GTCCGTTATCAACTTGAAAAAGTGGCACC






GTTCGCTACGAccca

GAGTCGGTGC



 


181
SpyCas9-
+
GTGTATGGGTCGTAGCGAACGTTTTAGAGCTAGAAATAG
29110
TAAAATGCCACTGAGAACTCGTTTTAGAG
29300



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCgctgccacaataccTCGGCCCTTCTCA

GTCCGTTATCAACTTGAAAAAGTGGCACC






GTTCGCTACGAccca

GAGTCGGTGC



 


182
SpyCas9-
−
TGGTCTTAGGAACTTTGCTGGTTTTAGAGCTAGAAATAGC
29111
ATTACTTACTGTTAATGGAAGTTTTAGAG
29301



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCgaactgagaagggcCGAGGTATTGTGG

GTCCGTTATCAACTTGAAAAAGTGGCACC






CAGCAAAGTTCctaa

GAGTCGGTGC



 


187
SauCas9
+
ttGGGTGTATGGGTCGTAGCGAAGTTTTAGTACTCTGGAAA
29112
taAAAAAGAAGTAAAATGCCACTGTTTTAG
29302





CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

TACTCTGGAAACAGAATCTACTAAAACAA






CGTCAACTTGTTGGCGAGActgccacaataccTCGGCCCTTCTCA

GGCAAAATGCCGTGTTTATCTCGTCAACT






GTTCGCTACGACccat

TGTTGGCGAGA



 


188
SauCas9KKH
+
GGGTGTATGGGTCGTAGCGAAGTTTTAGTACTCTGGAAA
29113
TAAAATGCCACTGAGAACTCTGTTTTAGT
29303





CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

ACTCTGGAAACAGAATCTACTAAAACAAG






CGTCAACTTGTTGGCGAGActgccacaataccTCGGCCCTTCTCA

GCAAAATGCCGTGTTTATCTCGTCAACTT






GTTCGCTACGACccat

GTTGGCGAGA



 


191
ScaCas9-
+
GGTGTATGGGTCGTAGCGAAGTTTTAGAGCTAGAAATAG
29114
ATGCCACTGAGAACTCTCTTGTTTTAGAG
29304



Sc++

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCctgccacaataccTCGGCCCTTCTCA

GTCCGTTATCAACTTGAAAAAGTGGCACC






GTTCGCTACGACccat

GAGTCGGTGC



 


192
SpyCas9-
+
GGTGTATGGGTCGTAGCGAAGTTTTAGAGCTAGAAATAG
29115
AAAATGCCACTGAGAACTCTGTTTTAGAG
29305



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCctgccacaataccTCGGCCCTTCTCA

GTCCGTTATCAACTTGAAAAAGTGGCACC






GTTCGCTACGACccat

GAGTCGGTGC



 


193
SpyCas9-
−
TTGGTCTTAGGAACTTTGCTGTTTTAGAGCTAGAAATAGC
29116
TTACTTACTGTTAATGGAATGTTTTAGAGC
29306



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

TAGAAATAGCAAGTTAAAATAAGGCTAGT






TGGCACCGAGTCGGTGCaactgagaagggcCGAGGTATTGTGGC

CCGTTATCAACTTGAAAAAGTGGCACCGA






AGCAAAGTTCCtaag

GTCGGTGC



 


194
BlatCas9
−
gtttTGGTCTTAGGAACTTTGCTGCTATAGTTCCTTACTGAA
29117
aaatTACTTACTGTTAATGGAATGCTATAGT
29307





AGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCGT

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATAT

AGGGCAACAGACCCGAGGCGTTGGGGAT






TCAAAATAATGACAGACGAGCACCTTGGAGCATTTATCT

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






CCGAGGTGCTaactgagaagggcCGAGGTATTGTGGCAGCAAA

ATATTCAAAATAATGACAGACGAGCACCT






GTTCCtaag

TGGAGCATTTATCTCCGAGGTGCT



 


195
BlatCas9
−
gtttTGGTCTTAGGAACTTTGCTGCTATAGTTCCTTACTGAA
29118
aaatTACTTACTGTTAATGGAATGCTATAGT
29308





AGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCGT

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATAT

AGGGCAACAGACCCGAGGCGTTGGGGAT






TCAAAATAATGACAGACGAGCACCTTGGAGCATTTATCT

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






CCGAGGTGCTaactgagaagggcCGAGGTATTGTGGCAGCAAA

ATATTCAAAATAATGACAGACGAGCACCT






GTTCCtaag

TGGAGCATTTATCTCCGAGGTGCT



 


198
SauCas9KKH
+
TGGGTGTATGGGTCGTAGCGAGTTTTAGTACTCTGGAAAC
29119
AAAATGCCACTGAGAACTCTCGTTTTAGT
29309





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

ACTCTGGAAACAGAATCTACTAAAACAAG






GTCAACTTGTTGGCGAGAtgccacaataccTCGGCCCTTCTCAG

GCAAAATGCCGTGTTTATCTCGTCAACTT






TTCGCTACGACCcata

GTTGGCGAGA



 


199
SpyCas9-
−
TTTGGTCTTAGGAACTTTGCGTTTTAGAGCTAGAAATAGC
29120
TACTTACTGTTAATGGAATCGTTTTAGAG
29310



NG

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCactgagaagggcCGAGGTATTGTGGC

GTCCGTTATCAACTTGAAAAAGTGGCACC






AGCAAAGTTCCTaaga

GAGTCGGTGC



 


203
SpyCas9-
−
TTTGGTCTTAGGAACTTTGCGTTTTAGAGCTAGAAATAGC
29121
TACTTACTGTTAATGGAATCGTTTTAGAG
29311



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCactgagaagggcCGAGGTATTGTGGC

GTCCGTTATCAACTTGAAAAAGTGGCACC






AGCAAAGTTCCTaaga

GAGTCGGTGC



 


204
SpyCas9-
+
GGGTGTATGGGTCGTAGCGAGTTTTAGAGCTAGAAATAG
29122
AAATGCCACTGAGAACTCTCGTTTTAGAG
29312



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCtgccacaataccTCGGCCCTTCTCAG

GTCCGTTATCAACTTGAAAAAGTGGCACC






TTCGCTACGACCcata

GAGTCGGTGC



 


208
ScaCas9-
−
TTTTGGTCTTAGGAACTTTGGTTTTAGAGCTAGAAATAGC
29123
TTACTTACTGTTAATGGAATGTTTTAGAGC
29313



Sc++

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

TAGAAATAGCAAGTTAAAATAAGGCTAGT






TGGCACCGAGTCGGTGCctgagaagggcCGAGGTATTGTGGCA

CCGTTATCAACTTGAAAAAGTGGCACCGA






GCAAAGTTCCTAagac

GTCGGTGC



 


209
SpyCas9-
−
TTTTGGTCTTAGGAACTTTGGTTTTAGAGCTAGAAATAGC
29124
ACTTACTGTTAATGGAATCAGTTTTAGAG
29314



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCctgagaagggcCGAGGTATTGTGGCA

GTCCGTTATCAACTTGAAAAAGTGGCACC






GCAAAGTTCCTAagac

GAGTCGGTGC



 


210
SpyCas9-
+
TGGGTGTATGGGTCGTAGCGGTTTTAGAGCTAGAAATAG
29125
AATGCCACTGAGAACTCTCTGTTTTAGAG
29315



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCgccacaataccTCGGCCCTTCTCAGT

GTCCGTTATCAACTTGAAAAAGTGGCACC






TCGCTACGACCCatac

GAGTCGGTGC



 


211
BlatCas9
−
tggtTTTGGTCTTAGGAACTTTGGCTATAGTTCCTTACTGAA
29126
aaatTACTTACTGTTAATGGAATGCTATAGT
29316





AGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCGT

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATAT

AGGGCAACAGACCCGAGGCGTTGGGGAT






TCAAAATAATGACAGACGAGCACCTTGGAGCATTTATCT

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






CCGAGGTGCTctgagaagggcCGAGGTATTGTGGCAGCAAAGT

ATATTCAAAATAATGACAGACGAGCACCT






TCCTAagac

TGGAGCATTTATCTCCGAGGTGCT



 


214
Nme2Cas9
−
tgTGGTTTTGGTCTTAGGAACTTTGTTGTAGCTCCCTTTCTC
29127
gtAAATTACTTACTGTTAATGGAAGTTGTA
29317





ATTTCGGAAACGAAATGAGAACCGTTGCTACAATAAGGC

GCTCCCTTTCTCATTTCGGAAACGAAATG






CGTCTGAAAAGATGTGCCGCAACGCTCTGCCCCTTAAAG

AGAACCGTTGCTACAATAAGGCCGTCTGA






CTTCTGCTTTAAGGGGCATCGTTTAtgagaagggcCGAGGTAT

AAAGATGTGCCGCAACGCTCTGCCCCTTA






TGTGGCAGCAAAGTTCCTAAgacc

AAGCTTCTGCTTTAAGGGGCATCGTTTA



 


215
SpyCas9-
+
TTGGGTGTATGGGTCGTAGCGTTTTAGAGCTAGAAATAG
29128
ATGCCACTGAGAACTCTCTTGTTTTAGAG
29318



SpRY

CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA

CTAGAAATAGCAAGTTAAAATAAGGCTA






GTGGCACCGAGTCGGTGCccacaataccTCGGCCCTTCTCAGT

GTCCGTTATCAACTTGAAAAAGTGGCACC






TCGCTACGACCCAtaca

GAGTCGGTGC



 


217
SpyCas9-
−
GTTTTGGTCTTAGGAACTTTGTTTTAGAGCTAGAAATAGC
29129
CTTACTGTTAATGGAATCAGGTTTTAGAG
29319



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCtgagaagggcCGAGGTATTGTGGCA

GTCCGTTATCAACTTGAAAAAGTGGCACC






GCAAAGTTCCTAAgacc

GAGTCGGTGC



 


218
BlatCas9
−
gtggTTTTGGTCTTAGGAACTTTGCTATAGTTCCTTACTGAA
29130
cttaCTGTTAATGGAATCAGCCAGCTATAGT
29320





AGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCGT

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATAT

AGGGCAACAGACCCGAGGCGTTGGGGAT






TCAAAATAATGACAGACGAGCACCTTGGAGCATTTATCT

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






CCGAGGTGCTtgagaagggcCGAGGTATTGTGGCAGCAAAGTT

ATATTCAAAATAATGACAGACGAGCACCT






CCTAAgacc

TGGAGCATTTATCTCCGAGGTGCT



 


221
SpyCas9-
+
TTTGGGTGTATGGGTCGTAGGTTTTAGAGCTAGAAATAGC
29131
TGCCACTGAGAACTCTCTTAGTTTTAGAG
29321



NG

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCcacaataccTCGGCCCTTCTCAGTTC

GTCCGTTATCAACTTGAAAAAGTGGCACC






GCTACGACCCATacac

GAGTCGGTGC



 


224
SpyCas9-
−
GGTTTTGGTCTTAGGAACTTGTTTTAGAGCTAGAAATAGC
29132
TACTTACTGTTAATGGAATCGTTTTAGAG
29322



NG

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCgagaagggcCGAGGTATTGTGGCAG

GTCCGTTATCAACTTGAAAAAGTGGCACC






CAAAGTTCCTAAGacca

GAGTCGGTGC



 


228
SpyCas9-
+
TTTGGGTGTATGGGTCGTAGGTTTTAGAGCTAGAAATAGC
29133
TGCCACTGAGAACTCTCTTAGTTTTAGAG
29323



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCcacaataccTCGGCCCTTCTCAGTTC

GTCCGTTATCAACTTGAAAAAGTGGCACC






GCTACGACCCATacac

GAGTCGGTGC



 


230
SpyCas9-
−
GGTTTTGGTCTTAGGAACTTGTTTTAGAGCTAGAAATAGC
29134
TTACTGTTAATGGAATCAGCGTTTTAGAG
29324



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCgagaagggcCGAGGTATTGTGGCAG

GTCCGTTATCAACTTGAAAAAGTGGCACC






CAAAGTTCCTAAGacca

GAGTCGGTGC



 


231
BlatCas9
+
tcctTTGGGTGTATGGGTCGTAGGCTATAGTTCCTTACTGAA
29135
aaaaTGCCACTGAGAACTCTCTTGCTATAGT
29325





AGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCGT

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATAT

AGGGCAACAGACCCGAGGCGTTGGGGAT






TCAAAATAATGACAGACGAGCACCTTGGAGCATTTATCT

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






CCGAGGTGCTcacaataccTCGGCCCTTCTCAGTTCGCTACGA

ATATTCAAAATAATGACAGACGAGCACCT






CCCATacac

TGGAGCATTTATCTCCGAGGTGCT



 


232
BlatCas9
+
tcctTTGGGTGTATGGGTCGTAGGCTATAGTTCCTTACTGAA
29136
aaaaTGCCACTGAGAACTCTCTTGCTATAGT
29326





AGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCGT

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATAT

AGGGCAACAGACCCGAGGCGTTGGGGAT






TCAAAATAATGACAGACGAGCACCTTGGAGCATTTATCT

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






CCGAGGTGCTcacaataccTCGGCCCTTCTCAGTTCGCTACGA

ATATTCAAAATAATGACAGACGAGCACCT






CCCATacac

TGGAGCATTTATCTCCGAGGTGCT



 


238
SauCas9
+
atCCTTTGGGTGTATGGGTCGTAGTTTTAGTACTCTGGAAA
29137
taAAAAAGAAGTAAAATGCCACTGTTTTAG
29327





CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

TACTCTGGAAACAGAATCTACTAAAACAA






CGTCAACTTGTTGGCGAGAacaataccTCGGCCCTTCTCAGTT

GGCAAAATGCCGTGTTTATCTCGTCAACT






CGCTACGACCCATAcacc

TGTTGGCGAGA



 


239
SauCas9KKH
+
CCTTTGGGTGTATGGGTCGTAGTTTTAGTACTCTGGAAAC
29138
AAATGCCACTGAGAACTCTCTGTTTTAGT
29328





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

ACTCTGGAAACAGAATCTACTAAAACAAG






GTCAACTTGTTGGCGAGAacaataccTCGGCCCTTCTCAGTTC

GCAAAATGCCGTGTTTATCTCGTCAACTT






GCTACGACCCATAcacc

GTTGGCGAGA



 


242
ScaCas9-
+
CTTTGGGTGTATGGGTCGTAGTTTTAGAGCTAGAAATAGC
29139
ATGCCACTGAGAACTCTCTTGTTTTAGAG
29329



Sc++

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCacaataccTCGGCCCTTCTCAGTTCG

GTCCGTTATCAACTTGAAAAAGTGGCACC






CTACGACCCATAcacc

GAGTCGGTGC



 


243
SpyCas9-
+
CTTTGGGTGTATGGGTCGTAGTTTTAGAGCTAGAAATAGC
29140
GCCACTGAGAACTCTCTTAAGTTTTAGAG
29330



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCacaataccTCGGCCCTTCTCAGTTCG

GTCCGTTATCAACTTGAAAAAGTGGCACC






CTACGACCCATAcacc

GAGTCGGTGC



 


246
ScaCas9-
−
TGGTTTTGGTCTTAGGAACTGTTTTAGAGCTAGAAATAGC
29141
TTACTTACTGTTAATGGAATGTTTTAGAGC
29331



Sc++

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

TAGAAATAGCAAGTTAAAATAAGGCTAGT






TGGCACCGAGTCGGTGCagaagggcCGAGGTATTGTGGCAGC

CCGTTATCAACTTGAAAAAGTGGCACCGA






AAAGTTCCTAAGAccaa

GTCGGTGC



 


247
SpyCas9-
−
TGGTTTTGGTCTTAGGAACTGTTTTAGAGCTAGAAATAGC
29142
TACTGTTAATGGAATCAGCCGTTTTAGAG
29332



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCagaagggcCGAGGTATTGTGGCAGC

GTCCGTTATCAACTTGAAAAAGTGGCACC






AAAGTTCCTAAGAccaa

GAGTCGGTGC



 


251
SauCas9KKH
+
TCCTTTGGGTGTATGGGTCGTGTTTTAGTACTCTGGAAAC
29143
AAATGCCACTGAGAACTCTCTGTTTTAGT
29333





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

ACTCTGGAAACAGAATCTACTAAAACAAG






GTCAACTTGTTGGCGAGAcaataccTCGGCCCTTCTCAGTTCG

GCAAAATGCCGTGTTTATCTCGTCAACTT






CTACGACCCATACaccc

GTTGGCGAGA



 


252
SpyCas9-
+
CCTTTGGGTGTATGGGTCGTGTTTTAGAGCTAGAAATAGC
29144
TGCCACTGAGAACTCTCTTAGTTTTAGAG
29334



NG

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCcaataccTCGGCCCTTCTCAGTTCG

GTCCGTTATCAACTTGAAAAAGTGGCACC






CTACGACCCATACaccc

GAGTCGGTGC



 


256
SpyCas9-
+
CCTTTGGGTGTATGGGTCGTGTTTTAGAGCTAGAAATAGC
29145
CCACTGAGAACTCTCTTAAGGTTTTAGAG
29335



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCcaataccTCGGCCCTTCTCAGTTCG

GTCCGTTATCAACTTGAAAAAGTGGCACC






CTACGACCCATACaccc

GAGTCGGTGC



 


257
SpyCas9-
−
GTGGTTTTGGTCTTAGGAACGTTTTAGAGCTAGAAATAGC
29146
ACTGTTAATGGAATCAGCCAGTTTTAGAG
29336



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCgaagggcCGAGGTATTGTGGCAGC

GTCCGTTATCAACTTGAAAAAGTGGCACC






AAAGTTCCTAAGACcaaa

GAGTCGGTGC



 


258
BlatCas9
−
cctgTGGTTTTGGTCTTAGGAACGCTATAGTTCCTTACTGAA
29147
cttaCTGTTAATGGAATCAGCCAGCTATAGT
29337





AGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCGT

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATAT

AGGGCAACAGACCCGAGGCGTTGGGGAT






TCAAAATAATGACAGACGAGCACCTTGGAGCATTTATCT

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






CCGAGGTGCTgaagggcCGAGGTATTGTGGCAGCAAAGTTC

ATATTCAAAATAATGACAGACGAGCACCT






CTAAGACcaaa

TGGAGCATTTATCTCCGAGGTGCT



 


264
ScaCas9-
+
TCCTTTGGGTGTATGGGTCGGTTTTAGAGCTAGAAATAGC
29148
ATGCCACTGAGAACTCTCTTGTTTTAGAG
29338



Sc++

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCaataccTCGGCCCTTCTCAGTTCGC

GTCCGTTATCAACTTGAAAAAGTGGCACC






TACGACCCATACAccca

GAGTCGGTGC



 


265
SpyCas9-
+
TCCTTTGGGTGTATGGGTCGGTTTTAGAGCTAGAAATAGC
29149
CACTGAGAACTCTCTTAAGAGTTTTAGAG
29339



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCaataccTCGGCCCTTCTCAGTTCGC

GTCCGTTATCAACTTGAAAAAGTGGCACC






TACGACCCATACAccca

GAGTCGGTGC



 


266
SpyCas9-
−
TGTGGTTTTGGTCTTAGGAAGTTTTAGAGCTAGAAATAGC
29150
CTGTTAATGGAATCAGCCAAGTTTTAGAG
29340



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCaagggcCGAGGTATTGTGGCAGCA

GTCCGTTATCAACTTGAAAAAGTGGCACC






AAGTTCCTAAGACCaaaa

GAGTCGGTGC



 


268
SauriCas9-
+
AATCCTTTGGGTGTATGGGTCGTTTTAGTACTCTGGAAAC
29151
AAATGCCACTGAGAACTCTCTGTTTTAGT
29341



KKH

AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

ACTCTGGAAACAGAATCTACTAAAACAAG






GTCAACTTGTTGGCGAGAataccTCGGCCCTTCTCAGTTCGC

GCAAAATGCCGTGTTTATCTCGTCAACTT






TACGACCCATACACccaa

GTTGGCGAGA



 


269
SpyCas9-
+
ATCCTTTGGGTGTATGGGTCGTTTTAGAGCTAGAAATAGC
29152
ACTGAGAACTCTCTTAAGACGTTTTAGAG
29342



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCataccTCGGCCCTTCTCAGTTCGCT

GTCCGTTATCAACTTGAAAAAGTGGCACC






ACGACCCATACACccaa

GAGTCGGTGC



 


270
SpyCas9-
−
CTGTGGTTTTGGTCTTAGGAGTTTTAGAGCTAGAAATAGC
29153
TGTTAATGGAATCAGCCAAAGTTTTAGAG
29343



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCagggcCGAGGTATTGTGGCAGCAA

GTCCGTTATCAACTTGAAAAAGTGGCACC






AGTTCCTAAGACCAaaac

GAGTCGGTGC



 


271
BlatCas9
+
tcaaTCCTTTGGGTGTATGGGTCGCTATAGTTCCTTACTGAA
29154
tgccACTGAGAACTCTCTTAAGAGCTATAGT
29344





AGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCGT

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATAT

AGGGCAACAGACCCGAGGCGTTGGGGAT






TCAAAATAATGACAGACGAGCACCTTGGAGCATTTATCT

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






CCGAGGTGCTataccTCGGCCCTTCTCAGTTCGCTACGACCC

ATATTCAAAATAATGACAGACGAGCACCT






ATACACccaa

TGGAGCATTTATCTCCGAGGTGCT



 


272
BlatCas9
+
tcaaTCCTTTGGGTGTATGGGTCGCTATAGTTCCTTACTGAA
29155
tgccACTGAGAACTCTCTTAAGAGCTATAGT
29345





AGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCGT

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATAT

AGGGCAACAGACCCGAGGCGTTGGGGAT






TCAAAATAATGACAGACGAGCACCTTGGAGCATTTATCT

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






CCGAGGTGCTataccTCGGCCCTTCTCAGTTCGCTACGACCC

ATATTCAAAATAATGACAGACGAGCACCT






ATACACccaa

TGGAGCATTTATCTCCGAGGTGCT



 


274
BlatCas9
+
tcaaTCCTTTGGGTGTATGGGTCGCTATAGTTCCTTACTGAA
29156
tgccACTGAGAACTCTCTTAAGAGCTATAGT
29346





AGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCGT

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATAT

AGGGCAACAGACCCGAGGCGTTGGGGAT






TCAAAATAATGACAGACGAGCACCTTGGAGCATTTATCT

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






CCGAGGTGCTataccTCGGCCCTTCTCAGTTCGCTACGACCC

ATATTCAAAATAATGACAGACGAGCACCT






ATACACccaa

TGGAGCATTTATCTCCGAGGTGCT



 


275
SauCas9KKH
+
CAATCCTTTGGGTGTATGGGTGTTTTAGTACTCTGGAAAC
29157
ACTCTCTTAAGACTACCTTTCGTTTTAGTA
29347





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

CTCTGGAAACAGAATCTACTAAAACAAGG






GTCAACTTGTTGGCGAGAtaccTCGGCCCTTCTCAGTTCGCT

CAAAATGCCGTGTTTATCTCGTCAACTTGT






ACGACCCATACACCcaaa

TGGCGAGA



 


276
SpyCas9-
+
AATCCTTTGGGTGTATGGGTGTTTTAGAGCTAGAAATAGC
29158
TGCCACTGAGAACTCTCTTAGTTTTAGAG
29348



NG

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCtaccTCGGCCCTTCTCAGTTCGCT

GTCCGTTATCAACTTGAAAAAGTGGCACC






ACGACCCATACACCcaaa

GAGTCGGTGC



 


280
SpyCas9-
+
AATCCTTTGGGTGTATGGGTGTTTTAGAGCTAGAAATAGC
29159
CTGAGAACTCTCTTAAGACTGTTTTAGAG
29349



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCtaccTCGGCCCTTCTCAGTTCGCT

GTCCGTTATCAACTTGAAAAAGTGGCACC






ACGACCCATACACCcaaa

GAGTCGGTGC



 


281
SpyCas9-
−
CCTGTGGTTTTGGTCTTAGGGTTTTAGAGCTAGAAATAGC
29160
GTTAATGGAATCAGCCAAAAGTTTTAGAG
29350



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCgggcCGAGGTATTGTGGCAGCAA

GTCCGTTATCAACTTGAAAAAGTGGCACC






AGTTCCTAAGACCAAaacc

GAGTCGGTGC



 


286
ScaCas9-
+
CAATCCTTTGGGTGTATGGGGTTTTAGAGCTAGAAATAGC
29161
ATGCCACTGAGAACTCTCTTGTTTTAGAG
29351



Sc++

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCaccTCGGCCCTTCTCAGTTCGCTA

GTCCGTTATCAACTTGAAAAAGTGGCACC






CGACCCATACACCCaaag

GAGTCGGTGC



 


287
SpyCas9-
+
CAATCCTTTGGGTGTATGGGGTTTTAGAGCTAGAAATAGC
29162
TGAGAACTCTCTTAAGACTAGTTTTAGAG
29352



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCaccTCGGCCCTTCTCAGTTCGCTA

GTCCGTTATCAACTTGAAAAAGTGGCACC






CGACCCATACACCCaaag

GAGTCGGTGC



 


288
SpyCas9-
−
GCCTGTGGTTTTGGTCTTAGGTTTTAGAGCTAGAAATAGC
29163
TTAATGGAATCAGCCAAAATGTTTTAGAG
29353



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCggcCGAGGTATTGTGGCAGCAAA

GTCCGTTATCAACTTGAAAAAGTGGCACC






GTTCCTAAGACCAAAacca

GAGTCGGTGC



 


293
SpyCas9-
−
AGCCTGTGGTTTTGGTCTTAGTTTTAGAGCTAGAAATAGC
29164
TGGAATCAGCCAAAATCTTAGTTTTAGAG
29354



NG

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCgcCGAGGTATTGTGGCAGCAAAG

GTCCGTTATCAACTTGAAAAAGTGGCACC






TTCCTAAGACCAAAAccac

GAGTCGGTGC



 


297
SpyCas9-
−
AGCCTGTGGTTTTGGTCTTAGTTTTAGAGCTAGAAATAGC
29165
TAATGGAATCAGCCAAAATCGTTTTAGAG
29355



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCgcCGAGGTATTGTGGCAGCAAAG

GTCCGTTATCAACTTGAAAAAGTGGCACC






TTCCTAAGACCAAAAccac

GAGTCGGTGC



 


298
SpyCas9-
+
TCAATCCTTTGGGTGTATGGGTTTTAGAGCTAGAAATAGC
29166
GAGAACTCTCTTAAGACTACGTTTTAGAG
29356



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCccTCGGCCCTTCTCAGTTCGCTAC

GTCCGTTATCAACTTGAAAAAGTGGCACC






GACCCATACACCCAaagg

GAGTCGGTGC



 


299
BlatCas9
−
tcaaGCCTGTGGTTTTGGTCTTAGCTATAGTTCCTTACTGAA
29167
gttaATGGAATCAGCCAAAATCTGCTATAGT
29357





AGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCGT

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATAT

AGGGCAACAGACCCGAGGCGTTGGGGAT






TCAAAATAATGACAGACGAGCACCTTGGAGCATTTATCT

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






CCGAGGTGCTgcCGAGGTATTGTGGCAGCAAAGTTCCTAA

ATATTCAAAATAATGACAGACGAGCACCT






GACCAAAAccac

TGGAGCATTTATCTCCGAGGTGCT



 


300
BlatCas9
−
tcaaGCCTGTGGTTTTGGTCTTAGCTATAGTTCCTTACTGAA
29168
gttaATGGAATCAGCCAAAATCTGCTATAGT
29358





AGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCGT

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATAT

AGGGCAACAGACCCGAGGCGTTGGGGAT






TCAAAATAATGACAGACGAGCACCTTGGAGCATTTATCT

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






CCGAGGTGCTgcCGAGGTATTGTGGCAGCAAAGTTCCTAA

ATATTCAAAATAATGACAGACGAGCACCT






GACCAAAAccac

TGGAGCATTTATCTCCGAGGTGCT



 


306
SauCas9
−
ctCAAGCCTGTGGTTTTGGTCTTGTTTTAGTACTCTGGAAA
29169
gaATCAGCCAAAATCTTAAGCTGGTTTTAG
29359





CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

TACTCTGGAAACAGAATCTACTAAAACAA






CGTCAACTTGTTGGCGAGAcCGAGGTATTGTGGCAGCAA

GGCAAAATGCCGTGTTTATCTCGTCAACT






AGTTCCTAAGACCAAAACcaca

TGTTGGCGAGA



 


307
SauCas9KKH
−
CAAGCCTGTGGTTTTGGTCTTGTTTTAGTACTCTGGAAAC
29170
TTAATGGAATCAGCCAAAATCGTTTTAGT
29360





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

ACTCTGGAAACAGAATCTACTAAAACAAG






GTCAACTTGTTGGCGAGAcCGAGGTATTGTGGCAGCAAA

GCAAAATGCCGTGTTTATCTCGTCAACTT






GTTCCTAAGACCAAAACcaca

GTTGGCGAGA



 


310
ScaCas9-
−
AAGCCTGTGGTTTTGGTCTTGTTTTAGAGCTAGAAATAGC
29171
ATGGAATCAGCCAAAATCTTGTTTTAGAG
29361



Sc++

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCcCGAGGTATTGTGGCAGCAAAG

GTCCGTTATCAACTTGAAAAAGTGGCACC






TTCCTAAGACCAAAACcaca

GAGTCGGTGC



 


311
SpyCas9
−
AAGCCTGTGGTTTTGGTCTTGTTTTAGAGCTAGAAATAGC
29172
CAGCCAAAATCTTAAGCTGCGTTTTAGAG
29362





AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCcCGAGGTATTGTGGCAGCAAAG

GTCCGTTATCAACTTGAAAAAGTGGCACC






TTCCTAAGACCAAAACcaca

GAGTCGGTGC



 


314
SpyCas9-
−
AAGCCTGTGGTTTTGGTCTTGTTTTAGAGCTAGAAATAGC
29173
AATGGAATCAGCCAAAATCTGTTTTAGAG
29363



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCcCGAGGTATTGTGGCAGCAAAG

GTCCGTTATCAACTTGAAAAAGTGGCACC






TTCCTAAGACCAAAACcaca

GAGTCGGTGC



 


315
SpyCas9-
+
CTCAATCCTTTGGGTGTATGGTTTTAGAGCTAGAAATAGC
29174
TGCCACTGAGAACTCTCTTAGTTTTAGAG
29364



NG

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCcTCGGCCCTTCTCAGTTCGCTAC

GTCCGTTATCAACTTGAAAAAGTGGCACC






GACCCATACACCCAAagga

GAGTCGGTGC



 


318
SpyCas9-
−
AAGCCTGTGGTTTTGGTCTTGTTTTAGAGCTAGAAATAGC
29175
TGGAATCAGCCAAAATCTTAGTTTTAGAG
29365



NG

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCcCGAGGTATTGTGGCAGCAAAG

GTCCGTTATCAACTTGAAAAAGTGGCACC






TTCCTAAGACCAAAACcaca

GAGTCGGTGC



 


322
SpyCas9-
+
CTCAATCCTTTGGGTGTATGGTTTTAGAGCTAGAAATAGC
29176
AGAACTCTCTTAAGACTACCGTTTTAGAG
29366



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCcTCGGCCCTTCTCAGTTCGCTAC

GTCCGTTATCAACTTGAAAAAGTGGCACC






GACCCATACACCCAAagga

GAGTCGGTGC



 


328
SauCas9
−
acTCAAGCCTGTGGTTTTGGTCTGTTTTAGTACTCTGGAAA
29177
gaATCAGCCAAAATCTTAAGCTGGTTTTAG
29367





CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

TACTCTGGAAACAGAATCTACTAAAACAA






CGTCAACTTGTTGGCGAGACGAGGTATTGTGGCAGCAAA

GGCAAAATGCCGTGTTTATCTCGTCAACT






GTTCCTAAGACCAAAACCacag

TGTTGGCGAGA



 


329
SauCas9KKH
−
TCAAGCCTGTGGTTTTGGTCTGTTTTAGTACTCTGGAAAC
29178
TTAATGGAATCAGCCAAAATCGTTTTAGT
29368





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

ACTCTGGAAACAGAATCTACTAAAACAAG






GTCAACTTGTTGGCGAGACGAGGTATTGTGGCAGCAAAG

GCAAAATGCCGTGTTTATCTCGTCAACTT






TTCCTAAGACCAAAACCacag

GTTGGCGAGA



 


330
SauriCas9
−
TCAAGCCTGTGGTTTTGGTCTGTTTTAGTACTCTGGAAAC
29179
ATCAGCCAAAATCTTAAGCTGGTTTTAGT
29369





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

ACTCTGGAAACAGAATCTACTAAAACAAG






GTCAACTTGTTGGCGAGACGAGGTATTGTGGCAGCAAAG

GCAAAATGCCGTGTTTATCTCGTCAACTT






TTCCTAAGACCAAAACCacag

GTTGGCGAGA



 


331
SauriCas9-
−
TCAAGCCTGTGGTTTTGGTCTGTTTTAGTACTCTGGAAAC
29180
TAATGGAATCAGCCAAAATCTGTTTTAGT
29370



KKH

AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

ACTCTGGAAACAGAATCTACTAAAACAAG






GTCAACTTGTTGGCGAGACGAGGTATTGTGGCAGCAAAG

GCAAAATGCCGTGTTTATCTCGTCAACTT






TTCCTAAGACCAAAACCacag

GTTGGCGAGA



 


334
ScaCas9-
+
CCTCAATCCTTTGGGTGTATGTTTTAGAGCTAGAAATAGC
29181
TTAAGACTACCTTTCTCCAAGTTTTAGAGC
29371



Sc++

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

TAGAAATAGCAAGTTAAAATAAGGCTAGT






TGGCACCGAGTCGGTGCTCGGCCCTTCTCAGTTCGCTACG

CCGTTATCAACTTGAAAAAGTGGCACCGA






ACCCATACACCCAAAggat

GTCGGTGC



 


335
SpyCas9
+
CCTCAATCCTTTGGGTGTATGTTTTAGAGCTAGAAATAGC
29182
TAAGACTACCTTTCTCCAAAGTTTTAGAG
29372





AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCTCGGCCCTTCTCAGTTCGCTACG

GTCCGTTATCAACTTGAAAAAGTGGCACC






ACCCATACACCCAAAggat

GAGTCGGTGC



 


338
SpyCas9-
+
CCTCAATCCTTTGGGTGTATGTTTTAGAGCTAGAAATAGC
29183
GAACTCTCTTAAGACTACCTGTTTTAGAG
29373



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCTCGGCCCTTCTCAGTTCGCTACG

GTCCGTTATCAACTTGAAAAAGTGGCACC






ACCCATACACCCAAAggat

GAGTCGGTGC



 


341
ScaCas9-
−
CAAGCCTGTGGTTTTGGTCTGTTTTAGAGCTAGAAATAGC
29184
ATGGAATCAGCCAAAATCTTGTTTTAGAG
29374



Sc++

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCCGAGGTATTGTGGCAGCAAAGT

GTCCGTTATCAACTTGAAAAAGTGGCACC






TCCTAAGACCAAAACCacag

GAGTCGGTGC



 


342
SpyCas9-
−
CAAGCCTGTGGTTTTGGTCTGTTTTAGAGCTAGAAATAGC
29185
ATGGAATCAGCCAAAATCTTGTTTTAGAG
29375



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCCGAGGTATTGTGGCAGCAAAGT

GTCCGTTATCAACTTGAAAAAGTGGCACC






TCCTAAGACCAAAACCacag

GAGTCGGTGC



 


343
SpyCas9-
+
CCTCAATCCTTTGGGTGTATGTTTTAGAGCTAGAAATAGC
29186
TGCCACTGAGAACTCTCTTAGTTTTAGAG
29376



NG

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCTCGGCCCTTCTCAGTTCGCTACG

GTCCGTTATCAACTTGAAAAAGTGGCACC






ACCCATACACCCAAAggat

GAGTCGGTGC



 


346
BlatCas9
+
agacCTCAATCCTTTGGGTGTATGCTATAGTTCCTTACTGAA
29187
tgagAACTCTCTTAAGACTACCTGCTATAGT
29377





AGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCGT

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATAT

AGGGCAACAGACCCGAGGCGTTGGGGAT






TCAAAATAATGACAGACGAGCACCTTGGAGCATTTATCT

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






CCGAGGTGCTTCGGCCCTTCTCAGTTCGCTACGACCCATA

ATATTCAAAATAATGACAGACGAGCACCT






CACCCAAAggat

TGGAGCATTTATCTCCGAGGTGCT



 


347
BlatCas9
+
agacCTCAATCCTTTGGGTGTATGCTATAGTTCCTTACTGAA
29188
tgagAACTCTCTTAAGACTACCTGCTATAGT
29378





AGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAGGCGT

TCCTTACTGAAAGGTAAGTTGCTATAGTA






TGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCCCCATAT

AGGGCAACAGACCCGAGGCGTTGGGGAT






TCAAAATAATGACAGACGAGCACCTTGGAGCATTTATCT

CGCCTAGCCCGTGTTTACGGGCTCTCCCC






CCGAGGTGCTTCGGCCCTTCTCAGTTCGCTACGACCCATA

ATATTCAAAATAATGACAGACGAGCACCT






CACCCAAAggat

TGGAGCATTTATCTCCGAGGTGCT



 


351
SauCas9KKH
−
CTCAAGCCTGTGGTTTTGGTCGTTTTAGTACTCTGGAAAC
29189
TTAATGGAATCAGCCAAAATCGTTTTAGT
29379





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

ACTCTGGAAACAGAATCTACTAAAACAAG






GTCAACTTGTTGGCGAGAGAGGTATTGTGGCAGCAAAGT

GCAAAATGCCGTGTTTATCTCGTCAACTT






TCCTAAGACCAAAACCAcagg

GTTGGCGAGA



 


352
SauriCas9
+
GACCTCAATCCTTTGGGTGTAGTTTTAGTACTCTGGAAAC
29190
CTTAAGACTACCTTTCTCCAAGTTTTAGTA
29380





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

CTCTGGAAACAGAATCTACTAAAACAAGG






GTCAACTTGTTGGCGAGACGGCCCTTCTCAGTTCGCTACG

CAAAATGCCGTGTTTATCTCGTCAACTTGT






ACCCATACACCCAAAGgatt

TGGCGAGA



 


353
SauriCas9-
+
GACCTCAATCCTTTGGGTGTAGTTTTAGTACTCTGGAAAC
29191
CTTAAGACTACCTTTCTCCAAGTTTTAGTA
29381



KKH

AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

CTCTGGAAACAGAATCTACTAAAACAAGG






GTCAACTTGTTGGCGAGACGGCCCTTCTCAGTTCGCTACG

CAAAATGCCGTGTTTATCTCGTCAACTTGT






ACCCATACACCCAAAGgatt

TGGCGAGA



 


354
SauriCas9-
−
CTCAAGCCTGTGGTTTTGGTCGTTTTAGTACTCTGGAAAC
29192
TAATGGAATCAGCCAAAATCTGTTTTAGT
29382



KKH

AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

ACTCTGGAAACAGAATCTACTAAAACAAG






GTCAACTTGTTGGCGAGAGAGGTATTGTGGCAGCAAAGT

GCAAAATGCCGTGTTTATCTCGTCAACTT






TCCTAAGACCAAAACCAcagg

GTTGGCGAGA



 


357
ScaCas9-
+
ACCTCAATCCTTTGGGTGTAGTTTTAGAGCTAGAAATAGC
29193
TTAAGACTACCTTTCTCCAAGTTTTAGAGC
29383



Sc++

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

TAGAAATAGCAAGTTAAAATAAGGCTAGT






TGGCACCGAGTCGGTGCCGGCCCTTCTCAGTTCGCTACGA

CCGTTATCAACTTGAAAAAGTGGCACCGA






CCCATACACCCAAAGgatt

GTCGGTGC



 


358
SpyCas9
+
ACCTCAATCCTTTGGGTGTAGTTTTAGAGCTAGAAATAGC
29194
TAAGACTACCTTTCTCCAAAGTTTTAGAG
29384





AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCCGGCCCTTCTCAGTTCGCTACGA

GTCCGTTATCAACTTGAAAAAGTGGCACC






CCCATACACCCAAAGgatt

GAGTCGGTGC



 


361
SpyCas9-
+
ACCTCAATCCTTTGGGTGTAGTTTTAGAGCTAGAAATAGC
29195
AACTCTCTTAAGACTACCTTGTTTTAGAGC
29385



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

TAGAAATAGCAAGTTAAAATAAGGCTAGT






TGGCACCGAGTCGGTGCCGGCCCTTCTCAGTTCGCTACGA

CCGTTATCAACTTGAAAAAGTGGCACCGA






CCCATACACCCAAAGgatt

GTCGGTGC



 


362
SpyCas9-
+
ACCTCAATCCTTTGGGTGTAGTTTTAGAGCTAGAAATAGC
29196
TAAGACTACCTTTCTCCAAAGTTTTAGAG
29386



NG

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCCGGCCCTTCTCAGTTCGCTACGA

GTCCGTTATCAACTTGAAAAAGTGGCACC






CCCATACACCCAAAGgatt

GAGTCGGTGC



 


365
SpyCas9-
−
TCAAGCCTGTGGTTTTGGTCGTTTTAGAGCTAGAAATAGC
29197
TGGAATCAGCCAAAATCTTAGTTTTAGAG
29387



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCGAGGTATTGTGGCAGCAAAGTT

GTCCGTTATCAACTTGAAAAAGTGGCACC






CCTAAGACCAAAACCAcagg

GAGTCGGTGC



 


366
St1Cas9
−
TCAAGCCTGTGGTTTTGGTCGTCTTTGTACTCTGGTACCA
29198
GTGTAAATTACTTACTGTTAGTCTTTGTAC
29388





GAAGCTACAAAGATAAGGCTTCATGCCGAAATCAACACC

TCTGGTACCAGAAGCTACAAAGATAAGGC






CTGTCATTTTATGGCAGGGTGTTTTGAGGTATTGTGGCAG

TTCATGCCGAAATCAACACCCTGTCATTTT






CAAAGTTCCTAAGACCAAAACCAcagg

ATGGCAGGGTGTTTT



 


369
SauCas9
+
caAGACCTCAATCCTTTGGGTGTGTTTTAGTACTCTGGAAA
29199
taAAAAAGAAGTAAAATGCCACTGTTTTAG
29389





CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

TACTCTGGAAACAGAATCTACTAAAACAA






CGTCAACTTGTTGGCGAGAGGCCCTTCTCAGTTCGCTACG

GGCAAAATGCCGTGTTTATCTCGTCAACT






ACCCATACACCCAAAGGattg

TGTTGGCGAGA



 


370
SauCas9KKH
+
AGACCTCAATCCTTTGGGTGTGTTTTAGTACTCTGGAAAC
29200
ACTCTCTTAAGACTACCTTTCGTTTTAGTA
29390





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

CTCTGGAAACAGAATCTACTAAAACAAGG






GTCAACTTGTTGGCGAGAGGCCCTTCTCAGTTCGCTACGA

CAAAATGCCGTGTTTATCTCGTCAACTTGT






CCCATACACCCAAAGGattg

TGGCGAGA



 


371
SauCas9
+
caAGACCTCAATCCTTTGGGTGTGTTTTAGTACTCTGGAAA
29201
taAAAAAGAAGTAAAATGCCACTGTTTTAG
29391





CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT

TACTCTGGAAACAGAATCTACTAAAACAA






CGTCAACTTGTTGGCGAGAGGCCCTTCTCAGTTCGCTACG

GGCAAAATGCCGTGTTTATCTCGTCAACT






ACCCATACACCCAAAGGattg

TGTTGGCGAGA



 


372
SauCas9KKH
+
AGACCTCAATCCTTTGGGTGTGTTTTAGTACTCTGGAAAC
29202
ACTCTCTTAAGACTACCTTTCGTTTTAGTA
29392





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

CTCTGGAAACAGAATCTACTAAAACAAGG






GTCAACTTGTTGGCGAGAGGCCCTTCTCAGTTCGCTACGA

CAAAATGCCGTGTTTATCTCGTCAACTTGT






CCCATACACCCAAAGGattg

TGGCGAGA



 


375
SauCas9KKH
−
ACTCAAGCCTGTGGTTTTGGTGTTTTAGTACTCTGGAAAC
29203
AATCAGCCAAAATCTTAAGCTGTTTTAGT
29393





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

ACTCTGGAAACAGAATCTACTAAAACAAG






GTCAACTTGTTGGCGAGAAGGTATTGTGGCAGCAAAGTT

GCAAAATGCCGTGTTTATCTCGTCAACTT






CCTAAGACCAAAACCACaggc

GTTGGCGAGA



 


376
SauriCas9
+
AGACCTCAATCCTTTGGGTGTGTTTTAGTACTCTGGAAAC
29204
CTTAAGACTACCTTTCTCCAAGTTTTAGTA
29394





AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

CTCTGGAAACAGAATCTACTAAAACAAGG






GTCAACTTGTTGGCGAGAGGCCCTTCTCAGTTCGCTACGA

CAAAATGCCGTGTTTATCTCGTCAACTTGT






CCCATACACCCAAAGGattg

TGGCGAGA



 


377
SauriCas9-
+
AGACCTCAATCCTTTGGGTGTGTTTTAGTACTCTGGAAAC
29205
CTTAAGACTACCTTTCTCCAAGTTTTAGTA
29395



KKH

AGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTC

CTCTGGAAACAGAATCTACTAAAACAAGG






GTCAACTTGTTGGCGAGAGGCCCTTCTCAGTTCGCTACGA

CAAAATGCCGTGTTTATCTCGTCAACTTGT






CCCATACACCCAAAGGattg

TGGCGAGA



 


380
ScaCas9-
+
GACCTCAATCCTTTGGGTGTGTTTTAGAGCTAGAAATAGC
29206
TTAAGACTACCTTTCTCCAAGTTTTAGAGC
29396



Sc++

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

TAGAAATAGCAAGTTAAAATAAGGCTAGT






TGGCACCGAGTCGGTGCGGCCCTTCTCAGTTCGCTACGAC

CCGTTATCAACTTGAAAAAGTGGCACCGA






CCATACACCCAAAGGattg

GTCGGTGC



 


381
SpyCas9-
+
GACCTCAATCCTTTGGGTGTGTTTTAGAGCTAGAAATAGC
29207
ACTCTCTTAAGACTACCTTTGTTTTAGAGC
29397



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

TAGAAATAGCAAGTTAAAATAAGGCTAGT






TGGCACCGAGTCGGTGCGGCCCTTCTCAGTTCGCTACGAC

CCGTTATCAACTTGAAAAAGTGGCACCGA






CCATACACCCAAAGGattg

GTCGGTGC



 


382
SpyCas9-
−
CTCAAGCCTGTGGTTTTGGTGTTTTAGAGCTAGAAATAGC
29208
GGAATCAGCCAAAATCTTAAGTTTTAGAG
29398



SpRY

AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG

CTAGAAATAGCAAGTTAAAATAAGGCTA






TGGCACCGAGTCGGTGCAGGTATTGTGGCAGCAAAGTTC

GTCCGTTATCAACTTGAAAAAGTGGCACC






CTAAGACCAAAACCACaggc

GAGTCGGTGC
















TABLE 4B







Exemplary template RNA sequences and second nick gRNA spacer sequences


Table 4B provides design of RNA components of gene modifying systems for correcting the pathogenic R261Q, mutation in PAH. The gRNA


spacers from Table 1B were filtered, e.g., filtered by occurrence within 15 nt of the desired editing location and use of a Tier 1 Cas


enzyme. For each gRNA ID, this table details the sequence of a complete template RNA, optional second-nick gRNA, and Cas variant for use in


a Cas-RT fusion gene modifying polypeptide. For exemplification, PBS sequences and post-edit homology regions (after the location of the


edit) are set to 12 nt and 30 nt, respectively. Additionally, a second-nick gRNA is selected with preference for a distance near 100 nt


from the first nick and a first preference for a design resulting in a PAM-in system, as described elsewhere in this application.

















SEQ

SEQ



Cas


ID

ID


ID
species
strand
Template RNA
NO
second-nick gRNA
NO
















1
Nme2Cas9
−
tcTTGGGTGGCCTGGCCTTCCAAGGTTGTAG
29399
gcAGCAGGAAAAGATGGCGCTCATGTTGTAGCTCCCT
29576





CTCCCTTTCTCATTTCGGAAACGAAATGAGA

TTCTCATTTCGGAAACGAAATGAGAACCGTTGCTACA






ACCGTTGCTACAATAAGGCCGTCTGAAAAG

ATAAGGCCGTCTGAAAAGATGTGCCGCAACGCTCTGC






ATGTGCCGCAACGCTCTGCCCCTTAAAGCTT

CCCTTAAAGCTTCTGCTTTAAGGGGCATCGTTTA






CTGCTTTAAGGGGCATCGTTTAgtctgatgtactgtg








tgcagtggaagacTCGGAAGGCCaggc





 


2
SpyCas9-
−
GGGTGGCCTGGCCTTCCAAGGTTTTAGAGC
29400
AGGAAAAGATGGCGCTCATTGTTTTAGAGCTAGAAAT
29577



SpRY

TAGAAATAGCAAGTTAAAATAAGGCTAGTC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






CGTTATCAACTTGAAAAAGTGGCACCGAGT

AAAAGTGGCACCGAGTCGGTGC






CGGTGCgtctgatgtactgtgtgcagtggaagacTCGGAAG








GCCaggc





 


3
BlatCas9
−
cttgGGTGGCCTGGCCTTCCAAGGCTATAGTT
29401
agcaGGAAAAGATGGCGCTCATTGCTATAGTTCCTTAC
29578





CCTTACTGAAAGGTAAGTTGCTATAGTAAG

TGAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCC






GGCAACAGACCCGAGGCGTTGGGGATCGCC

GAGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCT






TAGCCCGTGTTTACGGGCTCTCCCCATATTC

CTCCCCATATTCAAAATAATGACAGACGAGCACCTTG






AAAATAATGACAGACGAGCACCTTGGAGCA

GAGCATTTATCTCCGAGGTGCT






TTTATCTCCGAGGTGCTgtctgatgtactgtgtgcagtgg








aagacTCGGAAGGCCaggc





 


4
SauCas9KKH
+
CTGTGTGCAGTGGAAGACTTGGTTTTAGTAC
29402
CTGACTCAGTGGTGATGAGCTGTTTTAGTACTCTGGA
29579





TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAcgggatttcttgggtggcctggccttcCGAGTCTTC








CActgc





 


5
SauriCas9-
+
CTGTGTGCAGTGGAAGACTTGGTTTTAGTAC
29403
TGACTCAGTGGTGATGAGCTTGTTTTAGTACTCTGGA
29580



KKH

TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAcgggatttcttgggtggcctggccttcCGAGTCTTC








CActgc





 


8
SpyCas9-
+
TGTGTGCAGTGGAAGACTTGGTTTTAGAGC
29404
GGTGATGAGCTTTGAGTTTTGTTTTAGAGCTAGAAAT
29581



SpRY

TAGAAATAGCAAGTTAAAATAAGGCTAGTC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






CGTTATCAACTTGAAAAAGTGGCACCGAGT

AAAAGTGGCACCGAGTCGGTGC






CGGTGCcgggatttcttgggtggcctggccttcCGAGTCTT








CCActgc





 


10
SpyCas9-
−
TGGGTGGCCTGGCCTTCCAAGTTTTAGAGCT
29405
GGAAAAGATGGCGCTCATTGGTTTTAGAGCTAGAAAT
29582



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCtctgatgtactgtgtgcagtggaagacTCGGAAGGC








CAggcc





 


13
SauCas9KKH
+
ACTGTGTGCAGTGGAAGACTTGTTTTAGTAC
29406
CTGACTCAGTGGTGATGAGCTGTTTTAGTACTCTGGA
29583





TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAgggatttcttgggtggcctggccttcCGAGTCTTCC








ACtgca





 


14
SpyCas9-
−
TTGGGTGGCCTGGCCTTCCAGTTTTAGAGCT
29407
GGAAAAGATGGCGCTCATTGGTTTTAGAGCTAGAAAT
29584



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCctgatgtactgtgtgcagtggaagacTCGGAAGGC








CAGgcca





 


17
SpyCas9-
+
CTGTGTGCAGTGGAAGACTTGTTTTAGAGCT
29408
CTCAGTGGTGATGAGCTTTGGTTTTAGAGCTAGAAAT
29585



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCgggatttcttgggtggcctggccttcCGAGTCTTCC








ACtgca





 


21
SpyCas9-
−
TTGGGTGGCCTGGCCTTCCAGTTTTAGAGCT
29409
GAAAAGATGGCGCTCATTGTGTTTTAGAGCTAGAAAT
29586



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCctgatgtactgtgtgcagtggaagacTCGGAAGGC








CAGgcca





 


23
SpyCas9-
+
CTGTGTGCAGTGGAAGACTTGTTTTAGAGCT
29410
GTGATGAGCTTTGAGTTTTCGTTTTAGAGCTAGAAAT
29587



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCgggatttcttgggtggcctggccttcCGAGTCTTCC








ACtgca





 


29
SauCas9
+
tgTACTGTGTGCAGTGGAAGACTGTTTTAGT
29411
ctCTGACTCAGTGGTGATGAGCTGTTTTAGTACTCTGG
29588





ACTCTGGAAACAGAATCTACTAAAACAAGG

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT






CAAAATGCCGTGTTTATCTCGTCAACTTGTT

TTATCTCGTCAACTTGTTGGCGAGA






GGCGAGAggatttcttgggtggcctggccttcCGAGTCTT








CCACTgcac





 


30
SauCas9KKH
+
TACTGTGTGCAGTGGAAGACTGTTTTAGTAC
29412
CTGACTCAGTGGTGATGAGCTGTTTTAGTACTCTGGA
29589





TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAggatttcttgggtggcctggccttcCGAGTCTTCC








ACTgcac





 


33
ScaCas9-
−
CTTGGGTGGCCTGGCCTTCCGTTTTAGAGCT
29413
AAAGATGGCGCTCATTGTGCGTTTTAGAGCTAGAAAT
29590



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCtgatgtactgtgtgcagtggaagacTCGGAAGGCC








AGGccac





 


34
SpyCas9-
−
CTTGGGTGGCCTGGCCTTCCGTTTTAGAGCT
29414
AAAAGATGGCGCTCATTGTGGTTTTAGAGCTAGAAAT
29591



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCtgatgtactgtgtgcagtggaagacTCGGAAGGCC








AGGccac





 


37
ScaCas9-
−
ACTGTGTGCAGTGGAAGACTGTTTTAGAGC
29415
ACTCAGTGGTGATGAGCTTTGTTTTAGAGCTAGAAAT
29592



Sc++

TAGAAATAGCAAGTTAAAATAAGGCTAGTC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






CGTTATCAACTTGAAAAAGTGGCACCGAGT

AAAAGTGGCACCGAGTCGGTGC






CGGTGCggatttcttgggtggcctggccttcCGAGTCTTCC








ACTgcac





 


38
SpyCas9
+
ACTGTGTGCAGTGGAAGACTGTTTTAGAGC
29416
TCCTAGTGCCTCTGACTCAGGTTTTAGAGCTAGAAAT
29593





TAGAAATAGCAAGTTAAAATAAGGCTAGTC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






CGTTATCAACTTGAAAAAGTGGCACCGAGT

AAAAGTGGCACCGAGTCGGTGC






CGGTGCggatttcttgggtggcctggccttcCGAGTCTTCC








ACTgcac





 


41
SpyCas9-
+
ACTGTGTGCAGTGGAAGACTGTTTTAGAGC
29417
TGATGAGCTTTGAGTTTTCTGTTTTAGAGCTAGAAAT
29594



SpRY

TAGAAATAGCAAGTTAAAATAAGGCTAGTC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






CGTTATCAACTTGAAAAAGTGGCACCGAGT

AAAAGTGGCACCGAGTCGGTGC






CGGTGCggatttcttgggtggcctggccttcCGAGTCTTCC








ACTgcac





 


42
SpyCas9-
+
ACTGTGTGCAGTGGAAGACTGTTTTAGAGC
29418
CTCAGTGGTGATGAGCTTTGGTTTTAGAGCTAGAAAT
29595



NG

TAGAAATAGCAAGTTAAAATAAGGCTAGTC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






CGTTATCAACTTGAAAAAGTGGCACCGAGT

AAAAGTGGCACCGAGTCGGTGC






CGGTGCggatttcttgggtggcctggccttcCGAGTCTTCC








ACTgcac





 


45
BlatCas9
−
tttcTTGGGTGGCCTGGCCTTCCGCTATAGTTC
29419
ggaaAAGATGGCGCTCATTGTGCGCTATAGTTCCTTACT
29596





CTTACTGAAAGGTAAGTTGCTATAGTAAGG

GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG






GCAACAGACCCGAGGCGTTGGGGATCGCCT

AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC






AGCCCGTGTTTACGGGCTCTCCCCATATTCA

TCCCCATATTCAAAATAATGACAGACGAGCACCTTGG






AAATAATGACAGACGAGCACCTTGGAGCAT

AGCATTTATCTCCGAGGTGCT






TTATCTCCGAGGTGCTtgatgtactgtgtgcagtggaaga








cTCGGAAGGCCAGGccac





 


51
SauCas9
+
atGTACTGTGTGCAGTGGAAGACGTTTTAGT
29420
ctCTGACTCAGTGGTGATGAGCTGTTTTAGTACTCTGG
29597





ACTCTGGAAACAGAATCTACTAAAACAAGG

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT






CAAAATGCCGTGTTTATCTCGTCAACTTGTT

TTATCTCGTCAACTTGTTGGCGAGA






GGCGAGAgatttcttgggtggcctggccttcCGAGTCTTC








CACTGcaca





 


52
SauCas9KKH
+
GTACTGTGTGCAGTGGAAGACGTTTTAGTA
29421
AGTTTTCTTTCTTCTTTTCATGTTTTAGTACTCTGGAA
29598





CTCTGGAAACAGAATCTACTAAAACAAGGC

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTA






AAAATGCCGTGTTTATCTCGTCAACTTGTTG

TCTCGTCAACTTGTTGGCGAGA






GCGAGAgatttcttgggtggcctggccttcCGAGTCTTCC








ACTGcaca





 


53
SauriCas9
+
GTACTGTGTGCAGTGGAAGACGTTTTAGTA
29422
TTCTTTTCATCCCAGCTTGCAGTTTTAGTACTCTGGAA
29599





CTCTGGAAACAGAATCTACTAAAACAAGGC

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTA






AAAATGCCGTGTTTATCTCGTCAACTTGTTG

TCTCGTCAACTTGTTGGCGAGA






GCGAGAgatttcttgggtggcctggccttcCGAGTCTTCC








ACTGcaca





 


54
SauriCas9-
+
GTACTGTGTGCAGTGGAAGACGTTTTAGTA
29423
TGACTCAGTGGTGATGAGCTTGTTTTAGTACTCTGGA
29600



KKH

CTCTGGAAACAGAATCTACTAAAACAAGGC

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAAATGCCGTGTTTATCTCGTCAACTTGTTG

ATCTCGTCAACTTGTTGGCGAGA






GCGAGAgatttcttgggtggcctggccttcCGAGTCTTCC








ACTGcaca





 


55
SauriCas9-
−
TTCTTGGGTGGCCTGGCCTTCGTTTTAGTAC
29424
AAAAGATGGCGCTCATTGTGCGTTTTAGTACTCTGGA
29601



KKH

TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAgatgtactgtgtgcagtggaagacTCGGAAGGCC








AGGCcacc





 


60
ScaCas9-
+
TACTGTGTGCAGTGGAAGACGTTTTAGAGC
29425
ACTCAGTGGTGATGAGCTTTGTTTTAGAGCTAGAAAT
29602



Sc++

TAGAAATAGCAAGTTAAAATAAGGCTAGTC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






CGTTATCAACTTGAAAAAGTGGCACCGAGT

AAAAGTGGCACCGAGTCGGTGC






CGGTGCgatttcttgggtggcctggccttcCGAGTCTTCC








ACTGcaca





 


61
SpyCas9-
+
TACTGTGTGCAGTGGAAGACGTTTTAGAGC
29426
GATGAGCTTTGAGTTTTCTTGTTTTAGAGCTAGAAAT
29603



SpRY

TAGAAATAGCAAGTTAAAATAAGGCTAGTC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






CGTTATCAACTTGAAAAAGTGGCACCGAGT

AAAAGTGGCACCGAGTCGGTGC






CGGTGCgatttcttgggtggcctggccttcCGAGTCTTCC








ACTGcaca





 


62
SpyCas9-
−
TCTTGGGTGGCCTGGCCTTCGTTTTAGAGCT
29427
AAAGATGGCGCTCATTGTGCGTTTTAGAGCTAGAAAT
29604



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCgatgtactgtgtgcagtggaagacTCGGAAGGCC








AGGCcacc





 


65
SauCas9KKH
−
TTTCTTGGGTGGCCTGGCCTTGTTTTAGTAC
29428
AAGATGGCGCTCATTGTGCCTGTTTTAGTACTCTGGA
29605





TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAatgtactgtgtgcagtggaagacTCGGAAGGCCA








GGCCaccc





 


66
SauCas9KKH
+
TGTACTGTGTGCAGTGGAAGAGTTTTAGTA
29429
AGTTTTCTTTCTTCTTTTCATGTTTTAGTACTCTGGAA
29606





CTCTGGAAACAGAATCTACTAAAACAAGGC

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTA






AAAATGCCGTGTTTATCTCGTCAACTTGTTG

TCTCGTCAACTTGTTGGCGAGA






GCGAGAatttcttgggtggcctggccttcCGAGTCTTCCA








CTGCacac





 


67
SauCas9KKH
−
TTTCTTGGGTGGCCTGGCCTTGTTTTAGTAC
29430
AAGATGGCGCTCATTGTGCCTGTTTTAGTACTCTGGA
29607





TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAatgtactgtgtgcagtggaagacTCGGAAGGCCA








GGCCaccc





 


70
SpyCas9-
+
GTACTGTGTGCAGTGGAAGAGTTTTAGAGC
29431
ATGAGCTTTGAGTTTTCTTTGTTTTAGAGCTAGAAATA
29608



SpRY

TAGAAATAGCAAGTTAAAATAAGGCTAGTC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






CGTTATCAACTTGAAAAAGTGGCACCGAGT

AAAGTGGCACCGAGTCGGTGC






CGGTGCatttcttgggtggcctggccttcCGAGTCTTCCA








CTGCacac





 


71
SpyCas9-
−
TTCTTGGGTGGCCTGGCCTTGTTTTAGAGCT
29432
AAGATGGCGCTCATTGTGCCGTTTTAGAGCTAGAAAT
29609



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCatgtactgtgtgcagtggaagacTCGGAAGGCCA








GGCCaccc





 


73
SauCas9KKH
−
ATTTCTTGGGTGGCCTGGCCTGTTTTAGTAC
29433
AAGATGGCGCTCATTGTGCCTGTTTTAGTACTCTGGA
29610





TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAtgtactgtgtgcagtggaagacTCGGAAGGCCA








GGCCAccca





 


74
SpyCas9-
+
TGTACTGTGTGCAGTGGAAGGTTTTAGAGC
29434
TGAGCTTTGAGTTTTCTTTCGTTTTAGAGCTAGAAATA
29611



SpRY

TAGAAATAGCAAGTTAAAATAAGGCTAGTC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






CGTTATCAACTTGAAAAAGTGGCACCGAGT

AAAGTGGCACCGAGTCGGTGC






CGGTGCtttcttgggtggcctggccttcCGAGTCTTCCAC








TGCAcaca





 


75
SpyCas9-
−
TTTCTTGGGTGGCCTGGCCTGTTTTAGAGCT
29435
AGATGGCGCTCATTGTGCCTGTTTTAGAGCTAGAAAT
29612



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCtgtactgtgtgcagtggaagacTCGGAAGGCCA








GGCCAccca





 


78
SpyCas9-
+
ATGTACTGTGTGCAGTGGAAGTTTTAGAGC
29436
GAGCTTTGAGTTTTCTTTCTGTTTTAGAGCTAGAAATA
29613



SpRY

TAGAAATAGCAAGTTAAAATAAGGCTAGTC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






CGTTATCAACTTGAAAAAGTGGCACCGAGT

AAAGTGGCACCGAGTCGGTGC






CGGTGCttcttgggtggcctggccttcCGAGTCTTCCAC








TGCACacag





 


79
SpyCas9-
−
ATTTCTTGGGTGGCCTGGCCGTTTTAGAGCT
29437
GATGGCGCTCATTGTGCCTGGTTTTAGAGCTAGAAAT
29614



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCgtactgtgtgcagtggaagacTCGGAAGGCCAG








GCCACccaa





 


81
SpyCas9-
+
GATGTACTGTGTGCAGTGGAGTTTTAGAGC
29438
TTCTTTCTTCTTTTCATCCCGTTTTAGAGCTAGAAATA
29615



NG

TAGAAATAGCAAGTTAAAATAAGGCTAGTC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






CGTTATCAACTTGAAAAAGTGGCACCGAGT

AAAGTGGCACCGAGTCGGTGC






CGGTGCtcttgggtggcctggccttcCGAGTCTTCCACT








GCACAcagt





 


85
SpyCas9-
+
GATGTACTGTGTGCAGTGGAGTTTTAGAGC
29439
AGCTTTGAGTTTTCTTTCTTGTTTTAGAGCTAGAAATA
29616



SpRY

TAGAAATAGCAAGTTAAAATAAGGCTAGTC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






CGTTATCAACTTGAAAAAGTGGCACCGAGT

AAAGTGGCACCGAGTCGGTGC






CGGTGCtcttgggtggcctggccttcCGAGTCTTCCACT








GCACAcagt





 


86
SpyCas9-
−
GATTTCTTGGGTGGCCTGGCGTTTTAGAGCT
29440
ATGGCGCTCATTGTGCCTGGGTTTTAGAGCTAGAAAT
29617



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCtactgtgtgcagtggaagacTCGGAAGGCCAG








GCCACCcaag





 


87
BlatCas9
−
cgggATTTCTTGGGTGGCCTGGCGCTATAGTT
29441
aaagATGGCGCTCATTGTGCCTGGCTATAGTTCCTTACT
29618





CCTTACTGAAAGGTAAGTTGCTATAGTAAG

GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG






GGCAACAGACCCGAGGCGTTGGGGATCGCC

AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC






TAGCCCGTGTTTACGGGCTCTCCCCATATTC

TCCCCATATTCAAAATAATGACAGACGAGCACCTTGG






AAAATAATGACAGACGAGCACCTTGGAGCA

AGCATTTATCTCCGAGGTGCT






TTTATCTCCGAGGTGCTtactgtgtgcagtggaagacT








CGGAAGGCCAGGCCACCcaag





 


88
BlatCas9
−
cgggATTTCTTGGGTGGCCTGGCGCTATAGTT
29442
aaagATGGCGCTCATTGTGCCTGGCTATAGTTCCTTACT
29619





CCTTACTGAAAGGTAAGTTGCTATAGTAAG

GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG






GGCAACAGACCCGAGGCGTTGGGGATCGCC

AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC






TAGCCCGTGTTTACGGGCTCTCCCCATATTC

TCCCCATATTCAAAATAATGACAGACGAGCACCTTGG






AAAATAATGACAGACGAGCACCTTGGAGCA

AGCATTTATCTCCGAGGTGCT






TTTATCTCCGAGGTGCTtactgtgtgcagtggaagacT








CGGAAGGCCAGGCCACCcaag





 


91
Nme2Cas9
−
ctCGGGATTTCTTGGGTGGCCTGGGTTGTAG
29443
gcAGCAGGAAAAGATGGCGCTCATGTTGTAGCTCCCT
29620





CTCCCTTTCTCATTTCGGAAACGAAATGAGA

TTCTCATTTCGGAAACGAAATGAGAACCGTTGCTACA






ACCGTTGCTACAATAAGGCCGTCTGAAAAG

ATAAGGCCGTCTGAAAAGATGTGCCGCAACGCTCTGC






ATGTGCCGCAACGCTCTGCCCCTTAAAGCTT

CCCTTAAAGCTTCTGCTTTAAGGGGCATCGTTTA






CTGCTTTAAGGGGCATCGTTTAactgtgtgcagtgg








aagacTCGGAAGGCCAGGCCACCCaaga





 


94
ScaCas9-
+
TGATGTACTGTGTGCAGTGGGTTTTAGAGCT
29444
TTTCTTTCTTCTTTTCATCCGTTTTAGAGCTAGAAATA
29621



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCcttgggtggcctggccttcCGAGTCTTCCACTG








CACACagta





 


95
SpyCas9-
+
TGATGTACTGTGTGCAGTGGGTTTTAGAGCT
29445
GCTTTGAGTTTTCTTTCTTCGTTTTAGAGCTAGAAATA
29622



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCcttgggtggcctggccttcCGAGTCTTCCACTG








CACACagta





 


96
SpyCas9-
−
GGATTTCTTGGGTGGCCTGGGTTTTAGAGCT
29446
TGGCGCTCATTGTGCCTGGCGTTTTAGAGCTAGAAAT
29623



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCactgtgtgcagtggaagacTCGGAAGGCCAGG








CCACCCaaga





 


97
BlatCas9
+
gtctGATGTACTGTGTGCAGTGGGCTATAGTT
29447
tgagCTTTGAGTTTTCTTTCTTCGCTATAGTTCCTTACTG
29624





CCTTACTGAAAGGTAAGTTGCTATAGTAAG

AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGA






GGCAACAGACCCGAGGCGTTGGGGATCGCC

GGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCT






TAGCCCGTGTTTACGGGCTCTCCCCATATTC

CCCCATATTCAAAATAATGACAGACGAGCACCTTGGA






AAAATAATGACAGACGAGCACCTTGGAGCA

GCATTTATCTCCGAGGTGCT






TTTATCTCCGAGGTGCTcttgggtggcctggccttcCG








AGTCTTCCACTGCACACagta





 


98
BlatCas9
−
tcggGATTTCTTGGGTGGCCTGGGCTATAGTT
29448
aaagATGGCGCTCATTGTGCCTGGCTATAGTTCCTTACT
29625





CCTTACTGAAAGGTAAGTTGCTATAGTAAG

GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG






GGCAACAGACCCGAGGCGTTGGGGATCGCC

AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC






TAGCCCGTGTTTACGGGCTCTCCCCATATTC

TCCCCATATTCAAAATAATGACAGACGAGCACCTTGG






AAAATAATGACAGACGAGCACCTTGGAGCA

AGCATTTATCTCCGAGGTGCT






TTTATCTCCGAGGTGCTactgtgtgcagtggaagacTC








GGAAGGCCAGGCCACCCaaga





 


99
BlatCas9
−
tcggGATTTCTTGGGTGGCCTGGGCTATAGTT
29449
aaagATGGCGCTCATTGTGCCTGGCTATAGTTCCTTACT
29626





CCTTACTGAAAGGTAAGTTGCTATAGTAAG

GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG






GGCAACAGACCCGAGGCGTTGGGGATCGCC

AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC






TAGCCCGTGTTTACGGGCTCTCCCCATATTC

TCCCCATATTCAAAATAATGACAGACGAGCACCTTGG






AAAATAATGACAGACGAGCACCTTGGAGCA

AGCATTTATCTCCGAGGTGCT






TTTATCTCCGAGGTGCTactgtgtgcagtggaagacTC








GGAAGGCCAGGCCACCCaaga





 


100
BlatCas9
+
gtctGATGTACTGTGTGCAGTGGGCTATAGTT
29450
tgagCTTTGAGTTTTCTTTCTTCGCTATAGTTCCTTACTG
29627





CCTTACTGAAAGGTAAGTTGCTATAGTAAG

AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGA






GGCAACAGACCCGAGGCGTTGGGGATCGCC

GGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCT






TAGCCCGTGTTTACGGGCTCTCCCCATATTC

CCCCATATTCAAAATAATGACAGACGAGCACCTTGGA






AAAATAATGACAGACGAGCACCTTGGAGCA

GCATTTATCTCCGAGGTGCT






TTTATCTCCGAGGTGCTcttgggtggcctggccttcCG








AGTCTTCCACTGCACACagta





 


101
BlatCas9
−
tcggGATTTCTTGGGTGGCCTGGGCTATAGTT
29451
aaagATGGCGCTCATTGTGCCTGGCTATAGTTCCTTACT
29628





CCTTACTGAAAGGTAAGTTGCTATAGTAAG

GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG






GGCAACAGACCCGAGGCGTTGGGGATCGCC

AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC






TAGCCCGTGTTTACGGGCTCTCCCCATATTC

TCCCCATATTCAAAATAATGACAGACGAGCACCTTGG






AAAATAATGACAGACGAGCACCTTGGAGCA

AGCATTTATCTCCGAGGTGCT






TTTATCTCCGAGGTGCTactgtgtgcagtggaagacTC








GGAAGGCCAGGCCACCCaaga





 


106
SauCas9KKH
+
TCTGATGTACTGTGTGCAGTGGTTTTAGTAC
29452
AGTTTTCTTTCTTCTTTTCATGTTTTAGTACTCTGGAA
29629





TCTGGAAACAGAATCTACTAAAACAAGGCA

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTA






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

TCTCGTCAACTTGTTGGCGAGA






CGAGAttgggtggcctggccttcCGAGTCTTCCACTG








CACACAgtac





 


107
SauriCas9-
+
TCTGATGTACTGTGTGCAGTGGTTTTAGTAC
29453
GTTTTCTTTCTTCTTTTCATCGTTTTAGTACTCTGGAAA
29630



KKH

TCTGGAAACAGAATCTACTAAAACAAGGCA

CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

CTCGTCAACTTGTTGGCGAGA






CGAGAttgggtggcctggccttcCGAGTCTTCCACTG








CACACAgtac





 


110
SpyCas9-
+
CTGATGTACTGTGTGCAGTGGTTTTAGAGCT
29454
CTTTGAGTTTTCTTTCTTCTGTTTTAGAGCTAGAAATA
29631



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCttgggtggcctggccttcCGAGTCTTCCACTGC








ACACAgtac





 


112
SpyCas9-
−
GGGATTTCTTGGGTGGCCTGGTTTTAGAGCT
29455
GGCGCTCATTGTGCCTGGCAGTTTTAGAGCTAGAAAT
29632



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCctgtgtgcagtggaagacTCGGAAGGCCAGGC








CACCCAagaa





 


115
SauCas9KKH
+
GTCTGATGTACTGTGTGCAGTGTTTTAGTAC
29456
AGTTTTCTTTCTTCTTTTCATGTTTTAGTACTCTGGAA
29633





TCTGGAAACAGAATCTACTAAAACAAGGCA

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTA






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

TCTCGTCAACTTGTTGGCGAGA






CGAGAtgggtggcctggccttcCGAGTCTTCCACTGC








ACACAGtaca





 


116
SpyCas9-
+
TCTGATGTACTGTGTGCAGTGTTTTAGAGCT
29457
TTCTTTCTTCTTTTCATCCCGTTTTAGAGCTAGAAATA
29634



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCtgggtggcctggccttcCGAGTCTTCCACTGC








ACACAGtaca





 


119
SpyCas9-
−
CGGGATTTCTTGGGTGGCCTGTTTTAGAGCT
29458
CGCTCATTGTGCCTGGCAACGTTTTAGAGCTAGAAAT
29635



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCtgtgtgcagtggaagacTCGGAAGGCCAGGC








CACCCAAgaaa





 


123
SpyCas9-
+
TCTGATGTACTGTGTGCAGTGTTTTAGAGCT
29459
TTTGAGTTTTCTTTCTTCTTGTTTTAGAGCTAGAAATA
29636



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCtgggtggcctggccttcCGAGTCTTCCACTGC








ACACAGtaca





 


125
SpyCas9-
−
CGGGATTTCTTGGGTGGCCTGTTTTAGAGCT
29460
GCGCTCATTGTGCCTGGCAAGTTTTAGAGCTAGAAAT
29637



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCtgtgtgcagtggaagacTCGGAAGGCCAGGC








CACCCAAgaaa





 


132
SauCas9
+
caTGTCTGATGTACTGTGTGCAGGTTTTAGTA
29461
ctCTGACTCAGTGGTGATGAGCTGTTTTAGTACTCTGG
29638





CTCTGGAAACAGAATCTACTAAAACAAGGC

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT






AAAATGCCGTGTTTATCTCGTCAACTTGTTG

TTATCTCGTCAACTTGTTGGCGAGA






GCGAGAgggtggcctggccttcCGAGTCTTCCACTG








CACACAGTacat





 


133
SauCas9KKH
+
TGTCTGATGTACTGTGTGCAGGTTTTAGTAC
29462
AGTTTTCTTTCTTCTTTTCATGTTTTAGTACTCTGGAA
29639





TCTGGAAACAGAATCTACTAAAACAAGGCA

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTA






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

TCTCGTCAACTTGTTGGCGAGA






CGAGAgggtggcctggccttcCGAGTCTTCCACTGC








ACACAGTacat





 


136
ScaCas9-
+
GTCTGATGTACTGTGTGCAGGTTTTAGAGCT
29463
TTTCTTTCTTCTTTTCATCCGTTTTAGAGCTAGAAATA
29640



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCgggtggcctggccttcCGAGTCTTCCACTGC








ACACAGTacat





 


137
SpyCas9
+
GTCTGATGTACTGTGTGCAGGTTTTAGAGCT
29464
CTTTTCATCCCAGCTTGCACGTTTTAGAGCTAGAAAT
29641





AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCgggtggcctggccttcCGAGTCTTCCACTGC








ACACAGTacat





 


140
SpyCas9-
+
GTCTGATGTACTGTGTGCAGGTTTTAGAGCT
29465
TTGAGTTTTCTTTCTTCTTTGTTTTAGAGCTAGAAATA
29642



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCgggtggcctggccttcCGAGTCTTCCACTGC








ACACAGTacat





 


143
ScaCas9-
−
TCGGGATTTCTTGGGTGGCCGTTTTAGAGCT
29466
CGCTCATTGTGCCTGGCAACGTTTTAGAGCTAGAAAT
29643



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCgtgtgcagtggaagacTCGGAAGGCCAGGCC








ACCCAAGaaat





 


144
SpyCas9
−
TCGGGATTTCTTGGGTGGCCGTTTTAGAGCT
29467
CGCTCATTGTGCCTGGCAACGTTTTAGAGCTAGAAAT
29644





AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCgtgtgcagtggaagacTCGGAAGGCCAGGCC








ACCCAAGaaat





 


147
SpyCas9-
−
TCGGGATTTCTTGGGTGGCCGTTTTAGAGCT
29468
CGCTCATTGTGCCTGGCAACGTTTTAGAGCTAGAAAT
29645



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCgtgtgcagtggaagacTCGGAAGGCCAGGCC








ACCCAAGaaat





 


148
SpyCas9-
+
GTCTGATGTACTGTGTGCAGGTTTTAGAGCT
29469
TTCTTTCTTCTTTTCATCCCGTTTTAGAGCTAGAAATA
29646



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCgggtggcctggccttcCGAGTCTTCCACTGC








ACACAGTacat





 


151
SpyCas9-
−
TCGGGATTTCTTGGGTGGCCGTTTTAGAGCT
29470
CGCTCATTGTGCCTGGCAACGTTTTAGAGCTAGAAAT
29647



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCgtgtgcagtggaagacTCGGAAGGCCAGGCC








ACCCAAGaaat





 


154
BlatCas9
−
ctctCGGGATTTCTTGGGTGGCCGCTATAGTTC
29471
gcgcTCATTGTGCCTGGCAACTGGCTATAGTTCCTTACT
29648





CTTACTGAAAGGTAAGTTGCTATAGTAAGG

GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG






GCAACAGACCCGAGGCGTTGGGGATCGCCT

AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC






AGCCCGTGTTTACGGGCTCTCCCCATATTCA

TCCCCATATTCAAAATAATGACAGACGAGCACCTTGG






AAATAATGACAGACGAGCACCTTGGAGCAT

AGCATTTATCTCCGAGGTGCT






TTATCTCCGAGGTGCTgtgtgcagtggaagacTCGG








AAGGCCAGGCCACCCAAGaaat





 


160
Nme2Cas9
−
tcCTCTCGGGATTTCTTGGGTGGCGTTGTAGC
29472
gcAGCAGGAAAAGATGGCGCTCATGTTGTAGCTCCCT
29649





TCCCTTTCTCATTTCGGAAACGAAATGAGA

TTCTCATTTCGGAAACGAAATGAGAACCGTTGCTACA






ACCGTTGCTACAATAAGGCCGTCTGAAAAG

ATAAGGCCGTCTGAAAAGATGTGCCGCAACGCTCTGC






ATGTGCCGCAACGCTCTGCCCCTTAAAGCTT

CCCTTAAAGCTTCTGCTTTAAGGGGCATCGTTTA






CTGCTTTAAGGGGCATCGTTTAtgtgcagtggaaga








cTCGGAAGGCCAGGCCACCCAAGAaatc





 


161
SauCas9
+
ccATGTCTGATGTACTGTGTGCAGTTTTAGTA
29473
ctCTGACTCAGTGGTGATGAGCTGTTTTAGTACTCTGG
29650





CTCTGGAAACAGAATCTACTAAAACAAGGC

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT






AAAATGCCGTGTTTATCTCGTCAACTTGTTG

TTATCTCGTCAACTTGTTGGCGAGA






GCGAGAggtggcctggccttcCGAGTCTTCCACTGC








ACACAGTAcatc





 


162
SauCas9KKH
+
ATGTCTGATGTACTGTGTGCAGTTTTAGTAC
29474
AGTTTTCTTTCTTCTTTTCATGTTTTAGTACTCTGGAA
29651





TCTGGAAACAGAATCTACTAAAACAAGGCA

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTA






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

TCTCGTCAACTTGTTGGCGAGA






CGAGAggtggcctggccttcCGAGTCTTCCACTGCA








CACAGTAcatc





 


163
SauriCas9
+
ATGTCTGATGTACTGTGTGCAGTTTTAGTAC
29475
TTCTTTTCATCCCAGCTTGCAGTTTTAGTACTCTGGAA
29652





TCTGGAAACAGAATCTACTAAAACAAGGCA

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTA






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

TCTCGTCAACTTGTTGGCGAGA






CGAGAggtggcctggccttcCGAGTCTTCCACTGCA








CACAGTAcatc





 


164
SauriCas9-
+
ATGTCTGATGTACTGTGTGCAGTTTTAGTAC
29476
GTTTTCTTTCTTCTTTTCATCGTTTTAGTACTCTGGAAA
29653



KKH

TCTGGAAACAGAATCTACTAAAACAAGGCA

CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

CTCGTCAACTTGTTGGCGAGA






CGAGAggtggcctggccttcCGAGTCTTCCACTGCA








CACAGTAcatc





 


165
SauriCas9
−
TCTCGGGATTTCTTGGGTGGCGTTTTAGTAC
29477
GGCGCTCATTGTGCCTGGCAAGTTTTAGTACTCTGGA
29654





TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAtgtgcagtggaagacTCGGAAGGCCAGGCC








ACCCAAGAaatc





 


166
SauriCas9-
−
TCTCGGGATTTCTTGGGTGGCGTTTTAGTAC
29478
GCTCATTGTGCCTGGCAACTGGTTTTAGTACTCTGGA
29655



KKH

TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAtgtgcagtggaagacTCGGAAGGCCAGGCC








ACCCAAGAaatc





 


169
ScaCas9-
+
TGTCTGATGTACTGTGTGCAGTTTTAGAGCT
29479
TTTCTTTCTTCTTTTCATCCGTTTTAGAGCTAGAAATA
29656



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCggtggcctggccttcCGAGTCTTCCACTGCA








CACAGTAcatc





 


170
SpyCas9-
+
TGTCTGATGTACTGTGTGCAGTTTTAGAGCT
29480
TGAGTTTTCTTTCTTCTTTTGTTTTAGAGCTAGAAATA
29657



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCggtggcctggccttcCGAGTCTTCCACTGCA








CACAGTAcatc





 


173
ScaCas9-
−
CTCGGGATTTCTTGGGTGGCGTTTTAGAGCT
29481
CGCTCATTGTGCCTGGCAACGTTTTAGAGCTAGAAAT
29658



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCtgtgcagtggaagacTCGGAAGGCCAGGCC








ACCCAAGAaatc





 


174
SpyCas9-
−
CTCGGGATTTCTTGGGTGGCGTTTTAGAGCT
29482
GCTCATTGTGCCTGGCAACTGTTTTAGAGCTAGAAAT
29659



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCtgtgcagtggaagacTCGGAAGGCCAGGCC








ACCCAAGAaatc





 


175
BlatCas9
−
cctcTCGGGATTTCTTGGGTGGCGCTATAGTT
29483
gcgcTCATTGTGCCTGGCAACTGGCTATAGTTCCTTACT
29660





CCTTACTGAAAGGTAAGTTGCTATAGTAAG

GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG






GGCAACAGACCCGAGGCGTTGGGGATCGCC

AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC






TAGCCCGTGTTTACGGGCTCTCCCCATATTC

TCCCCATATTCAAAATAATGACAGACGAGCACCTTGG






AAAATAATGACAGACGAGCACCTTGGAGCA

AGCATTTATCTCCGAGGTGCT






TTTATCTCCGAGGTGCTtgtgcagtggaagacTCGG








AAGGCCAGGCCACCCAAGAaatc





 


176
BlatCas9
−
cctcTCGGGATTTCTTGGGTGGCGCTATAGTT
29484
gcgcTCATTGTGCCTGGCAACTGGCTATAGTTCCTTACT
29661





CCTTACTGAAAGGTAAGTTGCTATAGTAAG

GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG






GGCAACAGACCCGAGGCGTTGGGGATCGCC

AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC






TAGCCCGTGTTTACGGGCTCTCCCCATATTC

TCCCCATATTCAAAATAATGACAGACGAGCACCTTGG






AAAATAATGACAGACGAGCACCTTGGAGCA

AGCATTTATCTCCGAGGTGCT






TTTATCTCCGAGGTGCTtgtgcagtggaagacTCGG








AAGGCCAGGCCACCCAAGAaatc





 


179
SauCas9KKH
+
CATGTCTGATGTACTGTGTGCGTTTTAGTAC
29485
AGTTTTCTTTCTTCTTTTCATGTTTTAGTACTCTGGAA
29662





TCTGGAAACAGAATCTACTAAAACAAGGCA

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTA






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

TCTCGTCAACTTGTTGGCGAGA






CGAGAgtggcctggccttcCGAGTCTTCCACTGCAC








ACAGTACatca





 


180
SauCas9KKH
−
CTCTCGGGATTTCTTGGGTGGGTTTTAGTAC
29486
CGCTCATTGTGCCTGGCAACTGTTTTAGTACTCTGGA
29663





TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAgtgcagtggaagacTCGGAAGGCCAGGCCA








CCCAAGAAatcc





 


181
SpyCas9-
+
ATGTCTGATGTACTGTGTGCGTTTTAGAGCT
29487
TTCTTTCTTCTTTTCATCCCGTTTTAGAGCTAGAAATA
29664



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCgtggcctggccttcCGAGTCTTCCACTGCAC








ACAGTACatca





 


185
SpyCas9-
+
ATGTCTGATGTACTGTGTGCGTTTTAGAGCT
29488
GAGTTTTCTTTCTTCTTTTCGTTTTAGAGCTAGAAATA
29665



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCgtggcctggccttcCGAGTCTTCCACTGCAC








ACAGTACatca





 


186
SpyCas9-
−
TCTCGGGATTTCTTGGGTGGGTTTTAGAGCT
29489
CTCATTGTGCCTGGCAACTGGTTTTAGAGCTAGAAAT
29666



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCgtgcagtggaagacTCGGAAGGCCAGGCCA








CCCAAGAAatcc





 


189
ScaCas9-
+
CATGTCTGATGTACTGTGTGGTTTTAGAGCT
29490
TTTCTTTCTTCTTTTCATCCGTTTTAGAGCTAGAAATA
29667



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCtggcctggccttcCGAGTCTTCCACTGCACA








CAGTACAtcag





 


190
SpyCas9-
+
CATGTCTGATGTACTGTGTGGTTTTAGAGCT
29491
AGTTTTCTTTCTTCTTTTCAGTTTTAGAGCTAGAAATA
29668



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCtggcctggccttcCGAGTCTTCCACTGCACA








CAGTACAtcag





 


191
SpyCas9-
−
CTCTCGGGATTTCTTGGGTGGTTTTAGAGCT
29492
TCATTGTGCCTGGCAACTGGGTTTTAGAGCTAGAAAT
29669



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCtgcagtggaagacTCGGAAGGCCAGGCCA








CCCAAGAAAtccc





 


193
SauriCas9-
+
TCCATGTCTGATGTACTGTGTGTTTTAGTAC
29493
GTTTTCTTTCTTCTTTTCATCGTTTTAGTACTCTGGAAA
29670



KKH

TCTGGAAACAGAATCTACTAAAACAAGGCA

CAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

CTCGTCAACTTGTTGGCGAGA






CGAGAggcctggccttcCGAGTCTTCCACTGCACA








CAGTACATcaga





 


194
SpyCas9-
−
CCTCTCGGGATTTCTTGGGTGTTTTAGAGCT
29494
CATTGTGCCTGGCAACTGGTGTTTTAGAGCTAGAAAT
29671



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCgcagtggaagacTCGGAAGGCCAGGCCAC








CCAAGAAATcccg





 


198
SpyCas9-
−
CCTCTCGGGATTTCTTGGGTGTTTTAGAGCT
29495
CATTGTGCCTGGCAACTGGTGTTTTAGAGCTAGAAAT
29672



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCgcagtggaagacTCGGAAGGCCAGGCCAC








CCAAGAAATcccg





 


199
SpyCas9-
+
CCATGTCTGATGTACTGTGTGTTTTAGAGCT
29496
GTTTTCTTTCTTCTTTTCATGTTTTAGAGCTAGAAATA
29673



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCggcctggccttcCGAGTCTTCCACTGCACA








CAGTACATcaga





 


203
SauCas9KKH
+
ATCCATGTCTGATGTACTGTGGTTTTAGTAC
29497
AGTTTTCTTTCTTCTTTTCATGTTTTAGTACTCTGGAA
29674





TCTGGAAACAGAATCTACTAAAACAAGGCA

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTA






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

TCTCGTCAACTTGTTGGCGAGA






CGAGAgcctggccttcCGAGTCTTCCACTGCACAC








AGTACATCagac





 


204
SauCas9KKH
+
ATCCATGTCTGATGTACTGTGGTTTTAGTAC
29498
AGTTTTCTTTCTTCTTTTCATGTTTTAGTACTCTGGAA
29675





TCTGGAAACAGAATCTACTAAAACAAGGCA

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTA






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

TCTCGTCAACTTGTTGGCGAGA






CGAGAgcctggccttcCGAGTCTTCCACTGCACAC








AGTACATCagac





 


209
ScaCas9-
−
TCCTCTCGGGATTTCTTGGGGTTTTAGAGCT
29499
TTGTGCCTGGCAACTGGTAGGTTTTAGAGCTAGAAAT
29676



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCcagtggaagacTCGGAAGGCCAGGCCACC








CAAGAAATCccga





 


210
SpyCas9
−
TCCTCTCGGGATTTCTTGGGGTTTTAGAGCT
29500
TGTGCCTGGCAACTGGTAGCGTTTTAGAGCTAGAAAT
29677





AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCcagtggaagacTCGGAAGGCCAGGCCACC








CAAGAAATCccga





 


213
SpyCas9-
−
TCCTCTCGGGATTTCTTGGGGTTTTAGAGCT
29501
ATTGTGCCTGGCAACTGGTAGTTTTAGAGCTAGAAAT
29678



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCcagtggaagacTCGGAAGGCCAGGCCACC








CAAGAAATCccga





 


214
SpyCas9-
+
TCCATGTCTGATGTACTGTGGTTTTAGAGCT
29502
TTCTTTCTTCTTTTCATCCCGTTTTAGAGCTAGAAATA
29679



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCgcctggccttcCGAGTCTTCCACTGCACAC








AGTACATCagac





 


217
SpyCas9-
−
TCCTCTCGGGATTTCTTGGGGTTTTAGAGCT
29503
CATTGTGCCTGGCAACTGGTGTTTTAGAGCTAGAAAT
29680



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCcagtggaagacTCGGAAGGCCAGGCCACC








CAAGAAATCccga





 


221
SpyCas9-
+
TCCATGTCTGATGTACTGTGGTTTTAGAGCT
29504
TTTTCTTTCTTCTTTTCATCGTTTTAGAGCTAGAAATA
29681



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCgcctggccttcCGAGTCTTCCACTGCACAC








AGTACATCagac





 


222
BlatCas9
−
ctttCCTCTCGGGATTTCTTGGGGCTATAGTTC
29505
gcgcTCATTGTGCCTGGCAACTGGCTATAGTTCCTTACT
29682





CTTACTGAAAGGTAAGTTGCTATAGTAAGG

GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG






GCAACAGACCCGAGGCGTTGGGGATCGCCT

AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC






AGCCCGTGTTTACGGGCTCTCCCCATATTCA

TCCCCATATTCAAAATAATGACAGACGAGCACCTTGG






AAATAATGACAGACGAGCACCTTGGAGCAT

AGCATTTATCTCCGAGGTGCT






TTATCTCCGAGGTGCTcagtggaagacTCGGAAG








GCCAGGCCACCCAAGAAATCccga





 


223
BlatCas9
−
ctttCCTCTCGGGATTTCTTGGGGCTATAGTTC
29506
gcgcTCATTGTGCCTGGCAACTGGCTATAGTTCCTTACT
29683





CTTACTGAAAGGTAAGTTGCTATAGTAAGG

GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG






GCAACAGACCCGAGGCGTTGGGGATCGCCT

AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC






AGCCCGTGTTTACGGGCTCTCCCCATATTCA

TCCCCATATTCAAAATAATGACAGACGAGCACCTTGG






AAATAATGACAGACGAGCACCTTGGAGCAT

AGCATTTATCTCCGAGGTGCT






TTATCTCCGAGGTGCTcagtggaagacTCGGAAG








GCCAGGCCACCCAAGAAATCccga





 


226
Nme2Cas9
−
tgCTTTCCTCTCGGGATTTCTTGGGTTGTAGC
29507
gcAGCAGGAAAAGATGGCGCTCATGTTGTAGCTCCCT
29684





TCCCTTTCTCATTTCGGAAACGAAATGAGA

TTCTCATTTCGGAAACGAAATGAGAACCGTTGCTACA






ACCGTTGCTACAATAAGGCCGTCTGAAAAG

ATAAGGCCGTCTGAAAAGATGTGCCGCAACGCTCTGC






ATGTGCCGCAACGCTCTGCCCCTTAAAGCTT

CCCTTAAAGCTTCTGCTTTAAGGGGCATCGTTTA






CTGCTTTAAGGGGCATCGTTTAagtggaagacTC








GGAAGGCCAGGCCACCCAAGAAATCCcgag





 


227
SauriCas9
−
TTTCCTCTCGGGATTTCTTGGGTTTTAGTAC
29508
ATTGTGCCTGGCAACTGGTAGGTTTTAGTACTCTGGA
29685





TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAagtggaagacTCGGAAGGCCAGGCCACC








CAAGAAATCCcgag





 


228
SauriCas9-
−
TTTCCTCTCGGGATTTCTTGGGTTTTAGTAC
29509
ATTGTGCCTGGCAACTGGTAGGTTTTAGTACTCTGGA
29686



KKH

TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAagtggaagacTCGGAAGGCCAGGCCACC








CAAGAAATCCcgag





 


231
ScaCas9-
+
ATCCATGTCTGATGTACTGTGTTTTAGAGCT
29510
TTTCTTTCTTCTTTTCATCCGTTTTAGAGCTAGAAATA
29687



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCcctggccttcCGAGTCTTCCACTGCACAC








AGTACATCAgaca





 


232
SpyCas9-
+
ATCCATGTCTGATGTACTGTGTTTTAGAGCT
29511
TTTCTTTCTTCTTTTCATCCGTTTTAGAGCTAGAAATA
29688



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCcctggccttcCGAGTCTTCCACTGCACAC








AGTACATCAgaca





 


235
ScaCas9-
−
TTCCTCTCGGGATTTCTTGGGTTTTAGAGCT
29512
TTGTGCCTGGCAACTGGTAGGTTTTAGAGCTAGAAAT
29689



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCagtggaagacTCGGAAGGCCAGGCCACC








CAAGAAATCCcgag





 


236
SpyCas9-
−
TTCCTCTCGGGATTTCTTGGGTTTTAGAGCT
29513
TTGTGCCTGGCAACTGGTAGGTTTTAGAGCTAGAAAT
29690



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCagtggaagacTCGGAAGGCCAGGCCACC








CAAGAAATCCcgag





 


237
BlatCas9
−
gcttTCCTCTCGGGATTTCTTGGGCTATAGTTC
29514
gcgcTCATTGTGCCTGGCAACTGGCTATAGTTCCTTACT
29691





CTTACTGAAAGGTAAGTTGCTATAGTAAGG

GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG






GCAACAGACCCGAGGCGTTGGGGATCGCCT

AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC






AGCCCGTGTTTACGGGCTCTCCCCATATTCA

TCCCCATATTCAAAATAATGACAGACGAGCACCTTGG






AAATAATGACAGACGAGCACCTTGGAGCAT

AGCATTTATCTCCGAGGTGCT






TTATCTCCGAGGTGCTagtggaagacTCGGAAGG








CCAGGCCACCCAAGAAATCCcgag





 


238
BlatCas9
−
gcttTCCTCTCGGGATTTCTTGGGCTATAGTTC
29515
gcgcTCATTGTGCCTGGCAACTGGCTATAGTTCCTTACT
29692





CTTACTGAAAGGTAAGTTGCTATAGTAAGG

GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG






GCAACAGACCCGAGGCGTTGGGGATCGCCT

AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC






AGCCCGTGTTTACGGGCTCTCCCCATATTCA

TCCCCATATTCAAAATAATGACAGACGAGCACCTTGG






AAATAATGACAGACGAGCACCTTGGAGCAT

AGCATTTATCTCCGAGGTGCT






TTATCTCCGAGGTGCTagtggaagacTCGGAAGG








CCAGGCCACCCAAGAAATCCcgag





 


239
SauCas9KKH
−
CTTTCCTCTCGGGATTTCTTGGTTTTAGTACT
29516
TTGTGCCTGGCAACTGGTAGCGTTTTAGTACTCTGGA
29693





CTGGAAACAGAATCTACTAAAACAAGGCAA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AATGCCGTGTTTATCTCGTCAACTTGTTGGC

ATCTCGTCAACTTGTTGGCGAGA






GAGAgtggaagacTCGGAAGGCCAGGCCACCCA








AGAAATCCCgaga





 


240
SpyCas9-
+
GATCCATGTCTGATGTACTGGTTTTAGAGCT
29517
TTCTTTCTTCTTTTCATCCCGTTTTAGAGCTAGAAATA
29694



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCctggccttcCGAGTCTTCCACTGCACACA








GTACATCAGacat





 


243
SpyCas9-
−
TTTCCTCTCGGGATTTCTTGGTTTTAGAGCT
29518
TGTGCCTGGCAACTGGTAGCGTTTTAGAGCTAGAAAT
29695



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCgtggaagacTCGGAAGGCCAGGCCACCC








AAGAAATCCCgaga





 


247
SpyCas9-
+
GATCCATGTCTGATGTACTGGTTTTAGAGCT
29519
TTCTTTCTTCTTTTCATCCCGTTTTAGAGCTAGAAATA
29696



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCctggccttcCGAGTCTTCCACTGCACACA








GTACATCAGacat





 


249
SpyCas9-
−
TTTCCTCTCGGGATTTCTTGGTTTTAGAGCT
29520
TGTGCCTGGCAACTGGTAGCGTTTTAGAGCTAGAAAT
29697



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCgtggaagacTCGGAAGGCCAGGCCACCC








AAGAAATCCCgaga





 


250
BlatCas9
+
ttggATCCATGTCTGATGTACTGGCTATAGTTC
29521
gagtTTTCTTTCTTCTTTTCATCGCTATAGTTCCTTACTG
29698





CTTACTGAAAGGTAAGTTGCTATAGTAAGG

AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGA






GCAACAGACCCGAGGCGTTGGGGATCGCCT

GGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCT






AGCCCGTGTTTACGGGCTCTCCCCATATTCA

CCCCATATTCAAAATAATGACAGACGAGCACCTTGGA






AAATAATGACAGACGAGCACCTTGGAGCAT

GCATTTATCTCCGAGGTGCT






TTATCTCCGAGGTGCTctggccttcCGAGTCTTCC








ACTGCACACAGTACATCAGacat





 


251
BlatCas9
+
ttggATCCATGTCTGATGTACTGGCTATAGTTC
29522
gagtTTTCTTTCTTCTTTTCATCGCTATAGTTCCTTACTG
29699





CTTACTGAAAGGTAAGTTGCTATAGTAAGG

AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGA






GCAACAGACCCGAGGCGTTGGGGATCGCCT

GGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCT






AGCCCGTGTTTACGGGCTCTCCCCATATTCA

CCCCATATTCAAAATAATGACAGACGAGCACCTTGGA






AAATAATGACAGACGAGCACCTTGGAGCAT

GCATTTATCTCCGAGGTGCT






TTATCTCCGAGGTGCTctggccttcCGAGTCTTCC








ACTGCACACAGTACATCAGacat





 


254
ScaCas9-
+
GGATCCATGTCTGATGTACTGTTTTAGAGCT
29523
TTTCTTTCTTCTTTTCATCCGTTTTAGAGCTAGAAATA
29700



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCtggccttcCGAGTCTTCCACTGCACACAG








TACATCAGAcatg





 


255
SpyCas9-
+
GGATCCATGTCTGATGTACTGTTTTAGAGCT
29524
TCTTTCTTCTTTTCATCCCAGTTTTAGAGCTAGAAATA
29701



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCtggccttcCGAGTCTTCCACTGCACACAG








TACATCAGAcatg





 


258
ScaCas9-
−
CTTTCCTCTCGGGATTTCTTGTTTTAGAGCT
29525
TGTGCCTGGCAACTGGTAGCGTTTTAGAGCTAGAAAT
29702



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCtggaagacTCGGAAGGCCAGGCCACCCA








AGAAATCCCGagag





 


259
SpyCas9
−
CTTTCCTCTCGGGATTTCTTGTTTTAGAGCT
29526
TGTGCCTGGCAACTGGTAGCGTTTTAGAGCTAGAAAT
29703





AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCtggaagacTCGGAAGGCCAGGCCACCCA








AGAAATCCCGagag





 


262
SpyCas9-
−
CTTTCCTCTCGGGATTTCTTGTTTTAGAGCT
29527
GTGCCTGGCAACTGGTAGCTGTTTTAGAGCTAGAAAT
29704



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCtggaagacTCGGAAGGCCAGGCCACCCA








AGAAATCCCGagag





 


263
SpyCas9-
−
CTTTCCTCTCGGGATTTCTTGTTTTAGAGCT
29528
GTGCCTGGCAACTGGTAGCTGTTTTAGAGCTAGAAAT
29705



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCtggaagacTCGGAAGGCCAGGCCACCCA








AGAAATCCCGagag





 


267
SauriCas9
−
TGCTTTCCTCTCGGGATTTCTGTTTTAGTACT
29529
GTGCCTGGCAACTGGTAGCTGGTTTTAGTACTCTGGA
29706





CTGGAAACAGAATCTACTAAAACAAGGCAA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AATGCCGTGTTTATCTCGTCAACTTGTTGGC

ATCTCGTCAACTTGTTGGCGAGA






GAGAggaagacTCGGAAGGCCAGGCCACCCAA








GAAATCCCGAgagg





 


268
SauriCas9-
−
TGCTTTCCTCTCGGGATTTCTGTTTTAGTACT
29530
GTGCCTGGCAACTGGTAGCTGGTTTTAGTACTCTGGA
29707



KKH

CTGGAAACAGAATCTACTAAAACAAGGCAA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AATGCCGTGTTTATCTCGTCAACTTGTTGGC

ATCTCGTCAACTTGTTGGCGAGA






GAGAggaagacTCGGAAGGCCAGGCCACCCAA








GAAATCCCGAgagg





 


271
ScaCas9-
−
GCTTTCCTCTCGGGATTTCTGTTTTAGAGCT
29531
TGCCTGGCAACTGGTAGCTGGTTTTAGAGCTAGAAAT
29708



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCggaagacTCGGAAGGCCAGGCCACCCA








AGAAATCCCGAgagg





 


272
SpyCas9
−
GCTTTCCTCTCGGGATTTCTGTTTTAGAGCT
29532
GCCTGGCAACTGGTAGCTGGGTTTTAGAGCTAGAAAT
29709





AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCggaagacTCGGAAGGCCAGGCCACCCA








AGAAATCCCGAgagg





 


275
SpyCas9-
−
GCTTTCCTCTCGGGATTTCTGTTTTAGAGCT
29533
TGCCTGGCAACTGGTAGCTGGTTTTAGAGCTAGAAAT
29710



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCggaagacTCGGAAGGCCAGGCCACCCA








AGAAATCCCGAgagg





 


276
SpyCas9-
+
TGGATCCATGTCTGATGTACGTTTTAGAGCT
29534
TTCTTTCTTCTTTTCATCCCGTTTTAGAGCTAGAAATA
29711



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCggccttcCGAGTCTTCCACTGCACACAG








TACATCAGACatgg





 


279
SpyCas9-
−
GCTTTCCTCTCGGGATTTCTGTTTTAGAGCT
29535
GTGCCTGGCAACTGGTAGCTGTTTTAGAGCTAGAAAT
29712



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCggaagacTCGGAAGGCCAGGCCACCCA








AGAAATCCCGAgagg





 


283
SpyCas9-
+
TGGATCCATGTCTGATGTACGTTTTAGAGCT
29536
CTTTCTTCTTTTCATCCCAGGTTTTAGAGCTAGAAATA
29713



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCggccttcCGAGTCTTCCACTGCACACAG








TACATCAGACatgg





 


286
SauCas9
−
gcCTGCTTTCCTCTCGGGATTTCGTTTTAGTA
29537
ttGTGCCTGGCAACTGGTAGCTGGTTTTAGTACTCTGG
29714





CTCTGGAAACAGAATCTACTAAAACAAGGC

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT






AAAATGCCGTGTTTATCTCGTCAACTTGTTG

TTATCTCGTCAACTTGTTGGCGAGA






GCGAGAgaagacTCGGAAGGCCAGGCCACCCA








AGAAATCCCGAGagga





 


287
SauCas9KKH
−
CTGCTTTCCTCTCGGGATTTCGTTTTAGTAC
29538
GTGCCTGGCAACTGGTAGCTGGTTTTAGTACTCTGGA
29715





TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAgaagacTCGGAAGGCCAGGCCACCCAA








GAAATCCCGAGagga





 


288
SauCas9
−
gcCTGCTTTCCTCTCGGGATTTCGTTTTAGTA
29539
ttGTGCCTGGCAACTGGTAGCTGGTTTTAGTACTCTGG
29716





CTCTGGAAACAGAATCTACTAAAACAAGGC

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT






AAAATGCCGTGTTTATCTCGTCAACTTGTTG

TTATCTCGTCAACTTGTTGGCGAGA






GCGAGAgaagacTCGGAAGGCCAGGCCACCCA








AGAAATCCCGAGagga





 


289
SauCas9KKH
−
CTGCTTTCCTCTCGGGATTTCGTTTTAGTAC
29540
GTGCCTGGCAACTGGTAGCTGGTTTTAGTACTCTGGA
29717





TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAgaagacTCGGAAGGCCAGGCCACCCAA








GAAATCCCGAGagga





 


292
SauriCas9
−
CTGCTTTCCTCTCGGGATTTCGTTTTAGTAC
29541
GTGCCTGGCAACTGGTAGCTGGTTTTAGTACTCTGGA
29718





TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAgaagacTCGGAAGGCCAGGCCACCCAA








GAAATCCCGAGagga





 


293
SauriCas9-
−
CTGCTTTCCTCTCGGGATTTCGTTTTAGTAC
29542
GTGCCTGGCAACTGGTAGCTGGTTTTAGTACTCTGGA
29719



KKH

TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAgaagacTCGGAAGGCCAGGCCACCCAA








GAAATCCCGAGagga





 


296
ScaCas9-
+
TTGGATCCATGTCTGATGTAGTTTTAGAGCT
29543
TTTCTTCTTTTCATCCCAGCGTTTTAGAGCTAGAAATA
29720



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCgccttcCGAGTCTTCCACTGCACACAGT








ACATCAGACAtgga





 


297
SpyCas9-
+
TTGGATCCATGTCTGATGTAGTTTTAGAGCT
29544
TTTCTTCTTTTCATCCCAGCGTTTTAGAGCTAGAAATA
29721



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCgccttcCGAGTCTTCCACTGCACACAGT








ACATCAGACAtgga





 


300
ScaCas9-
−
TGCTTTCCTCTCGGGATTTCGTTTTAGAGCT
29545
GCCTGGCAACTGGTAGCTGGGTTTTAGAGCTAGAAAT
29722



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCgaagacTCGGAAGGCCAGGCCACCCAA








GAAATCCCGAGagga





 


301
SpyCas9-
−
TGCTTTCCTCTCGGGATTTCGTTTTAGAGCT
29546
GCCTGGCAACTGGTAGCTGGGTTTTAGAGCTAGAAAT
29723



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCgaagacTCGGAAGGCCAGGCCACCCAA








GAAATCCCGAGagga





 


302
SauCas9KKH
−
CCTGCTTTCCTCTCGGGATTTGTTTTAGTAC
29547
CCTGGCAACTGGTAGCTGGAGGTTTTAGTACTCTGGA
29724





TCTGGAAACAGAATCTACTAAAACAAGGCA

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT






AAATGCCGTGTTTATCTCGTCAACTTGTTGG

ATCTCGTCAACTTGTTGGCGAGA






CGAGAaagacTCGGAAGGCCAGGCCACCCAA








GAAATCCCGAGAggaa





 


303
SpyCas9-
+
CTTGGATCCATGTCTGATGTGTTTTAGAGCT
29548
TTCTTCTTTTCATCCCAGCTGTTTTAGAGCTAGAAATA
29725



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCccttcCGAGTCTTCCACTGCACACAGTA








CATCAGACATggat





 


304
SpyCas9-
−
CTGCTTTCCTCTCGGGATTTGTTTTAGAGCT
29549
CCTGGCAACTGGTAGCTGGAGTTTTAGAGCTAGAAAT
29726



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCaagacTCGGAAGGCCAGGCCACCCAA








GAAATCCCGAGAggaa





 


305
SpyCas9-
+
GCTTGGATCCATGTCTGATGGTTTTAGAGCT
29550
TCTTCTTTTCATCCCAGCTTGTTTTAGAGCTAGAAATA
29727



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCcttcCGAGTCTTCCACTGCACACAGTA








CATCAGACATGgatc





 


306
SpyCas9-
−
CCTGCTTTCCTCTCGGGATTGTTTTAGAGCT
29551
CTGGCAACTGGTAGCTGGAGGTTTTAGAGCTAGAAAT
29728



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCagacTCGGAAGGCCAGGCCACCCAAG








AAATCCCGAGAGgaaa





 


308
SpyCas9-
+
GGCTTGGATCCATGTCTGATGTTTTAGAGCT
29552
CTTCTTTTCATCCCAGCTTGGTTTTAGAGCTAGAAATA
29729



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCttcCGAGTCTTCCACTGCACACAGTAC








ATCAGACATGGatcc





 


309
SpyCas9-
−
GCCTGCTTTCCTCTCGGGATGTTTTAGAGCT
29553
TGGCAACTGGTAGCTGGAGGGTTTTAGAGCTAGAAAT
29730



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCgacTCGGAAGGCCAGGCCACCCAAGA








AATCCCGAGAGGaaag





 


310
SpyCas9-
+
GGGCTTGGATCCATGTCTGAGTTTTAGAGCT
29554
CTTTTCATCCCAGCTTGCACGTTTTAGAGCTAGAAAT
29731



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCtcCGAGTCTTCCACTGCACACAGTAC








ATCAGACATGGAtcca





 


314
SpyCas9-
+
GGGCTTGGATCCATGTCTGAGTTTTAGAGCT
29555
TTCTTTTCATCCCAGCTTGCGTTTTAGAGCTAGAAATA
29732



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCtcCGAGTCTTCCACTGCACACAGTAC








ATCAGACATGGAtcca





 


315
SpyCas9-
−
GGCCTGCTTTCCTCTCGGGAGTTTTAGAGCT
29556
GGCAACTGGTAGCTGGAGGAGTTTTAGAGCTAGAAA
29733



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAAGTGGCACCGAGTCGGTGC






GGTGCacTCGGAAGGCCAGGCCACCCAAGA








AATCCCGAGAGGAaagc





 


316
BlatCas9
+
catgGGCTTGGATCCATGTCTGAGCTATAGTT
29557
tcttTCTTCTTTTCATCCCAGCTGCTATAGTTCCTTACTG
29734





CCTTACTGAAAGGTAAGTTGCTATAGTAAG

AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGA






GGCAACAGACCCGAGGCGTTGGGGATCGCC

GGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCT






TAGCCCGTGTTTACGGGCTCTCCCCATATTC

CCCCATATTCAAAATAATGACAGACGAGCACCTTGGA






AAAATAATGACAGACGAGCACCTTGGAGCA

GCATTTATCTCCGAGGTGCT






TTTATCTCCGAGGTGCTtcCGAGTCTTCCACT








GCACACAGTACATCAGACATGGAtcca





 


317
BlatCas9
+
catgGGCTTGGATCCATGTCTGAGCTATAGTT
29558
tcttTCTTCTTTTCATCCCAGCTGCTATAGTTCCTTACTG
29735





CCTTACTGAAAGGTAAGTTGCTATAGTAAG

AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGA






GGCAACAGACCCGAGGCGTTGGGGATCGCC

GGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCT






TAGCCCGTGTTTACGGGCTCTCCCCATATTC

CCCCATATTCAAAATAATGACAGACGAGCACCTTGGA






AAAATAATGACAGACGAGCACCTTGGAGCA

GCATTTATCTCCGAGGTGCT






TTTATCTCCGAGGTGCTtcCGAGTCTTCCACT








GCACACAGTACATCAGACATGGAtcca





 


321
ScaCas9-
+
TGGGCTTGGATCCATGTCTGGTTTTAGAGCT
29559
TCTTTTCATCCCAGCTTGCAGTTTTAGAGCTAGAAATA
29736



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCcCGAGTCTTCCACTGCACACAGTACA








TCAGACATGGATccaa





 


322
SpyCas9-
+
TGGGCTTGGATCCATGTCTGGTTTTAGAGCT
29560
TCTTTTCATCCCAGCTTGCAGTTTTAGAGCTAGAAATA
29737



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCcCGAGTCTTCCACTGCACACAGTACA








TCAGACATGGATccaa





 


323
SpyCas9-
−
TGGCCTGCTTTCCTCTCGGGGTTTTAGAGCT
29561
GCAACTGGTAGCTGGAGGACGTTTTAGAGCTAGAAAT
29738



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCcTCGGAAGGCCAGGCCACCCAAGAA








ATCCCGAGAGGAAagca





 


324
BlatCas9
−
ggctGGCCTGCTTTCCTCTCGGGGCTATAGTT
29562
ctggCAACTGGTAGCTGGAGGACGCTATAGTTCCTTACT
29739





CCTTACTGAAAGGTAAGTTGCTATAGTAAG

GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG






GGCAACAGACCCGAGGCGTTGGGGATCGCC

AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC






TAGCCCGTGTTTACGGGCTCTCCCCATATTC

TCCCCATATTCAAAATAATGACAGACGAGCACCTTGG






AAAATAATGACAGACGAGCACCTTGGAGCA

AGCATTTATCTCCGAGGTGCT






TTTATCTCCGAGGTGCTcTCGGAAGGCCAGG








CCACCCAAGAAATCCCGAGAGGAAagca





 


325
BlatCas9
−
ggctGGCCTGCTTTCCTCTCGGGGCTATAGTT
29563
ctggCAACTGGTAGCTGGAGGACGCTATAGTTCCTTACT
29740





CCTTACTGAAAGGTAAGTTGCTATAGTAAG

GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG






GGCAACAGACCCGAGGCGTTGGGGATCGCC

AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC






TAGCCCGTGTTTACGGGCTCTCCCCATATTC

TCCCCATATTCAAAATAATGACAGACGAGCACCTTGG






AAAATAATGACAGACGAGCACCTTGGAGCA

AGCATTTATCTCCGAGGTGCT






TTTATCTCCGAGGTGCTcTCGGAAGGCCAGG








CCACCCAAGAAATCCCGAGAGGAAagca





 


327
SpyCas9-
+
ATGGGCTTGGATCCATGTCTGTTTTAGAGCT
29564
CTTTTCATCCCAGCTTGCACGTTTTAGAGCTAGAAAT
29741



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCCGAGTCTTCCACTGCACACAGTACA








TCAGACATGGATCcaag





 


329
SpyCas9-
−
CTGGCCTGCTTTCCTCTCGGGTTTTAGAGCT
29565
CAACTGGTAGCTGGAGGACAGTTTTAGAGCTAGAAAT
29742



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCTCGGAAGGCCAGGCCACCCAAGAAA








TCCCGAGAGGAAAgcag





 


332
SpyCas9-
+
CATGGGCTTGGATCCATGTCGTTTTAGAGCT
29566
TTTTCATCCCAGCTTGCACTGTTTTAGAGCTAGAAATA
29743



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCGAGTCTTCCACTGCACACAGTACAT








CAGACATGGATCCaagc





 


335
SpyCas9-
−
GCTGGCCTGCTTTCCTCTCGGTTTTAGAGCT
29567
GCAACTGGTAGCTGGAGGACGTTTTAGAGCTAGAAAT
29744



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCCGGAAGGCCAGGCCACCCAAGAAAT








CCCGAGAGGAAAGcagg





 


339
SpyCas9-
+
CATGGGCTTGGATCCATGTCGTTTTAGAGCT
29568
TTTTCATCCCAGCTTGCACTGTTTTAGAGCTAGAAATA
29745



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAGTGGCACCGAGTCGGTGC






GGTGCGAGTCTTCCACTGCACACAGTACAT








CAGACATGGATCCaagc





 


341
SpyCas9-
−
GCTGGCCTGCTTTCCTCTCGGTTTTAGAGCT
29569
AACTGGTAGCTGGAGGACAGGTTTTAGAGCTAGAAA
29746



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAAGTGGCACCGAGTCGGTGC






GGTGCCGGAAGGCCAGGCCACCCAAGAAAT








CCCGAGAGGAAAGcagg





 


350
ScaCas9-
+
ACATGGGCTTGGATCCATGTGTTTTAGAGCT
29570
CTTTTCATCCCAGCTTGCACGTTTTAGAGCTAGAAAT
29747



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCAGTCTTCCACTGCACACAGTACATC








AGACATGGATCCAagcc





 


351
SpyCas9-
+
ACATGGGCTTGGATCCATGTGTTTTAGAGCT
29571
TTTCATCCCAGCTTGCACTGGTTTTAGAGCTAGAAAT
29748



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCAGTCTTCCACTGCACACAGTACATC








AGACATGGATCCAagcc





 


354
ScaCas9-
−
GGCTGGCCTGCTTTCCTCTCGTTTTAGAGCT
29572
GTAGCTGGAGGACAGTACTCGTTTTAGAGCTAGAAAT
29749



Sc++

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCGGAAGGCCAGGCCACCCAAGAAATC








CCGAGAGGAAAGCaggc





 


355
SpyCas9
−
GGCTGGCCTGCTTTCCTCTCGTTTTAGAGCT
29573
TAGCTGGAGGACAGTACTCAGTTTTAGAGCTAGAAAT
29750





AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCGGAAGGCCAGGCCACCCAAGAAATC








CCGAGAGGAAAGCaggc





 


358
SpyCas9-
−
GGCTGGCCTGCTTTCCTCTCGTTTTAGAGCT
29574
ACTGGTAGCTGGAGGACAGTGTTTTAGAGCTAGAAAT
29751



SpRY

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCGGAAGGCCAGGCCACCCAAGAAATC








CCGAGAGGAAAGCaggc





 


359
SpyCas9-
−
GGCTGGCCTGCTTTCCTCTCGTTTTAGAGCT
29575
GCAACTGGTAGCTGGAGGACGTTTTAGAGCTAGAAAT
29752



NG

AGAAATAGCAAGTTAAAATAAGGCTAGTCC

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA






GTTATCAACTTGAAAAAGTGGCACCGAGTC

AAAAGTGGCACCGAGTCGGTGC






GGTGCGGAAGGCCAGGCCACCCAAGAAATC








CCGAGAGGAAAGCaggc
















TABLE 4C







Exemplary template RNA sequences and second nick gRNA spacer sequences


Table 4C provides design of RNA components of gene modifying systems for correcting the pathogenic R243Q, mutation in PAH. The gRNA


spacers from Table 1C were filtered, e.g., filtered by occurrence within 15 nt of the desired editing location and use of a Tier 1 Cas


enzyme. For each gRNA ID, this table details the sequence of a complete template RNA, optional second-nick gRNA, and Cas variant for use


in a Cas-RT fusion gene modifying polypeptide. For exemplification, PBS sequences and post-edit homology regions (after the location of


the edit) are set to 12 nt and 30 nt, respectively. Additionally, a second-nick gRNA is selected with preference for a distance near 100 nt


from the first nick and a first preference for a design resulting in a PAM-in system, as described elsewhere in this application.

















SEQ

SEQ



Cas


ID

ID


ID
species
strand
Template RNA
NO
second-nick gRNA
NO
















3
ScaCas9-
−
CACTGGTTTCCGCCTCCAACGTTTTAGAGCTAGAAAT
29753
CACGGTTCGGGGGTATACATGTTTTAGAGCTA
29938



Sc++

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCcccgagaggaaagcaggcc

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






agccacaggTCGGAGGCGGaaac





 


4
SpyCas9-
−
CACTGGTTTCCGCCTCCAACGTTTTAGAGCTAGAAAT
29754
ACGGTTCGGGGGTATACATGGTTTTAGAGCTA
29939



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCcccgagaggaaagcaggcc

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






agccacaggTCGGAGGCGGaaac





 


5
SauriCas9
+
AAGCAGGCCAGCCACAGGTTGGTTTTAGTACTCTGG
29755
TGTGTACTACTCCACTACCTAGTTTTAGTACTC
29940





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAatcccagcttgcactggttt

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






ccgcctcCGACCTGTGGCtggc





 


6
SauriCas9-
+
AAGCAGGCCAGCCACAGGTTGGTTTTAGTACTCTGG
29756
ATGTGTACTACTCCACTACCTGTTTTAGTACTC
29941



KKH

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAatcccagcttgcactggttt

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






ccgcctcCGACCTGTGGCtggc





 


9
ScaCas9-
+
AGCAGGCCAGCCACAGGTTGGTTTTAGAGCTAGAAA
29757
GTGTACTACTCCACTACCTAGTTTTAGAGCTAG
29942



Sc++

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCatcccagcttgcactggttt

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






ccgcctcCGACCTGTGGCtggc





 


10
SpyCas9-
+
AGCAGGCCAGCCACAGGTTGGTTTTAGAGCTAGAAA
29758
CAGTTATGTGTACTACTCCAGTTTTAGAGCTAG
29943



SpRY

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCatcccagcttgcactggttt

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






ccgcctcCGACCTGTGGCtggc





 


11
SpyCas9-
−
GCACTGGTTTCCGCCTCCAAGTTTTAGAGCTAGAAAT
29759
CGGTTCGGGGGTATACATGGGTTTTAGAGCTA
29944



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCccgagaggaaagcaggcca

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






gccacaggTCGGAGGCGGAaacc





 


12
BlatCas9
+
gaaaGCAGGCCAGCCACAGGTTGGCTATAGTTCCTTAC
29760
gggcAGTTATGTGTACTACTCCAGCTATAGTTCC
29945





TGAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCC

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






GAGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGC

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






TCTCCCCATATTCAAAATAATGACAGACGAGCACCT

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






TGGAGCATTTATCTCCGAGGTGCTatcccagcttgcactggtttcc

GACAGACGAGCACCTTGGAGCATTTATCTCCG






gcctcCGACCTGTGGCtggc

AGGTGCT



 


13
BlatCas9
+
gaaaGCAGGCCAGCCACAGGTTGGCTATAGTTCCTTAC
29761
gggcAGTTATGTGTACTACTCCAGCTATAGTTCC
29946





TGAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCC

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






GAGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGC

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






TCTCCCCATATTCAAAATAATGACAGACGAGCACCT

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






TGGAGCATTTATCTCCGAGGTGCTatcccagcttgcactggtttcc

GACAGACGAGCACCTTGGAGCATTTATCTCCG






gcctcCGACCTGTGGCtggc

AGGTGCT



 


14
SauCas9KKH
+
AAAGCAGGCCAGCCACAGGTTGTTTTAGTACTCTGG
29762
GTTATGTGTACTACTCCACTAGTTTTAGTACTC
29947





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAtcccagcttgcactggtttc

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






cgcctcCGACCTGTGGCTggcc





 


15
SauriCas9-
+
AAAGCAGGCCAGCCACAGGTTGTTTTAGTACTCTGG
29763
ATGTGTACTACTCCACTACCTGTTTTAGTACTC
29948



KKH

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAtcccagcttgcactggtttc

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






cgcctcCGACCTGTGGCTggcc





 


17
SpyCas9-
+
AAGCAGGCCAGCCACAGGTTGTTTTAGAGCTAGAAA
29764
TGTACTACTCCACTACCTAAGTTTTAGAGCTAG
29949



NG

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCtcccagcttgcactggtttc

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






cgcctcCGACCTGTGGCTggcc





 


21
SpyCas9-
+
AAGCAGGCCAGCCACAGGTTGTTTTAGAGCTAGAAA
29765
AGTTATGTGTACTACTCCACGTTTTAGAGCTAG
29950



SpRY

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCtcccagcttgcactggtttc

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






cgcctcCGACCTGTGGCTggcc





 


22
SpyCas9-
−
TGCACTGGTTTCCGCCTCCAGTTTTAGAGCTAGAAAT
29766
GGTTCGGGGGTATACATGGGGTTTTAGAGCTA
29951



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCcgagaggaaagcaggccag

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






ccacaggTCGGAGGCGGAAacca





 


25
SauCas9
+
agGAAAGCAGGCCAGCCACAGGTGTTTTAGTACTCTG
29767
gcCTAGCGTCAAAGCCTATGTCCGTTTTAGTAC
29952





GAAACAGAATCTACTAAAACAAGGCAAAATGCCGT

TCTGGAAACAGAATCTACTAAAACAAGGCAAA






GTTTATCTCGTCAACTTGTTGGCGAGAcccagcttgcactggt

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGAG






ttccgcctcCGACCTGTGGCTGgcct

A



 


26
SauCas9KKH
+
GAAAGCAGGCCAGCCACAGGTGTTTTAGTACTCTGG
29768
GTTATGTGTACTACTCCACTAGTTTTAGTACTC
29953





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAcccagcttgcactggtttcc

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






gcctcCGACCTGTGGCTGgcct





 


29
ScaCas9-
+
AAAGCAGGCCAGCCACAGGTGTTTTAGAGCTAGAAA
29769
GTGTACTACTCCACTACCTAGTTTTAGAGCTAG
29954



Sc++

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCcccagcttgcactggtttcc

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






gcctcCGACCTGTGGCTGgcct





 


30
SpyCas9
+
AAAGCAGGCCAGCCACAGGTGTTTTAGAGCTAGAAA
29770
TGTACTACTCCACTACCTAAGTTTTAGAGCTAG
29955





TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCcccagcttgcactggtttcc

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






gcctcCGACCTGTGGCTGgcct





 


33
SpyCas9-
+
AAAGCAGGCCAGCCACAGGTGTTTTAGAGCTAGAAA
29771
GTTATGTGTACTACTCCACTGTTTTAGAGCTAG
29956



SpRY

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCcccagcttgcactggtttcc

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






gcctcCGACCTGTGGCTGgcct





 


34
SpyCas9-
+
AAAGCAGGCCAGCCACAGGTGTTTTAGAGCTAGAAA
29772
TGTACTACTCCACTACCTAAGTTTTAGAGCTAG
29957



NG

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCcccagcttgcactggtttcc

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






gcctcCGACCTGTGGCTGgcct





 


37
SpyCas9-
−
TTGCACTGGTTTCCGCCTCCGTTTTAGAGCTAGAAAT
29773
GTTCGGGGGTATACATGGGCGTTTTAGAGCTA
29958



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCgagaggaaagcaggccagc

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






cacaggTCGGAGGCGGAAAccag





 


42
SauCas9
+
gaGGAAAGCAGGCCAGCCACAGGGTTTTAGTACTCTG
29774
gcCTAGCGTCAAAGCCTATGTCCGTTTTAGTAC
29959





GAAACAGAATCTACTAAAACAAGGCAAAATGCCGT

TCTGGAAACAGAATCTACTAAAACAAGGCAAA






GTTTATCTCGTCAACTTGTTGGCGAGAccagcttgcactggttt

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGAG






ccgcctcCGACCTGTGGCTGGcctg

A



 


43
SauCas9KKH
+
GGAAAGCAGGCCAGCCACAGGGTTTTAGTACTCTGG
29775
GTTATGTGTACTACTCCACTAGTTTTAGTACTC
29960





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAccagcttgcactggtttccg

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






cctcCGACCTGTGGCTGGcctg





 


44
SauriCas9
+
GGAAAGCAGGCCAGCCACAGGGTTTTAGTACTCTGG
29776
TGTGTACTACTCCACTACCTAGTTTTAGTACTC
29961





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAccagcttgcactggtttccg

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






cctcCGACCTGTGGCTGGcctg





 


45
SauriCas9-
+
GGAAAGCAGGCCAGCCACAGGGTTTTAGTACTCTGG
29777
ATGTGTACTACTCCACTACCTGTTTTAGTACTC
29962



KKH

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAccagcttgcactggtttccg

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






cctcCGACCTGTGGCTGGcctg





 


48
ScaCas9-
+
GAAAGCAGGCCAGCCACAGGGTTTTAGAGCTAGAA
29778
GTGTACTACTCCACTACCTAGTTTTAGAGCTAG
29963



Sc++

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCccagcttgcactggtttcc

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






gcctcCGACCTGTGGCTGGcctg





 


49
SpyCas9-
+
GAAAGCAGGCCAGCCACAGGGTTTTAGAGCTAGAA
29779
TTATGTGTACTACTCCACTAGTTTTAGAGCTAG
29964



SpRY

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCccagcttgcactggtttcc

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






gcctcCGACCTGTGGCTGGcctg





 


50
SpyCas9-
−
CTTGCACTGGTTTCCGCCTCGTTTTAGAGCTAGAAAT
29780
TTCGGGGGTATACATGGGCTGTTTTAGAGCTA
29965



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCagaggaaagcaggccagcc

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






acaggTCGGAGGCGGAAACcagt





 


51
BlatCas9
−
cagcTTGCACTGGTTTCCGCCTCGCTATAGTTCCTTACT
29781
ggttCGGGGGTATACATGGGCTTGCTATAGTTCC
29966





GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCT

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCCCCATATTCAAAATAATGACAGACGAGCACCTT

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






GGAGCATTTATCTCCGAGGTGCTagaggaaagcaggccagcca

GACAGACGAGCACCTTGGAGCATTTATCTCCG






caggTCGGAGGCGGAAACcagt

AGGTGCT



 


52
BlatCas9
−
cagcTTGCACTGGTTTCCGCCTCGCTATAGTTCCTTACT
29782
ggttCGGGGGTATACATGGGCTTGCTATAGTTCC
29967





GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCT

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCCCCATATTCAAAATAATGACAGACGAGCACCTT

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






GGAGCATTTATCTCCGAGGTGCTagaggaaagcaggccagcca

GACAGACGAGCACCTTGGAGCATTTATCTCCG






caggTCGGAGGCGGAAACcagt

AGGTGCT



 


57
Nme2Cas9
−
ccCAGCTTGCACTGGTTTCCGCCTGTTGTAGCTCCCTT
29783
cgGTTCGGGGGTATACATGGGCTTGTTGTAGCT
29968





TCTCATTTCGGAAACGAAATGAGAACCGTTGCTACA

CCCTTTCTCATTTCGGAAACGAAATGAGAACC






ATAAGGCCGTCTGAAAAGATGTGCCGCAACGCTCTG

GTTGCTACAATAAGGCCGTCTGAAAAGATGTG






CCCCTTAAAGCTTCTGCTTTAAGGGGCATCGTTTAgag

CCGCAACGCTCTGCCCCTTAAAGCTTCTGCTTT






gaaagcaggccagccacaggTCGGAGGCGGAAACCagtg

AAGGGGCATCGTTTA



 


58
SauCas9KKH
+
AGGAAAGCAGGCCAGCCACAGGTTTTAGTACTCTGG
29784
TTATGTGTACTACTCCACTACGTTTTAGTACTC
29969





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAcagcttgcactggtttccgc

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






ctcCGACCTGTGGCTGGCctgc





 


59
SpyCas9-
+
GGAAAGCAGGCCAGCCACAGGTTTTAGAGCTAGAA
29785
TATGTGTACTACTCCACTACGTTTTAGAGCTAG
29970



SpRY

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCcagcttgcactggtttcc

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






gcctcCGACCTGTGGCTGGCctgc





 


60
SpyCas9-
−
GCTTGCACTGGTTTCCGCCTGTTTTAGAGCTAGAAAT
29786
TCGGGGGTATACATGGGCTTGTTTTAGAGCTA
29971



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCgaggaaagcaggccagcca

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






caggTCGGAGGCGGAAACCagtg





 


61
BlatCas9
−
ccagCTTGCACTGGTTTCCGCCTGCTATAGTTCCTTACT
29787
ggttCGGGGGTATACATGGGCTTGCTATAGTTCC
29972





GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCT

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCCCCATATTCAAAATAATGACAGACGAGCACCTT

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






GGAGCATTTATCTCCGAGGTGCTgaggaaagcaggccagccac

GACAGACGAGCACCTTGGAGCATTTATCTCCG






aggTCGGAGGCGGAAACCagtg

AGGTGCT



 


 


62
BlatCas9
−
ccagCTTGCACTGGTTTCCGCCTGCTATAGTTCCTTACT
29788
ggttCGGGGGTATACATGGGCTTGCTATAGTTCC
29973





GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCT

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCCCCATATTCAAAATAATGACAGACGAGCACCTT

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






GGAGCATTTATCTCCGAGGTGCTgaggaaagcaggccagccac

GACAGACGAGCACCTTGGAGCATTTATCTCCG






aggTCGGAGGCGGAAACCagtg

AGGTGCT



 


64
SauCas9KKH
−
CAGCTTGCACTGGTTTCCGCCGTTTTAGTACTCTGGA
29789
GGTTCGGGGGTATACATGGGCGTTTTAGTACT
29974





AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






TATCTCGTCAACTTGTTGGCGAGAaggaaagcaggccagccac

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGAG






aggTCGGAGGCGGAAACCAgtgc

A



 


65
SpyCas9-
+
AGGAAAGCAGGCCAGCCACAGTTTTAGAGCTAGAA
29790
TGTACTACTCCACTACCTAAGTTTTAGAGCTAG
29975



NG

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCagcttgcactggtttccg

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






cctcCGACCTGTGGCTGGCCtgct





 


69
SpyCas9-
+
AGGAAAGCAGGCCAGCCACAGTTTTAGAGCTAGAA
29791
ATGTGTACTACTCCACTACCGTTTTAGAGCTAG
29976



SpRY

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCagcttgcactggtttccg

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






cctcCGACCTGTGGCTGGCCtgct





 


70
SpyCas9-
−
AGCTTGCACTGGTTTCCGCCGTTTTAGAGCTAGAAAT
29792
CGGGGGTATACATGGGCTTGGTTTTAGAGCTA
29977



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCaggaaagcaggccagccac

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






aggTCGGAGGCGGAAACCAgtgc





 


76
ScaCas9-
+
GAGGAAAGCAGGCCAGCCACGTTTTAGAGCTAGAA
29793
GTGTACTACTCCACTACCTAGTTTTAGAGCTAG
29978



Sc++

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCgcttgcactggtttccgc

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






ctcCGACCTGTGGCTGGCCTgctt





 


77
SpyCas9
+
GAGGAAAGCAGGCCAGCCACGTTTTAGAGCTAGAA
29794
TGTACTACTCCACTACCTAAGTTTTAGAGCTAG
29979





ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCgcttgcactggtttccgc

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






ctcCGACCTGTGGCTGGCCTgctt





 


80
SpyCas9-
+
GAGGAAAGCAGGCCAGCCACGTTTTAGAGCTAGAA
29795
TGTGTACTACTCCACTACCTGTTTTAGAGCTAG
29980



SpRY

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCgcttgcactggtttccgc

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






ctcCGACCTGTGGCTGGCCTgctt





 


81
SpyCas9-
+
GAGGAAAGCAGGCCAGCCACGTTTTAGAGCTAGAA
29796
TGTACTACTCCACTACCTAAGTTTTAGAGCTAG
29981



NG

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCgcttgcactggtttccgc

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






ctcCGACCTGTGGCTGGCCTgctt





 


84
SpyCas9-
−
CAGCTTGCACTGGTTTCCGCGTTTTAGAGCTAGAAAT
29797
GGGGGTATACATGGGCTTGGGTTTTAGAGCTA
29982



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCggaaagcaggccagccaca

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






ggTCGGAGGCGGAAACCAGtgca





 


86
SauriCas9
+
GAGAGGAAAGCAGGCCAGCCAGTTTTAGTACTCTGG
29798
TGTGTACTACTCCACTACCTAGTTTTAGTACTC
29983





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGActtgcactggtttccgcctc

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






CGACCTGTGGCTGGCCTGcttt





 


87
SauriCas9-
+
GAGAGGAAAGCAGGCCAGCCAGTTTTAGTACTCTGG
29799
TGTGTACTACTCCACTACCTAGTTTTAGTACTC
29984



KKH

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGActtgcactggtttccgcctc

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






CGACCTGTGGCTGGCCTGcttt





 


90
ScaCas9-
+
AGAGGAAAGCAGGCCAGCCAGTTTTAGAGCTAGAA
29800
GTGTACTACTCCACTACCTAGTTTTAGAGCTAG
29985



Sc++

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCcttgcactggtttccgcct

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






cCGACCTGTGGCTGGCCTGcttt





 


91
SpyCas9-
+
AGAGGAAAGCAGGCCAGCCAGTTTTAGAGCTAGAA
29801
GTGTACTACTCCACTACCTAGTTTTAGAGCTAG
29986



SpRY

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCcttgcactggtttccgcct

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






cCGACCTGTGGCTGGCCTGcttt





 


92
SpyCas9-
−
CCAGCTTGCACTGGTTTCCGGTTTTAGAGCTAGAAAT
29802
GGGGTATACATGGGCTTGGAGTTTTAGAGCTA
29987



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCgaaagcaggccagccacag

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






gTCGGAGGCGGAAACCAGTgcaa





 


93
BlatCaS9
−
atccCAGCTTGCACTGGTTTCCGGCTATAGTTCCTTACT
29803
gttcGGGGGTATACATGGGCTTGGCTATAGTTCC
29988





GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCT

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCCCCATATTCAAAATAATGACAGACGAGCACCTT

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






GGAGCATTTATCTCCGAGGTGCTgaaagcaggccagccacagg

GACAGACGAGCACCTTGGAGCATTTATCTCCG






TCGGAGGCGGAAACCAGTgcaa

AGGTGCT



 


96
Nme2Cas9
−
tcATCCCAGCTTGCACTGGTTTCCGTTGTAGCTCCCTT
29804
cgGTTCGGGGGTATACATGGGCTTGTTGTAGCT
29989





TCTCATTTCGGAAACGAAATGAGAACCGTTGCTACA

CCCTTTCTCATTTCGGAAACGAAATGAGAACC






ATAAGGCCGTCTGAAAAGATGTGCCGCAACGCTCTG

GTTGCTACAATAAGGCCGTCTGAAAAGATGTG






CCCCTTAAAGCTTCTGCTTTAAGGGGCATCGTTTAaaa

CCGCAACGCTCTGCCCCTTAAAGCTTCTGCTTT






gcaggccagccacaggTCGGAGGCGGAAACCAGTGcaag

AAGGGGCATCGTTTA



 


97
PpnCas9
+
tccCGAGAGGAAAGCAGGCCAGCCGTTGTAGCTCCCT
29805
ctaGCGTCAAAGCCTATGTCCCTGGTTGTAGCTC
29990





TTTTCATTTCGCGAAAGCGAAATGAAAAACGTTGTT

CCTTTTTCATTTCGCGAAAGCGAAATGAAAAA






ACAATAAGAGATGAATTTCTCGCAAAGCTCTGCCTC

CGTTGTTACAATAAGAGATGAATTTCTCGCAA






TTGAAATTTCGGTTTCAAGAGGCATCttgcactggtttccgcct

AGCTCTGCCTCTTGAAATTTCGGTTTCAAGAGG






cCGACCTGTGGCTGGCCTGCtttc

CATC



 


98
SauCas9KKH
+
CGAGAGGAAAGCAGGCCAGCCGTTTTAGTACTCTGG
29806
ATGTGTACTACTCCACTACCTGTTTTAGTACTC
29991





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAttgcactggtttccgcctcC

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






GACCTGTGGCTGGCCTGCtttc





 


99
SauCas9KKH
+
CGAGAGGAAAGCAGGCCAGCCGTTTTAGTACTCTGG
29807
ATGTGTACTACTCCACTACCTGTTTTAGTACTC
29992





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAttgcactggtttccgcctcC

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






GACCTGTGGCTGGCCTGCtttc





 


102
SauriCas9-
+
CGAGAGGAAAGCAGGCCAGCCGTTTTAGTACTCTGG
29808
TGTGTACTACTCCACTACCTAGTTTTAGTACTC
29993



KKH

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAttgcactggtttccgcctcC

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






GACCTGTGGCTGGCCTGCtttc





 


103
SpyCas9-
+
GAGAGGAAAGCAGGCCAGCCGTTTTAGAGCTAGAA
29809
TGTACTACTCCACTACCTAAGTTTTAGAGCTAG
29994



SpRY

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCttgcactggtttccgcctc

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CGACCTGTGGCTGGCCTGCtttc





 


104
SpyCas9-
−
CCCAGCTTGCACTGGTTTCCGTTTTAGAGCTAGAAAT
29810
GGGTATACATGGGCTTGGATGTTTTAGAGCTA
29995



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCaaagcaggccagccacagg

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






TCGGAGGCGGAAACCAGTGcaag





 


105
BlatCas9
−
catcCCAGCTTGCACTGGTTTCCGCTATAGTTCCTTACT
29811
ggggTATACATGGGCTTGGATCCGCTATAGTTCC
29996





GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCT

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCCCCATATTCAAAATAATGACAGACGAGCACCTT

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






GGAGCATTTATCTCCGAGGTGCTaaagcaggccagccacaggT

GACAGACGAGCACCTTGGAGCATTTATCTCCG






CGGAGGCGGAAACCAGTGcaag

AGGTGCT



 


106
BlatCas9
−
catcCCAGCTTGCACTGGTTTCCGCTATAGTTCCTTACT
29812
ggggTATACATGGGCTTGGATCCGCTATAGTTCC
29997





GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCT

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCCCCATATTCAAAATAATGACAGACGAGCACCTT

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






GGAGCATTTATCTCCGAGGTGCTaaagcaggccagccacaggT

GACAGACGAGCACCTTGGAGCATTTATCTCCG






CGGAGGCGGAAACCAGTGcaag

AGGTGCT



 


107
BlatCas9
−
catcCCAGCTTGCACTGGTTTCCGCTATAGTTCCTTACT
29813
ggggTATACATGGGCTTGGATCCGCTATAGTTCC
29998





GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCT

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCCCCATATTCAAAATAATGACAGACGAGCACCTT

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






GGAGCATTTATCTCCGAGGTGCTaaagcaggccagccacaggT

GACAGACGAGCACCTTGGAGCATTTATCTCCG






CGGAGGCGGAAACCAGTGcaag

AGGTGCT



 


108
SauCas9KKH
+
CCGAGAGGAAAGCAGGCCAGCGTTTTAGTACTCTGG
29814
ATGTGTACTACTCCACTACCTGTTTTAGTACTC
29999





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAtgcactggtttccgcctcC

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






GACCTGTGGCTGGCCTGCTttcc





 


109
SpyCas9-
−
TCCCAGCTTGCACTGGTTTCGTTTTAGAGCTAGAAAT
29815
TATACATGGGCTTGGATCCAGTTTTAGAGCTA
30000



NG

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCaagcaggccagccacaggT

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CGGAGGCGGAAACCAGTGCaagc





 


112
SpyCas9-
+
CGAGAGGAAAGCAGGCCAGCGTTTTAGAGCTAGAA
29816
GTACTACTCCACTACCTAAAGTTTTAGAGCTAG
30001



SpRY

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCtgcactggtttccgcctc

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CGACCTGTGGCTGGCCTGCTttcc





 


114
SpyCas9-
−
TCCCAGCTTGCACTGGTTTCGTTTTAGAGCTAGAAAT
29817
GGTATACATGGGCTTGGATCGTTTTAGAGCTA
30002



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCaagcaggccagccacaggT

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CGGAGGCGGAAACCAGTGCaagc





 


121
ScaCas9-
−
ATCCCAGCTTGCACTGGTTTGTTTTAGAGCTAGAAAT
29818
GTATACATGGGCTTGGATCCGTTTTAGAGCTA
30003



Sc++

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCagcaggccagccacaggT

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CGGAGGCGGAAACCAGTGCAagct





 


122
SpyCas9-
−
ATCCCAGCTTGCACTGGTTTGTTTTAGAGCTAGAAAT
29819
GTATACATGGGCTTGGATCCGTTTTAGAGCTA
30004



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCagcaggccagccacaggT

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CGGAGGCGGAAACCAGTGCAagct





 


123
SpyCas9-
+
CCGAGAGGAAAGCAGGCCAGGTTTTAGAGCTAGAA
29820
TACTACTCCACTACCTAAAGGTTTTAGAGCTAG
30005



SpRY

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCgcactggtttccgcctcC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GACCTGTGGCTGGCCTGCTTtcct





 


124
BlatCas9
−
ttcaTCCCAGCTTGCACTGGTTTGCTATAGTTCCTTACT
29821
ggggTATACATGGGCTTGGATCCGCTATAGTTCC
30006





GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCT

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCCCCATATTCAAAATAATGACAGACGAGCACCTT

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






GGAGCATTTATCTCCGAGGTGCTagcaggccagccacaggTC

GACAGACGAGCACCTTGGAGCATTTATCTCCG






GGAGGCGGAAACCAGTGCAagct

AGGTGCT



 


126
Nme2Cas9
−
ttTTCATCCCAGCTTGCACTGGTTGTTGTAGCTCCCTTT
29822
cgGTTCGGGGGTATACATGGGCTTGTTGTAGCT
30007





CTCATTTCGGAAACGAAATGAGAACCGTTGCTACAA

CCCTTTCTCATTTCGGAAACGAAATGAGAACC






TAAGGCCGTCTGAAAAGATGTGCCGCAACGCTCTGC

GTTGCTACAATAAGGCCGTCTGAAAAGATGTG






CCCTTAAAGCTTCTGCTTTAAGGGGCATCGTTTAgcag

CCGCAACGCTCTGCCCCTTAAAGCTTCTGCTTT






gccagccacaggTCGGAGGCGGAAACCAGTGCAAgctg

AAGGGGCATCGTTTA



 


127
SpyCas9-
+
CCCGAGAGGAAAGCAGGCCAGTTTTAGAGCTAGAA
29823
ACTACTCCACTACCTAAAGGGTTTTAGAGCTA
30008



SpRY

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






TGAAAAAGTGGCACCGAGTCGGTGCcactggtttccgcctcC

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GACCTGTGGCTGGCCTGCTTTcctc





 


128
SpyCas9-
−
CATCCCAGCTTGCACTGGTTGTTTTAGAGCTAGAAAT
29824
TATACATGGGCTTGGATCCAGTTTTAGAGCTA
30009



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCgcaggccagccacaggTC

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GGAGGCGGAAACCAGTGCAAgctg





 


129
BlatCas9
+
aatcCCGAGAGGAAAGCAGGCCAGCTATAGTTCCTTAC
29825
tgtaCTACTCCACTACCTAAAGGGCTATAGTTCC
30010





TGAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCC

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






GAGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGC

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






TCTCCCCATATTCAAAATAATGACAGACGAGCACCT

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






TGGAGCATTTATCTCCGAGGTGCTcactggtttccgcctcCGA

GACAGACGAGCACCTTGGAGCATTTATCTCCG






CCTGTGGCTGGCCTGCTTTcctc

AGGTGCT



 


130
BlatCas9
+
aatcCCGAGAGGAAAGCAGGCCAGCTATAGTTCCTTAC
29826
tgtaCTACTCCACTACCTAAAGGGCTATAGTTCC
30011





TGAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCC

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






GAGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGC

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






TCTCCCCATATTCAAAATAATGACAGACGAGCACCT

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






TGGAGCATTTATCTCCGAGGTGCTcactggtttccgcctcCGA

GACAGACGAGCACCTTGGAGCATTTATCTCCG






CCTGTGGCTGGCCTGCTTTcctc

AGGTGCT



 


131
BlatCas9
−
tttcATCCCAGCTTGCACTGGTTGCTATAGTTCCTTACT
29827
ggggTATACATGGGCTTGGATCCGCTATAGTTCC
30012





GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCT

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCCCCATATTCAAAATAATGACAGACGAGCACCTT

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






GGAGCATTTATCTCCGAGGTGCTgcaggccagccacaggTCG

GACAGACGAGCACCTTGGAGCATTTATCTCCG






GAGGCGGAAACCAGTGCAAgctg

AGGTGCT



 


132
SpyCas9-
+
TCCCGAGAGGAAAGCAGGCCGTTTTAGAGCTAGAAA
29828
GTACTACTCCACTACCTAAAGTTTTAGAGCTAG
30013



NG

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCactggtttccgcctcCGA

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CCTGTGGCTGGCCTGCTTTCctct





 


136
SpyCas9-
+
TCCCGAGAGGAAAGCAGGCCGTTTTAGAGCTAGAAA
29829
CTACTCCACTACCTAAAGGTGTTTTAGAGCTAG
30014



SpRY

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCactggtttccgcctcCGA

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CCTGTGGCTGGCCTGCTTTCctct





 


137
SpyCas9-
−
TCATCCCAGCTTGCACTGGTGTTTTAGAGCTAGAAAT
29830
ATACATGGGCTTGGATCCATGTTTTAGAGCTA
30015



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCcaggccagccacaggTCG

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GAGGCGGAAACCAGTGCAAGctgg





 


141
ScaCas9-
+
ATCCCGAGAGGAAAGCAGGCGTTTTAGAGCTAGAAA
29831
TCCACTACCTAAAGGTCTCCGTTTTAGAGCTAG
30016



Sc++

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCctggtttccgcctcCGA

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CCTGTGGCTGGCCTGCTTTCCtctc





 


142
SpyCas9-
+
ATCCCGAGAGGAAAGCAGGCGTTTTAGAGCTAGAAA
29832
TACTCCACTACCTAAAGGTCGTTTTAGAGCTAG
30017



SpRY

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCctggtttccgcctcCGA

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CCTGTGGCTGGCCTGCTTTCCtctc





 


143
SpyCas9-
−
TTCATCCCAGCTTGCACTGGGTTTTAGAGCTAGAAAT
29833
TACATGGGCTTGGATCCATGGTTTTAGAGCTA
30018



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCaggccagccacaggTCG

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GAGGCGGAAACCAGTGCAAGCtggg





 


144
BlatCas9
+
gaaaTCCCGAGAGGAAAGCAGGCGCTATAGTTCCTTA
29834
tgtaCTACTCCACTACCTAAAGGGCTATAGTTCC
30019





CTGAAAGGTAAGTTGCTATAGTAAGGGCAACAGACC

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






CGAGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGG

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCTCCCCATATTCAAAATAATGACAGACGAGCACC

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






TTGGAGCATTTATCTCCGAGGTGCTctggtttccgcctcCGA

GACAGACGAGCACCTTGGAGCATTTATCTCCG






CCTGTGGCTGGCCTGCTTTCCtctc

AGGTGCT



 


145
BlatCas9
−
ctttTCATCCCAGCTTGCACTGGGCTATAGTTCCTTACT
29835
ggggTATACATGGGCTTGGATCCGCTATAGTTCC
30020





GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCT

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCCCCATATTCAAAATAATGACAGACGAGCACCTT

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






GGAGCATTTATCTCCGAGGTGCTaggccagccacaggTCGG

GACAGACGAGCACCTTGGAGCATTTATCTCCG






AGGCGGAAACCAGTGCAAGCtggg

AGGTGCT



 


148
Nme2Cas9
+
aaGAAATCCCGAGAGGAAAGCAGGGTTGTAGCTCCC
29836
taCTCCACTACCTAAAGGTCTCCTGTTGTAGCTC
30021





TTTCTCATTTCGGAAACGAAATGAGAACCGTTGCTA

CCTTTCTCATTTCGGAAACGAAATGAGAACCG






CAATAAGGCCGTCTGAAAAGATGTGCCGCAACGCTC

TTGCTACAATAAGGCCGTCTGAAAAGATGTGC






TGCCCCTTAAAGCTTCTGCTTTAAGGGGCATCGTTTA

CGCAACGCTCTGCCCCTTAAAGCTTCTGCTTTA






tggtttccgcctcCGACCTGTGGCTGGCCTGCTTTCCTctcg

AGGGGCATCGTTTA



 


149
Nme2Cas9
−
ttCTTTTCATCCCAGCTTGCACTGGTTGTAGCTCCCTTT
29837
cgGTTCGGGGGTATACATGGGCTTGTTGTAGCT
30022





CTCATTTCGGAAACGAAATGAGAACCGTTGCTACAA

CCCTTTCTCATTTCGGAAACGAAATGAGAACC






TAAGGCCGTCTGAAAAGATGTGCCGCAACGCTCTGC

GTTGCTACAATAAGGCCGTCTGAAAAGATGTG






CCCTTAAAGCTTCTGCTTTAAGGGGCATCGTTTAggcc

CCGCAACGCTCTGCCCCTTAAAGCTTCTGCTTT






agccacaggTCGGAGGCGGAAACCAGTGCAAGCTggga

AAGGGGCATCGTTTA



 


150
SauriCas9-
+
AAATCCCGAGAGGAAAGCAGGGTTTTAGTACTCTGG
29838
ACTCCACTACCTAAAGGTCTCGTTTTAGTACTC
30023



KKH

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAtggtttccgcctcCGAC

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






CTGTGGCTGGCCTGCTTTCCTctcg





 


151
SpyCas9-
+
AATCCCGAGAGGAAAGCAGGGTTTTAGAGCTAGAA
29839
ACTCCACTACCTAAAGGTCTGTTTTAGAGCTAG
30024



SpRY

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCtggtttccgcctcCGA

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CCTGTGGCTGGCCTGCTTTCCTctcg





 


152
SpyCas9-
−
TTTCATCCCAGCTTGCACTGGTTTTAGAGCTAGAAAT
29840
ACATGGGCTTGGATCCATGTGTTTTAGAGCTA
30025



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCggccagccacaggTCGG

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






AGGCGGAAACCAGTGCAAGCTggga





 


153
BlatCas9
+
agaaATCCCGAGAGGAAAGCAGGGCTATAGTTCCTTA
29841
actcCACTACCTAAAGGTCTCCTGCTATAGTTCC
30026





CTGAAAGGTAAGTTGCTATAGTAAGGGCAACAGACC

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






CGAGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGG

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCTCCCCATATTCAAAATAATGACAGACGAGCACC

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






TTGGAGCATTTATCTCCGAGGTGCTtggtttccgcctcCGAC

GACAGACGAGCACCTTGGAGCATTTATCTCCG






CTGTGGCTGGCCTGCTTTCCTctcg

AGGTGCT



 


154
BlatCas9
−
tcttTTCATCCCAGCTTGCACTGGCTATAGTTCCTTACT
29842
catgGGCTTGGATCCATGTCTGAGCTATAGTTCC
30027





GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCG

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






AGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCT

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCCCCATATTCAAAATAATGACAGACGAGCACCTT

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






GGAGCATTTATCTCCGAGGTGCTggccagccacaggTCGG

GACAGACGAGCACCTTGGAGCATTTATCTCCG






AGGCGGAAACCAGTGCAAGCTggga

AGGTGCT



 


155
SauCas9KKH
+
GAAATCCCGAGAGGAAAGCAGGTTTTAGTACTCTGG
29843
TACTCCACTACCTAAAGGTCTGTTTTAGTACTC
30028





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAggtttccgcctcCGAC

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






CTGTGGCTGGCCTGCTTTCCTCtcgg





 


156
SpyCas9-
−
TTTTCATCCCAGCTTGCACTGTTTTAGAGCTAGAAAT
29844
CATGGGCTTGGATCCATGTCGTTTTAGAGCTAG
30029



NG

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAAGTGGCACCGAGTCGGTGCgccagccacaggTCGGA

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GGCGGAAACCAGTGCAAGCTGggat





 


160
SpyCas9-
−
TTTTCATCCCAGCTTGCACTGTTTTAGAGCTAGAAAT
29845
CATGGGCTTGGATCCATGTCGTTTTAGAGCTAG
30030



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAAGTGGCACCGAGTCGGTGCgccagccacaggTCGGA

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GGCGGAAACCAGTGCAAGCTGggat





 


161
SpyCas9-
+
AAATCCCGAGAGGAAAGCAGGTTTTAGAGCTAGAA
29846
CTCCACTACCTAAAGGTCTCGTTTTAGAGCTAG
30031



SpRY

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCggtttccgcctcCGAC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CTGTGGCTGGCCTGCTTTCCTCtcgg





 


167
ScaCas9-
−
CTTTTCATCCCAGCTTGCACGTTTTAGAGCTAGAAAT
29847
TGGGCTTGGATCCATGTCTGGTTTTAGAGCTAG
30032



Sc++

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAAGTGGCACCGAGTCGGTGCccagccacaggTCGGA

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GGCGGAAACCAGTGCAAGCTGGgatg





 


168
SpyCas9
−
CTTTTCATCCCAGCTTGCACGTTTTAGAGCTAGAAAT
29848
TTCGGGGGTATACATGGGCTGTTTTAGAGCTA
30033





AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCccagccacaggTCGGA

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GGCGGAAACCAGTGCAAGCTGGgatg





 


171
SpyCas9-
−
CTTTTCATCCCAGCTTGCACGTTTTAGAGCTAGAAAT
29849
ATGGGCTTGGATCCATGTCTGTTTTAGAGCTAG
30034



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAAGTGGCACCGAGTCGGTGCccagccacaggTCGGA

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GGCGGAAACCAGTGCAAGCTGGgatg





 


172
SpyCas9-
−
GAAATCCCGAGAGGAAAGCAGTTTTAGAGCTAGAA
29850
CCACTACCTAAAGGTCTCCTGTTTTAGAGCTAG
30035



NG

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCgtttccgcctcCGAC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CTGTGGCTGGCCTGCTTTCCTCTcggg





 


175
SpyCas9-
−
CTTTTCATCCCAGCTTGCACGTTTTAGAGCTAGAAAT
29851
CATGGGCTTGGATCCATGTCGTTTTAGAGCTAG
30036



NG

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAAGTGGCACCGAGTCGGTGCccagccacaggTCGGA

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GGCGGAAACCAGTGCAAGCTGGgatg





 


179
SpyCas9-
+
GAAATCCCGAGAGGAAAGCAGTTTTAGAGCTAGAA
29852
TCCACTACCTAAAGGTCTCCGTTTTAGAGCTAG
30037



SpRY

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCgtttccgcctcCGAC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CTGTGGCTGGCCTGCTTTCCTCTcggg





 


183
SauriCas9
−
TTCTTTTCATCCCAGCTTGCAGTTTTAGTACTCTGGA
29853
ATGTCTGATGTACTGTGTGCAGTTTTAGTACTC
30038





AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TATCTCGTCAACTTGTTGGCGAGAcagccacaggTCGGAG

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






GCGGAAACCAGTGCAAGCTGGGatga





 


184
SauriCas9-
−
TTCTTTTCATCCCAGCTTGCAGTTTTAGTACTCTGGA
29854
TCCATGTCTGATGTACTGTGTGTTTTAGTACTC
30039



KKH

AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TATCTCGTCAACTTGTTGGCGAGAcagccacaggTCGGAG

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






GCGGAAACCAGTGCAAGCTGGGatga





 


187
ScaCas9-
+
AGAAATCCCGAGAGGAAAGCGTTTTAGAGCTAGAA
29855
CACTACCTAAAGGTCTCCTAGTTTTAGAGCTAG
30040



Sc++

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCtttccgcctcCGACC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






TGTGGCTGGCCTGCTTTCCTCTCggga





 


188
SpyCas9
+
AGAAATCCCGAGAGGAAAGCGTTTTAGAGCTAGAA
29856
TGTACTACTCCACTACCTAAGTTTTAGAGCTAG
30041





ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCtttccgcctcCGACC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






TGTGGCTGGCCTGCTTTCCTCTCggga





 


191
SpyCas9-
+
AGAAATCCCGAGAGGAAAGCGTTTTAGAGCTAGAA
29857
CCACTACCTAAAGGTCTCCTGTTTTAGAGCTAG
30042



SpRY

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCtttccgcctcCGACC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






TGTGGCTGGCCTGCTTTCCTCTCggga





 


194
ScaCas9-
−
TCTTTTCATCCCAGCTTGCAGTTTTAGAGCTAGAAAT
29858
TGGGCTTGGATCCATGTCTGGTTTTAGAGCTAG
30043



Sc++

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAAGTGGCACCGAGTCGGTGCcagccacaggTCGGAG

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GCGGAAACCAGTGCAAGCTGGGatga





 


195
SpyCas9-
−
TCTTTTCATCCCAGCTTGCAGTTTTAGAGCTAGAAAT
29859
TGGGCTTGGATCCATGTCTGGTTTTAGAGCTAG
30044



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAAGTGGCACCGAGTCGGTGCcagccacaggTCGGAG

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GCGGAAACCAGTGCAAGCTGGGatga





 


196
SpyCas9-
+
AGAAATCCCGAGAGGAAAGCGTTTTAGAGCTAGAA
29860
CCACTACCTAAAGGTCTCCTGTTTTAGAGCTAG
30045



NG

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCtttccgcctcCGACC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






TGTGGCTGGCCTGCTTTCCTCTCggga





 


199
BlatCas9
+
ccaaGAAATCCCGAGAGGAAAGCGCTATAGTTCCTTA
29861
actcCACTACCTAAAGGTCTCCTGCTATAGTTCC
30046





CTGAAAGGTAAGTTGCTATAGTAAGGGCAACAGACC

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






CGAGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGG

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCTCCCCATATTCAAAATAATGACAGACGAGCACC

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






TTGGAGCATTTATCTCCGAGGTGCTtttccgcctcCGACCT

GACAGACGAGCACCTTGGAGCATTTATCTCCG






GTGGCTGGCCTGCTTTCCTCTCggga

AGGTGCT



 


203
Nme2Cas9
+
acCCAAGAAATCCCGAGAGGAAAGGTTGTAGCTCCCT
29862
taCTCCACTACCTAAAGGTCTCCTGTTGTAGCTC
30047





TTCTCATTTCGGAAACGAAATGAGAACCGTTGCTAC

CCTTTCTCATTTCGGAAACGAAATGAGAACCG






AATAAGGCCGTCTGAAAAGATGTGCCGCAACGCTCT

TTGCTACAATAAGGCCGTCTGAAAAGATGTGC






GCCCCTTAAAGCTTCTGCTTTAAGGGGCATCGTTTAtt

CGCAACGCTCTGCCCCTTAAAGCTTCTGCTTTA






ccgcctcCGACCTGTGGCTGGCCTGCTTTCCTCTCGggat

AGGGGCATCGTTTA



 


204
PpnCas9
−
tttCTTCTTTTCATCCCAGCTTGCGTTGTAGCTCCCTTTT
29863
aacTGGTAGCTGGAGGACAGTACTGTTGTAGCT
30048





TCATTTCGCGAAAGCGAAATGAAAAACGTTGTTACA

CCCTTTTTCATTTCGCGAAAGCGAAATGAAAA






ATAAGAGATGAATTTCTCGCAAAGCTCTGCCTCTTG

ACGTTGTTACAATAAGAGATGAATTTCTCGCA






AAATTTCGGTTTCAAGAGGCATCagccacaggTCGGAGG

AAGCTCTGCCTCTTGAAATTTCGGTTTCAAGAG






CGGAAACCAGTGCAAGCTGGGAtgaa

GCATC



 


205
SauCas9KKH
−
CTTCTTTTCATCCCAGCTTGCGTTTTAGTACTCTGGA
29864
ATACATGGGCTTGGATCCATGGTTTTAGTACTC
30049





AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TATCTCGTCAACTTGTTGGCGAGAagccacaggTCGGAG

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






GCGGAAACCAGTGCAAGCTGGGAtgaa





 


206
SauCas9KKH
−
CTTCTTTTCATCCCAGCTTGCGTTTTAGTACTCTGGA
29865
ATACATGGGCTTGGATCCATGGTTTTAGTACTC
30050





AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TATCTCGTCAACTTGTTGGCGAGAagccacaggTCGGAG

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






GCGGAAACCAGTGCAAGCTGGGAtgaa





 


207
SauriCas9
+
CAAGAAATCCCGAGAGGAAAGGTTTTAGTACTCTGG
29866
TGTGTACTACTCCACTACCTAGTTTTAGTACTC
30051





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAttccgcctcCGACCT

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






GTGGCTGGCCTGCTTTCCTCTCGggat





 


208
SauriCas9-
+
CAAGAAATCCCGAGAGGAAAGGTTTTAGTACTCTGG
29867
ACTCCACTACCTAAAGGTCTCGTTTTAGTACTC
30052



KKH

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAttccgcctcCGACCT

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






GTGGCTGGCCTGCTTTCCTCTCGggat





 


211
ScaCas9-
+
AAGAAATCCCGAGAGGAAAGGTTTTAGAGCTAGAA
29868
CACTACCTAAAGGTCTCCTAGTTTTAGAGCTAG
30053



Sc++

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCttccgcctcCGACCT

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GTGGCTGGCCTGCTTTCCTCTCGggat





 


212
SpyCas9-
+
AAGAAATCCCGAGAGGAAAGGTTTTAGAGCTAGAA
29869
CACTACCTAAAGGTCTCCTAGTTTTAGAGCTAG
30054



SpRY

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






TGAAAAAGTGGCACCGAGTCGGTGCttccgcctcCGACCT

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GTGGCTGGCCTGCTTTCCTCTCGggat





 


213
SpyCas9-
−
TTCTTTTCATCCCAGCTTGCGTTTTAGAGCTAGAAAT
29870
GGGCTTGGATCCATGTCTGAGTTTTAGAGCTA
30055



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






AAAAAGTGGCACCGAGTCGGTGCagccacaggTCGGAG

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GCGGAAACCAGTGCAAGCTGGGAtgaa





 


214
BlatCas9
+
cccaAGAAATCCCGAGAGGAAAGGCTATAGTTCCTTA
29871
ctccACTACCTAAAGGTCTCCTAGCTATAGTTCC
30056





CTGAAAGGTAAGTTGCTATAGTAAGGGCAACAGACC

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






CGAGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGG

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCTCCCCATATTCAAAATAATGACAGACGAGCACC

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






TTGGAGCATTTATCTCCGAGGTGCTttccgcctcCGACCT

GACAGACGAGCACCTTGGAGCATTTATCTCCG






GTGGCTGGCCTGCTTTCCTCTCGggat

AGGTGCT



 


215
BlatCas9
+
cccaAGAAATCCCGAGAGGAAAGGCTATAGTTCCTTA
29872
ctccACTACCTAAAGGTCTCCTAGCTATAGTTCC
30057





CTGAAAGGTAAGTTGCTATAGTAAGGGCAACAGACC

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






CGAGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGG

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCTCCCCATATTCAAAATAATGACAGACGAGCACC

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






TTGGAGCATTTATCTCCGAGGTGCTttccgcctcCGACCT

GACAGACGAGCACCTTGGAGCATTTATCTCCG






GTGGCTGGCCTGCTTTCCTCTCGggat

AGGTGCT



 


217
SauCas9KKH
+
CCAAGAAATCCCGAGAGGAAAGTTTTAGTACTCTGG
29873
ACCTAAAGGTCTCCTAGTGCCGTTTTAGTACTC
30058





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAtccgcctcCGACCTG

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






TGGCTGGCCTGCTTTCCTCTCGGgatt





 


218
SauriCas9-
+
CCAAGAAATCCCGAGAGGAAAGTTTTAGTACTCTGG
29874
ACTCCACTACCTAAAGGTCTCGTTTTAGTACTC
30059



KKH

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAtccgcctcCGACCTG

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






TGGCTGGCCTGCTTTCCTCTCGGgatt





 


219
SpyCas9-
−
CTTCTTTTCATCCCAGCTTGGTTTTAGAGCTAGAAAT
29875
GGCTTGGATCCATGTCTGATGTTTTAGAGCTAG
30060



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAAGTGGCACCGAGTCGGTGCgccacaggTCGGAGG

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CGGAAACCAGTGCAAGCTGGGATgaaa





 


220
SpyCas9-
+
CAAGAAATCCCGAGAGGAAAGTTTTAGAGCTAGAA
29876
ACTACCTAAAGGTCTCCTAGGTTTTAGAGCTA
30061



SpRY

ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






TGAAAAAGTGGCACCGAGTCGGTGCtccgcctcCGACCT

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GTGGCTGGCCTGCTTTCCTCTCGGgatt





 


224
SauCas9KKH
+
CCCAAGAAATCCCGAGAGGAAGTTTTAGTACTCTGG
29877
ACCTAAAGGTCTCCTAGTGCCGTTTTAGTACTC
30062





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAccgcctcCGACCTG

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






TGGCTGGCCTGCTTTCCTCTCGGGattt





 


225
SpyCas9-
+
CCAAGAAATCCCGAGAGGAAGTTTTAGAGCTAGAAA
29878
ACTACCTAAAGGTCTCCTAGGTTTTAGAGCTA
30063



NG

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






GAAAAAGTGGCACCGAGTCGGTGCccgcctcCGACCTG

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






TGGCTGGCCTGCTTTCCTCTCGGGattt





 


229
SpyCas9-
+
CCAAGAAATCCCGAGAGGAAGTTTTAGAGCTAGAAA
29879
CTACCTAAAGGTCTCCTAGTGTTTTAGAGCTAG
30064



SpRY

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCccgcctcCGACCTG

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






TGGCTGGCCTGCTTTCCTCTCGGGattt





 


230
SpyCas9-
−
TCTTCTTTTCATCCCAGCTTGTTTTAGAGCTAGAAAT
29880
GCTTGGATCCATGTCTGATGGTTTTAGAGCTAG
30065



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAAGTGGCACCGAGTCGGTGCccacaggTCGGAGGC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GGAAACCAGTGCAAGCTGGGATGaaaa





 


236
ScaCas9-
+
CCCAAGAAATCCCGAGAGGAGTTTTAGAGCTAGAAA
29881
CACTACCTAAAGGTCTCCTAGTTTTAGAGCTAG
30066



Sc++

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCcgcctcCGACCTGT

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GGCTGGCCTGCTTTCCTCTCGGGAtttc





 


237
SpyCas9-
+
CCCAAGAAATCCCGAGAGGAGTTTTAGAGCTAGAAA
29882
TACCTAAAGGTCTCCTAGTGGTTTTAGAGCTAG
30067



SpRY

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCcgcctcCGACCTGT

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GGCTGGCCTGCTTTCCTCTCGGGAtttc





 


238
SpyCas9-
−
TTCTTCTTTTCATCCCAGCTGTTTTAGAGCTAGAAAT
29883
TGGATCCATGTCTGATGTACGTTTTAGAGCTAG
30068



NG

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAAGTGGCACCGAGTCGGTGCcacaggTCGGAGGC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GGAAACCAGTGCAAGCTGGGATGAaaag





 


242
SpyCas9-
−
TTCTTCTTTTCATCCCAGCTGTTTTAGAGCTAGAAAT
29884
CTTGGATCCATGTCTGATGTGTTTTAGAGCTAG
30069



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAAGTGGCACCGAGTCGGTGCcacaggTCGGAGGC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GGAAACCAGTGCAAGCTGGGATGAaaag





 


243
BlatCas9
−
tcttTCTTCTTTTCATCCCAGCTGCTATAGTTCCTTACTG
29885
catgGGCTTGGATCCATGTCTGAGCTATAGTTCC
30070





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGA

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






GGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






TCCCCATATTCAAAATAATGACAGACGAGCACCTTG

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






GAGCATTTATCTCCGAGGTGCTcacaggTCGGAGGCGG

GACAGACGAGCACCTTGGAGCATTTATCTCCG






AAACCAGTGCAAGCTGGGATGAaaag

AGGTGCT



 


244
BlatCas9
−
tcttTCTTCTTTTCATCCCAGCTGCTATAGTTCCTTACTG
29886
catgGGCTTGGATCCATGTCTGAGCTATAGTTCC
30071





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGA

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






GGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






TCCCCATATTCAAAATAATGACAGACGAGCACCTTG

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






GAGCATTTATCTCCGAGGTGCTcacaggTCGGAGGCGG

GACAGACGAGCACCTTGGAGCATTTATCTCCG






AAACCAGTGCAAGCTGGGATGAaaag

AGGTGCT



 


249
SauriCas9-
+
CACCCAAGAAATCCCGAGAGGGTTTTAGTACTCTGG
29887
ACTCCACTACCTAAAGGTCTCGTTTTAGTACTC
30072



KKH

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAgcctcCGACCTGTG

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






GCTGGCCTGCTTTCCTCTCGGGATttct





 


254
ScaCas9-
−
TTTCTTCTTTTCATCCCAGCGTTTTAGAGCTAGAAAT
29888
TTGGATCCATGTCTGATGTAGTTTTAGAGCTAG
30073



Sc++

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAAGTGGCACCGAGTCGGTGCacaggTCGGAGGCG

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GAAACCAGTGCAAGCTGGGATGAAaaga





 


255
SpyCas9-
−
TTTCTTCTTTTCATCCCAGCGTTTTAGAGCTAGAAAT
29889
TTGGATCCATGTCTGATGTAGTTTTAGAGCTAG
30074



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAAGTGGCACCGAGTCGGTGCacaggTCGGAGGCG

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GAAACCAGTGCAAGCTGGGATGAAaaga





 


256
SpyCas9-
+
ACCCAAGAAATCCCGAGAGGGTTTTAGAGCTAGAAA
29890
ACCTAAAGGTCTCCTAGTGCGTTTTAGAGCTA
30075



SpRY

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

GAAATAGCAAGTTAAAATAAGGCTAGTCCGTT






GAAAAAGTGGCACCGAGTCGGTGCgcctcCGACCTGTG

ATCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GCTGGCCTGCTTTCCTCTCGGGATttct





 


257
BlatCas9
+
gccaCCCAAGAAATCCCGAGAGGGCTATAGTTCCTTA
29891
ccacTACCTAAAGGTCTCCTAGTGCTATAGTTCC
30076





CTGAAAGGTAAGTTGCTATAGTAAGGGCAACAGACC

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






CGAGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGG

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCTCCCCATATTCAAAATAATGACAGACGAGCACC

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






TTGGAGCATTTATCTCCGAGGTGCTgcctcCGACCTGTG

GACAGACGAGCACCTTGGAGCATTTATCTCCG






GCTGGCCTGCTTTCCTCTCGGGATttct

AGGTGCT



 


258
BlatCas9
+
gccaCCCAAGAAATCCCGAGAGGGCTATAGTTCCTTA
29892
ccacTACCTAAAGGTCTCCTAGTGCTATAGTTCC
30077





CTGAAAGGTAAGTTGCTATAGTAAGGGCAACAGACC

TTACTGAAAGGTAAGTTGCTATAGTAAGGGCA






CGAGGCGTTGGGGATCGCCTAGCCCGTGTTTACGGG

ACAGACCCGAGGCGTTGGGGATCGCCTAGCCC






CTCTCCCCATATTCAAAATAATGACAGACGAGCACC

GTGTTTACGGGCTCTCCCCATATTCAAAATAAT






TTGGAGCATTTATCTCCGAGGTGCTgcctcCGACCTGTG

GACAGACGAGCACCTTGGAGCATTTATCTCCG






GCTGGCCTGCTTTCCTCTCGGGATttct

AGGTGCT



 


260
SauCas9KKH
+
CCACCCAAGAAATCCCGAGAGGTTTTAGTACTCTGG
29893
ACCTAAAGGTCTCCTAGTGCCGTTTTAGTACTC
30078





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAcctcCGACCTGTG

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






GCTGGCCTGCTTTCCTCTCGGGATTtctt





 


261
SpyCas9-
+
CACCCAAGAAATCCCGAGAGGTTTTAGAGCTAGAAA
29894
CCTAAAGGTCTCCTAGTGCCGTTTTAGAGCTAG
30079



SpRY

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCcctcCGACCTGTG

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GCTGGCCTGCTTTCCTCTCGGGATTtctt





 


263
SpyCas9-
−
CTTTCTTCTTTTCATCCCAGGTTTTAGAGCTAGAAAT
29895
TGGATCCATGTCTGATGTACGTTTTAGAGCTAG
30080



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAAGTGGCACCGAGTCGGTGCcaggTCGGAGGCGG

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






AAACCAGTGCAAGCTGGGATGAAAagaa





 


264
BlatCas9
−
tttcTTTCTTCTTTTCATCCCAGGCTATAGTTCCTTACTG
29896
ttggATCCATGTCTGATGTACTGGCTATAGTTCCT
30081





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGA

TACTGAAAGGTAAGTTGCTATAGTAAGGGCAA






GGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC

CAGACCCGAGGCGTTGGGGATCGCCTAGCCCG






TCCCCATATTCAAAATAATGACAGACGAGCACCTTG

TGTTTACGGGCTCTCCCCATATTCAAAATAATG






GAGCATTTATCTCCGAGGTGCTcaggTCGGAGGCGGA

ACAGACGAGCACCTTGGAGCATTTATCTCCGA






AACCAGTGCAAGCTGGGATGAAAagaa

GGTGCT



 


269
SauCas9KKH
+
GCCACCCAAGAAATCCCGAGAGTTTTAGTACTCTGG
29897
ACCTAAAGGTCTCCTAGTGCCGTTTTAGTACTC
30082





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGActcCGACCTGTGG

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






CTGGCCTGCTTTCCTCTCGGGATTTcttg





 


270
SpyCas9-
+
CCACCCAAGAAATCCCGAGAGTTTTAGAGCTAGAAA
29898
TAAAGGTCTCCTAGTGCCTCGTTTTAGAGCTAG
30083



NG

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCctcCGACCTGTGG

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CTGGCCTGCTTTCCTCTCGGGATTTcttg





 


274
SpyCas9-
+
CCACCCAAGAAATCCCGAGAGTTTTAGAGCTAGAAA
29899
CTAAAGGTCTCCTAGTGCCTGTTTTAGAGCTAG
30084



SpRY

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCctcCGACCTGTGG

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CTGGCCTGCTTTCCTCTCGGGATTTcttg





 


275
SpyCas9-
−
TCTTTCTTCTTTTCATCCCAGTTTTAGAGCTAGAAAT
29900
GGATCCATGTCTGATGTACTGTTTTAGAGCTAG
30085



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAAGTGGCACCGAGTCGGTGCaggTCGGAGGCGG

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






AAACCAGTGCAAGCTGGGATGAAAAgaag





 


281
SauCas9
+
caGGCCACCCAAGAAATCCCGAGGTTTTAGTACTCTG
29901
ctAGTGCCTCTGACTCAGTGGTGGTTTTAGTACT
30086





GAAACAGAATCTACTAAAACAAGGCAAAATGCCGT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






GTTTATCTCGTCAACTTGTTGGCGAGAtcCGACCTGTG

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGAG






GCTGGCCTGCTTTCCTCTCGGGATTTCttgg

A



 


282
SauCas9KKH
+
GGCCACCCAAGAAATCCCGAGGTTTTAGTACTCTGG
29902
ACCTAAAGGTCTCCTAGTGCCGTTTTAGTACTC
30087





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAtcCGACCTGTGGC

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






TGGCCTGCTTTCCTCTCGGGATTTCttgg





 


285
ScaCas9-
+
GCCACCCAAGAAATCCCGAGGTTTTAGAGCTAGAAA
29903
CTAAAGGTCTCCTAGTGCCTGTTTTAGAGCTAG
30088



Sc++

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCtcCGACCTGTGGC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






TGGCCTGCTTTCCTCTCGGGATTTCttgg





 


286
SpyCas9
+
GCCACCCAAGAAATCCCGAGGTTTTAGAGCTAGAAA
29904
TCCTAGTGCCTCTGACTCAGGTTTTAGAGCTAG
30089





TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCtcCGACCTGTGGC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






TGGCCTGCTTTCCTCTCGGGATTTCttgg





 


289
SpyCas9-
+
GCCACCCAAGAAATCCCGAGGTTTTAGAGCTAGAAA
29905
TAAAGGTCTCCTAGTGCCTCGTTTTAGAGCTAG
30090



SpRY

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCtcCGACCTGTGGC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






TGGCCTGCTTTCCTCTCGGGATTTCttgg





 


290
SpyCas9-
+
GCCACCCAAGAAATCCCGAGGTTTTAGAGCTAGAAA
29906
TAAAGGTCTCCTAGTGCCTCGTTTTAGAGCTAG
30091



NG

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCtcCGACCTGTGGC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






TGGCCTGCTTTCCTCTCGGGATTTCttgg





 


293
SpyCas9-
−
TTCTTTCTTCTTTTCATCCCGTTTTAGAGCTAGAAATA
29907
GATCCATGTCTGATGTACTGGTTTTAGAGCTAG
30092



NG

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAGTGGCACCGAGTCGGTGCggTCGGAGGCGGAA

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






ACCAGTGCAAGCTGGGATGAAAAGaaga





 


297
SpyCas9-
−
TTCTTTCTTCTTTTCATCCCGTTTTAGAGCTAGAAATA
29908
GATCCATGTCTGATGTACTGGTTTTAGAGCTAG
30093



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAGTGGCACCGAGTCGGTGCggTCGGAGGCGGAA

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






ACCAGTGCAAGCTGGGATGAAAAGaaga





 


303
SauCas9
+
ccAGGCCACCCAAGAAATCCCGAGTTTTAGTACTCTG
29909
ctAGTGCCTCTGACTCAGTGGTGGTTTTAGTACT
30094





GAAACAGAATCTACTAAAACAAGGCAAAATGCCGT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






GTTTATCTCGTCAACTTGTTGGCGAGAcCGACCTGTG

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGAG






GCTGGCCTGCTTTCCTCTCGGGATTTCTtggg

A



 


304
SauCaS9KKH
+
AGGCCACCCAAGAAATCCCGAGTTTTAGTACTCTGG
29910
AAGGTCTCCTAGTGCCTCTGAGTTTTAGTACTC
30095





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAcCGACCTGTGGC

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






TGGCCTGCTTTCCTCTCGGGATTTCTtggg





 


305
SauriCas9
+
AGGCCACCCAAGAAATCCCGAGTTTTAGTACTCTGG
29911
TCTCCTAGTGCCTCTGACTCAGTTTTAGTACTC
30096





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAcCGACCTGTGGC

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






TGGCCTGCTTTCCTCTCGGGATTTCTtggg





 


306
SauriCas9-
+
AGGCCACCCAAGAAATCCCGAGTTTTAGTACTCTGG
29912
AGGTCTCCTAGTGCCTCTGACGTTTTAGTACTC
30097



KKH

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAcCGACCTGTGGC

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






TGGCCTGCTTTCCTCTCGGGATTTCTtggg





 


309
ScaCas9-
+
GGCCACCCAAGAAATCCCGAGTTTTAGAGCTAGAAA
29913
CTAAAGGTCTCCTAGTGCCTGTTTTAGAGCTAG
30098



Sc++

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCcCGACCTGTGGC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






TGGCCTGCTTTCCTCTCGGGATTTCTtggg





 


310
SpyCas9-
+
GGCCACCCAAGAAATCCCGAGTTTTAGAGCTAGAAA
29914
AAAGGTCTCCTAGTGCCTCTGTTTTAGAGCTAG
30099



SpRY

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCcCGACCTGTGGC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






TGGCCTGCTTTCCTCTCGGGATTTCTtggg





 


313
ScaCas9-
−
TTTCTTTCTTCTTTTCATCCGTTTTAGAGCTAGAAATA
29915
ATCCATGTCTGATGTACTGTGTTTTAGAGCTAG
30100



Sc++

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAGTGGCACCGAGTCGGTGCgTCGGAGGCGGAAA

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CCAGTGCAAGCTGGGATGAAAAGAagaa





 


314
SpyCas9-
−
TTTCTTTCTTCTTTTCATCCGTTTTAGAGCTAGAAATA
29916
ATCCATGTCTGATGTACTGTGTTTTAGAGCTAG
30101



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAGTGGCACCGAGTCGGTGCgTCGGAGGCGGAAA

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CCAGTGCAAGCTGGGATGAAAAGAagaa





 


315
St1Cas9
+
GGCCACCCAAGAAATCCCGAGTCTTTGTACTCTGGT
29917
NAGTCTTTGTACTCTGGTACCAGAAGCTACAA
30102





ACCAGAAGCTACAAAGATAAGGCTTCATGCCGAAAT

AGATAAGGCTTCATGCCGAAATCAACACCCTG






CAACACCCTGTCATTTTATGGCAGGGTGTTTTcCGAC

TCATTTTATGGCAGGGTGTTTT






CTGTGGCTGGCCTGCTTTCCTCTCGGGATTTCTtggg





 


319
SauCas9KKH
+
CAGGCCACCCAAGAAATCCCGGTTTTAGTACTCTGG
29918
AAGGTCTCCTAGTGCCTCTGAGTTTTAGTACTC
30103





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGACGACCTGTGGCT

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






GGCCTGCTTTCCTCTCGGGATTTCTTgggt





 


320
SauriCas9-
+
CAGGCCACCCAAGAAATCCCGGTTTTAGTACTCTGG
29919
AGGTCTCCTAGTGCCTCTGACGTTTTAGTACTC
30104



KKH

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGACGACCTGTGGCT

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






GGCCTGCTTTCCTCTCGGGATTTCTTgggt





 


322
SauriCas9-
−
GTTTTCTTTCTTCTTTTCATCGTTTTAGTACTCTGGAA
29920
TCCATGTCTGATGTACTGTGTGTTTTAGTACTC
30105



KKH

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






ATCTCGTCAACTTGTTGGCGAGATCGGAGGCGGAAA

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






CCAGTGCAAGCTGGGATGAAAAGAAgaaa





 


323
SpyCas9-
+
AGGCCACCCAAGAAATCCCGGTTTTAGAGCTAGAAA
29921
TAAAGGTCTCCTAGTGCCTCGTTTTAGAGCTAG
30106



NG

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCCGACCTGTGGCT

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GGCCTGCTTTCCTCTCGGGATTTCTTgggt





 


327
SpyCas9-
+
AGGCCACCCAAGAAATCCCGGTTTTAGAGCTAGAAA
29922
AAGGTCTCCTAGTGCCTCTGGTTTTAGAGCTAG
30107



SpRY

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCCGACCTGTGGCT

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GGCCTGCTTTCCTCTCGGGATTTCTTgggt





 


328
SpyCas9-
−
TTTTCTTTCTTCTTTTCATCGTTTTAGAGCTAGAAATA
29923
TCCATGTCTGATGTACTGTGGTTTTAGAGCTAG
30108



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAGTGGCACCGAGTCGGTGCTCGGAGGCGGAAA

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CCAGTGCAAGCTGGGATGAAAAGAAgaaa





 


329
St1Cas9
+
AGGCCACCCAAGAAATCCCGGTCTTTGTACTCTGGT
29924
NAGTCTTTGTACTCTGGTACCAGAAGCTACAA
30109





ACCAGAAGCTACAAAGATAAGGCTTCATGCCGAAAT

AGATAAGGCTTCATGCCGAAATCAACACCCTG






CAACACCCTGTCATTTTATGGCAGGGTGTTTTCGACC

TCATTTTATGGCAGGGTGTTTT






TGTGGCTGGCCTGCTTTCCTCTCGGGATTTCTTgggt





 


330
BlatCas9
−
gagtTTTCTTTCTTCTTTTCATCGCTATAGTTCCTTACTG
29925
ttggATCCATGTCTGATGTACTGGCTATAGTTCCT
30110





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGA

TACTGAAAGGTAAGTTGCTATAGTAAGGGCAA






GGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC

CAGACCCGAGGCGTTGGGGATCGCCTAGCCCG






TCCCCATATTCAAAATAATGACAGACGAGCACCTTG

TGTTTACGGGCTCTCCCCATATTCAAAATAATG






GAGCATTTATCTCCGAGGTGCTTCGGAGGCGGAAAC

ACAGACGAGCACCTTGGAGCATTTATCTCCGA






CAGTGCAAGCTGGGATGAAAAGAAgaaa

GGTGCT



 


331
BlatCas9
−
gagtTTTCTTTCTTCTTTTCATCGCTATAGTTCCTTACTG
29926
ttggATCCATGTCTGATGTACTGGCTATAGTTCCT
30111





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGA

TACTGAAAGGTAAGTTGCTATAGTAAGGGCAA






GGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTC

CAGACCCGAGGCGTTGGGGATCGCCTAGCCCG






TCCCCATATTCAAAATAATGACAGACGAGCACCTTG

TGTTTACGGGCTCTCCCCATATTCAAAATAATG






GAGCATTTATCTCCGAGGTGCTTCGGAGGCGGAAAC

ACAGACGAGCACCTTGGAGCATTTATCTCCGA






CAGTGCAAGCTGGGATGAAAAGAAgaaa

GGTGCT



 


336
SauCas9
+
ggCCAGGCCACCCAAGAAATCCCGTTTTAGTACTCTG
29927
ctAGTGCCTCTGACTCAGTGGTGGTTTTAGTACT
30112





GAAACAGAATCTACTAAAACAAGGCAAAATGCCGT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






GTTTATCTCGTCAACTTGTTGGCGAGAGACCTGTGGC

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGAG






TGGCCTGCTTTCCTCTCGGGATTTCTTGggtg

A



 


337
SauCas9KKH
+
CCAGGCCACCCAAGAAATCCCGTTTTAGTACTCTGG
29928
AAGGTCTCCTAGTGCCTCTGAGTTTTAGTACTC
30113





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAGACCTGTGGCTG

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






GCCTGCTTTCCTCTCGGGATTTCTTGggtg





 


338
SauCas9KKH
−
AGTTTTCTTTCTTCTTTTCATGTTTTAGTACTCTGGAA
29929
ATCCATGTCTGATGTACTGTGGTTTTAGTACTC
30114





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






ATCTCGTCAACTTGTTGGCGAGACGGAGGCGGAAAC

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






CAGTGCAAGCTGGGATGAAAAGAAGaaag





 


341
ScaCas9-
+
CAGGCCACCCAAGAAATCCCGTTTTAGAGCTAGAAA
29930
GTCTCCTAGTGCCTCTGACTGTTTTAGAGCTAG
30115



Sc++

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCGACCTGTGGCTG

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GCCTGCTTTCCTCTCGGGATTTCTTGggtg





 


342
SpyCas9-
−
CAGGCCACCCAAGAAATCCCGTTTTAGAGCTAGAAA
29931
AGGTCTCCTAGTGCCTCTGAGTTTTAGAGCTAG
30116



SpRY

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCGACCTGTGGCTG

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






GCCTGCTTTCCTCTCGGGATTTCTTGggtg





 


343
SpyCas9-
−
GTTTTCTTTCTTCTTTTCATGTTTTAGAGCTAGAAATA
29932
CCATGTCTGATGTACTGTGTGTTTTAGAGCTAG
30117



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAGTGGCACCGAGTCGGTGCCGGAGGCGGAAAC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CAGTGCAAGCTGGGATGAAAAGAAGaaag





 


350
SauCas9KKH
+
GCCAGGCCACCCAAGAAATCCGTTTTAGTACTCTGG
29933
AAGGTCTCCTAGTGCCTCTGAGTTTTAGTACTC
30118





AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAACCTGTGGCTGG

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






CCTGCTTTCCTCTCGGGATTTCTTGGgtgg





 


351
SauriCas9-
+
GCCAGGCCACCCAAGAAATCCGTTTTAGTACTCTGG
29934
AGGTCTCCTAGTGCCTCTGACGTTTTAGTACTC
30119



KKH

AAACAGAATCTACTAAAACAAGGCAAAATGCCGTGT

TGGAAACAGAATCTACTAAAACAAGGCAAAAT






TTATCTCGTCAACTTGTTGGCGAGAACCTGTGGCTGG

GCCGTGTTTATCTCGTCAACTTGTTGGCGAGA






CCTGCTTTCCTCTCGGGATTTCTTGGgtgg





 


353
SpyCas9-
+
CCAGGCCACCCAAGAAATCCGTTTTAGAGCTAGAAA
29935
TCTCCTAGTGCCTCTGACTCGTTTTAGAGCTAG
30120



NG

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCACCTGTGGCTGG

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CCTGCTTTCCTCTCGGGATTTCTTGGgtgg





 


357
SpyCas9-
+
CCAGGCCACCCAAGAAATCCGTTTTAGAGCTAGAAA
29936
GGTCTCCTAGTGCCTCTGACGTTTTAGAGCTAG
30121



SpRY

TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






GAAAAAGTGGCACCGAGTCGGTGCACCTGTGGCTGG

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CCTGCTTTCCTCTCGGGATTTCTTGGgtgg





 


358
SpyCas9-
−
AGTTTTCTTTCTTCTTTTCAGTTTTAGAGCTAGAAAT
29937
CATGTCTGATGTACTGTGTGGTTTTAGAGCTAG
30122



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG

AAATAGCAAGTTAAAATAAGGCTAGTCCGTTA






AAAAAGTGGCACCGAGTCGGTGCGGAGGCGGAAAC

TCAACTTGAAAAAGTGGCACCGAGTCGGTGC






CAGTGCAAGCTGGGATGAAAAGAAGAaaga
















TABLE 4D







Exemplary template RNA sequences and second nick gRNA spacer sequences


Table 4D provides design of RNA components of gene modifying systems for correcting the pathogenic IVS10-11G>A, mutation in PAH. The


gRNA spacers from Table 1D were filtered, e.g., filtered by occurrence within 15 nt of the desired editing location and use of a Tier 1


Cas enzyme. For each gRNA ID, this table details the sequence of a complete template RNA, optional second-nick gRNA, and Cas variant for use


in a Cas-RT fusion gene modifying polypeptide. For exemplification, PBS sequences and post-edit homology regions (after the location of the


edit) are set to 12 nt and 30 nt, respectively. Additionally, a second-nick gRNA is selected with preference for a distance near 100 nt from


the first nick and a first preference for a design resulting in a PAM-in system, as described elsewhere in this application.

















SEQ

SEQ



Cas


ID

ID


ID
species
strand
Template RNA
NO
second-nick gRNA
NO
















1
SpyCas9-
−
ATAATAACTTTTCACTTAGGGTTTTAGAGCTAGAAATA
30123
TCTCTGCCACGTAATAGAGGGTTTTAGAGCTA
30272



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCgcttctctgataagcagtactgtaggccC

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






CAAGTGAAAagtt

GC



 


2
SauCas9KKH
+
TAAGCAGTACTGTAGGCCCTAGTTTTAGTACTCTGGAA
30124
GCATTTGGGCTGTGATGTAGAGTTTTAGTACT
30273





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAatttaacagtgataataacttttcactTG

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






GGGCCTACAgtac

GA



 


3
SpyCas9-
−
GATAATAACTTTTCACTTAGGTTTTAGAGCTAGAAATA
30125
TCTCTGCCACGTAATAGAGGGTTTTAGAGCTA
30274



NG

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCcttctctgataagcagtactgtaggccC

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






CAAGTGAAAAgtta

GC



 


6
SpyCas9-
+
AAGCAGTACTGTAGGCCCTAGTTTTAGAGCTAGAAATA
30126
ATTTGGGCTGTGATGTAGAAGTTTTAGAGCTA
30275



NG

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCatttaacagtgataataacttttcactTG

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






GGGCCTACAgtac

GC



 


10
SpyCas9-
−
GATAATAACTTTTCACTTAGGTTTTAGAGCTAGAAATA
30127
CTCTGCCACGTAATAGAGGGGTTTTAGAGCTA
30276



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCcttctctgataagcagtactgtaggccC

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






CAAGTGAAAAgtta

GC



 


12
SpyCas9-
+
AAGCAGTACTGTAGGCCCTAGTTTTAGAGCTAGAAATA
30128
TTTGGGCTGTGATGTAGAAGGTTTTAGAGCTA
30277



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCatttaacagtgataataacttttcactTG

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






GGGCCTACAgtac

GC



 


16
ScaCas9-
−
TGATAATAACTTTTCACTTAGTTTTAGAGCTAGAAATA
30129
TCTGCCACGTAATAGAGGGGGTTTTAGAGCTA
30278



Sc++

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCttctctgataagcagtactgtaggccCC

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






AAGTGAAAAGttat

GC



 


17
SpyCas9
−
TGATAATAACTTTTCACTTAGTTTTAGAGCTAGAAATA
30130
CTGCCACGTAATAGAGGGGCGTTTTAGAGCTA
30279





GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCttctctgataagcagtactgtaggccCC

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






AAGTGAAAAGttat

GC



 


20
SpyCas9-
−
TGATAATAACTTTTCACTTAGTTTTAGAGCTAGAAATA
30131
TCTGCCACGTAATAGAGGGGGTTTTAGAGCTA
30280



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCttctctgataagcagtactgtaggccCC

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






AAGTGAAAAGttat

GC



 


23
ScaCas9-
+
TAAGCAGTACTGTAGGCCCTGTTTTAGAGCTAGAAATA
30132
GGGCTGTGATGTAGAAGGAAGTTTTAGAGCT
30281



Sc++

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCtttaacagtgataataacttttcactTGG

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






GGCCTACAGtact

GC



 


24
SpyCas9-
+
TAAGCAGTACTGTAGGCCCTGTTTTAGAGCTAGAAATA
30133
TTGGGCTGTGATGTAGAAGGGTTTTAGAGCTA
30282



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCtttaacagtgataataacttttcactTGG

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






GGCCTACAGtact

GC



 


25
SpyCas9-
−
TGATAATAACTTTTCACTTAGTTTTAGAGCTAGAAATA
30134
CTGCCACGTAATAGAGGGGCGTTTTAGAGCTA
30283



NG

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCttctctgataagcagtactgtaggccCC

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






AAGTGAAAAGttat

GC



 


28
BlatCas9
−
cagtGATAATAACTTTTCACTTAGCTATAGTTCCTTACTG
30135
ctctGCCACGTAATAGAGGGGCTGCTATAGTTC
30284





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTttctctgataagcagtactgtaggccCCAAG

AATGACAGACGAGCACCTTGGAGCATTTATCT






TGAAAAGttat

CCGAGGTGCT



 


31
Nme2Cas9
−
aaCAGTGATAATAACTTTTCACTTGTTGTAGCTCCCTTTC
30136
tcTGCCACGTAATAGAGGGGCTGGGTTGTAGC
30285





TCATTTCGGAAACGAAATGAGAACCGTTGCTACAATAA

TCCCTTTCTCATTTCGGAAACGAAATGAGAAC






GGCCGTCTGAAAAGATGTGCCGCAACGCTCTGCCCCTT

CGTTGCTACAATAAGGCCGTCTGAAAAGATGT






AAAGCTTCTGCTTTAAGGGGCATCGTTTAtctctgataagcagta

GCCGCAACGCTCTGCCCCTTAAAGCTTCTGCT






ctgtaggccCCAAGTGAAAAGTtatt

TTAAGGGGCATCGTTTA



 


32
SauriCas9
−
AGTGATAATAACTTTTCACTTGTTTTAGTACTCTGGAA
30137
CTCTGCCACGTAATAGAGGGGGTTTTAGTACT
30286





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAtctctgataagcagtactgtaggccCC

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






AAGTGAAAAGTtatt

GA



 


33
SauriCas9-
−
AGTGATAATAACTTTTCACTTGTTTTAGTACTCTGGAA
30138
CTCTGCCACGTAATAGAGGGGGTTTTAGTACT
30287



KKH

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAtctctgataagcagtactgtaggccCC

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






AAGTGAAAAGTtatt

GA



 


34
SauriCas9-
+
GATAAGCAGTACTGTAGGCCCGTTTTAGTACTCTGGAA
30139
TGGGCTGTGATGTAGAAGGAAGTTTTAGTACT
30288



KKH

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAttaacagtgataataacttttcactTGG

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






GGCCTACAGTactg

GA



 


39
ScaCas9-
−
GTGATAATAACTTTTCACTTGTTTTAGAGCTAGAAATA
30140
CTGCCACGTAATAGAGGGGCGTTTTAGAGCTA
30289



Sc++

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCtctctgataagcagtactgtaggccCC

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






AAGTGAAAAGTtatt

GC



 


40
SpyCas9
−
GTGATAATAACTTTTCACTTGTTTTAGAGCTAGAAATA
30141
CTGCCACGTAATAGAGGGGCGTTTTAGAGCTA
30290





GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCtctctgataagcagtactgtaggccCC

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






AAGTGAAAAGTtatt

GC



 


43
SpyCas9-
−
GTGATAATAACTTTTCACTTGTTTTAGAGCTAGAAATA
30142
CTGCCACGTAATAGAGGGGCGTTTTAGAGCTA
30291



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCtctctgataagcagtactgtaggccCC

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






AAGTGAAAAGTtatt

GC



 


44
SpyCas9-
−
GTGATAATAACTTTTCACTTGTTTTAGAGCTAGAAATA
30143
CTGCCACGTAATAGAGGGGCGTTTTAGAGCTA
30292



NG

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCtctctgataagcagtactgtaggccCC

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






AAGTGAAAAGTtatt

GC



 


47
SpyCas9-
+
ATAAGCAGTACTGTAGGCCCGTTTTAGAGCTAGAAATA
30144
TGGGCTGTGATGTAGAAGGAGTTTTAGAGCTA
30293



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCttaacagtgataataacttttcactTGG

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






GGCCTACAGTactg

GC



 


48
BlatCas9
−
acagTGATAATAACTTTTCACTTGCTATAGTTCCTTACTG
30145
ctctGCCACGTAATAGAGGGGCTGCTATAGTTC
30294





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTtctctgataagcagtactgtaggccCCAAG

AATGACAGACGAGCACCTTGGAGCATTTATCT






TGAAAAGTtatt

CCGAGGTGCT



 


49
BlatCas9
−
acagTGATAATAACTTTTCACTTGCTATAGTTCCTTACTG
30146
ctctGCCACGTAATAGAGGGGCTGCTATAGTTC
30295





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTtctctgataagcagtactgtaggccCCAAG

AATGACAGACGAGCACCTTGGAGCATTTATCT






TGAAAAGTtatt

CCGAGGTGCT



 


52
SauCas9
−
aaCAGTGATAATAACTTTTCACTGTTTTAGTACTCTGGA
30147
tcTCTGCCACGTAATAGAGGGGCGTTTTAGTAC
30296





AACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTT

TCTGGAAACAGAATCTACTAAAACAAGGCAA






ATCTCGTCAACTTGTTGGCGAGActctgataagcagtactgtaggcc

AATGCCGTGTTTATCTCGTCAACTTGTTGGCG






CCAAGTGAAAAGTTatta

AGA



 


53
SauCas9KKH
−
CAGTGATAATAACTTTTCACTGTTTTAGTACTCTGGAA
30148
TCTGCCACGTAATAGAGGGGCGTTTTAGTACT
30297





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGActctgataagcagtactgtaggccCC

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






AAGTGAAAAGTTatta

GA



 


54
SauCas9KKH
+
TGATAAGCAGTACTGTAGGCCGTTTTAGTACTCTGGAA
30149
TGGGCTGTGATGTAGAAGGAAGTTTTAGTACT
30298





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAtaacagtgataataacttttcactTGG

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






GGCCTACAGTActgc

GA



 


55
SauCas9KKH
+
TGATAAGCAGTACTGTAGGCCGTTTTAGTACTCTGGAA
30150
TGGGCTGTGATGTAGAAGGAAGTTTTAGTACT
30299





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAtaacagtgataataacttttcactTGG

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






GGCCTACAGTActgc

GA



 


58
SauriCas9
−
CAGTGATAATAACTTTTCACTGTTTTAGTACTCTGGAA
30151
CTCTGCCACGTAATAGAGGGGGTTTTAGTACT
30300





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGActctgataagcagtactgtaggccCC

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






AAGTGAAAAGTTatta

GA



 


59
SauriCas9-
−
CAGTGATAATAACTTTTCACTGTTTTAGTACTCTGGAA
30152
CTCTGCCACGTAATAGAGGGGGTTTTAGTACT
30301



KKH

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGActctgataagcagtactgtaggccCC

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






AAGTGAAAAGTTatta

GA



 


62
ScaCas9-
−
AGTGATAATAACTTTTCACTGTTTTAGAGCTAGAAATA
30153
CTGCCACGTAATAGAGGGGCGTTTTAGAGCTA
30302



Sc++

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCctctgataagcagtactgtaggccCCA

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






AGTGAAAAGTTatta

GC



 


63
SpyCas9-
−
AGTGATAATAACTTTTCACTGTTTTAGAGCTAGAAATA
30154
TGCCACGTAATAGAGGGGCTGTTTTAGAGCTA
30303



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCctctgataagcagtactgtaggccCCA

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






AGTGAAAAGTTatta

GC



 


64
SpyCas9-
+
GATAAGCAGTACTGTAGGCCGTTTTAGAGCTAGAAATA
30155
GGGCTGTGATGTAGAAGGAAGTTTTAGAGCT
30304



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCtaacagtgataataacttttcactTGGG

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






GCCTACAGTActgc

GC



 


67
SauCas9KKH
+
CTGATAAGCAGTACTGTAGGCGTTTTAGTACTCTGGAA
30156
GGGCTGTGATGTAGAAGGAATGTTTTAGTACT
30305





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAaacagtgataataacttttcactTGGG

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






GCCTACAGTACtgct

GA



 


68
SauCas9KKH
−
ACAGTGATAATAACTTTTCACGTTTTAGTACTCTGGAA
30157
TCTGCCACGTAATAGAGGGGCGTTTTAGTACT
30306





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAtctgataagcagtactgtaggccCCA

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






AGTGAAAAGTTAttat

GA



 


69
SauriCas9-
−
ACAGTGATAATAACTTTTCACGTTTTAGTACTCTGGAA
30158
CTCTGCCACGTAATAGAGGGGGTTTTAGTACT
30307



KKH

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAtctgataagcagtactgtaggccCCA

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






AGTGAAAAGTTAttat

GA



 


70
SpyCas9-
+
TGATAAGCAGTACTGTAGGCGTTTTAGAGCTAGAAATA
30159
GGCTGTGATGTAGAAGGAATGTTTTAGAGCTA
30308



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCaacagtgataataacttttcactTGGG

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






GCCTACAGTACtgct

GC



 


71
SpyCas9-
−
CAGTGATAATAACTTTTCACGTTTTAGAGCTAGAAATA
30160
GCCACGTAATAGAGGGGCTGGTTTTAGAGCT
30309



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCtctgataagcagtactgtaggccCCA

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






AGTGAAAAGTTAttat

GC



 


74
SauCas9KKH
−
AACAGTGATAATAACTTTTCAGTTTTAGTACTCTGGAA
30161
TCTGCCACGTAATAGAGGGGCGTTTTAGTACT
30310





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGActgataagcagtactgtaggccCCA

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






AGTGAAAAGTTATtatc

GA



 


75
SpyCas9-
+
CTGATAAGCAGTACTGTAGGGTTTTAGAGCTAGAAATA
30162
GCTGTGATGTAGAAGGAATCGTTTTAGAGCTA
30311



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCacagtgataataacttttcactTGGGG

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






CCTACAGTACTgctt

GC



 


76
SpyCas9-
−
ACAGTGATAATAACTTTTCAGTTTTAGAGCTAGAAATA
30163
CCACGTAATAGAGGGGCTGGGTTTTAGAGCT
30312



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCctgataagcagtactgtaggccCCAA

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






GTGAAAAGTTATtatc

GC



 


77
SpyCas9-
+
TCTGATAAGCAGTACTGTAGGTTTTAGAGCTAGAAATA
30164
CTGTGATGTAGAAGGAATCGGTTTTAGAGCTA
30313



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCcagtgataataacttttcactTGGGG

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






CCTACAGTACTGctta

GC



 


78
SpyCas9-
−
AACAGTGATAATAACTTTTCGTTTTAGAGCTAGAAATA
30165
CACGTAATAGAGGGGCTGGAGTTTTAGAGCT
30314



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCtgataagcagtactgtaggccCCAA

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






GTGAAAAGTTATTatca

GC



 


79
SpyCas9-
+
CTCTGATAAGCAGTACTGTAGTTTTAGAGCTAGAAATA
30166
TGTGATGTAGAAGGAATCGGGTTTTAGAGCTA
30315



NG

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCagtgataataacttttcactTGGGGC

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






CTACAGTACTGCttat

GC



 


83
SpyCas9-
−
CTCTGATAAGCAGTACTGTAGTTTTAGAGCTAGAAATA
30167
TGTGATGTAGAAGGAATCGGGTTTTAGAGCTA
30316



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCagtgataataacttttcactTGGGGC

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






CTACAGTACTGCttat

GC



 


84
SpyCas9-
−
TAACAGTGATAATAACTTTTGTTTTAGAGCTAGAAATA
30168
ACGTAATAGAGGGGCTGGAAGTTTTAGAGCT
30317



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCgataagcagtactgtaggccCCAAG

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






TGAAAAGTTATTAtcac

GC



 


85
BlatCas9
+
cttcTCTGATAAGCAGTACTGTAGCTATAGTTCCTTACTG
30169
gcatTTGGGCTGTGATGTAGAAGGCTATAGTTC
30318





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTagtgataataacttttcactTGGGGCCTA

AATGACAGACGAGCACCTTGGAGCATTTATCT






CAGTACTGCttat

CCGAGGTGCT



 


86
BlatCas9
+
cttcTCTGATAAGCAGTACTGTAGCTATAGTTCCTTACTG
30170
gcatTTGGGCTGTGATGTAGAAGGCTATAGTTC
30319





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTagtgataataacttttcactTGGGGCCTA

AATGACAGACGAGCACCTTGGAGCATTTATCT






CAGTACTGCttat

CCGAGGTGCT



 


87
BlatCas9
+
cttcTCTGATAAGCAGTACTGTAGCTATAGTTCCTTACTG
30171
gcatTTGGGCTGTGATGTAGAAGGCTATAGTTC
30320





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTagtgataataacttttcactTGGGGCCTA

AATGACAGACGAGCACCTTGGAGCATTTATCT






CAGTACTGCttat

CCGAGGTGCT



 


90
Nme2Cas9
−
ggCTTCTCTGATAAGCAGTACTGTGTTGTAGCTCCCTTT
30172
ggTGAGATGAGAGAAGGGGCACAAGTTGTAG
30321





CTCATTTCGGAAACGAAATGAGAACCGTTGCTACAATA

CTCCCTTTCTCATTTCGGAAACGAAATGAGAA






AGGCCGTCTGAAAAGATGTGCCGCAACGCTCTGCCCCT

CCGTTGCTACAATAAGGCCGTCTGAAAAGAT






TAAAGCTTCTGCTTTAAGGGGCATCGTTTAgtgataataactttt

GTGCCGCAACGCTCTGCCCCTTAAAGCTTCTG






cactTGGGGCCTACAGTACTGCTtatc

CTTTAAGGGGCATCGTTTA



 


93
ScaCas9-
+
TCTCTGATAAGCAGTACTGTGTTTTAGAGCTAGAAATA
30173
GTGATGTAGAAGGAATCGGGGTTTTAGAGCT
30322



Sc++

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCgtgataataacttttcactTGGGGCC

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






TACAGTACTGCTtatc

GC



 


94
SpyCas9
+
TCTCTGATAAGCAGTACTGTGTTTTAGAGCTAGAAATA
30174
CTGTGATGTAGAAGGAATCGGTTTTAGAGCTA
30323





GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCgtgataataacttttcactTGGGGCC

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






TACAGTACTGCTtatc

GC



 


97
SpyCas9-
+
TCTCTGATAAGCAGTACTGTGTTTTAGAGCTAGAAATA
30175
GTGATGTAGAAGGAATCGGGGTTTTAGAGCT
30324



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCgtgataataacttttcactTGGGGCC

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






TACAGTACTGCTtatc

GC



 


98
SpyCas9-
+
TCTCTGATAAGCAGTACTGTGTTTTAGAGCTAGAAATA
30176
TGTGATGTAGAAGGAATCGGGTTTTAGAGCTA
30325



NG

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCgtgataataacttttcactTGGGGCC

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






TACAGTACTGCTtatc

GC



 


101
SpyCas9-
−
TTAACAGTGATAATAACTTTGTTTTAGAGCTAGAAATA
30177
CGTAATAGAGGGGCTGGAACGTTTTAGAGCT
30326



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCataagcagtactgtaggccCCAAGT

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






GAAAAGTTATTATcact

GC



 


102
BlatCas9
+
gcttCTCTGATAAGCAGTACTGTGCTATAGTTCCTTACTG
30178
gcatTTGGGCTGTGATGTAGAAGGCTATAGTTC
30327





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTgtgataataacttttcactTGGGGCCTAC

AATGACAGACGAGCACCTTGGAGCATTTATCT






AGTACTGCTtatc

CCGAGGTGCT



 


103
BlatCas9
+
gcttCTCTGATAAGCAGTACTGTGCTATAGTTCCTTACTG
30179
gcatTTGGGCTGTGATGTAGAAGGCTATAGTTC
30328





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTgtgataataacttttcactTGGGGCCTAC

AATGACAGACGAGCACCTTGGAGCATTTATCT






AGTACTGCTtatc

CCGAGGTGCT



 


106
Nme2Cas9
+
tgGCTTCTCTGATAAGCAGTACTGGTTGTAGCTCCCTTT
30180
ggTGAGATGAGAGAAGGGGCACAAGTTGTAG
30329





CTCATTTCGGAAACGAAATGAGAACCGTTGCTACAATA

CTCCCTTTCTCATTTCGGAAACGAAATGAGAA






AGGCCGTCTGAAAAGATGTGCCGCAACGCTCTGCCCCT

CCGTTGCTACAATAAGGCCGTCTGAAAAGAT






TAAAGCTTCTGCTTTAAGGGGCATCGTTTAtgataataacttttc

GTGCCGCAACGCTCTGCCCCTTAAAGCTTCTG






actTGGGGCCTACAGTACTGCTTatca

CTTTAAGGGGCATCGTTTA



 


107
SauriCas9
+
CTTCTCTGATAAGCAGTACTGGTTTTAGTACTCTGGAA
30181
GGCTGTGATGTAGAAGGAATCGTTTTAGTACT
30330





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAtgataataacttttcactTGGGGCC

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






TACAGTACTGCTTatca

GA



 


108
SauriCas9-
+
CTTCTCTGATAAGCAGTACTGGTTTTAGTACTCTGGAA
30182
GTGATGTAGAAGGAATCGGGGGTTTTAGTACT
30331



KKH

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAtgataataacttttcactTGGGGCC

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






TACAGTACTGCTTatca

GA



 


111
ScaCas9-
+
TTCTCTGATAAGCAGTACTGGTTTTAGAGCTAGAAATA
30183
GTGATGTAGAAGGAATCGGGGTTTTAGAGCT
30332



Sc++

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCtgataataacttttcactTGGGGCCT

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






ACAGTACTGCTTatca

GC



 


112
SpyCas9-
+
TTCTCTGATAAGCAGTACTGGTTTTAGAGCTAGAAATA
30184
TGATGTAGAAGGAATCGGGGGTTTTAGAGCT
30333



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCtgataataacttttcactTGGGGCCT

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






ACAGTACTGCTTatca

GC



 


113
SpyCas9-
−
TTTAACAGTGATAATAACTTGTTTTAGAGCTAGAAATA
30185
GTAATAGAGGGGCTGGAACTGTTTTAGAGCT
30334



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCtaagcagtactgtaggccCCAAGT

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






GAAAAGTTATTATCactg

GC



 


114
BlatCas9
−
tgatTTAACAGTGATAATAACTTGCTATAGTTCCTTACTG
30186
cgtaATAGAGGGGCTGGAACTCCGCTATAGTTC
30335





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTtaagcagtactgtaggccCCAAGTGAA

AATGACAGACGAGCACCTTGGAGCATTTATCT






AAGTTATTATCactg

CCGAGGTGCT



 


115
BlatCas9
+
ggctTCTCTGATAAGCAGTACTGGCTATAGTTCCTTACTG
30187
gcatTTGGGCTGTGATGTAGAAGGCTATAGTTC
30336





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTtgataataacttttcactTGGGGCCTAC

AATGACAGACGAGCACCTTGGAGCATTTATCT






AGTACTGCTTatca

CCGAGGTGCT



 


116
BlatCas9
−
tgatTTAACAGTGATAATAACTTGCTATAGTTCCTTACTG
30188
cgtaATAGAGGGGCTGGAACTCCGCTATAGTTC
30337





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTtaagcagtactgtaggccCCAAGTGAA

AATGACAGACGAGCACCTTGGAGCATTTATCT






AAGTTATTATCactg

CCGAGGTGCT



 


119
SauCas9KKH
+
GCTTCTCTGATAAGCAGTACTGTTTTAGTACTCTGGAA
30189
GTGATGTAGAAGGAATCGGGGGTTTTAGTACT
30338





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAgataataacttttcactTGGGGCC

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






TACAGTACTGCTTAtcag

GA



 


120
SauriCas9-
+
GCTTCTCTGATAAGCAGTACTGTTTTAGTACTCTGGAA
30190
GTGATGTAGAAGGAATCGGGGGTTTTAGTACT
30339



KKH

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAgataataacttttcactTGGGGCC

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






TACAGTACTGCTTAtcag

GA



 


121
SpyCas9-
+
CTTCTCTGATAAGCAGTACTGTTTTAGAGCTAGAAATA
30191
GATGTAGAAGGAATCGGGGTGTTTTAGAGCT
30340



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCgataataacttttcactTGGGGCCT

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






ACAGTACTGCTTAtcag

GC



 


122
SpyCas9-
−
ATTTAACAGTGATAATAACTGTTTTAGAGCTAGAAATA
30192
TAATAGAGGGGCTGGAACTCGTTTTAGAGCTA
30341



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCaagcagtactgtaggccCCAAGTG

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






AAAAGTTATTATCActgt

GC



 


125
SauCas9KKH
+
GGCTTCTCTGATAAGCAGTACGTTTTAGTACTCTGGAA
30193
ATGTAGAAGGAATCGGGGTGAGTTTTAGTACT
30342





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAataataacttttcactTGGGGCCT

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






ACAGTACTGCTTATcaga

GA



 


126
SpyCas9-
+
GCTTCTCTGATAAGCAGTACGTTTTAGAGCTAGAAATA
30194
ATGTAGAAGGAATCGGGGTGGTTTTAGAGCT
30343



NG

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCataataacttttcactTGGGGCCTA

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






CAGTACTGCTTATcaga

GC



 


130
SpyCas9-
+
GCTTCTCTGATAAGCAGTACGTTTTAGAGCTAGAAATA
30195
ATGTAGAAGGAATCGGGGTGGTTTTAGAGCT
30344



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCataataacttttcactTGGGGCCTA

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






CAGTACTGCTTATcaga

GC



 


131
SpyCas9-
−
GATTTAACAGTGATAATAACGTTTTAGAGCTAGAAATA
30196
AATAGAGGGGCTGGAACTCCGTTTTAGAGCT
30345



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCagcagtactgtaggccCCAAGTG

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






AAAAGTTATTATCACtgtt

GC



 


132
BlatCas9
−
cctgATTTAACAGTGATAATAACGCTATAGTTCCTTACTG
30197
cgtaATAGAGGGGCTGGAACTCCGCTATAGTTC
30346





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTagcagtactgtaggccCCAAGTGAAA

AATGACAGACGAGCACCTTGGAGCATTTATCT






AGTTATTATCACtgtt

CCGAGGTGCT



 


136
ScaCas9-
+
GGCTTCTCTGATAAGCAGTAGTTTTAGAGCTAGAAATA
30198
GTAGAAGGAATCGGGGTGAGGTTTTAGAGCT
30347



Sc++

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCtaataacttttcactTGGGGCCTA

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






CAGTACTGCTTATCagag

GC



 


137
SpyCas9-
+
GGCTTCTCTGATAAGCAGTAGTTTTAGAGCTAGAAATA
30199
TGTAGAAGGAATCGGGGTGAGTTTTAGAGCT
30348



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCtaataacttttcactTGGGGCCTA

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






CAGTACTGCTTATCagag

GC



 


138
SpyCas9-
−
TGATTTAACAGTGATAATAAGTTTTAGAGCTAGAAATA
30200
ATAGAGGGGCTGGAACTCCGGTTTTAGAGCT
30349



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCgcagtactgtaggccCCAAGTGA

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






AAAGTTATTATCACTgtta

GC



 


139
SpyCas9-
+
TGGCTTCTCTGATAAGCAGTGTTTTAGAGCTAGAAATA
30201
GTAGAAGGAATCGGGGTGAGGTTTTAGAGCT
30350



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCaataacttttcactTGGGGCCTAC

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






AGTACTGCTTATCAgaga

GC



 


140
SpyCas9-
−
CTGATTTAACAGTGATAATAGTTTTAGAGCTAGAAATA
30202
TAGAGGGGCTGGAACTCCGTGTTTTAGAGCTA
30351



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCcagtactgtaggccCCAAGTGAA

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






AAGTTATTATCACTGttaa

GC



 


141
SpyCas9-
+
TTGGCTTCTCTGATAAGCAGGTTTTAGAGCTAGAAATA
30203
TAGAAGGAATCGGGGTGAGAGTTTTAGAGCT
30352



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCataacttttcactTGGGGCCTAC

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






AGTACTGCTTATCAGagaa

GC



 


142
SpyCas9-
−
CCTGATTTAACAGTGATAATGTTTTAGAGCTAGAAATA
30204
AGAGGGGCTGGAACTCCGTGGTTTTAGAGCT
30353



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCagtactgtaggccCCAAGTGAA

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






AAGTTATTATCACTGTtaaa

GC



 


145
SpyCas9-
−
TCCTGATTTAACAGTGATAAGTTTTAGAGCTAGAAATA
30205
GAGGGGCTGGAACTCCGTGAGTTTTAGAGCT
30354



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCgtactgtaggccCCAAGTGAAA

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






AGTTATTATCACTGTTaaat

GC



 


146
SpyCas9-
+
TTTGGCTTCTCTGATAAGCAGTTTTAGAGCTAGAAATA
30206
AGAAGGAATCGGGGTGAGATGTTTTAGAGCT
30355



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCtaacttttcactTGGGGCCTACA

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






GTACTGCTTATCAGAgaag

GC



 


150
SpyCas9-
+
CTTTGGCTTCTCTGATAAGCGTTTTAGAGCTAGAAATA
30207
GAAGGAATCGGGGTGAGATGGTTTTAGAGCT
30356



NG

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCaacttttcactTGGGGCCTACA

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






GTACTGCTTATCAGAGaagc

GC



 


154
SpyCas9-
+
CTTTGGCTTCTCTGATAAGCGTTTTAGAGCTAGAAATA
30208
GAAGGAATCGGGGTGAGATGGTTTTAGAGCT
30357



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCaacttttcactTGGGGCCTACA

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






GTACTGCTTATCAGAGaagc

GC



 


155
SpyCas9-
−
ATCCTGATTTAACAGTGATAGTTTTAGAGCTAGAAATA
30209
AGGGGCTGGAACTCCGTGACGTTTTAGAGCTA
30358



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCtactgtaggccCCAAGTGAAA

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






AGTTATTATCACTGTTAaatc

GC



 


156
BlatCas9
+
aagcTTTGGCTTCTCTGATAAGCGCTATAGTTCCTTACTG
30210
ggaaTCGGGGTGAGATGAGAGAAGCTATAGTTC
30359





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTaacttttcactTGGGGCCTACAGTA

AATGACAGACGAGCACCTTGGAGCATTTATCT






CTGCTTATCAGAGaagc

CCGAGGTGCT



 


157
BlatCas9
−
ctgaTCCTGATTTAACAGTGATAGCTATAGTTCCTTACTG
30211
cgtaATAGAGGGGCTGGAACTCCGCTATAGTTC
30360





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTtactgtaggccCCAAGTGAAAAGT

AATGACAGACGAGCACCTTGGAGCATTTATCT






TATTATCACTGTTAaatc

CCGAGGTGCT



 


158
BlatCas9
+
aagcTTTGGCTTCTCTGATAAGCGCTATAGTTCCTTACTG
30212
ggaaTCGGGGTGAGATGAGAGAAGCTATAGTTC
30361





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTaacttttcactTGGGGCCTACAGTA

AATGACAGACGAGCACCTTGGAGCATTTATCT






CTGCTTATCAGAGaagc

CCGAGGTGCT



 


159
BlatCas9
−
ctgaTCCTGATTTAACAGTGATAGCTATAGTTCCTTACTG
30213
cgtaATAGAGGGGCTGGAACTCCGCTATAGTTC
30362





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTtactgtaggccCCAAGTGAAAAGT

AATGACAGACGAGCACCTTGGAGCATTTATCT






TATTATCACTGTTAaatc

CCGAGGTGCT



 


164
SauCas9KKH
−
TGATCCTGATTTAACAGTGATGTTTTAGTACTCTGGAA
30214
GGGGCTGGAACTCCGTGACAGGTTTTAGTACT
30363





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAactgtaggccCCAAGTGAAA

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






AGTTATTATCACTGTTAAatca

GA



 


167
ScaCas9-
+
GCTTTGGCTTCTCTGATAAGGTTTTAGAGCTAGAAATA
30215
AAGGAATCGGGGTGAGATGAGTTTTAGAGCT
30364



Sc++

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCacttttcactTGGGGCCTACAG

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






TACTGCTTATCAGAGAagcc

GC



 


168
SpyCas9-
+
GCTTTGGCTTCTCTGATAAGGTTTTAGAGCTAGAAATA
30216
AAGGAATCGGGGTGAGATGAGTTTTAGAGCT
30365



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCacttttcactTGGGGCCTACAG

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






TACTGCTTATCAGAGAagcc

GC



 


169
SpyCas9-
−
GATCCTGATTTAACAGTGATGTTTTAGAGCTAGAAATA
30217
GGGGCTGGAACTCCGTGACAGTTTTAGAGCTA
30366



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCactgtaggccCCAAGTGAAAA

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






GTTATTATCACTGTTAAatca

GC



 


173
SauriCas9-
+
AAGCTTTGGCTTCTCTGATAAGTTTTAGTACTCTGGAA
30218
AGGAATCGGGGTGAGATGAGAGTTTTAGTAC
30367



KKH

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

TCTGGAAACAGAATCTACTAAAACAAGGCAA






CTCGTCAACTTGTTGGCGAGActtttcactTGGGGCCTACAG

AATGCCGTGTTTATCTCGTCAACTTGTTGGCG






TACTGCTTATCAGAGAAgcca

AGA



 


174
SpyCas9-
−
TGATCCTGATTTAACAGTGAGTTTTAGAGCTAGAAATA
30219
GGGCTGGAACTCCGTGACAGGTTTTAGAGCTA
30368



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCctgtaggccCCAAGTGAAAA

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






GTTATTATCACTGTTAAAtcag

GC



 


175
SpyCas9-
+
AGCTTTGGCTTCTCTGATAAGTTTTAGAGCTAGAAATA
30220
AGGAATCGGGGTGAGATGAGGTTTTAGAGCT
30369



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCcttttcactTGGGGCCTACAGT

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






ACTGCTTATCAGAGAAgcca

GC



 


179
SauCas9KKH
+
GAAGCTTTGGCTTCTCTGATAGTTTTAGTACTCTGGAA
30221
AGGAATCGGGGTGAGATGAGAGTTTTAGTAC
30370





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

TCTGGAAACAGAATCTACTAAAACAAGGCAA






CTCGTCAACTTGTTGGCGAGAttttcactTGGGGCCTACAGT

AATGCCGTGTTTATCTCGTCAACTTGTTGGCG






ACTGCTTATCAGAGAAGccaa

AGA



 


180
SauCas9KKH
+
GAAGCTTTGGCTTCTCTGATAGTTTTAGTACTCTGGAA
30222
AGGAATCGGGGTGAGATGAGAGTTTTAGTAC
30371





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

TCTGGAAACAGAATCTACTAAAACAAGGCAA






CTCGTCAACTTGTTGGCGAGAttttcactTGGGGCCTACAGT

AATGCCGTGTTTATCTCGTCAACTTGTTGGCG






ACTGCTTATCAGAGAAGccaa

AGA



 


183
SpyCas9-
−
AAGCTTTGGCTTCTCTGATAGTTTTAGAGCTAGAAATA
30223
AGGAATCGGGGTGAGATGAGGTTTTAGAGCT
30372



NG

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCttttcactTGGGGCCTACAGT

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






ACTGCTTATCAGAGAAGccaa

GC



 


187
SpyCas9-
+
AAGCTTTGGCTTCTCTGATAGTTTTAGAGCTAGAAATA
30224
GGAATCGGGGTGAGATGAGAGTTTTAGAGCT
30373



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCttttcactTGGGGCCTACAGT

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






ACTGCTTATCAGAGAAGccaa

GC



 


188
SpyCas9-
−
CTGATCCTGATTTAACAGTGGTTTTAGAGCTAGAAATA
30225
GGCTGGAACTCCGTGACAGTGTTTTAGAGCTA
30374



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCtgtaggccCCAAGTGAAAAG

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






TTATTATCACTGTTAAATcagg

GC



 


191
SauCas9KKH
−
TACTGATCCTGATTTAACAGTGTTTTAGTACTCTGGAA
30226
GGGGCTGGAACTCCGTGACAGGTTTTAGTACT
30375





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAgtaggccCCAAGTGAAAAG

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






TTATTATCACTGTTAAATCagga

GA



 


192
SauCas9KKH
−
TACTGATCCTGATTTAACAGTGTTTTAGTACTCTGGAA
30227
GGGGCTGGAACTCCGTGACAGGTTTTAGTACT
30376





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAgtaggccCCAAGTGAAAAG

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






TTATTATCACTGTTAAATCagga

GA



 


195
ScaCas9-
+
GAAGCTTTGGCTTCTCTGATGTTTTAGAGCTAGAAATA
30228
GAATCGGGGTGAGATGAGAGGTTTTAGAGCT
30377



Sc++

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCtttcactTGGGGCCTACAGTA

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






CTGCTTATCAGAGAAGCcaaa

GC



 


196
SpyCas9-
+
GAAGCTTTGGCTTCTCTGATGTTTTAGAGCTAGAAATA
30229
GAATCGGGGTGAGATGAGAGGTTTTAGAGCT
30378



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCtttcactTGGGGCCTACAGTA

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






CTGCTTATCAGAGAAGCcaaa

GC



 


197
SpyCas9-
−
ACTGATCCTGATTTAACAGTGTTTTAGAGCTAGAAATA
30230
GCTGGAACTCCGTGACAGTGGTTTTAGAGCTA
30379



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCgtaggccCCAAGTGAAAAGT

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






TATTATCACTGTTAAATCagga

GC



 


201
SauriCas9-
+
GAGAAGCTTTGGCTTCTCTGAGTTTTAGTACTCTGGAA
30231
GAATCGGGGTGAGATGAGAGAGTTTTAGTAC
30380



KKH

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

TCTGGAAACAGAATCTACTAAAACAAGGCAA






CTCGTCAACTTGTTGGCGAGAttcactTGGGGCCTACAGTA

AATGCCGTGTTTATCTCGTCAACTTGTTGGCG






CTGCTTATCAGAGAAGCCaaag

AGA



 


204
SpyCas9-
−
TACTGATCCTGATTTAACAGGTTTTAGAGCTAGAAATA
30232
GGGCTGGAACTCCGTGACAGGTTTTAGAGCTA
30381



NG

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCtaggccCCAAGTGAAAAGT

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






TATTATCACTGTTAAATCAggat

GC



 


207
SpyCas9-
+
AGAAGCTTTGGCTTCTCTGAGTTTTAGAGCTAGAAATA
30233
AATCGGGGTGAGATGAGAGAGTTTTAGAGCT
30382



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCttcactTGGGGCCTACAGTA

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






CTGCTTATCAGAGAAGCCaaag

GC



 


209
SpyCas9-
−
TACTGATCCTGATTTAACAGGTTTTAGAGCTAGAAATA
30234
CTGGAACTCCGTGACAGTGTGTTTTAGAGCTA
30383



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCtaggccCCAAGTGAAAAGT

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






TATTATCACTGTTAAATCAggat

GC



 


210
BlatCas9
+
gggaGAAGCTTTGGCTTCTCTGAGCTATAGTTCCTTACTG
30235
ggaaTCGGGGTGAGATGAGAGAAGCTATAGTTC
30384





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTttcactTGGGGCCTACAGTACTG

AATGACAGACGAGCACCTTGGAGCATTTATCT






CTTATCAGAGAAGCCaaag

CCGAGGTGCT



 


211
BlatCas9
+
gggaGAAGCTTTGGCTTCTCTGAGCTATAGTTCCTTACTG
30236
ggaaTCGGGGTGAGATGAGAGAAGCTATAGTTC
30385





AAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGAG

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTCC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCATATTCAAAATAATGACAGACGAGCACCTTGGAGC

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






ATTTATCTCCGAGGTGCTttcactTGGGGCCTACAGTACTG

AATGACAGACGAGCACCTTGGAGCATTTATCT






CTTATCAGAGAAGCCaaag

CCGAGGTGCT



 


215
SauCas9KKH
+
GGAGAAGCTTTGGCTTCTCTGGTTTTAGTACTCTGGAA
30237
GAATCGGGGTGAGATGAGAGAGTTTTAGTAC
30386





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

TCTGGAAACAGAATCTACTAAAACAAGGCAA






CTCGTCAACTTGTTGGCGAGAtcactTGGGGCCTACAGTA

AATGCCGTGTTTATCTCGTCAACTTGTTGGCG






CTGCTTATCAGAGAAGCCAaagc

AGA



 


218
ScaCas9-
−
ATACTGATCCTGATTTAACAGTTTTAGAGCTAGAAATA
30238
AACTCCGTGACAGTGTAATTGTTTTAGAGCTA
30387



Sc++

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCaggccCCAAGTGAAAAGTT

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






ATTATCACTGTTAAATCAGgatc

GC



 


219
SpyCas9-
−
ATACTGATCCTGATTTAACAGTTTTAGAGCTAGAAATA
30239
TGGAACTCCGTGACAGTGTAGTTTTAGAGCTA
30388



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCaggccCCAAGTGAAAAGTT

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






ATTATCACTGTTAAATCAGgatc

GC



 


220
SpyCas9-
+
GAGAAGCTTTGGCTTCTCTGGTTTTAGAGCTAGAAATA
30240
ATCGGGGTGAGATGAGAGAAGTTTTAGAGCT
30389



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCtcactTGGGGCCTACAGTAC

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






TGCTTATCAGAGAAGCCAaagc

GC



 


223
SauCas9KKH
+
GGGAGAAGCTTTGGCTTCTCTGTTTTAGTACTCTGGAA
30241
GAATCGGGGTGAGATGAGAGAGTTTTAGTAC
30390





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

TCTGGAAACAGAATCTACTAAAACAAGGCAA






CTCGTCAACTTGTTGGCGAGAcactTGGGGCCTACAGTA

AATGCCGTGTTTATCTCGTCAACTTGTTGGCG






CTGCTTATCAGAGAAGCCAAagct

AGA



 


224
SauCas9KKH
−
GAATACTGATCCTGATTTAACGTTTTAGTACTCTGGAA
30242
GGAACTCCGTGACAGTGTAATGTTTTAGTACT
30391





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAggccCCAAGTGAAAAGTT

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






ATTATCACTGTTAAATCAGGatca

GA



 


225
SauCas9KKH
−
GAATACTGATCCTGATTTAACGTTTTAGTACTCTGGAA
30243
GGAACTCCGTGACAGTGTAATGTTTTAGTACT
30392





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAggccCCAAGTGAAAAGTT

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






ATTATCACTGTTAAATCAGGatca

GA



 


226
SpyCas9-
−
AATACTGATCCTGATTTAACGTTTTAGAGCTAGAAATA
30244
ACTCCGTGACAGTGTAATTTGTTTTAGAGCTA
30393



NG

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCggccCCAAGTGAAAAGTT

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






ATTATCACTGTTAAATCAGGatca

GC



 


229
SpyCas9-
+
GGAGAAGCTTTGGCTTCTCTGTTTTAGAGCTAGAAATA
30245
TCGGGGTGAGATGAGAGAAGGTTTTAGAGCT
30394



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCcactTGGGGCCTACAGTAC

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






TGCTTATCAGAGAAGCCAAagct

GC



 


232
SpyCas9-
−
AATACTGATCCTGATTTAACGTTTTAGAGCTAGAAATA
30246
GGAACTCCGTGACAGTGTAAGTTTTAGAGCTA
30395



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCggccCCAAGTGAAAAGTT

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






ATTATCACTGTTAAATCAGGatca

GC



 


236
ScaCas9-
−
GAATACTGATCCTGATTTAAGTTTTAGAGCTAGAAATA
30247
AACTCCGTGACAGTGTAATTGTTTTAGAGCTA
30396



Sc++

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCgccCCAAGTGAAAAGTTA

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






TTATCACTGTTAAATCAGGAtcag

GC



 


237
SpyCas9-
−
GAATACTGATCCTGATTTAAGTTTTAGAGCTAGAAATA
30248
GAACTCCGTGACAGTGTAATGTTTTAGAGCTA
30397



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCgccCCAAGTGAAAAGTTA

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






TTATCACTGTTAAATCAGGAtcag

GC



 


238
SpyCas9-
+
GGGAGAAGCTTTGGCTTCTCGTTTTAGAGCTAGAAATA
30249
CGGGGTGAGATGAGAGAAGGGTTTTAGAGCT
30398



NG

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCactTGGGGCCTACAGTACT

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






GCTTATCAGAGAAGCCAAAgctt

GC



 


242
SpyCas9-
+
GGGAGAAGCTTTGGCTTCTCGTTTTAGAGCTAGAAATA
30250
CGGGGTGAGATGAGAGAAGGGTTTTAGAGCT
30399



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCactTGGGGCCTACAGTACT

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






GCTTATCAGAGAAGCCAAAgctt

GC



 


247
SauriCas9-
−
GGGAATACTGATCCTGATTTAGTTTTAGTACTCTGGAA
30251
GAACTCCGTGACAGTGTAATTGTTTTAGTACT
30400



KKH

ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAccCCAAGTGAAAAGTTA

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






TTATCACTGTTAAATCAGGATcagt

GA



 


250
ScaCas9-
+
GGGGAGAAGCTTTGGCTTCTGTTTTAGAGCTAGAAATA
30252
TCGGGGTGAGATGAGAGAAGGTTTTAGAGCT
30401



Sc++

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCctTGGGGCCTACAGTACT

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






GCTTATCAGAGAAGCCAAAGcttc

GC



 


251
SpyCas9-
−
GGGGAGAAGCTTTGGCTTCTGTTTTAGAGCTAGAAATA
30253
GGGGTGAGATGAGAGAAGGGGTTTTAGAGCT
30402



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCctTGGGGCCTACAGTACT

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






GCTTATCAGAGAAGCCAAAGcttc

GC



 


252
SpyCas9-
−
GGAATACTGATCCTGATTTAGTTTTAGAGCTAGAAATA
30254
AACTCCGTGACAGTGTAATTGTTTTAGAGCTA
30403



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCcCCCAAGTGAAAAGTTAT

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






TATCACTGTTAAATCAGGATcagt

GC



 


255
SauCas9KKH
+
AGGGGGAGAAGCTTTGGCTTCGTTTTAGTACTCTGGAA
30255
GGGGTGAGATGAGAGAAGGGGGTTTTAGTAC
30404





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

TCTGGAAACAGAATCTACTAAAACAAGGCAA






CTCGTCAACTTGTTGGCGAGAUTGGGGCCTACAGTACT

AATGCCGTGTTTATCTCGTCAACTTGTTGGCG






GCTTATCAGAGAAGCCAAAGCttct

AGA



 


256
SauCas9KKH
−
AGGGAATACTGATCCTGATTTGTTTTAGTACTCTGGAA
30256
GAACTCCGTGACAGTGTAATTGTTTTAGTACT
30405





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAcCCAAGTGAAAAGTTAT

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






TATCACTGTTAAATCAGGATCagta

GA



 


257
SauCas9KKH
+
AGGGGGAGAAGCTTTGGCTTCGTTTTAGTACTCTGGAA
30257
GGGGTGAGATGAGAGAAGGGGGTTTTAGTAC
30406





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

TCTGGAAACAGAATCTACTAAAACAAGGCAA






CTCGTCAACTTGTTGGCGAGAtTGGGGCCTACAGTACT

AATGCCGTGTTTATCTCGTCAACTTGTTGGCG






GCTTATCAGAGAAGCCAAAGCttct

AGA



 


258
SauCas9KKH
−
AGGGAATACTGATCCTGATTTGTTTTAGTACTCTGGAA
30258
GAACTCCGTGACAGTGTAATTGTTTTAGTACT
30407





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAcCCAAGTGAAAAGTTAT

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






TATCACTGTTAAATCAGGATCagta

GA



 


261
SpyCas9-
−
GGGAATACTGATCCTGATTTGTTTTAGAGCTAGAAATA
30259
ACTCCGTGACAGTGTAATTTGTTTTAGAGCTA
30408



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCcCCAAGTGAAAAGTTATT

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






ATCACTGTTAAATCAGGATCagta

GC



 


262
SpyCas9-
+
GGGGGAGAAGCTTTGGCTTCGTTTTAGAGCTAGAAATA
30260
GGGTGAGATGAGAGAAGGGGGTTTTAGAGCT
30409



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCtTGGGGCCTACAGTACTG

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






CTTATCAGAGAAGCCAAAGCttct

GC



 


264
SpyCas9-
−
AGGGAATACTGATCCTGATTGTTTTAGAGCTAGAAATA
30261
CTCCGTGACAGTGTAATTTTGTTTTAGAGCTA
30410



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCCCAAGTGAAAAGTTATT

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






ATCACTGTTAAATCAGGATCAgtat

GC



 


265
SpyCas9-
+
AGGGGGAGAAGCTTTGGCTTGTTTTAGAGCTAGAAATA
30262
GGTGAGATGAGAGAAGGGGCGTTTTAGAGCT
30411



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAGTGGCACCGAGTCGGTGCTGGGGCCTACAGTACTG

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






CTTATCAGAGAAGCCAAAGCTtctc

GC



 


269
SpyCas9-
+
CAGGGGGAGAAGCTTTGGCTGTTTTAGAGCTAGAAAT
30263
GTGAGATGAGAGAAGGGGCAGTTTTAGAGCT
30412



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAAGTGGCACCGAGTCGGTGCGGGGCCTACAGTACTG

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






CTTATCAGAGAAGCCAAAGCTTctcc

GC



 


270
SpyCas9-
−
CAGGGAATACTGATCCTGATGTTTTAGAGCTAGAAATA
30264
TCCGTGACAGTGTAATTTTGGTTTTAGAGCTA
30413



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCCAAGTGAAAAGTTATTA

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






TCACTGTTAAATCAGGATCAGtatt

GC



 


271
BlatCas9
−
cagcAGGGAATACTGATCCTGATGCTATAGTTCCTTACT
30265
actcCGTGACAGTGTAATTTTGGGCTATAGTTCC
30414





GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGA

TTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCCATATTCAAAATAATGACAGACGAGCACCTTGGAG

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






CATTTATCTCCGAGGTGCTCAAGTGAAAAGTTATTATC

AATGACAGACGAGCACCTTGGAGCATTTATCT






ACTGTTAAATCAGGATCAGtatt

CCGAGGTGCT



 


272
BlatCas9
−
cagcAGGGAATACTGATCCTGATGCTATAGTTCCTTACT
30266
actcCGTGACAGTGTAATTTTGGGCTATAGTTCC
30415





GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGA

TTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCCATATTCAAAATAATGACAGACGAGCACCTTGGAG

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






CATTTATCTCCGAGGTGCTCAAGTGAAAAGTTATTATC

AATGACAGACGAGCACCTTGGAGCATTTATCT






ACTGTTAAATCAGGATCAGtatt

CCGAGGTGCT



 


273
SauCas9KKH
−
AGCAGGGAATACTGATCCTGAGTTTTAGTACTCTGGAA
30267
CTCCGTGACAGTGTAATTTTGGTTTTAGTACT
30416





ACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT

CTGGAAACAGAATCTACTAAAACAAGGCAAA






CTCGTCAACTTGTTGGCGAGAAAGTGAAAAGTTATTAT

ATGCCGTGTTTATCTCGTCAACTTGTTGGCGA






CACTGTTAAATCAGGATCAGTattc

GA



 


274
SpyCas9-
+
CCAGGGGGAGAAGCTTTGGCGTTTTAGAGCTAGAAAT
30268
TGAGATGAGAGAAGGGGCACGTTTTAGAGCT
30417



SpRY

AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG






AAAGTGGCACCGAGTCGGTGCGGGCCTACAGTACTGC

TTATCAACTTGAAAAAGTGGCACCGAGTCGGT






TTATCAGAGAAGCCAAAGCTTCtccc

GC



 


275
SpyCas9-
−
GCAGGGAATACTGATCCTGAGTTTTAGAGCTAGAAATA
30269
CCGTGACAGTGTAATTTTGGGTTTTAGAGCTA
30418



SpRY

GCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT






AAGTGGCACCGAGTCGGTGCAAGTGAAAAGTTATTAT

TATCAACTTGAAAAAGTGGCACCGAGTCGGT






CACTGTTAAATCAGGATCAGTattc

GC



 


276
BlatCas9
+
gctcCAGGGGGAGAAGCTTTGGCGCTATAGTTCCTTACT
30270
gtgaGATGAGAGAAGGGGCACAAGCTATAGTTC
30419





GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGA

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCCATATTCAAAATAATGACAGACGAGCACCTTGGAG

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






CATTTATCTCCGAGGTGCTGGGCCTACAGTACTGCTTA

AATGACAGACGAGCACCTTGGAGCATTTATCT






TCAGAGAAGCCAAAGCTTCtccc

CCGAGGTGCT



 


277
BlatCas9
+
gctcCAGGGGGAGAAGCTTTGGCGCTATAGTTCCTTACT
30271
gtgaGATGAGAGAAGGGGCACAAGCTATAGTTC
30420





GAAAGGTAAGTTGCTATAGTAAGGGCAACAGACCCGA

CTTACTGAAAGGTAAGTTGCTATAGTAAGGGC






GGCGTTGGGGATCGCCTAGCCCGTGTTTACGGGCTCTC

AACAGACCCGAGGCGTTGGGGATCGCCTAGC






CCCATATTCAAAATAATGACAGACGAGCACCTTGGAG

CCGTGTTTACGGGCTCTCCCCATATTCAAAAT






CATTTATCTCCGAGGTGCTGGGCCTACAGTACTGCTTA

AATGACAGACGAGCACCTTGGAGCATTTATCT






TCAGAGAAGCCAAAGCTTCtccc

CCGAGGTGCT









Capital letters indicate “core nucleotides” while lower case letters indicate “flanking nucleotides.” Herein, when an RNA sequence (e.g., a template RNA sequence) is said to comprise a particular sequence (e.g., a sequence of Table 4A, Table 4B, Table 4C, or Table 4D or a portion thereof) that comprises thymine (T), it is of course understood that the RNA sequence may (and frequently does) comprise uracil (U) in place of T. For instance, the RNA sequence may comprise U at every position shown as T in the sequence in Table 4A, Table 4B, Table 4C, or Table 4D. More specifically, the present disclosure provides an RNA sequence according to every template sequence shown in Table 4A, Table 4B, Table 4C, or Table 4D, wherein the RNA sequence has a U in place of each T in the sequence of Table 4A, Table 4B, Table 4C, or Table 4D.
In some embodiments, the systems and methods provided herein may comprise a template sequence listed in any of Tables 5A-5F. Tables 5A-5F provide exemplary template RNA sequences (column 2) designed to be paired with a gene modifying polypeptide to correct a mutation in the PAH gene. The templates in Tables 5A-5F are meant to exemplify the total sequence of: (1) gRNA spacer (e.g., for targeting for first strand nick), (2) gRNA scaffold, (3) RT (heterologous object sequence) sequence, and (4) PBS sequence (e.g., for initiating TPRT at first strand nick).







Lengthy table referenced here




US12544458-20260210-T00002


Please refer to the end of the specification for access instructions.













Lengthy table referenced here




US12544458-20260210-T00003


Please refer to the end of the specification for access instructions.













Lengthy table referenced here




US12544458-20260210-T00004


Please refer to the end of the specification for access instructions.













Lengthy table referenced here




US12544458-20260210-T00005


Please refer to the end of the specification for access instructions.













Lengthy table referenced here




US12544458-20260210-T00006


Please refer to the end of the specification for access instructions.













Lengthy table referenced here




US12544458-20260210-T00007


Please refer to the end of the specification for access instructions.






In some embodiments, the systems and methods provided herein may comprise a template sequence listed in Table 6A. Table 6A provides exemplary template RNA sequences (column 4) and second-nick gRNA spacer sequences (column 3) designed to be paired with a gene modifying polypeptide to correct a R408W mutation in the PAH gene.







TABLE 6A







Exemplary second nick gRNA sequences


Table 6A provides spacer sequences for second-strand targeting gRNAs and relevant characteristics. Second-nick gRNAs in this table


are designed to be used in combination with template RNAs comprising the particular spacers noted in Column 6. In some embodiments, a


second-nick gRNA is selected with preference for a distance of less than or equal to 100 nt from the first nick (i.e., the nick specified


by the template RNA). In some embodiments, a second-nick gRNA is selected with a preference for a PAM-in orientation with the template RNA


of the gene modifying system, as described elsewhere in this application.

















Exemplary



Second-nick



Compatible


Name
spacer
SEQ ID NO
Sequence
SEQ ID NO
Spacer





hPKU_ngRNA_1
TTACTTCTTTTT
37082
TTACTTCTTTTTTAGGAACAGTTTTAGAGCTA
37124
hPKU6


7−
TAGGAACA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT







TATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_2
TGGCATTTTACT
37083
TGGCATTTTACTTCTTTTTTGTTTTAGAGCTAG
37125
hPKU6


4−
TCTTTTTT

AAATAGCAAGTTAAAATAAGGCTAGTCCGTT







ATCAACTTGAAAAAGTGGCACCGAGTCGGTG







CTTTT




 


hPKU_ngRNA_4
AGTCTTAAGAG
37084
AGTCTTAAGAGAGTTCTCAGGTTTTAGAGCTA
37126
hPKU6


4−
AGTTCTCAG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT







TATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_6
GAAGGGCACCA
37085
GAAGGGCACCATTTGGAGAAGTTTTAGAGCT
37127
hPKU6


8−
TTTGGAGAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG







TTATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_7
CTTGAGTGAAG
37086
CTTGAGTGAAGGGCACCATTGTTTTAGAGCTA
37128
hPKU6


5−
GGCACCATT

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT







TATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_5
TAAGACTACCT
37087
TAAGACTACCTTTCTCCAAAGTTTTAGAGCTA
37129
hPKU1, hPKU2,


7+
TTCTCCAAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT

hPKU3, hPKU4,





TATCAACTTGAAAAAGTGGCACCGAGTCGGT

hPKU5





GCTTTT




 


hPKU_ngRNA_8
AAACCACAGGC
37088
AAACCACAGGCTTGAGTGAAGTTTTAGAGCT
37130
hPKU6


5−
TTGAGTGAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG







TTATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_8
AAAACCACAGG
37089
AAAACCACAGGCTTGAGTGAGTTTTAGAGCT
37131
hPKU6


6−
CTTGAGTGA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG







TTATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_9
GTTCCTAAGAC
37090
GTTCCTAAGACCAAAACCACGTTTTAGAGCTA
37132
hPKU6


8−
CAAAACCAC

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT







TATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_7
GTGCCCTTCACT
37091
GTGCCCTTCACTCAAGCCTGGTTTTAGAGCTA
37133
hPKU1, hPKU2,


9+
CAAGCCTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT

hPKU3, hPKU4,





TATCAACTTGAAAAAGTGGCACCGAGTCGGT

hPKU5





GCTTTT




 


hPKU_ngRNA_8
TTCACTCAAGC
37092
TTCACTCAAGCCTGTGGTTTGTTTTAGAGCTA
37134
hPKU1, hPKU2,


5+
CTGTGGTTT

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT

hPKU3, hPKU4,





TATCAACTTGAAAAAGTGGCACCGAGTCGGT

hPKU5





GCTTTT




 


hPKU_ngRNA_9
AAGCCTGTGGT
37093
AAGCCTGTGGTTTTGGTCTTGTTTTAGAGCTA
37135
hPKU1, hPKU2,


2+
TTTGGTCTT

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT

hPKU3, hPKU4,





TATCAACTTGAAAAAGTGGCACCGAGTCGGT

hPKU5





GCTTTT




 


hPKU_ngRNA_1
GTCCAAGACCT
37094
GTCCAAGACCTCAATCCTTTGTTTTAGAGCTA
37136
hPKU6


70−
CAATCCTTT

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT







TATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_1
TGTCCAAGACC
37095
TGTCCAAGACCTCAATCCTTGTTTTAGAGCTA
37137
hPKU6


71−
TCAATCCTT

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT







TATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_1
GCTACGACCCA
37096
GCTACGACCCATACACCCAAGTTTTAGAGCTA
37138
hPKU1, hPKU2,


52+
TACACCCAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT

hPKU3, hPKU4,





TATCAACTTGAAAAAGTGGCACCGAGTCGGT

hPKU5





GCTTTT




 


hPKU_ngRNA_1
CCCATACACCC
37097
CCCATACACCCAAAGGATTGGTTTTAGAGCTA
37139
hPKU1, hPKU2,


59+
AAAGGATTG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT

hPKU3, hPKU4,





TATCAACTTGAAAAAGTGGCACCGAGTCGGT

hPKU5





GCTTTT




 


hPKU_ngRNA_1
CACCCAAAGGA
37098
CACCCAAAGGATTGAGGTCTGTTTTAGAGCTA
37140
hPKU1, hPKU2,


65+
TTGAGGTCT

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT

hPKU3, hPKU4,





TATCAACTTGAAAAAGTGGCACCGAGTCGGT

hPKU5





GCTTTT




 


hPKU_ngRNA_1
AGCCAAAATCT
37099
AGCCAAAATCTTAAGCTGCTGTTTTAGAGCTA
37141
hPKU6


97−
TAAGCTGCT

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT







TATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_1
CAGCCAAAATC
37100
CAGCCAAAATCTTAAGCTGCGTTTTAGAGCTA
37142
hPKU6


98−
TTAAGCTGC

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT







TATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_1
TACCCAGCAGC
37101
TACCCAGCAGCTTAAGATTTGTTTTAGAGCTA
37143
hPKU1, hPKU2,


92+
TTAAGATTT

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT

hPKU3, hPKU4,





TATCAACTTGAAAAAGTGGCACCGAGTCGGT

hPKU5





GCTTTT




 


hPKU_ngRNA_2
TGTAAATTACTT
37102
TGTAAATTACTTACTGTTAAGTTTTAGAGCTA
37144
hPKU6


24−
ACTGTTAA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT







TATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_2
AGAAACCGAGT
37103
AGAAACCGAGTGGCCTCGTAGTTTTAGAGCTA
37145
hPKU6


47−
GGCCTCGTA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT







TATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_2
TAAGTAATTTA
37104
TAAGTAATTTACACCTTACGGTTTTAGAGCTA
37146
hPKU1, hPKU2,


31+
CACCTTACG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT

hPKU3, hPKU4,





TATCAACTTGAAAAAGTGGCACCGAGTCGGT

hPKU5





GCTTTT




 


hPKU_ngRNA_2
TCGATTACTGA
37105
TCGATTACTGAGAAACCGAGGTTTTAGAGCTA
37147
hPKU6


57−
GAAACCGAG

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT







TATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_2
TTACACCTTACG
37106
TTACACCTTACGAGGCCACTGTTTTAGAGCTA
37148
hPKU1, hPKU2,


39+
AGGCCACT

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT

hPKU3, hPKU4,





TATCAACTTGAAAAAGTGGCACCGAGTCGGT

hPKU5





GCTTTT




 


hPKU_ngRNA_2
TTTTTCCTATGG
37107
TTTTTCCTATGGCGATGGTAGTTTTAGAGCTA
37149
hPKU6


91−
CGATGGTA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT







TATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_2
ATTTTTCCTATG
37108
ATTTTTCCTATGGCGATGGTGTTTTAGAGCTA
37150
hPKU6


92−
GCGATGGT

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT







TATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_2
TATTATTTTTCC
37109
TATTATTTTTCCTATGGCGAGTTTTAGAGCTA
37151
hPKU6


96−
TATGGCGA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT







TATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_3
TAAATTTATTAT
37110
TAAATTTATTATTTTTCCTAGTTTTAGAGCTAG
37152
hPKU6


02−
TTTTCCTA

AAATAGCAAGTTAAAATAAGGCTAGTCCGTT







ATCAACTTGAAAAAGTGGCACCGAGTCGGTG







CTTTT




 


hPKU_ngRNA_2
TCTTTCCCTACC
37111
TCTTTCCCTACCATCGCCATGTTTTAGAGCTA
37153
hPKU1, hPKU2,


83+
ATCGCCAT

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT

hPKU3, hPKU4,





TATCAACTTGAAAAAGTGGCACCGAGTCGGT

hPKU5





GCTTTT




 


hPKU_ngRNA_3
TTTATTGAAATA
37112
TTTATTGAAATATTTAATTAGTTTTAGAGCTA
37154
hPKU1, hPKU2,


18+
TTTAATTA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT

hPKU3, hPKU4,





TATCAACTTGAAAAAGTGGCACCGAGTCGGT

hPKU5





GCTTTT




 


hPKU_ngRNA3b
TGAGAAGGGCC
37113
TGAGAAGGGCCGAGGTATTGGTTTTAGAGCT
37155
hPKU6


128-wt
GAGGTATTG

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG







TTATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA3b
TAGCGAACTGA
37114
TAGCGAACTGAGAAGGGCCGGTTTTAGAGCT
37156
hPKU6


136-mut
GAAGGGCCA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG







TTATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA3b
TAGCGAACTGA
37115
TAGCGAACTGAGAAGGGCCGGTTTTAGAGCT
37157
hPKU6


136-wt
GAAGGGCCG

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG







TTATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA3b
TGAGAAGGGCC
37116
TGAGAAGGGCCAAGGTATTGGTTTTAGAGCT
37158
hPKU6


128-mut
AAGGTATTG

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG







TTATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA3b
TAGCGAACTGA
37117
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCT
37159
hPKU6


136-mut
GAAGGGCCA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG







TTATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA3b
TAGCGAACTGA
37118
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCT
37160
hPKU6


136-wt
GAAGGGCCG

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG







TTATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_1
ACTTTGCTGCCA
37119
ACTTTGCTGCCACAATACCTGTTTTAGAGCTA
37161
hPKU1, hPKU2,


16+
CAATACCT

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT

hPKU3, hPKU4,





TATCAACTTGAAAAAGTGGCACCGAGTCGGT

hPKU5





GCTTTT




 


hPKU_ngRNA_1
GGGTCGTAGCG
37120
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCT
37162
hPKU6


42−
AACTGAGAA

AGAAATAGCAAGTTAAAATAAGGCTAGTCCG







TTATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_1
TGGGTCGTAGC
37121
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTA
37163
hPKU6


43−
GAACTGAGA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT







TATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_1
CCTCAATCCTTT
37122
CCTCAATCCTTTGGGTGTATGTTTTAGAGCTA
37164
hPKU6


62−
GGGTGTAT

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT







TATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT




 


hPKU_ngRNA_1
ACCTCAATCCTT
37123
ACCTCAATCCTTTGGGTGTAGTTTTAGAGCTA
37165
hPKU6


63−
TGGGTGTA

GAAATAGCAAGTTAAAATAAGGCTAGTCCGT







TATCAACTTGAAAAAGTGGCACCGAGTCGGT







GCTTTT









The template RNA sequences shown in Tables 1-4, 5A-5F, and 6A may be customized depending on the cell being targeted. For example, in some embodiments it is desired to inactivate a PAM sequence upon editing (e.g., using a “PAM-kill” modification) to decrease the potential for further gene editing (e.g., by Cas retargeting) following the initial edit. Consequently, certain template RNAs described herein are designed to write a mutation (e.g., a substitution) into the PAM of the target site, such that upon editing, the PAM site will be mutated to a sequence no longer recognized by the gene modifying polypeptide. Thus, a mutation region within the heterologous object sequence of the template RNA may comprise a PAM-kill sequence. Without wishing to be bound by theory, in some embodiments, a PAM-kill sequence prevents re-engagement of the gene modifying polypeptide upon completion of a genetic modification, or decreases re-engagement relative to a template RNA lacking a PAM-kill sequence. In some embodiments, a PAM-kill sequence does not alter the amino acid sequence encoded by a gene, e.g., the PAM-kill sequence results in a silent mutation. In other embodiments, it is desired to leave the PAM sequence intact (no PAM-kill).
Similarly, in some embodiments, to decrease the potential for further gene editing (e.g., by Cas retargeting) following the initial edit, it may be desirable to alter the first three nucleotides of the RT template sequence via a “seed-kill” motif. Consequently, certain template RNAs described herein are designed to write a mutation (e.g., a substitution) into the portion of the target site corresponding to the first three nucleotides of the RT template sequence, such that upon editing, the target site will be mutated to a sequence with lower homology to the RT template sequence. Thus, a mutation region within the heterologous object sequence of the template RNA may comprise a seed-kill sequence. Without wishing to be bound by theory, in some embodiments, a seed-kill sequence prevents re-engagement of the gene modifying polypeptide upon completion of a genetic modification, or decreases re-engagement relative to an otherwise similar template RNA lacking a seed-kill sequence. In some embodiments, a seed-kill sequence does not alter the amino acid sequence encoded by a gene, e.g., the seed-kill sequence results in a silent mutation. In other embodiments, it is desired to leave the seed region intact, and a seed-kill sequence is not used.
In further embodiments, to optimize or improve gene editing efficiency, it may be desirable to evade the target cell's mismatch repair or nucleotide repair pathways or to bias the target cell's repair pathways toward preservation of the edited strand. In some embodiments, multiple silent mutations (for example, silent substitutions) may be introduced within the RT template sequence to evade the target cell's mismatch repair or nucleotide repair pathways or to bias the target cell's repair pathways toward preservation of the edited strand.
Table 7A provides exemplary silent mutations for various positions within the PAH gene for use with a template to correct a R408W mutation.







TABLE 7A







Exemplary Silent Mutation Codons for the PAH Gene


for Templates to Correct a R408W mutation







Amino Acid










Position





(Including
WT
WT


the Initial
Amino
Co-










Methionine)
Acid
don
ALL CODONS


















401
N
AAC
AAT
AAC






402
F
TTT
TTT
TTC


403
A
GCT
GCT
GCC
GCA
GCG


404
A
GCC
GCT
GCC
GCA
GCG


405
T
ACA
ACT
ACC
ACA
ACG


406
I
ATA
ATA
ATT
ATC


407
P
CCT
CCT
CCC
CCA
CCG


409
P
CCC
CCT
CCC
CCA
CCG


410
F
TTC
TTT
TTC


411
S
TCA
TCT
TCC
TCA
TCG
AGT
AGC


412
V
GTT
GTT
GTC
GTA
GTG


413
R
CGC
CGT
CGC
CGA
CGG
AGA
AGG


414
Y
TAC
TAT
TAC


415
D
GAC
GAT
GAC


416
P
CCA
CCT
CCC
CCA
CCG


417
Y
TAC
TAT
TAC


418
T
ACC
ACT
ACC
ACA
ACG


419
Q
CAA
CAA
CAG


420
R
AGG
CGT
CGC
CGA
CGG
AGA
AGG


421
I
ATT
ATA
ATT
ATC


422
E
GAG
GAA
GAG


423
V
GTC
GTT
GTC
GTA
GTG


424
L
TTG
TTA
TTG
CTT
CTC
CTA
CTG


425
D
GAC
GAT
GAC


426
N
AAT
AAT
AAC


427
T
ACC
ACT
ACC
ACA
ACG


428
Q
CAG
CAA
CAG


429
Q
CAG
CAA
CAG


430
L
CTT
TTA
TTG
CTT
CTC
CTA
CTG


431
K
AAG
AAA
AAG


432
I
ATT
ATA
ATT
ATC


433
L
TTG
TTA
TTG
CTT
CTC
CTA
CTG


434
A
GCT
GCT
GCC
GCA
GCG


435
D
GAT
GAT
GAC


436
S
TCC
TCT
TCC
TCA
TCG
AGT
AGC


437
I
ATT
ATA
ATT
ATC


438
N
AAC
AAT
AAC









Table 7B provides exemplary silent mutations for various positions within the PAH gene for use with a template to correct a R261Q mutation.







TABLE 7B







Exemplary Silent Mutation Codons for the PAH Gene


for Templates to Correct a R261Q mutation







Amino Acid










Position





(Including
WT
WT


the Initial
Amino
Co-










Methionine)
Acid
don
All Codons


















237
C
TGC
TGT
TGC






238
T
ACT
ACT
ACC
ACA
ACG


239
G
GGT
GGT
GGC
GGA
GGG


240
F
TTC
TTT
TTC


241
R
CGC
CGT
CGC
CGA
CGG
AGA
AGG


242
L
CTC
TTA
TTG
CTT
CTC
CTA
CTG


243
R
CGA
CGT
CGC
CGA
CGG
AGA
AGG


244
P
CCT
CCT
CCC
CCA
CCG


245
V
GTG
GTT
GTC
GTA
GTG


246
A
GCT
GCT
GCC
GCA
GCG


247
G
GGC
GGT
GGC
GGA
GGG


248
L
CTG
TTA
TTG
CTT
CTC
CTA
CTG


249
L
CTT
TTA
TTG
CTT
CTC
CTA
CTG


250
S
TCC
TCT
TCC
TCA
TCG
AGT
AGC


251
S
TCT
TCT
TCC
TCA
TCG
AGT
AGC


252
R
CGG
CGT
CGC
CGA
CGG
AGA
AGG


253
D
GAT
GAT
GAC


254
F
TTC
TTT
TTC


255
L
TTG
TTA
TTG
CTT
CTC
CTA
CTG


256
G
GGT
GGT
GGC
GGA
GGG


257
G
GGC
GGT
GGC
GGA
GGG


258
L
CTG
TTA
TTG
CTT
CTC
CTA
CTG


259
A
GCC
GCT
GCC
GCA
GCG


260
F
TTC
TTT
TTC


262
V
GTC
GTT
GTC
GTA
GTG


263
F
TTC
TTT
TTC


264
H
CAC
CAT
CAC


265
C
TGC
TGT
TGC


266
T
ACA
ACT
ACC
ACA
ACG


267
Q
CAG
CAA
CAG


268
Y
TAC
TAT
TAC


269
I
ATC
ATA
ATT
ATC


270
R
AGA
CGT
CGC
CGA
CGG
AGA
AGG


271
H
CAT
CAT
CAC


272
G
GGA
GGT
GGC
GGA
GGG


273
S
TCC
TCT
TCC
TCA
TCG
AGT
AGC


274
K
AAG
AAA
AAG


275
P
CCC
CCT
CCC
CCA
CCG


276
M
ATG
ATG


277
Y
TAT
TAT
TAC


278
T
ACC
ACT
ACC
ACA
ACG


279
P
CCC
CCT
CCC
CCA
CCG


280
E
GAA
GAA
GAG









Table 7C provides exemplary silent mutations for various positions within the PAH gene for use with a template to correct a R243Q mutation.







TABLE 7C







Exemplary Silent Mutation Codons for the 


PAH Gene for Templates to Correct a R243Q mutation















Amino Acid










Position










(Including
WT 









the Initial
Amino

















Methionine)
Acid
WT Codon
ALL CODONS


















237
C
TGC
TGT
TGC






238
T
ACT
ACT
ACC
ACA
ACG




239
G
GGT
GGT
GGC
GGA
GGG




240
F
TTC
TTT
TTC






241
R
CGC
CGT
CGC
CGA
CGG
AGA
AGG


242
L
CTC
TTA
TTG
CTT
CTC
CTA
CTG


244
P
CCT
CCT
CCC
CCA
CCG




245
V
GTG
GTT
GTC
GTA
GTG




246
A
GCT
GCT
GCC
GCA
GCG




247
G
GGC
GGT
GGC
GGA
GGG




248
L
CTG
TTA
TTG
CTT
CTC
CTA
CTG


249
L
CTT
TTA
TTG
CTT
CTC
CTA
CTG


250
S
TCC
TCT
TCC
TCA
TCG
AGT
AGC


251
S
TCT
TCT
TCC
TCA
TCG
AGT
AGC


252
R
CGG
CGT
CGC
CGA
CGG
AGA
AGG


253
D
GAT
GAT
GAC






254
F
TTC
TTT
TTC






255
L
TTG
TTA
TTG
CTT
CTC
CTA
CTG


256
G
GGT
GGT
GGC
GGA
GGG




257
G
GGC
GGT
GGC
GGA
GGG




258
L
CTG
TTA
TTG
CTT
CTC
CTA
CTG


259
A
GCC
GCT
GCC
GCA
GCG




260
F
TTC
TTT
TTC






261
R
CGA
CGT
CGC
CGA
CGG
AGA
AGG


262
V
GTC
GTT
GTC
GTA
GTG




263
F
TTC
TTT
TTC






264
H
CAC
CAT
CAC






265
C
TGC
TGT
TGC






266
T
ACA
ACT
ACC
ACA
ACG




267
Q
CAG
CAA
CAG






268
Y
TAC
TAT
TAC






269
I
ATC
ATA
ATT
ATC





270
R
AGA
CGT
CGC
CGA
CGG
AGA
AGG


271
H
CAT
CAT
CAC






272
G
GGA
GGT
GGC
GGA
GGG




273
S
TCC
TCT
TCC
TCA
TCG
AGT
AGC


274
K
AAG
AAA
AAG






275
P
CCC
CCT
CCC
CCA
CCG




276
M
ATG
ATG







277
Y
TAT
TAT
TAC






278
T
ACC
ACT
ACC
ACA
ACG




279
P
CCC
CCT
CCC
CCA
CCG




280
E
GAA
GAA
GAG









In some embodiments, the template RNA comprises one or more silent mutations.
It should be understood that the silent mutations illustrated in Tables 7A-7C may be used individually or combined in any manner in a template RNA sequence described herein. In some embodiments, the template RNA comprises a sequence having one or more silent substitutions as shown in Table E6 or E6A.
In some embodiments, the template RNA comprises a sequence listed in any one of Tables 8A-8D. Tables 8A-8D provide exemplary template RNA sequences comprising one or more silent substitutions (column 2) designed to be paired with a gene modifying polypeptide to correct a mutation in the PAH gene. The templates in Tables 5A-5F are meant to exemplify the total sequence of: (1) gRNA spacer (e.g., for targeting for first strand nick), (2) gRNA scaffold, (3) RT (heterologous object sequence) sequence, and (4) PBS sequence (e.g., for initiating TPRT at first strand nick).







TABLE 8A







Exemplary template RNA sequences


Table 8A provides design of exemplary components of gene modifying systems for correcting the 


pathogenic R408W mutation in PAH to the wild-type form. This table details the sequence of a complete 


template RNA comprising one or more silent substitutions described in Table 7A for use in a Cas-RT 


fusion gene modifying polypeptide. Templates in this table employ the hPKU3 spacer TGGGTCGTAGCGAACTGAGA


(SEQ ID NO: 16102).











SEQ




ID


Name
tgRNA sequence
NO





hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36542


P10_sub0
ACCGAGTCGGTGCaatacctCggcccttctcagttcgcta



 


hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36543


P10_sub1
ACCGAGTCGGTGCaataccgCggcccttctcagttcgcta



 


hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36544


P10_sub2
ACCGAGTCGGTGCaatccctCggcccttctcagttcgcta



 


hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36545


P10_sub4
ACCGAGTCGGTGCaatacctCgccccttctcagttcgcta



 


hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36546


P10_sub7
ACCGAGTCGGTGCaataccgCgccccttctcagttcgcta



 


hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36547


P10_sub8
ACCGAGTCGGTGCaataccgCgcccattctcagttcgcta



 


hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36548


P8_sub0
ACCGAGTCGGTGCaatacctCggcccttctcagttcgc



 


hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36549


P8_sub1
ACCGAGTCGGTGCaataccgCggcccttctcagttcgc



 


hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36550


P8_sub2
ACCGAGTCGGTGCaatccctCggcccttctcagttcgc



 


hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36551


P8_sub4
ACCGAGTCGGTGCaatacctCgccccttctcagttcgc



 


hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36552


P8_sub7
ACCGAGTCGGTGCaataccgCgccccttctcagttcgc



 


hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36553


P8_sub8
ACCGAGTCGGTGCaataccgCgcccattctcagttcgc



 


hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36554


P9_sub0
ACCGAGTCGGTGCaatacctCggcccttctcagttcgct



 


hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36555


P9_sub1
ACCGAGTCGGTGCaataccgCggcccttctcagttcgct



 


hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36556


P9_sub2
ACCGAGTCGGTGCaatccctCggcccttctcagttcgct



 


hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36557


P9_sub4
ACCGAGTCGGTGCaatacctCgccccttctcagttcgct



 


hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36558


P9_sub7
ACCGAGTCGGTGCaataccgCgccccttctcagttcgct



 


hPKU3_R17_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36559


P9_sub8
ACCGAGTCGGTGCaataccgCgcccattctcagttcgct



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36560


P10_sub0
ACCGAGTCGGTGCacaatacctCggcccttctcagttcgcta



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36561


P10_sub1
ACCGAGTCGGTGCacaataccgCggcccttctcagttcgcta



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36562


P10_sub2
ACCGAGTCGGTGCacaatccctCggcccttctcagttcgcta



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36563


P10_sub4
ACCGAGTCGGTGCacaatacctCgccccttctcagttcgcta



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36564


P10_sub7
ACCGAGTCGGTGCacaataccgCgccccttctcagttcgcta



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36565


P10_sub8
ACCGAGTCGGTGCacaataccgCgcccattctcagttcgcta



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36566


P8_sub0
ACCGAGTCGGTGCacaatacctCggcccttctcagttcgc



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36567


P8_sub1
ACCGAGTCGGTGCacaataccgCggcccttctcagttcgc



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36568


P8_sub2
ACCGAGTCGGTGCacaatccctCggcccttctcagttcgc



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36569


P8_sub4
ACCGAGTCGGTGCacaatacctCgccccttctcagttcgc



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36570


P8_sub7
ACCGAGTCGGTGCacaataccgCgccccttctcagttcgc



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36571


P8_sub8
ACCGAGTCGGTGCacaataccgCgcccattctcagttcgc



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36572


P9_sub0
ACCGAGTCGGTGCacaatacctCggcccttctcagttcgct



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36573


P9_sub1
ACCGAGTCGGTGCacaataccgCggcccttctcagttcgct



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36574


P9_sub2
ACCGAGTCGGTGCacaatccctCggcccttctcagttcgct



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36575


P9_sub4
ACCGAGTCGGTGCacaatacctCgccccttctcagttcgct



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36576


P9_sub7
ACCGAGTCGGTGCacaataccgCgccccttctcagttcgct



 


hPKU3_R19_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36577


P9_sub8
ACCGAGTCGGTGCacaataccgCgcccattctcagttcgct



 


hPKU3_R21_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36578


P10_sub0
ACCGAGTCGGTGCccacaatacctCggcccttctcagttcgcta



 


hPKU3_R21_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36579


P10_sub1
ACCGAGTCGGTGCccacaataccgCggcccttctcagttcgcta



 


hPKU3_R21_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36580


P10_sub2
ACCGAGTCGGTGCccacaatccctCggcccttctcagttcgcta



 


hPKU3_R21_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36581


P10_sub4
ACCGAGTCGGTGCccacaatacctCgccccttctcagttcgcta



 


hPKU3_R21_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36582


P10_sub7
ACCGAGTCGGTGCccacaataccgCgccccttctcagttcgcta



 


hPKU3_R21_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36583


P10_sub8
ACCGAGTCGGTGCccacaataccgCgcccattctcagttcgcta



 


hPKU3_R21_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36584


P8_sub0
ACCGAGTCGGTGCccacaatacctCggcccttctcagttcgc



 


hPKU3_R21_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36585


P8_sub1
ACCGAGTCGGTGCccacaataccgCggcccttctcagttcgc



 


hPKU3_R21_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36586


P8_sub2
ACCGAGTCGGTGCccacaatccctCggcccttctcagttcgc



 


hPKU3_R21_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36587


P8_sub4
ACCGAGTCGGTGCccacaatacctCgccccttctcagttcgc



 


hPKU3_R21
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36588


P8_sub7
ACCGAGTCGGTGCccacaataccgCgccccttctcagttcgc



 


hPKU3_R21_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36589


P8_sub8
ACCGAGTCGGTGCccacaataccgCgcccattctcagttcgc



 


hPKU3_R21_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36590


P9_sub0
ACCGAGTCGGTGCccacaatacctCggcccttctcagttcgct



 


hPKU3_R21_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36591


P9_sub1
ACCGAGTCGGTGCccacaataccgCggcccttctcagttcgct



 


hPKU3_R21_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36592


P9_sub2
ACCGAGTCGGTGCccacaatccctCggcccttctcagttcgct



 


hPKU3_R21_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36593


P9_sub4
ACCGAGTCGGTGCccacaatacctCgccccttctcagttcgct



 


hPKU3_R21_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36594


P9_sub7
ACCGAGTCGGTGCccacaataccgCgccccttctcagttcgct



 


hPKU3_R21_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36595


P9_sub8
ACCGAGTCGGTGCccacaataccgCgcccattctcagttcgct



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36596


P10_sub0
ACCGAGTCGGTGCtgccacaatacctCggcccttctcagttcgcta



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36597


P10_sub1
ACCGAGTCGGTGCtgccacaataccgCggcccttctcagttcgcta



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36598


P10_sub2
ACCGAGTCGGTGCtgccacaatccctCggcccttctcagttcgcta



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36599


P10_sub4
ACCGAGTCGGTGCtgccacaatacctCgccccttctcagttcgcta



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36600


P10_sub7
ACCGAGTCGGTGCtgccacaataccgCgccccttctcagttcgcta



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36601


P10_sub8
ACCGAGTCGGTGCtgccacaataccgCgcccattctcagttcgcta



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36602


P8_sub0
ACCGAGTCGGTGCtgccacaatacctCggcccttctcagttcgc



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36603


P8_sub1
ACCGAGTCGGTGCtgccacaataccgCggcccttctcagttcgc



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36604


P8_sub2
ACCGAGTCGGTGCtgccacaatccctCggcccttctcagttcgc



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36605


P8_sub4
ACCGAGTCGGTGCtgccacaatacctCgccccttctcagttcgc



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36606


P8_sub7
ACCGAGTCGGTGCtgccacaataccgCgccccttctcagttcgc



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36607


P8_sub8
ACCGAGTCGGTGCtgccacaataccgCgcccattctcagttcgc



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36608


P9_sub0
ACCGAGTCGGTGCtgccacaatacctCggcccttctcagttcgct



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36609


P9_sub1
ACCGAGTCGGTGCtgccacaataccgCggcccttctcagttcgct



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36610


P9_sub2
ACCGAGTCGGTGCtgccacaatccctCggcccttctcagttcgct



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36611


P9_sub4
ACCGAGTCGGTGCtgccacaatacctCgccccttctcagttcgct



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36612


P9_sub7
ACCGAGTCGGTGCtgccacaataccgCgccccttctcagttcgct



 


hPKU3_R23_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36613


P9_sub8
ACCGAGTCGGTGCtgccacaataccgCgcccattctcagttcgct



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36614


P10_sub0
ACCGAGTCGGTGCgctgccacaatacctCggcccttctcagttcgcta



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36615


P10_sub1
ACCGAGTCGGTGCgctgccacaataccgCggcccttctcagttcgcta



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36616


P10_sub2
ACCGAGTCGGTGCgctgccacaatccctCggcccttctcagttcgcta



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36617


P10_sub4
ACCGAGTCGGTGCgctgccacaatacctCgccccttctcagttcgcta



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36618


P10_sub7
ACCGAGTCGGTGCgctgccacaataccgCgccccttctcagttcgcta



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36619


P10_sub8
ACCGAGTCGGTGCgctgccacaataccgCgcccattctcagttcgcta



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36620


P8_sub0
ACCGAGTCGGTGCgctgccacaatacctCggcccttctcagttcgc



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36621


P8_sub1
ACCGAGTCGGTGCgctgccacaataccgCggcccttctcagttcgc



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36622


P8_sub2
ACCGAGTCGGTGCgctgccacaatccctCggcccttctcagttcgc



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36623


P8_sub4
ACCGAGTCGGTGCgctgccacaatacctCgccccttctcagttcgc



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36624


P8_sub7
ACCGAGTCGGTGCgctgccacaataccgCgccccttctcagttcgc



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36625


P8_sub8
ACCGAGTCGGTGCgctgccacaataccgCgcccattctcagttcgc



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36626


P9_sub0
ACCGAGTCGGTGCgctgccacaatacctCggcccttctcagttcgct



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36627


P9_sub1
ACCGAGTCGGTGCgctgccacaataccgCggcccttctcagttcgct



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36628


P9_sub2
ACCGAGTCGGTGCgctgccacaatccctCggcccttctcagttcgct



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36629


P9_sub4
ACCGAGTCGGTGCgctgccacaatacctCgccccttctcagttcgct



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36630


P9_sub7
ACCGAGTCGGTGCgctgccacaataccgCgccccttctcagttcgct



 


hPKU3_R25_
TGGGTCGTAGCGAACTGAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36631


P9_sub8
ACCGAGTCGGTGCgctgccacaataccgCgcccattctcagttcgct
















TABLE 8B







Exemplary template RNA sequences


Table 8B provides design of exemplary DNA components of gene modifying systems for correcting the 


pathogenic R408W mutation in PAH to the wild-type form. This table details the sequence of a complete 


template RNA comprising one or more silent substitutions described in Table 7A for use in a Cas-RT


fusion gene modifying polypeptide. Templates in this table employ the hPKU4 spacer GGGTCGTAGCGAACTGAGAA


(SEQ ID NO: 16084).











SEQ




ID


Name
tgRNA sequence
NO





hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36632


P10_sub0
ACCGAGTCGGTGCaatacctCggcccttctcagttcgct



 


hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36633


P10_sub1
ACCGAGTCGGTGCaataccgCggcccttctcagttcgct



 


hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36634


P10_sub4
ACCGAGTCGGTGCaatacctCgccccttctcagttcgct



 


hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36635


P10_sub5
ACCGAGTCGGTGCaatacctCgcccattctcagttcgct



 


hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36636


P10_sub7
ACCGAGTCGGTGCaataccgCgccccttctcagttcgct



 


hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36637


P10_sub8
ACCGAGTCGGTGCaataccgCgcccattctcagttcgct



 


hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36638


P8_sub0
ACCGAGTCGGTGCaatacctCggcccttctcagttcg



 


hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36639


P8_sub1
ACCGAGTCGGTGCaataccgCggcccttctcagttcg



 


hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36640


P8_sub4
ACCGAGTCGGTGCaatacctCgccccttctcagttcg



 


hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36641


P8_sub5
ACCGAGTCGGTGCaatacctCgcccattctcagttcg



 


hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36642


P8_sub7
ACCGAGTCGGTGCaataccgCgccccttctcagttcg



 


hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36643


P8_sub8
ACCGAGTCGGTGCaataccgCgcccattctcagttcg



 


hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36644


P9_sub0
ACCGAGTCGGTGCaatacctCggcccttctcagttcgc



 


hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36645


P9_sub1
ACCGAGTCGGTGCaataccgCggcccttctcagttcgc



 


hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36646


P9_sub4
ACCGAGTCGGTGCaatacctCgccccttctcagttcgc



 


hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36647


P9_sub5
ACCGAGTCGGTGCaatacctCgcccattctcagttcgc



 


hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36648


P9_sub7
ACCGAGTCGGTGCaataccgCgccccttctcagttcgc



 


hPKU4_R16_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36649


P9_sub8
ACCGAGTCGGTGCaataccgCgcccattctcagttcgc



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36650


P10_sub0
ACCGAGTCGGTGCacaatacctCggcccttctcagttcgct



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36651


P10_sub1
ACCGAGTCGGTGCacaataccgCggcccttctcagttcgct



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36652


P10_sub4
ACCGAGTCGGTGCacaatacctCgccccttctcagttcgct



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36653


P10_sub5
ACCGAGTCGGTGCacaatacctCgcccattctcagttcgct



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36654


P10_sub7
ACCGAGTCGGTGCacaataccgCgccccttctcagttcgct



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36655


P10_sub8
ACCGAGTCGGTGCacaataccgCgcccattctcagttcgct



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36656


P8_sub0
ACCGAGTCGGTGCacaatacctCggcccttctcagttcg



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36657


P8_sub1
ACCGAGTCGGTGCacaataccgCggcccttctcagttcg



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36658


P8_sub4
ACCGAGTCGGTGCacaatacctCgccccttctcagttcg



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36659


P8_sub5
ACCGAGTCGGTGCacaatacctCgcccattctcagttcg



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36660


P8_sub7
ACCGAGTCGGTGCacaataccgCgccccttctcagttcg



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36661


P8_sub8
ACCGAGTCGGTGCacaataccgCgcccattctcagttcg



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36662


P9_sub0
ACCGAGTCGGTGCacaatacctCggcccttctcagttcgc



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36663


P9_sub1
ACCGAGTCGGTGCacaataccgCggcccttctcagttcgc



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36664


P9_sub4
ACCGAGTCGGTGCacaatacctCgccccttctcagttcgc



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36665


P9_sub5
ACCGAGTCGGTGCacaatacctCgcccattctcagttcgc



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36666


P9_sub7
ACCGAGTCGGTGCacaataccgCgccccttctcagttcgc



 


hPKU4_R18_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36667


P9_sub8
ACCGAGTCGGTGCacaataccgCgcccattctcagttcgc



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36668


P10_sub0
ACCGAGTCGGTGCccacaatacctCggcccttctcagttcgct



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36669


P10_sub1
ACCGAGTCGGTGCccacaataccgCggcccttctcagttcgct



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36670


P10_sub4
ACCGAGTCGGTGCccacaatacctCgccccttctcagttcgct



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36671


P10_sub5
ACCGAGTCGGTGCccacaatacctCgcccattctcagttcgct



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36672


P10_sub7
ACCGAGTCGGTGCccacaataccgCgccccttctcagttcgct



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36673


P10_sub8
ACCGAGTCGGTGCccacaataccgCgcccattctcagttcgct



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36674


P8_sub0
ACCGAGTCGGTGCccacaatacctCggcccttctcagttcg



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36675


P8_sub1
ACCGAGTCGGTGCccacaataccgCggcccttctcagttcg



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36676


P8_sub4
ACCGAGTCGGTGCccacaatacctCgccccttctcagttcg



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36677


P8_sub5
ACCGAGTCGGTGCccacaatacctCgcccattctcagttcg



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36678


P8_sub7
ACCGAGTCGGTGCccacaataccgCgccccttctcagttcg



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36679


P8_sub8
ACCGAGTCGGTGCccacaataccgCgcccattctcagttcg



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36680


P9_sub0
ACCGAGTCGGTGCccacaatacctCggcccttctcagttcgc



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36681


P9_sub1
ACCGAGTCGGTGCccacaataccgCggcccttctcagttcgc



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36682


P9_sub4
ACCGAGTCGGTGCccacaatacctCgccccttctcagttcgc



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36683


P9_sub5
ACCGAGTCGGTGCccacaatacctCgcccattctcagttcgc



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36684


P9_sub7
ACCGAGTCGGTGCccacaataccgCgccccttctcagttcgc



 


hPKU4_R20_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36685


P9_sub8
ACCGAGTCGGTGCccacaataccgCgcccattctcagttcgc



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36686


P10_sub0
ACCGAGTCGGTGCtgccacaatacctCggcccttctcagttcgct



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36687


P10_sub1
ACCGAGTCGGTGCtgccacaataccgCggcccttctcagttcgct



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36688


P10_sub4
ACCGAGTCGGTGCtgccacaatacctCgccccttctcagttcgct



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36689


P10_sub5
ACCGAGTCGGTGCtgccacaatacctCgcccattctcagttcgct



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36690


P10_sub7
ACCGAGTCGGTGCtgccacaataccgCgccccttctcagttcgct



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36691


P10_sub8
ACCGAGTCGGTGCtgccacaataccgCgcccattctcagttcgct



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36692


P8_sub0
ACCGAGTCGGTGCtgccacaatacctCggcccttctcagttcg



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36693


P8_sub1
ACCGAGTCGGTGCtgccacaataccgCggcccttctcagttcg



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36694


P8_sub4
ACCGAGTCGGTGCtgccacaatacctCgccccttctcagttcg



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36695


P8_sub5
ACCGAGTCGGTGCtgccacaatacctCgcccattctcagttcg



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36696


P8_sub7
ACCGAGTCGGTGCtgccacaataccgCgccccttctcagttcg



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36697


P8_sub8
ACCGAGTCGGTGCtgccacaataccgCgcccattctcagttcg



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36698


P9_sub0
ACCGAGTCGGTGCtgccacaatacctCggcccttctcagttcgc



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36699


P9_sub1
ACCGAGTCGGTGCtgccacaataccgCggcccttctcagttcgc



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36700


P9_sub4
ACCGAGTCGGTGCtgccacaatacctCgccccttctcagttcgc



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36701


P9_sub5
ACCGAGTCGGTGCtgccacaatacctCgcccattctcagttcgc



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36702


P9_sub7
ACCGAGTCGGTGCtgccacaataccgCgccccttctcagttcgc



 


hPKU4_R22_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36703


P9_sub8
ACCGAGTCGGTGCtgccacaataccgCgcccattctcagttcgc



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36704


P10_sub0
ACCGAGTCGGTGCgctgccacaatacctCggcccttctcagttcgct



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36705


P10_sub1
ACCGAGTCGGTGCgctgccacaataccgCggcccttctcagttcgct



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36706


P10_sub4
ACCGAGTCGGTGCgctgccacaatacctCgccccttctcagttcgct



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36707


P10_sub5
ACCGAGTCGGTGCgctgccacaatacctCgcccattctcagttcgct



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36708


P10_sub7
ACCGAGTCGGTGCgctgccacaataccgCgccccttctcagttcgct



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36709


P10_sub8
ACCGAGTCGGTGCgctgccacaataccgCgcccattctcagttcgct



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36710


P8_sub0
ACCGAGTCGGTGCgctgccacaatacctCggcccttctcagttcg



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36711


P8_sub1
ACCGAGTCGGTGCgctgccacaataccgCggcccttctcagttcg



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36712


P8_sub4
ACCGAGTCGGTGCgctgccacaatacctCgccccttctcagttcg



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36713


P8_sub5
ACCGAGTCGGTGCgctgccacaatacctCgcccattctcagttcg



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36714


P8_sub7
ACCGAGTCGGTGCgctgccacaataccgCgccccttctcagttcg



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36715


P8_sub8
ACCGAGTCGGTGCgctgccacaataccgCgcccattctcagttcg



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36716


P9_sub0
ACCGAGTCGGTGCgctgccacaatacctCggcccttctcagttcgc



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36717


P9_sub1
ACCGAGTCGGTGCgctgccacaataccgCggcccttctcagttcgc



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36718


P9_sub4
ACCGAGTCGGTGCgctgccacaatacctCgccccttctcagttcgc



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36719


P9_sub5
ACCGAGTCGGTGCgctgccacaatacctCgcccattctcagttcgc



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36720


P9_sub7
ACCGAGTCGGTGCgctgccacaataccgCgccccttctcagttcgc



 


hPKU4_R24_
GGGTCGTAGCGAACTGAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36721


P9_sub8
ACCGAGTCGGTGCgctgccacaataccgCgcccattctcagttcgc
















TABLE 8C







Exemplary template RNA sequences


Table 8C provides design of exemplary DNA components of gene modifying systems for correcting the


pathogenic R408W mutation in PAH to the wild-type form. This table details the sequence of a complete


template RNA comprising one or more silent substitutions described in Table 7A for use in a Cas-RT


fusion gene modifying polypeptide. Templates in this table employ the hPKU5 spacer


TAGCGAACTGAGAAGGGCCA (SEQ ID NO: 16011).











SEQ




ID


Name
tgRNA sequence
NO





hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36722


P10R10_
GTCGGTGCaataccgCgccccttctcag



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36723


P10R12_
GTCGGTGCacaataccgCgccccttctcag



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36724


P10R14_
GTCGGTGCccacaataccgCgccccttctcag



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36725


P10R16_
GTCGGTGCtgccacaataccgCgccccttctcag



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36726


P10R18_
GTCGGTGCgctgccacaataccgCgccccttctcag



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36727


P10R20_
GTCGGTGCttgctgccacaataccgCgccccttctcag



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36728


P10R22_
GTCGGTGCctttgctgccacaataccgCgccccttctcag



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36729


P10R24_
GTCGGTGCaactttgctgccacaataccgCgccccttctcag



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36730


P10R8_
GTCGGTGCtaccgCgccccttctcag



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36731


P11R10_
GTCGGTGCaataccgCgccccttctcagt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36732


P11R12_
GTCGGTGCacaataccgCgccccttctcagt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36733


P11R14_
GTCGGTGCccacaataccgCgccccttctcagt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36734


P11R16_
GTCGGTGCtgccacaataccgCgccccttctcagt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36735


P11R18_
GTCGGTGCgctgccacaataccgCgccccttctcagt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36736


P11R20_
GTCGGTGCttgctgccacaataccgCgccccttctcagt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36737


P11R22_
GTCGGTGCctttgctgccacaataccgCgccccttctcagt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36738


P11R24_
GTCGGTGCaactttgctgccacaataccgCgccccttctcagt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36739


P11R8_
GTCGGTGCtaccgCgccccttctcagt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36740


P12R10_
GTCGGTGCaataccgCgccccttctcagtt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36741


P12R12_
GTCGGTGCacaataccgCgccccttctcagtt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36742


P12R14_
GTCGGTGCccacaataccgCgccccttctcagtt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36743


P12R16_
GTCGGTGCtgccacaataccgCgccccttctcagtt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36744


P12R18_
GTCGGTGCgctgccacaataccgCgccccttctcagtt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36745


P12R20_
GTCGGTGCttgctgccacaataccgCgccccttctcagtt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36746


P12R22_
GTCGGTGCctttgctgccacaataccgCgccccttctcagtt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36747


P12R24_
GTCGGTGCaactttgctgccacaataccgCgccccttctcagtt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36748


P12R8_
GTCGGTGCtaccgCgccccttctcagtt



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36749


P13R10_
GTCGGTGCaataccgCgccccttctcagttc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36750


P13R12_
GTCGGTGCacaataccgCgccccttctcagttc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36751


P13R14_
GTCGGTGCccacaataccgCgccccttctcagttc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36752


P13R16_
GTCGGTGCtgccacaataccgCgccccttctcagttc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36753


P13R18_
GTCGGTGCgctgccacaataccgCgccccttctcagttc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36754


P13R20_
GTCGGTGCttgctgccacaataccgCgccccttctcagttc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36755


P13R22_
GTCGGTGCctttgctgccacaataccgCgccccttctcagttc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36756


P13R24_
GTCGGTGCaactttgctgccacaataccgCgccccttctcagttc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36757


P13R8_
GTCGGTGCtaccgCgccccttctcagttc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36758


P14R10_
GTCGGTGCaataccgCgccccttctcagttcg



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36759


P14R12_
GTCGGTGCacaataccgCgccccttctcagttcg



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36760


P14R14_
GTCGGTGCccacaataccgCgccccttctcagttcg



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36761


P14R16_
GTCGGTGCtgccacaataccgCgccccttctcagttcg



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36762


P14R18_
GTCGGTGCgctgccacaataccgCgccccttctcagttcg



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36763


P14R20_
GTCGGTGCttgctgccacaataccgCgccccttctcagttcg



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36764


P14R22_
GTCGGTGCctttgctgccacaataccgCgccccttctcagttcg



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36765


P14R24_
GTCGGTGCaactttgctgccacaataccgCgccccttctcagttcg



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36766


P14R8_
GTCGGTGCtaccgCgccccttctcagttcg



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36767


P15R10_
GTCGGTGCaataccgCgccccttctcagttcgc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36768


P15R12_
GTCGGTGCacaataccgCgccccttctcagttcgc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36769


P15R14_
GTCGGTGCccacaataccgCgccccttctcagttcgc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36770


P15R16_
GTCGGTGCtgccacaataccgCgccccttctcagttcgc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36771


P15R18_
GTCGGTGCgctgccacaataccgCgccccttctcagttcgc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36772


P15R20_
GTCGGTGCttgctgccacaataccgCgccccttctcagttcgc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36773


P15R22_
GTCGGTGCctttgctgccacaataccgCgccccttctcagttcgc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36774


P15R24_
GTCGGTGCaactttgctgccacaataccgCgccccttctcagttcgc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36775


P15R8_
GTCGGTGCtaccgCgccccttctcagttcgc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36776


P16R10_
GTCGGTGCaataccgCgccccttctcagttcgct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36777


P16R12_
GTCGGTGCacaataccgCgccccttctcagttcgct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36778


P16R14_
GTCGGTGCccacaataccgCgccccttctcagttcgct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36779


P16R16_
GTCGGTGCtgccacaataccgCgccccttctcagttcgct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36780


P16R18_
GTCGGTGCgctgccacaataccgCgccccttctcagttcgct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36781


P16R20_
GTCGGTGCttgctgccacaataccgCgccccttctcagttcgct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36782


P16R22_
GTCGGTGCctttgctgccacaataccgCgccccttctcagttcgct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36783


P16R24_
GTCGGTGCaactttgctgccacaataccgCgccccttctcagttcgct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36784


P16R8_
GTCGGTGCtaccgCgccccttctcagttcgct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36785


P7R10_
GTCGGTGCaataccgCgccccttct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36786


P7R12_
GTCGGTGCacaataccgCgccccttct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36787


P7R14_
GTCGGTGCccacaataccgCgccccttct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36788


P7R16_
GTCGGTGCtgccacaataccgCgccccttct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36789


P7R18_
GTCGGTGCgctgccacaataccgCgccccttct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36790


P7R20_
GTCGGTGCttgctgccacaataccgCgccccttct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36791


P7R22_
GTCGGTGCctttgctgccacaataccgCgccccttct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36792


P7R24_
GTCGGTGCaactttgctgccacaataccgCgccccttct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36793


P7R8_
GTCGGTGCtaccgCgccccttct



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36794


P8R10_
GTCGGTGCaataccgCgccccttctc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36795


P8R12_
GTCGGTGCacaataccgCgccccttctc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36796


P8R14_
GTCGGTGCccacaataccgCgccccttctc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36797


P8R16_
GTCGGTGCtgccacaataccgCgccccttctc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36798


P8R18_
GTCGGTGCgctgccacaataccgCgccccttctc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36799


P8R20_
GTCGGTGCttgctgccacaataccgCgccccttctc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36800


P8R22_
GTCGGTGCctttgctgccacaataccgCgccccttctc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36801


P8R24_
GTCGGTGCaactttgctgccacaataccgCgccccttctc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36802


P8R8_
GTCGGTGCtaccgCgccccttctc



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36803


P9R10_
GTCGGTGCaataccgCgccccttctca



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36804


P9R12_
GTCGGTGCacaataccgCgccccttctca



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36805


P9R14_
GTCGGTGCccacaataccgCgccccttctca



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36806


P9R16_
GTCGGTGCtgccacaataccgCgccccttctca



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36807


P9R18_
GTCGGTGCgctgccacaataccgCgccccttctca



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36808


P9R20_
GTCGGTGCttgctgccacaataccgCgccccttctca



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36809


P9R22_
GTCGGTGCctttgctgccacaataccgCgccccttctca



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36810


P9R24_
GTCGGTGCaactttgctgccacaataccgCgccccttctca



sub5




 


hPKU5_
tagcgaactgagaagggccAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA
36811


P9R8_
GTCGGTGCtaccgCgccccttctca



sub5




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36812


R12_P10_
ACCGAGTCGGTGCacaatacctCggcccttctcag



sub0




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36813


R12_P10_
ACCGAGTCGGTGCacaataccgCggcccttctcag



sub1




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36814


R12_P10_
ACCGAGTCGGTGCacaatccctCggcccttctcag



sub2




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36815


R12_P10_
ACCGAGTCGGTGCacaatcccgCggcccttctcag



sub3




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36816


R12_P10_
ACCGAGTCGGTGCacaatacctCgccccttctcag



sub4




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36817


R12_P10_
ACCGAGTCGGTGCacgatacctCggcccttctcag



sub7




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36818


R12_P8_
ACCGAGTCGGTGCacaatacctCggcccttctc



sub0




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36819


R12_P8_
ACCGAGTCGGTGCacaataccgCggcccttctc



sub1




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36820


R12_P8_
ACCGAGTCGGTGCacaatccctCggcccttctc



sub2




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36821


R12_P8_
ACCGAGTCGGTGCacaatcccgCggcccttctc



sub3




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36822


R12_P8_
ACCGAGTCGGTGCacaatacctCgccccttctc



sub4




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36823


R12_P8_
ACCGAGTCGGTGCacgatacctCggcccttctc



sub7




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36824


R12_P9_
ACCGAGTCGGTGCacaatacctCggcccttctca



sub0




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36825


R12_P9_
ACCGAGTCGGTGCacaataccgCggcccttctca



sub1




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36826


R12_P9_
ACCGAGTCGGTGCacaatccctCggcccttctca



sub2




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36827


R12_P9_
ACCGAGTCGGTGCacaatcccgCggcccttctca



sub3




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36828


R12_P9_
ACCGAGTCGGTGCacaatacctCgccccttctca



sub4




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36829


R12_P9_
ACCGAGTCGGTGCacgatacctCggcccttctca



sub7




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36830


R14_P10_
ACCGAGTCGGTGCccacaatacctCggcccttctcag



sub0




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36831


R14_P10_
ACCGAGTCGGTGCccacaataccgCggcccttctcag



sub1




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36832


R14_P10_
ACCGAGTCGGTGCccacaatccctCggcccttctcag



sub2




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36833


R14_P10_
ACCGAGTCGGTGCccacaatcccgCggcccttctcag



sub3




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36834


R14_P10_
ACCGAGTCGGTGCccacaatacctCgccccttctcag



sub4




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36835


R14_P10_
ACCGAGTCGGTGCccacgatacctCggcccttctcag



sub7




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36836


R14_P8_
ACCGAGTCGGTGCccacaatacctCggcccttctc



sub0




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36837


R14P8_
ACCGAGTCGGTGCccacaataccgCggcccttctc



sub1




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36838


R14_P8_
ACCGAGTCGGTGCccacaatccctCggcccttctc



sub2




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36839


R14_P8_
ACCGAGTCGGTGCccacaatcccgCggcccttctc



sub3




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36840


R14_P8_
ACCGAGTCGGTGCccacaatacctCgccccttctc



sub4




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36841


R14_P8_
ACCGAGTCGGTGCccacgatacctCggcccttctc



sub7




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36842


R14_P9_
ACCGAGTCGGTGCccacaatacctCggcccttctca



sub0




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36843


R14_P9_
ACCGAGTCGGTGCccacaataccgCggcccttctca



sub1




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36844


R14_P9_
ACCGAGTCGGTGCccacaatccctCggcccttctca



sub2




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36845


R14_P9_
ACCGAGTCGGTGCccacaatcccgCggcccttctca



sub3




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36846


R14_P9_
ACCGAGTCGGTGCccacaatacctCgccccttctca



sub4




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36847


R14_P9_
ACCGAGTCGGTGCccacgatacctCggcccttctca



sub7




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36848


R16_P10_
ACCGAGTCGGTGCtgccacaatacctCggcccttctcag



sub0




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36849


R16_P10_
ACCGAGTCGGTGCtgccacaataccgCggcccttctcag



sub1




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36850


R16P_10_
ACCGAGTCGGTGCtgccacaatccctCggcccttctcag



sub2




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36851


R16_P10_
ACCGAGTCGGTGCtgccacaatcccgCggcccttctcag



sub3




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36852


R16_P10_
ACCGAGTCGGTGCtgccacaatacctCgccccttctcag



sub4




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36853


R16_P10_
ACCGAGTCGGTGCtgccacgatacctCggcccttctcag



sub7




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36854


R16_P8_
ACCGAGTCGGTGCtgccacaatacctCggcccttctc



sub0




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36855


R16_P8_
ACCGAGTCGGTGCtgccacaataccgCggcccttctc



sub1




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36856


R16_P8_
ACCGAGTCGGTGCtgccacaatccctCggcccttctc



sub2




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36857


R16_P8_
ACCGAGTCGGTGCtgccacaatcccgCggcccttctc



sub3




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36858


R16_P8_
ACCGAGTCGGTGCtgccacaatacctCgccccttctc



sub4




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36859


R16_P8_
ACCGAGTCGGTGCtgccacgatacctCggcccttctc



sub7




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36860


R16_P9_
ACCGAGTCGGTGCtgccacaatacctCggcccttctca



sub0




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36861


R16_P9_
ACCGAGTCGGTGCtgccacaataccgCggcccttctca



sub1




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36862


R16_P9_
ACCGAGTCGGTGCtgccacaatccctCggcccttctca



sub2




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36863


R16_P9_
ACCGAGTCGGTGCtgccacaatcccgCggcccttctca



sub3




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36864


R16_P9_
ACCGAGTCGGTGCtgccacaatacctCgccccttctca



sub4




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36865


R16_P9_
ACCGAGTCGGTGCtgccacgatacctCggcccttctca



sub7




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36866


R18_P10_
ACCGAGTCGGTGCgctgccacaatacctCggcccttctcag



sub0




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36867


R18_P10_
ACCGAGTCGGTGCgctgccacaataccgCggcccttctcag



sub1




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36868


R18_P10_
ACCGAGTCGGTGCgctgccacaatccctCggcccttctcag



sub2




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36869


R18_P10_
ACCGAGTCGGTGCgctgccacaatcccgCggcccttctcag



sub3




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36870


R18_P10_
ACCGAGTCGGTGCgctgccacaatacctCgccccttctcag



sub4




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36871


R18_P10_
ACCGAGTCGGTGCgctgccacgatacctCggcccttctcag



sub7




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36872


R18_P8_
ACCGAGTCGGTGCgctgccacaatacctCggcccttctc



sub0




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36873


R18_P8_
ACCGAGTCGGTGCgctgccacaataccgCggcccttctc



sub1




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36874


R18_P8_
ACCGAGTCGGTGCgctgccacaatccctCggcccttctc



sub2




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36875


R18_P8_
ACCGAGTCGGTGCgctgccacaatcccgCggcccttctc



sub3




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36876


R18_P8_
ACCGAGTCGGTGCgctgccacaatacctCgccccttctc



sub4




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36877


R18_P8_
ACCGAGTCGGTGCgctgccacgatacctCggcccttctc



sub7




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36878


R18_P9_
ACCGAGTCGGTGCgctgccacaatacctCggcccttctca



sub0




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36879


R18_P9_
ACCGAGTCGGTGCgctgccacaataccgCggcccttctca



sub1




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36880


R18_P9_
ACCGAGTCGGTGCgctgccacaatccctCggcccttctca



sub2




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36881


R18_P9_
ACCGAGTCGGTGCgctgccacaatcccgCggcccttctca



sub3




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36882


R18_P9_
ACCGAGTCGGTGCgctgccacaatacctCgccccttctca



sub4




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36883


R18_P9_
ACCGAGTCGGTGCgctgccacgatacctCggcccttctca



sub7




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36884


R20_P10_
ACCGAGTCGGTGCttgctgccacaatacctCggcccttctcag



sub0




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36885


R20_P10_
ACCGAGTCGGTGCttgctgccacaataccgCggcccttctcag



sub1




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36886


R20_P10_
ACCGAGTCGGTGCttgctgccacaatccctCggcccttctcag



sub2




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36887


R20_P10_
ACCGAGTCGGTGCttgctgccacaatcccgCggcccttctcag



sub3




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36888


R20_P10_
ACCGAGTCGGTGCttgctgccacaatacctCgccccttctcag



sub4




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36889


R20_P10_
ACCGAGTCGGTGCttgctgccacgatacctCggcccttctcag



sub7




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36890


R20_P8_
ACCGAGTCGGTGCttgctgccacaatacctCggcccttctc



sub0




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36891


R20_P8_
ACCGAGTCGGTGCttgctgccacaataccgCggcccttctc



sub1




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36892


R20_P8_
ACCGAGTCGGTGCttgctgccacaatccctCggcccttctc



sub2




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36893


R20_P8_
ACCGAGTCGGTGCttgctgccacaatcccgCggcccttctc



sub3




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36894


R20_P8_
ACCGAGTCGGTGCttgctgccacaatacctCgccccttctc



sub4




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36895


R20_P8_
ACCGAGTCGGTGCttgctgccacgatacctCggcccttctc



sub7




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36896


R20_P9_
ACCGAGTCGGTGCttgctgccacaatacctCggcccttctca



sub0




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36897


R20_P9_
ACCGAGTCGGTGCttgctgccacaataccgCggcccttctca



sub1




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36898


R20_P9_
ACCGAGTCGGTGCttgctgccacaatccctCggcccttctca



sub2




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36899


R20_P9_
ACCGAGTCGGTGCttgctgccacaatcccgCggcccttctca



sub3




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36900


R20_P9_
ACCGAGTCGGTGCttgctgccacaatacctCgccccttctca



sub4




 


hPKU5_
TAGCGAACTGAGAAGGGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC
36901


R20_P9_
ACCGAGTCGGTGCttgctgccacgatacctCggcccttctca



sub7
















TABLE 8D







Exemplary template RNA sequences


Table 8D provides design of exemplary DNA components of gene modifying systems for correcting the pathogenic R408W mutation in PAH to


the wild-type form. This table details the sequence of a complete template RNA comprising one or more silent substitutions described in


Table 7A for use in a Cas-RT fusion gene modifying polypeptide. Templates in this table employ the hPKU6 spacer ACTTTGCTGCCACAATACCT


(SEQ ID NO: 16032).











SEQ ID


Name
tgRNA sequence
NO





hPKU6_P10R11
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36902


_sub3
AGTCGGTGCaagggacGgggtattgtggca



 


hPKU6_P10R13
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36903


_sub3
AGTCGGTGCagaagggacGgggtattgtggca



 


hPKU6_P10R15
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36904


_sub3
AGTCGGTGCtgagaagggacGgggtattgtggca



 


hPKU6_P10R17
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36905


_sub3
AGTCGGTGCactgagaagggacGgggtattgtggca



 


hPKU6_P10R19
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36906


_sub3
AGTCGGTGCgaactgagaagggacGgggtattgtggca



 


hPKU6_P10R21
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36907


_sub3
AGTCGGTGCgcgaactgagaagggacGgggtattgtggca



 


hPKU6_P10R23
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36908


_sub3
AGTCGGTGCtagcgaactgagaagggacGgggtattgtggca



 


hPKU6_P10R25
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36909


_sub3
AGTCGGTGCcgtagcgaactgagaagggacGgggtattgtggca



 


hPKU6_P10R9
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36910


_sub3
AGTCGGTGCgggacGgggtattgtggca



 


hPKU6_P11R11
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36911


_sub3
AGTCGGTGCaagggacGgggtattgtggcag



 


hPKU6_P11R13
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36912


_sub3
AGTCGGTGCagaagggacGgggtattgtggcag



 


hPKU6_P11R15
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36913


_sub3
AGTCGGTGCtgagaagggacGgggtattgtggcag



 


hPKU6_P11R17
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36914


_sub3
AGTCGGTGCactgagaagggacGgggtattgtggcag



 


hPKU6_P11R19
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36915


_sub3
AGTCGGTGCgaactgagaagggacGgggtattgtggcag



 


hPKU6_P11R21
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36916


_sub3
AGTCGGTGCgcgaactgagaagggacGgggtattgtggcag



 


hPKU6_P11R23
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36917


_sub3
AGTCGGTGCtagcgaactgagaagggacGgggtattgtggcag



 


hPKU6_P11R25
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36918


_sub3
AGTCGGTGCcgtagcgaactgagaagggacGgggtattgtggcag



 


hPKU6_P11R9
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36919


_sub3
AGTCGGTGCgggacGgggtattgtggcag



 


hPKU6_P12R11
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36920


_sub3
AGTCGGTGCaagggacGgggtattgtggcagc



 


hPKU6_P12R13
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36921


_sub3
AGTCGGTGCagaagggacGgggtattgtggcagc



 


hPKU6_P12R15
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36922


_sub3
AGTCGGTGCtgagaagggacGgggtattgtggcagc



 


hPKU6_P12R17
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36923


_sub3
AGTCGGTGCactgagaagggacGgggtattgtggcagc



 


hPKU6_P12R19
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36924


_sub3
AGTCGGTGCgaactgagaagggacGgggtattgtggcagc



 


hPKU6_P12R21
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36925


_sub3
AGTCGGTGCgcgaactgagaagggacGgggtattgtggcagc



 


hPKU6_P12R23
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36926


_sub3
AGTCGGTGCtagcgaactgagaagggacGgggtattgtggcagc



 


hPKU6_P12R25
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36927


_sub3
AGTCGGTGCcgtagcgaactgagaagggacGgggtattgtggcagc



 


hPKU6_P12R9
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36928


_sub3
AGTCGGTGCgggacGgggtattgtggcagc



 


hPKU6_P13R11
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36929


_sub3
AGTCGGTGCaagggacGgggtattgtggcagca



 


hPKU6_P13R13
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36930


_sub3
AGTCGGTGCagaagggacGgggtattgtggcagca



 


hPKU6_P13R15
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36931


_sub3
AGTCGGTGCtgagaagggacGgggtattgtggcagca



 


hPKU6_P13R17
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36932


_sub3
AGTCGGTGCactgagaagggacGgggtattgtggcagca



 


hPKU6_P13R19
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36933


_sub3
AGTCGGTGCgaactgagaagggacGgggtattgtggcagca



 


hPKU6_P13R21
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36934


_sub3
AGTCGGTGCgcgaactgagaagggacGgggtattgtggcagca



 


hPKU6_P13R23
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36935


_sub3
AGTCGGTGCtagcgaactgagaagggacGgggtattgtggcagca



 


hPKU6_P13R25
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36936


_sub3
AGTCGGTGCcgtagcgaactgagaagggacGgggtattgtggcagca



 


hPKU6_P13R9
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36937


_sub3
AGTCGGTGCgggacGgggtattgtggcagca



 


hPKU6_P14R11
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36938


_sub3
AGTCGGTGCaagggacGgggtattgtggcagcaa



 


hPKU6_P14R13
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36939


_sub3
AGTCGGTGCagaagggacGgggtattgtggcagcaa



 


hPKU6_P14R15
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36940


_sub3
AGTCGGTGCtgagaagggacGgggtattgtggcagcaa



 


hPKU6_P14R17
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36941


_sub3
AGTCGGTGCactgagaagggacGgggtattgtggcagcaa



 


hPKU6_P14R19
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36942


_sub3
AGTCGGTGCgaactgagaagggacGgggtattgtggcagcaa



 


hPKU6_P14R21
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36943


_sub3
AGTCGGTGCgcgaactgagaagggacGgggtattgtggcagcaa



 


hPKU6_P14R23
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36944


_sub3
AGTCGGTGCtagcgaactgagaagggacGgggtattgtggcagcaa



 


hPKU6_P14R25
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36945


_sub3
AGTCGGTGCcgtagcgaactgagaagggacGgggtattgtggcagcaa



 


hPKU6_P14R9
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36946


_sub3
AGTCGGTGCgggacGgggtattgtggcagcaa



 


hPKU6_P15R11
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36947


_sub3
AGTCGGTGCaagggacGgggtattgtggcagcaaa



 


hPKU6_P15R13
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36948


_sub3
AGTCGGTGCagaagggacGgggtattgtggcagcaaa



 


hPKU6_P15R15
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36949


_sub3
AGTCGGTGCtgagaagggacGgggtattgtggcagcaaa



 


hPKU6_P15R17
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36950


_sub3
AGTCGGTGCactgagaagggacGgggtattgtggcagcaaa



 


hPKU6_P15R19
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36951


_sub3
AGTCGGTGCgaactgagaagggacGgggtattgtggcagcaaa



 


hPKU6_P15R21
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36952


_sub3
AGTCGGTGCgcgaactgagaagggacGgggtattgtggcagcaaa



 


hPKU6_P15R23
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36953


_sub3
AGTCGGTGCtagcgaactgagaagggacGgggtattgtggcagcaaa



 


hPKU6_P15R25
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36954


_sub3
AGTCGGTGCcgtagcgaactgagaagggacGgggtattgtggcagcaaa



 


hPKU6_P15R9
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36955


_sub3
AGTCGGTGCgggacGgggtattgtggcagcaaa



 


hPKU6_P16R11
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36956


_sub3
AGTCGGTGCaagggacGgggtattgtggcagcaaag



 


hPKU6_P16R13
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36957


_sub3
AGTCGGTGCagaagggacGgggtattgtggcagcaaag



 


hPKU6_P16R15
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36958


_sub3
AGTCGGTGCtgagaagggacGgggtattgtggcagcaaag



 


hPKU6_P16R17
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36959


_sub3
AGTCGGTGCactgagaagggacGgggtattgtggcagcaaag



 


hPKU6_P16R19
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36960


_sub3
AGTCGGTGCgaactgagaagggacGgggtattgtggcagcaaag



 


hPKU6_P16R21
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36961


_sub3
AGTCGGTGCgcgaactgagaagggacGgggtattgtggcagcaaag



 


hPKU6_P16R23
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36962


_sub3
AGTCGGTGCtagcgaactgagaagggacGgggtattgtggcagcaaag



 


hPKU6_P16R9
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36963


_sub3
AGTCGGTGCgggacGgggtattgtggcagcaaag



 


hPKU6_P16R9
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36964


_sub3
AGTCGGTGCgggacGgggtattgtggcagcaaag



 


hPKU6_P7R11
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36965


_sub3
AGTCGGTGCaagggacGgggtattgtg



 


hPKU6_P7R13
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36966


_sub3
AGTCGGTGCagaagggacGgggtattgtg



 


hPKU6_P7R15
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36967


_sub3
AGTCGGTGCtgagaagggacGgggtattgtg



 


hPKU6_P7R17
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36968


_sub3
AGTCGGTGCactgagaagggacGgggtattgtg



 


hPKU6_P7R19
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36969


_sub3
AGTCGGTGCgaactgagaagggacGgggtattgtg



 


hPKU6_P7R21
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36970


_sub3
AGTCGGTGCgcgaactgagaagggacGgggtattgtg



 


hPKU6_P7R23
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36971


_sub3
AGTCGGTGCtagcgaactgagaagggacGgggtattgtg



 


hPKU6_P7R25
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36972


_sub3
AGTCGGTGCcgtagcgaactgagaagggacGgggtattgtg



 


hPKU6_P7R9
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36973


_sub3
AGTCGGTGCgggacGgggtattgtg



 


hPKU6_P8R11
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36974


_sub3
AGTCGGTGCaagggacGgggtattgtgg



 


hPKU6_P8R13
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36975


_sub3
AGTCGGTGCagaagggacGgggtattgtgg



 


hPKU6_P8R15
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36976


_sub3
AGTCGGTGCtgagaagggacGgggtattgtgg



 


hPKU6_P8R17
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36977


_sub3
AGTCGGTGCactgagaagggacGgggtattgtgg



 


hPKU6_P8R19
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36978


_sub3
AGTCGGTGCgaactgagaagggacGgggtattgtgg



 


hPKU6_P8R21
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36979


_sub3
AGTCGGTGCgcgaactgagaagggacGgggtattgtgg



 


hPKU6_P8R23
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36980


_sub3
AGTCGGTGCtagcgaactgagaagggacGgggtattgtgg



 


hPKU6_P8R25
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36981


_sub3
AGTCGGTGCcgtagcgaactgagaagggacGgggtattgtgg



 


hPKU6_P8R9
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36982


_sub3
AGTCGGTGCgggacGgggtattgtgg



 


hPKU6_P9R11
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36983


_sub3
AGTCGGTGCaagggacGgggtattgtggc



 


hPKU6_P9R13
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36984


_sub3
AGTCGGTGCagaagggacGgggtattgtggc



 


hPKU6_P9R15
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36985


_sub3
AGTCGGTGCtgagaagggacGgggtattgtggc



 


hPKU6_P9R17
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36986


_sub3
AGTCGGTGCactgagaagggacGgggtattgtggc



 


hPKU6_P9R19
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36987


_sub3
AGTCGGTGCgaactgagaagggacGgggtattgtggc



 


hPKU6_P9R21
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36988


_sub3
AGTCGGTGCgcgaactgagaagggacGgggtattgtggc



 


hPKU6_P9R23
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36989


_sub3
AGTCGGTGCtagcgaactgagaagggacGgggtattgtggc



 


hPKU6_P9R25
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36990


_sub3
AGTCGGTGCcgtagcgaactgagaagggacGgggtattgtggc



 


hPKU6_P9R9_
actttgctgccacaatacctGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCG
36991


_sub3
AGTCGGTGCgggacGgggtattgtggc



 


hPKU6_R12_P10
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
36992


_sub0
CACCGAGTCGGTGCgaagggccGaggtattgtggca



 


hPKU6_R12_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
36993


0_sub1
CACCGAGTCGGTGCgaagggccGgggtattgtggca



 


hPKU6_R12_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
36994


0_sub4
CACCGAGTCGGTGCgaacggccGgggtattgtggca



 


hPKU6_R12_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
36995


0_sub5
CACCGAGTCGGTGCgaacggacGgggtattgtggca



 


hPKU6_R12_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
36996


0_sub6
CACCGAGTCGGTGCgaagggccGcggtattgtggca



 


hPKU6_R12_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
36997


0_sub7
CACCGAGTCGGTGCgaagggacGcggtattgtggca



 


hPKU6_R12_P1
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
36998


1_sub0
CACCGAGTCGGTGCgaagggccGaggtattgtggcag



 


hPKU6_R12_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
36999


1_sub1
CACCGAGTCGGTGCgaagggccGgggtattgtggcag



 


hPKU6_R12_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37000


1_sub4
CACCGAGTCGGTGCgaacggccGgggtattgtggcag



 


hPKU6_R12_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37001


1_sub5
CACCGAGTCGGTGCgaacggacGgggtattgtggcag



 


hPKU6_R12_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37002


1_sub6
CACCGAGTCGGTGCgaagggccGcggtattgtggcag



 


hPKU6_R12_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37003


1_sub7
CACCGAGTCGGTGCgaagggacGcggtattgtggcag



 


hPKU6_R12_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37004


_sub0
CACCGAGTCGGTGCgaagggccGaggtattgtggc



 


hPKU6_R12_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37005


_sub1
CACCGAGTCGGTGCgaagggccGgggtattgtggc



 


hPKU6_R12_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37006


_sub4
CACCGAGTCGGTGCgaacggccGgggtattgtggc



 


hPKU6_R12_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37007


_sub5
CACCGAGTCGGTGCgaacggacGgggtattgtggc



 


hPKU6_R12_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37008


_sub6
CACCGAGTCGGTGCgaagggccGcggtattgtggc



 


hPKU6_R12_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37009


_sub7
CACCGAGTCGGTGCgaagggacGcggtattgtggc



 


hPKU6_R14_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37010


0_sub0
CACCGAGTCGGTGCgagaagggccGaggtattgtggca



 


hPKU6_R14_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37011


0_sub1
CACCGAGTCGGTGCgagaagggccGgggtattgtggca



 


hPKU6_R14_P1
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37012


0_sub4
CACCGAGTCGGTGCgagaacggccGgggtattgtggca



 


hPKU6_R14_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37013


0_sub5
CACCGAGTCGGTGCgagaacggacGgggtattgtggca



 


hPKU6_R14_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37014


0_sub6
CACCGAGTCGGTGCgagaagggccGcggtattgtggca



 


hPKU6_R14_P1
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37015


0_sub7
CACCGAGTCGGTGCgagaagggacGcggtattgtggca



 


hPKU6_R14_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37016


1 sub0
CACCGAGTCGGTGCgagaagggccGaggtattgtggcag



 


hPKU6_R14_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37017


1_sub1
CACCGAGTCGGTGCgagaagggccGgggtattgtggcag



 


hPKU6_R14_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37018


1 sub4
CACCGAGTCGGTGCgagaacggccGgggtattgtggcag



 


hPKU6_R14_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37019


1_sub5
CACCGAGTCGGTGCgagaacggacGgggtattgtggcag



 


hPKU6_R14_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37020


1_sub6
CACCGAGTCGGTGCgagaagggccGcggtattgtggcag



 


hPKU6_R14_P1
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37021


1 sub7
CACCGAGTCGGTGCgagaagggacGcggtattgtggcag



 


hPKU6_R14_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37022


_sub0
CACCGAGTCGGTGCgagaagggccGaggtattgtggc



 


hPKU6_R14_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37023


_sub1
CACCGAGTCGGTGCgagaagggccGgggtattgtggc



 


hPKU6_R14_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37024


_sub4
CACCGAGTCGGTGCgagaacggccGgggtattgtggc



 


hPKU6_R14_P9
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37025


_sub5
CACCGAGTCGGTGCgagaacggacGgggtattgtggc



 


hPKU6_R14_P9
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37026


_sub6
CACCGAGTCGGTGCgagaagggccGcggtattgtggc



 


hPKU6_R14_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37027


_sub7
CACCGAGTCGGTGCgagaagggacGcggtattgtggc



 


hPKU6_R16_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37028


0_sub0
CACCGAGTCGGTGCctgagaagggccGaggtattgtggca



 


hPKU6_R16_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37029


0_sub1
CACCGAGTCGGTGCctgagaagggccGgggtattgtggca



 


hPKU6_R16_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37030


0_sub4
CACCGAGTCGGTGCctgagaacggccGgggtattgtggca



 


hPKU6_R16_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37031


0_sub5
CACCGAGTCGGTGCctgagaacggacGgggtattgtggca



 


hPKU6_R16_P1
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37032


0_sub6
CACCGAGTCGGTGCctgagaagggccGcggtattgtggca



 


hPKU6_R16_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37033


0_sub7
CACCGAGTCGGTGCctgagaagggacGcggtattgtggca



 


hPKU6_R16_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37034


1_sub0
CACCGAGTCGGTGCctgagaagggccGaggtattgtggcag



 


hPKU6_R16_P1
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37035


1_sub1
CACCGAGTCGGTGCctgagaagggccGgggtattgtggcag



 


hPKU6_R16_P1
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37036


1_sub4
CACCGAGTCGGTGCctgagaacggccGgggtattgtggcag



 


hPKU6_R16_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37037


1_sub5
CACCGAGTCGGTGCctgagaacggacGgggtattgtggcag



 


hPKU6_R16_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37038


1_sub6
CACCGAGTCGGTGCctgagaagggccGcggtattgtggcag



 


hPKU6_R16_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37039


1_sub7
CACCGAGTCGGTGCctgagaagggacGcggtattgtggcag



 


hPKU6_R16_P9
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37040


_sub0
CACCGAGTCGGTGCctgagaagggccGaggtattgtggc



 


hPKU6_R16_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37041


_sub1
CACCGAGTCGGTGCctgagaagggccGgggtattgtggc



 


hPKU6_R16_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37042


_sub4
CACCGAGTCGGTGCctgagaacggccGgggtattgtggc



 


hPKU6_R16_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37043


_sub5
CACCGAGTCGGTGCctgagaacggacGgggtattgtggc



 


hPKU6_R16_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37044


_sub6
CACCGAGTCGGTGCctgagaagggccGcggtattgtggc



 


hPKU6_R16_P9
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37045


_sub7
CACCGAGTCGGTGCctgagaagggacGcggtattgtggc



 


hPKU6_R18_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37046


0_sub0
CACCGAGTCGGTGCaactgagaagggccGaggtattgtggca



 


hPKU6_R18_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37047


0_sub1
CACCGAGTCGGTGCaactgagaagggccGgggtattgtggca



 


hPKU6_R18_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37048


0_sub4
CACCGAGTCGGTGCaactgagaacggccGgggtattgtggca



 


hPKU6_R18_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37049


0_sub5
CACCGAGTCGGTGCaactgagaacggacGgggtattgtggca



 


hPKU6_R18_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37050


0_sub6
CACCGAGTCGGTGCaactgagaagggccGcggtattgtggca



 


hPKU6_R18_P1
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37051


0_sub7
CACCGAGTCGGTGCaactgagaagggacGcggtattgtggca



 


hPKU6_R18_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37052


1 sub0
CACCGAGTCGGTGCaactgagaagggccGaggtattgtggcag



 


hPKU6_R18_P1
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37053


1_sub1
CACCGAGTCGGTGCaactgagaagggccGgggtattgtggcag



 


hPKU6_R18_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37054


1_sub4
CACCGAGTCGGTGCaactgagaacggccGgggtattgtggcag



 


hPKU6_R18_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37055


1_sub5
CACCGAGTCGGTGCaactgagaacggacGgggtattgtggcag



 


hPKU6_R18_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37056


1_sub6
CACCGAGTCGGTGCaactgagaagggccGcggtattgtggcag



 


hPKU6_R18_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37057


1_sub7
CACCGAGTCGGTGCaactgagaagggacGcggtattgtggcag



 


hPKU6_R18_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37058


_sub0
CACCGAGTCGGTGCaactgagaagggccGaggtattgtggc



 


hPKU6_R18_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37059


_sub1
CACCGAGTCGGTGCaactgagaagggccGgggtattgtggc



 


hPKU6_R18_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37060


_sub4
CACCGAGTCGGTGCaactgagaacggccGgggtattgtggc



 


hPKU6_R18_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37061


_sub5
CACCGAGTCGGTGCaactgagaacggacGgggtattgtggc



 


hPKU6_R18_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37062


_sub6
CACCGAGTCGGTGCaactgagaagggccGcggtattgtggc



 


hPKU6_R18_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37063


_sub7
CACCGAGTCGGTGCaactgagaagggacGcggtattgtggc



 


hPKU6_R20_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37064


0_sub0
CACCGAGTCGGTGCcgaactgagaagggccGaggtattgtggca



 


hPKU6_R20_P1
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37065


0_sub1
CACCGAGTCGGTGCcgaactgagaagggccGgggtattgtggca



 


hPKU6_R20_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37066


0_sub4
CACCGAGTCGGTGCcgaactgagaacggccGgggtattgtggca



 


hPKU6_R20_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37067


0_sub5
CACCGAGTCGGTGCcgaactgagaacggacGgggtattgtggca



 


hPKU6_R20_P1
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37068


0_sub6
CACCGAGTCGGTGCcgaactgagaagggccGcggtattgtggca



 


hPKU6_R20_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37069


0_sub7
CACCGAGTCGGTGCcgaactgagaagggacGcggtattgtggca



 


hPKU6_R20_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37070


1_sub0
CACCGAGTCGGTGCcgaactgagaagggccGaggtattgtggcag



 


hPKU6_R20_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37071


1_sub1
CACCGAGTCGGTGCcgaactgagaagggccGgggtattgtggcag



 


hPKU6_R20_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37072


1 sub4
CACCGAGTCGGTGCcgaactgagaacggccGgggtattgtggcag



 


hPKU6_R20_P1
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37073


1_sub5
CACCGAGTCGGTGCcgaactgagaacggacGgggtattgtggcag



 


hPKU6_R20_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37074


1_sub6
CACCGAGTCGGTGCcgaactgagaagggccGcggtattgtggcag



 


hPKU6_R20_P1
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37075


1_sub7
CACCGAGTCGGTGCcgaactgagaagggacGcggtattgtggcag



 


hPKU6_R20_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37076


_sub0
CACCGAGTCGGTGCcgaactgagaagggccGaggtattgtggc



 


hPKU6_R20_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37077


_sub1
CACCGAGTCGGTGCcgaactgagaagggccGgggtattgtggc



 


hPKU6_R20_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37078


_sub4
CACCGAGTCGGTGCcgaactgagaacggccGgggtattgtggc



 


hPKU6_R20_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37079


_sub5
CACCGAGTCGGTGCcgaactgagaacggacGgggtattgtggc



 


hPKU6_R20_P9
ACTTTGCTGCCACAATACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37080


_sub6
CACCGAGTCGGTGCcgaactgagaagggccGcggtattgtggc



 


hPKU6_R20_P9
ACTTTGCTGCCACAATACCTGTITTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG
37081


_sub7
CACCGAGTCGGTGCcgaactgagaagggacGcggtattgtggc









gRNAs with Inducible Activity

In some embodiments, a gRNA described herein (e.g., a gRNA that is part of a template RNA or a gRNA used for second strand nicking) has inducible activity. Inducible activity may be achieved by the template nucleic acid, e.g., template RNA, further comprising (in addition to the gRNA) a blocking domain, wherein the sequence of a portion of or all of the blocking domain is at least partially complementary to a portion or all of the gRNA. The blocking domain is thus capable of hybridizing or substantially hybridizing to a portion of or all of the gRNA. In some embodiments, the blocking domain and inducibly active gRNA are disposed on the template nucleic acid, e.g., template RNA, such that the gRNA can adopt a first conformation where the blocking domain is hybridized or substantially hybridized to the gRNA, and a second conformation where the blocking domain is not hybridized or not substantially hybridized to the gRNA. In some embodiments, in the first conformation the gRNA is unable to bind to the gene modifying polypeptide (e.g., the template nucleic acid binding domain, DNA binding domain, or endonuclease domain (e.g., a CRISPR/Cas protein)) or binds with substantially decreased affinity compared to an otherwise similar template RNA lacking the blocking domain. In some embodiments, in the second conformation the gRNA is able to bind to the gene modifying polypeptide (e.g., the template nucleic acid binding domain, DNA binding domain, or endonuclease domain (e.g., a CRISPR/Cas protein)). In some embodiments, whether the gRNA is in the first or second conformation can influence whether the DNA binding or endonuclease activities of the gene modifying polypeptide (e.g., of the CRISPR/Cas protein the gene modifying polypeptide comprises) are active.
In some embodiments, the gRNA that coordinates the second nick has inducible activity. In some embodiments, the gRNA that coordinates the second nick is induced after the template is reverse transcribed. In some embodiments, hybridization of the gRNA to the blocking domain can be disrupted using an opener molecule. In some embodiments, an opener molecule comprises an agent that binds to a portion or all of the gRNA or blocking domain and inhibits hybridization of the gRNA to the blocking domain. In some embodiments, the opener molecule comprises a nucleic acid, e.g., comprising a sequence that is partially or wholly complementary to the gRNA, blocking domain, or both. By choosing or designing an appropriate opener molecule, providing the opener molecule can promote a change in the conformation of the gRNA such that it can associate with a CRISPR/Cas protein and provide the associated functions of the CRISPR/Cas protein (e.g., DNA binding and/or endonuclease activity). Without wishing to be bound by theory, providing the opener molecule at a selected time and/or location may allow for spatial and temporal control of the activity of the gRNA, CRISPR/Cas protein, or gene modifying system comprising the same. In some embodiments, the opener molecule is exogenous to the cell comprising the gene modifying polypeptide and or template nucleic acid. In some embodiments, the opener molecule comprises an endogenous agent (e.g., endogenous to the cell comprising the gene modifying polypeptide and or template nucleic acid comprising the gRNA and blocking domain). For example, an inducible gRNA, blocking domain, and opener molecule may be chosen such that the opener molecule is an endogenous agent expressed in a target cell or tissue, e.g., thereby ensuring activity of a gene modifying system in the target cell or tissue. As a further example, an inducible gRNA, blocking domain, and opener molecule may be chosen such that the opener molecule is absent or not substantially expressed in one or more non-target cells or tissues, e.g., thereby ensuring that activity of a gene modifying system does not occur or substantially occur in the one or more non-target cells or tissues, or occurs at a reduced level compared to a target cell or tissue. Exemplary blocking domains, opener molecules, and uses thereof are described in PCT App. Publication WO2020044039A1, which is incorporated herein by reference in its entirety. In some embodiments, the template nucleic acid, e.g., template RNA, may comprise one or more sequences or structures for binding by one or more components of a gene modifying polypeptide, e.g., by a reverse transcriptase or RNA binding domain, and a gRNA. In some embodiments, the gRNA facilitates interaction with the template nucleic acid binding domain (e.g., RNA binding domain) of the gene modifying polypeptide. In some embodiments, the gRNA directs the gene modifying polypeptide to the matching target sequence, e.g., in a target cell genome.
Circular RNAs and Ribozymes in Gene Modifying Systems
It is contemplated that it may be useful to employ circular and/or linear RNA states during the formulation, delivery, or gene modifying reaction within the target cell. Thus, in some embodiments of any of the aspects described herein, a gene modifying system comprises one or more circular RNAs (circRNAs). In some embodiments of any of the aspects described herein, a gene modifying system comprises one or more linear RNAs. In some embodiments, a nucleic acid as described herein (e.g., a template nucleic acid, a nucleic acid molecule encoding a gene modifying polypeptide, or both) is a circRNA. In some embodiments, a circular RNA molecule encodes the gene modifying polypeptide. In some embodiments, the circRNA molecule encoding the gene modifying polypeptide is delivered to a host cell. In some embodiments, a circular RNA molecule encodes a recombinase, e.g., as described herein. In some embodiments, the circRNA molecule encoding the recombinase is delivered to a host cell. In some embodiments, the circRNA molecule encoding the gene modifying polypeptide is linearized (e.g., in the host cell, e.g., in the nucleus of the host cell) prior to translation.
Circular RNAs (circRNAs) have been found to occur naturally in cells and have been found to have diverse functions, including both non-coding and protein coding roles in human cells. It has been shown that a circRNA can be engineered by incorporating a self-splicing intron into an RNA molecule (or DNA encoding the RNA molecule) that results in circularization of the RNA, and that an engineered circRNA can have enhanced protein production and stability (Wesselhoeft et al. Nature Communications 2018). In some embodiments, the gene modifying polypeptide is encoded as circRNA. In certain embodiments, the template nucleic acid is a DNA, such as a dsDNA or ssDNA. In certain embodiments, the circDNA comprises a template RNA.
In some embodiments, the circRNA comprises one or more ribozyme sequences. In some embodiments, the ribozyme sequence is activated for autocleavage, e.g., in a host cell, e.g., thereby resulting in linearization of the circRNA. In some embodiments, the ribozyme is activated when the concentration of magnesium reaches a sufficient level for cleavage, e.g., in a host cell. In some embodiments the circRNA is maintained in a low magnesium environment prior to delivery to the host cell. In some embodiments, the ribozyme is a protein-responsive ribozyme. In some embodiments, the ribozyme is a nucleic acid-responsive ribozyme. In some embodiments, the circRNA comprises a cleavage site. In some embodiments, the circRNA comprises a second cleavage site.
In some embodiments, the circRNA is linearized in the nucleus of a target cell. In some embodiments, linearization of a circRNA in the nucleus of a cell involves components present in the nucleus of the cell, e.g., to activate a cleavage event. In some embodiments, a ribozyme, e.g., a ribozyme from a B2 or ALU element, that is responsive to a nuclear element, e.g., a nuclear protein, e.g., a genome-interacting protein, e.g., an epigenetic modifier, e.g., EZH2, is incorporated into a circRNA, e.g., of a gene modifying system. In some embodiments, nuclear localization of the circRNA results in an increase in autocatalytic activity of the ribozyme and linearization of the circRNA.
In some embodiments, the ribozyme is heterologous to one or more of the other components of the gene modifying system. In some embodiments, an inducible ribozyme (e.g., in a circRNA as described herein) is created synthetically, for example, by utilizing a protein ligand-responsive aptamer design. A system for utilizing the satellite RNA of tobacco ringspot virus hammerhead ribozyme with an MS2 coat protein aptamer has been described (Kennedy et al. Nucleic Acids Res 42(19):12306-12321 (2014), incorporated herein by reference in its entirety) that results in activation of the ribozyme activity in the presence of the MS2 coat protein. In embodiments, such a system responds to protein ligand localized to the cytoplasm or the nucleus. In some embodiments the protein ligand is not MS2. Methods for generating RNA aptamers to target ligands have been described, for example, based on the systematic evolution of ligands by exponential enrichment (SELEX) (Tuerk and Gold, Science 249(4968):505-510 (1990); Ellington and Szostak, Nature 346(6287):818-822 (1990); the methods of each of which are incorporated herein by reference) and have, in some instances, been aided by in silico design (Bell et al. PNAS 117(15):8486-8493, the methods of which are incorporated herein by reference). Thus, in some embodiments, an aptamer for a target ligand is generated and incorporated into a synthetic ribozyme system, e.g., to trigger ribozyme-mediated cleavage and circRNA linearization, e.g., in the presence of the protein ligand. In some embodiments, circRNA linearization is triggered in the cytoplasm, e.g., using an aptamer that associates with a ligand in the cytoplasm. In some embodiments, circRNA linearization is triggered in the nucleus, e.g., using an aptamer that associates with a ligand in the nucleus. In embodiments, the ligand in the nucleus comprises an epigenetic modifier or a transcription factor. In some embodiments the ligand that triggers linearization is present at higher levels in on-target cells than off-target cells.
It is further contemplated that a nucleic acid-responsive ribozyme system can be employed for circRNA linearization. For example, biosensors that sense defined target nucleic acid molecules to trigger ribozyme activation are described, e.g., in Penchovsky (Biotechnology Advances 32(5):1015-1027 (2014), incorporated herein by reference). By these methods, a ribozyme naturally folds into an inactive state and is only activated in the presence of a defined target nucleic acid molecule (e.g., an RNA molecule). In some embodiments, a circRNA of a gene modifying system comprises a nucleic acid-responsive ribozyme that is activated in the presence of a defined target nucleic acid, e.g., an RNA, e.g., an mRNA, miRNA, guide RNA, gRNA, sgRNA, ncRNA, lncRNA, tRNA, snRNA, or mtRNA. In some embodiments the nucleic acid that triggers linearization is present at higher levels in on-target cells than off-target cells.
In some embodiments of any of the aspects herein, a gene modifying system incorporates one or more ribozymes with inducible specificity to a target tissue or target cell of interest, e.g., a ribozyme that is activated by a ligand or nucleic acid present at higher levels in a target tissue or target cell of interest. In some embodiments, the gene modifying system incorporates a ribozyme with inducible specificity to a subcellular compartment, e.g., the nucleus, nucleolus, cytoplasm, or mitochondria. In some embodiments, the ribozyme that is activated by a ligand or nucleic acid present at higher levels in the target subcellular compartment. In some embodiments, an RNA component of a gene modifying system is provided as circRNA, e.g., that is activated by linearization. In some embodiments, linearization of a circRNA encoding a gene modifying polypeptide activates the molecule for translation. In some embodiments, a signal that activates a circRNA component of a gene modifying system is present at higher levels in on-target cells or tissues, e.g., such that the system is specifically activated in these cells.
In some embodiments, an RNA component of a gene modifying system is provided as a circRNA that is inactivated by linearization. In some embodiments, a circRNA encoding the gene modifying polypeptide is inactivated by cleavage and degradation. In some embodiments, a circRNA encoding the gene modifying polypeptide is inactivated by cleavage that separates a translation signal from the coding sequence of the polypeptide. In some embodiments, a signal that inactivates a circRNA component of a gene modifying system is present at higher levels in off-target cells or tissues, such that the system is specifically inactivated in these cells.
Target Nucleic Acid Site
In some embodiments, after gene modification, the target site surrounding the edited sequence contains a limited number of insertions or deletions, for example, in less than about 50% or 10% of editing events, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. (2020) bioRxiv doi.org/10.1101/645903 (incorporated by reference herein in its entirety). In some embodiments, the target site does not show multiple consecutive editing events, e.g., head-to-tail or head-to-head duplications, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. bioRxiv doi.org/10.1101/645903 (2020) (incorporated herein by reference in its entirety). In some embodiments, the target site contains an integrated sequence corresponding to the template RNA. In some embodiments, the target site does not contain insertions resulting from endogenous RNA in more than about 1% or 10% of events, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. bioRxiv doi.org/10.1101/645903 (2020) (incorporated herein by reference in its entirety). In some embodiments, the target site contains the integrated sequence corresponding to the template RNA.
In certain aspects of the present invention, the host DNA-binding site integrated into by the gene modifying system can be in a gene, in an intron, in an exon, an ORF, outside of a coding region of any gene, in a regulatory region of a gene, or outside of a regulatory region of a gene. In other aspects, the polypeptide may bind to one or more than one host DNA sequence.
In some embodiments, a gene modifying system is used to edit a target locus in multiple alleles. In some embodiments, a gene modifying system is designed to edit a specific allele. For example, a gene modifying polypeptide may be directed to a specific sequence that is only present on one allele, e.g., comprises a template RNA with homology to a target allele, e.g., a gRNA or annealing domain, but not to a second cognate allele. In some embodiments, a gene modifying system can alter a haplotype-specific allele. In some embodiments, a gene modifying system that targets a specific allele preferentially targets that allele, e.g., has at least a 2, 4, 6, 8, or 10-fold preference for a target allele.
Second Strand Nicking
In some embodiments, a gene modifying system described herein comprises a nickase activity (e.g., in the gene modifying polypeptide) that nicks the first strand, and a nickase activity (e.g., in a polypeptide separate from the gene modifying polypeptide) that nicks the second strand of target DNA. As discussed herein, without wishing to be bound by theory, nicking of the first strand of the target site DNA is thought to provide a 3′ OH that can be used by an RT domain to reverse transcribe a sequence of a template RNA, e.g., a heterologous object sequence. Without wishing to be bound by theory, it is thought that introducing an additional nick to the second strand may bias the cellular DNA repair machinery to adopt the heterologous object sequence-based sequence more frequently than the original genomic sequence. In some embodiments, the additional nick to the second strand is made by the same endonuclease domain (e.g., nickase domain) as the nick to the first strand. In some embodiments, the same gene modifying polypeptide performs both the nick to the first strand and the nick to the second strand. In some embodiments, the gene modifying polypeptide comprises a CRISPR/Cas domain and the additional nick to the second strand is directed by an additional nucleic acid, e.g., comprising a second gRNA directing the CRISPR/Cas domain to nick the second strand. In other embodiments, the additional second strand nick is made by a different endonuclease domain (e.g., nickase domain) than the nick to the first strand. In some embodiments, that different endonuclease domain is situated in an additional polypeptide (e.g., a system of the invention further comprises the additional polypeptide), separate from the gene modifying polypeptide. In some embodiments, the additional polypeptide comprises an endonuclease domain (e.g., nickase domain) described herein. In some embodiments, the additional polypeptide comprises a DNA binding domain, e.g., described herein.
It is contemplated herein that the position at which the second strand nick occurs relative to the first strand nick may influence the extent to which one or more of: desired gene modifying DNA modifications are obtained, undesired double-strand breaks (DSBs) occur, undesired insertions occur, or undesired deletions occur. Without wishing to be bound by theory, second strand nicking may occur in two general orientations: inward nicks and outward nicks.
In some embodiments, in the inward nick orientation, the RT domain polymerizes (e.g., using the template RNA (e.g., the heterologous object sequence)) away from the second strand nick. In some embodiments, in the inward nick orientation, the location of the nick to the first strand and the location of the nick to the second strand are positioned between the first PAM site and second PAM site (e.g., in a scenario wherein both nicks are made by a polypeptide (e.g., a gene modifying polypeptide) comprising a CRISPR/Cas domain). When there are two PAMs on the outside and two nicks on the inside, this inward nick orientation can also be referred to as “PAM-out”. In some embodiments, in the inward nick orientation, the location of the nick to the first strand and the location of the nick to the second strand are between the sites where the polypeptide and the additional polypeptide bind to the target DNA. In some embodiments, in the inward nick orientation, the location of the nick to the second strand is positioned between the binding sites of the polypeptide and additional polypeptide, and the nick to the first strand is also located between the binding sites of the polypeptide and additional polypeptide. In some embodiments, in the inward nick orientation, the location of the nick to the first strand and the location of the nick to the second strand are positioned between the PAM site and the binding site of the second polypeptide which is at a distance from the target site.
An example of a gene modifying system that provides an inward nick orientation comprises a gene modifying polypeptide comprising a CRISPR/Cas domain, a template RNA comprising a gRNA that directs nicking of the target site DNA on the first strand, and an additional nucleic acid comprising an additional gRNA that directs nicking at a site a distance from the location of the first nick, wherein the location of the first nick and the location of the second nick are between the PAM sites of the sites to which the two gRNAs direct the gene modifying polypeptide. As a further example, another gene modifying system that provides an inward nick orientation comprises a gene modifying polypeptide comprising a zinc finger molecule and a first nickase domain wherein the zinc finger molecule binds to the target DNA in a manner that directs the first nickase domain to nick the first strand of the target site; an additional polypeptide comprising a CRISPR/Cas domain, and an additional nucleic acid comprising a gRNA that directs the additional polypeptide to nick a site a distance from the target site DNA on the second strand, wherein the location of the first nick and the location of the second nick are between the PAM site and the site to which the zinc finger molecule binds. As a further example, another gene modifying system that provides an inward nick orientation comprises a gene modifying polypeptide comprising a zinc finger molecule and a first nickase domain wherein the zinc finger molecule binds to the target DNA in a manner that directs the first nickase domain to nick the first strand of the target site; an additional polypeptide comprising a TAL effector molecule and a second nickase domain wherein the TAL effector molecule binds to a site a distance from the target site in a manner that directs the additional polypeptide to nick the second strand, wherein the location of the first nick and the location of the second nick are between the site to which the TAL effector molecule binds and the site to which the zinc finger molecule binds.
In some embodiments, in the outward nick orientation, the RT domain polymerizes (e.g., using the template RNA (e.g., the heterologous object sequence)) toward the second strand nick. In some embodiments, in the outward nick orientation when both the first and second nicks are made by a polypeptide comprising a CRISPR/Cas domain (e.g., a gene modifying polypeptide), the first PAM site and second PAM site are positioned between the location of the nick to the first strand and the location of the nick to the second strand. When there are two PAMs on the inside and two nicks on the outside, this outward nick orientation also can be referred to as “PAM-in”. In some embodiments, in the outward nick orientation, the polypeptide (e.g., the gene modifying polypeptide) and the additional polypeptide bind to sites on the target DNA between the location of the nick to the first strand and the location of the nick to the second. In some embodiments, in the outward nick orientation, the location of the nick to the second strand is positioned on the opposite side of the binding sites of the polypeptide and additional polypeptide relative to the location of the nick to the first strand. In some embodiments, in the outward orientation, the PAM site and the binding site of the second polypeptide which is at a distance from the target site are positioned between the location of the nick to the first strand and the location of the nick to the second strand.
An example of a gene modifying system that provides an outward nick orientation comprises a gene modifying polypeptide comprising a CRISPR/Cas domain, a template RNA comprising a gRNA that directs nicking of the target site DNA on the first strand, and an additional nucleic acid comprising an additional gRNA that directs nicking at a site a distance from the location of the first nick, wherein the location of the first nick and the location of the second nick are outside of the PAM sites of the sites to which the two gRNAs direct the gene modifying polypeptide (i.e., the PAM sites are between the location of the first nick and the location of the second nick). As a further example, another gene modifying system that provides an outward nick orientation comprises a gene modifying polypeptide comprising a zinc finger molecule and a first nickase domain wherein the zinc finger molecule binds to the target DNA in a manner that directs the first nickase domain to nick the first strand of the target site; an additional polypeptide comprising a CRISPR/Cas domain, and an additional nucleic acid comprising a gRNA that directs the additional polypeptide to nick a site a distance from the target site DNA on the second strand, wherein the location of the first nick and the location of the second nick are outside the PAM site and the site to which the zinc finger molecule binds (i.e., the PAM site and the site to which the zinc finger molecule binds are between the location of the first nick and the location of the second nick). As a further example, another gene modifying system that provides an outward nick orientation comprises a gene modifying polypeptide comprising a zinc finger molecule and a first nickase domain wherein the zinc finger molecule binds to the target DNA in a manner that directs the first nickase domain to nick the first strand of the target site; an additional polypeptide comprising a TAL effector molecule and a second nickase domain wherein the TAL effector molecule binds to a site a distance from the target site in a manner that directs the additional polypeptide to nick the second strand, wherein the location of the first nick and the location of the second nick are outside the site to which the TAL effector molecule binds and the site to which the zinc finger molecule binds (i.e., the site to which the TAL effector molecule binds and the site to which the zinc finger molecule binds are between the location of the first nick and the location of the second nick).
Without wishing to be bound by theory, it is thought that, for gene modifying systems where a second strand nick is provided, an outward nick orientation is preferred in some embodiments. As is described herein, an inward nick may produce a higher number of double-strand breaks (DSBs) than an outward nick orientation. DSBs may be recognized by the DSB repair pathways in the nucleus of a cell, which can result in undesired insertions and deletions. An outward nick orientation may provide a decreased risk of DSB formation, and a corresponding lower amount of undesired insertions and deletions. In some embodiments, undesired insertions and deletions are insertions and deletions not encoded by the heterologous object sequence, e.g., an insertion or deletion produced by the double-strand break repair pathway unrelated to the modification encoded by the heterologous object sequence. In some embodiments, a desired gene modification comprises a change to the target DNA (e.g., a substitution, insertion, or deletion) encoded by the heterologous object sequence (e.g., and achieved by the gene modifying writing the heterologous object sequence into the target site). In some embodiments, the first strand nick and the second strand nick are in an outward orientation.
In addition, the distance between the first strand nick and second strand nick may influence the extent to which one or more of: desired gene modifying system DNA modifications are obtained, undesired double-strand breaks (DSBs) occur, undesired insertions occur, or undesired deletions occur. Without wishing to be bound by theory, it is thought the second strand nick benefit, the biasing of DNA repair toward incorporation of the heterologous object sequence into the target DNA, increases as the distance between the first strand nick and second strand nick decreases. However, it is thought that the risk of DSB formation also increases as the distance between the first strand nick and second strand nick decreases. Correspondingly, it is thought that the number of undesired insertions and/or deletions may increase as the distance between the first strand nick and second strand nick decreases. In some embodiments, the distance between the first strand nick and second strand nick is chosen to balance the benefit of biasing DNA repair toward incorporation of the heterologous object sequence into the target DNA and the risk of DSB formation and of undesired deletions and/or insertions. In some embodiments, a system where the first strand nick and the second strand nick are at least a threshold distance apart has an increased level of desired gene modifying system modification outcomes, a decreased level of undesired deletions, and/or a decreased level of undesired insertions relative to an otherwise similar inward nick orientation system where the first nick and the second nick are less than the a threshold distance apart. In some embodiments the threshold distance(s) is given below.
In some embodiments, the first nick and the second nick are at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides apart. In some embodiments, the first nick and the second nick are no more than 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 250 nucleotides apart. In some embodiments, the first nick and the second nick are 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 110-200, 120-200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-200, 190-200, 20-190, 30-190, 40-190, 50-190, 60-190, 70-190, 80-190, 90-190, 100-190, 110-190, 120-190, 130-190, 140-190, 150-190, 160-190, 170-190, 180-190, 20-180, 30-180, 40-180, 50-180, 60-180, 70-180, 80-180, 90-180, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 170-180, 20-170, 30-170, 40-170, 50-170, 60-170, 70-170, 80-170, 90-170, 100-170, 110-170, 120-170, 130-170, 140-170, 150-170, 160-170, 20-160, 30-160, 40-160, 50-160, 60-160, 70-160, 80-160, 90-160, 100-160, 110-160, 120-160, 130-160, 140-160, 150-160, 20-150, 30-150, 40-150, 50-150, 60-150, 70-150, 80-150, 90-150, 100-150, 110-150, 120-150, 130-150, 140-150, 20-140, 30-140, 40-140, 50-140, 60-140, 70-140, 80-140, 90-140, 100-140, 110-140, 120-140, 130-140, 20-130, 30-130, 40-130, 50-130, 60-130, 70-130, 80-130, 90-130, 100-130, 110-130, 120-130, 20-120, 30-120, 40-120, 50-120, 60-120, 70-120, 80-120, 90-120, 100-120, 110-120, 20-110, 30-110, 40-110, 50-110, 60-110, 70-110, 80-110, 90-110, 100-110, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100, 20-90, 30-90, 40-90, 50-90, 60-90, 70-90, 80-90, 20-80, 30-80, 40-80, 50-80, 60-80, 70-80, 20-70, 30-70, 40-70, 50-70, 60-70, 20-60, 30-60, 40-60, 50-60, 20-50, 30-50, 40-50, 20-40, 30-40, or 20-30 nucleotides apart. In some embodiments, the first nick and the second nick are 40-100 nucleotides apart.
Without wishing to be bound by theory, it is thought that, for gene modifying systems where a second strand nick is provided and an inward nick orientation is selected, increasing the distance between the first strand nick and second strand nick may be preferred. As is described herein, an inward nick orientation may produce a higher number of DSBs than an outward nick orientation, and may result in a higher amount of undesired insertions and deletions than an outward nick orientation, but increasing the distance between the nicks may mitigate that increase in DSBs, undesired deletions, and/or undesired insertions. In some embodiments, an inward nick orientation wherein the first nick and the second nick are at least a threshold distance apart has an increased level of desired gene modifying system modification outcomes, a decreased level of undesired deletions, and/or a decreased level of undesired insertions relative to an otherwise similar inward nick orientation system where the first nick and the second nick are less than the a threshold distance apart. In some embodiments the threshold distance is given below.
In some embodiments, the first strand nick and the second strand nick are in an inward orientation. In some embodiments, the first strand nick and the second strand nick are in an inward orientation and the first strand nick and second strand nick are at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 350, 400, 450, or 500 nucleotides apart, e.g., at least 100 nucleotides apart, (and optionally no more than 500, 400, 300, 200, 190, 180, 170, 160, 150, 140, 130, or 120 nucleotides apart). In some embodiments, the first strand nick and the second strand nick are in an inward orientation and the first strand nick and second strand nick are 100-200, 110-200, 120-200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-200, 190-200, 100-190, 110-190, 120-190, 130-190, 140-190, 150-190, 160-190, 170-190, 180-190, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 170-180, 100-170, 110-170, 120-170, 130-170, 140-170, 150-170, 160-170, 100-160, 110-160, 120-160, 130-160, 140-160, 150-160, 100-150, 110-150, 120-150, 130-150, 140-150, 100-140, 110-140, 120-140, 130-140, 100-130, 110-130, 120-130, 100-120, 110-120, or 100-110 nucleotides apart.
Chemically Modified Nucleic Acids and Nucleic Acid End Features
A nucleic acid described herein (e.g., a template nucleic acid, e.g., a template RNA; or a nucleic acid (e.g., mRNA) encoding a gene modifying polypeptide; or a gRNA) can comprise unmodified or modified nucleobases. Naturally occurring RNAs are synthesized from four basic ribonucleotides: ATP, CTP, UTP and GTP, but may contain post-transcriptionally modified nucleotides. Further, approximately one hundred different nucleoside modifications have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197). An RNA can also comprise wholly synthetic nucleotides that do not occur in nature.
In some embodiments, the chemical modification is one provided in WO/2016/183482, US Pat. Pub. No. 20090286852, of International Application No. WO/2012/019168, WO/2012/045075, WO/2012/135805, WO/2012/158736, WO/2013/039857, WO/2013/039861, WO/2013/052523, WO/2013/090648, WO/2013/096709, WO/2013/101690, WO/2013/106496, WO/2013/130161, WO/2013/151669, WO/2013/151736, WO/2013/151672, WO/2013/151664, WO/2013/151665, WO/2013/151668, WO/2013/151671, WO/2013/151667, WO/2013/151670, WO/2013/151666, WO/2013/151663, WO/2014/028429, WO/2014/081507, WO/2014/093924, WO/2014/093574, WO/2014/113089, WO/2014/144711, WO/2014/144767, WO/2014/144039, WO/2014/152540, WO/2014/152030, WO/2014/152031, WO/2014/152027, WO/2014/152211, WO/2014/158795, WO/2014/159813, WO/2014/164253, WO/2015/006747, WO/2015/034928, WO/2015/034925, WO/2015/038892, WO/2015/048744, WO/2015/051214, WO/2015/051173, WO/2015/051169, WO/2015/058069, WO/2015/085318, WO/2015/089511, WO/2015/105926, WO/2015/164674, WO/2015/196130, WO/2015/196128, WO/2015/196118, WO/2016/011226, WO/2016/011222, WO/2016/011306, WO/2016/014846, WO/2016/022914, WO/2016/036902, WO/2016/077125, or WO/2016/077123, each of which is herein incorporated by reference in its entirety. It is understood that incorporation of a chemically modified nucleotide into a polynucleotide can result in the modification being incorporated into a nucleobase, the backbone, or both, depending on the location of the modification in the nucleotide. In some embodiments, the backbone modification is one provided in EP 2813570, which is herein incorporated by reference in its entirety. In some embodiments, the modified cap is one provided in US Pat. Pub. No. 20050287539, which is herein incorporated by reference in its entirety.
In some embodiments, the chemically modified nucleic acid (e.g., RNA, e.g., mRNA) comprises one or more of ARCA: anti-reverse cap analog (m27.3′-OGP3G), GP3G (Unmethylated Cap Analog), m7GP3G (Monomethylated Cap Analog), m32.2.7GP3G (Trimethylated Cap Analog), m5CTP (5′-methyl-cytidine triphosphate), m6ATP (N6-methyl-adenosine-5′-triphosphate), s2UTP (2-thio-uridine triphosphate), and Ψ (pseudouridine triphosphate).
In some embodiments, the chemically modified nucleic acid comprises a 5′ cap, e.g.: a 7-methylguanosine cap (e.g., a O-Me-m7G cap); a hypermethylated cap analog; an NAD+-derived cap analog (e.g., as described in Kiledjian, Trends in Cell Biology 28, 454-464 (2018)); or a modified, e.g., biotinylated, cap analog (e.g., as described in Bednarek et al., Phil Trans R Soc B 373, 20180167 (2018)).
In some embodiments, the chemically modified nucleic acid comprises a 3′ feature selected from one or more of: a polyA tail; a 16-nucleotide long stem-loop structure flanked by unpaired 5 nucleotides (e.g., as described by Mannironi et al., Nucleic Acid Research 17, 9113-9126 (1989)); a triple-helical structure (e.g., as described by Brown et al., PNAS 109, 19202-19207 (2012)); a tRNA, Y RNA, or vault RNA structure (e.g., as described by Labno et al., Biochemica et Biophysica Acta 1863, 3125-3147 (2016)); incorporation of one or more deoxyribonucleotide triphosphates (dNTPs), 2′O-Methylated NTPs, or phosphorothioate-NTPs; a single nucleotide chemical modification (e.g., oxidation of the 3′ terminal ribose to a reactive aldehyde followed by conjugation of the aldehyde-reactive modified nucleotide); or chemical ligation to another nucleic acid molecule.
In some embodiments, the nucleic acid (e.g., template nucleic acid) comprises one or more modified nucleotides, e.g., selected from dihydrouridine, inosine, 7-methylguanosine, 5-methylcytidine (5mC), 5′ Phosphate ribothymidine, 2′-O-methyl ribothymidine, 2′-O-ethyl ribothymidine, 2′-fluoro ribothymidine, C-5 propynyl-deoxycytidine (pdC), C-5 propynyl-deoxyuridine (pdU), C-5 propynyl-cytidine (pC), C-5 propynyl-uridine (pU), 5-methyl cytidine, 5-methyl uridine, 5-methyl deoxycytidine, 5-methyl deoxyuridine methoxy, 2,6-diaminopurine, 5′-Dimethoxytrityl-N4-ethyl-2′-deoxycytidine, C-5 propynyl-f-cytidine (pfC), C-5 propynyl-f-uridine (pfU), 5-methyl f-cytidine, 5-methyl f-uridine, C-5 propynyl-m-cytidine (pmC), C-5 propynyl-f-uridine (pmU), 5-methyl m-cytidine, 5-methyl m-uridine, LNA (locked nucleic acid), MGB (minor groove binder) pseudouridine (Ψ), 1-N-methylpseudouridine (1-Me-Ψ), or 5-methoxyuridine (5-MO-U).
In some embodiments, the nucleic acid comprises a backbone modification, e.g., a modification to a sugar or phosphate group in the backbone. In some embodiments, the nucleic acid comprises a nucleobase modification.
In some embodiments, the nucleic acid comprises one or more chemically modified nucleotides of Table 13, one or more chemical backbone modifications of Table 14, one or more chemically modified caps of Table 15. For instance, in some embodiments, the nucleic acid comprises two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or more) different types of chemical modifications. As an example, the nucleic acid may comprise two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or more) different types of modified nucleobases, e.g., as described herein, e.g., in Table 13. Alternatively or in combination, the nucleic acid may comprise two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or more) different types of backbone modifications, e.g., as described herein, e.g., in Table 14. Alternatively or in combination, the nucleic acid may comprise one or more modified cap, e.g., as described herein, e.g., in Table 15. For instance, in some embodiments, the nucleic acid comprises one or more type of modified nucleobase and one or more type of backbone modification; one or more type of modified nucleobase and one or more modified cap; one or more type of modified cap and one or more type of backbone modification; or one or more type of modified nucleobase, one or more type of backbone modification, and one or more type of modified cap.
In some embodiments, the nucleic acid comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or more) modified nucleobases. In some embodiments, all nucleobases of the nucleic acid are modified. In some embodiments, the nucleic acid is modified at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or more) positions in the backbone. In some embodiments, all backbone positions of the nucleic acid are modified.







TABLE 13





Modified nucleotides
















5-aza-uridine
N2-methyl-6-thio-guanosine


2-thio-5-aza-midine
N2,N2-dimethyl-6-thio-guanosine


2-thiouridine
pyridin-4-one ribonucleoside


4-thio-pseudouridine
2-thio-5-aza-uridine


2-thio-pseudouridine
2-thiomidine


5-hydroxyuridine
4-thio-pseudomidine


3-methyluridine
2-thio-pseudowidine


5-carboxymethyl-uridine
3-methylmidine


1-carboxymethyl-pseudouridine
1-propynyl-pseudomidine


5-propynyl-uridine
1-methyl-1-deaza-pseudomidine


1-propynyl-pseudouridine
2-thio-1-methyl-1-deaza-pseudouridine


5-taurinomethyluridine
4-methoxy-pseudomidine


1-taurinomethyl-pseudouridine
5′-O-(1-Thiophosphate)-Adenosine


5-taurinomethyl-2-thio-uridine
5′-O-(1-Thiophosphate)-Cytidine


1-taurinomethyl-4-thio-uridine
5′-O-(1-thiophosphate)-Guanosine


5-methyl-uridine
5′-O-(1-Thiophophate)-Uridine


1-methyl-pseudouridine
5′-O-(1-Thiophosphate)-Pseudouridine


4-thio-1-methyl-pseudouridine
2′-O-methyl-Adenosine


2-thio-1-methyl-pseudouridine
2′-O-methyl-Cytidine


1-methyl-1-deaza-pseudouridine
2′-O-methyl-Guanosine


2-thio-1-methyl-1-deaza-pseudomidine
2′-O-methyl-Uridine


dihydrouridine
2′-O-methyl-Pseudouridine


dihydropseudouridine
2′-O-methyl-Inosine


2-thio-dihydromidine
2-methyladenosine


2-thio-dihydropseudouridine
2-methylthio-N6-methyladenosine


2-methoxyuridine
2-methylthio-N6 isopentenyladenosine


2-methoxy-4-thio-uridine
2-methylthio-N6-(cis-


4-methoxy-pseudouridine
hydroxyisopentenyl)adenosine


4-methoxy-2-thio-pseudouridine
N6-methyl-N6-threonylcarbamoyladenosine


5-aza-cytidine
N6-hydroxynorvalylcarbamoyladenosine


pseudoisocytidine
2-methylthio-N6-hydroxynorvalyl


3-methyl-cytidine
carbamoyladenosine


N4-acetylcytidine
2′-O-ribosyladenosine (phosphate)


5-formylcytidine
1,2′-O-dimethylinosine


N4-methylcytidine
5,2′-O-dimethylcytidine


5-hydroxymethylcytidine
N4-acetyl-2′-O-methylcytidine


1-methyl-pseudoisocytidine
Lysidine


pyrrolo-cytidine
7-methylguanosine


pyrrolo-pseudoisocytidine
N2,2′-O-dimethylguanosine


2-thio-cytidine
N2,N2,2′-O-trimethylguanosine


2-thio-5-methyl-cytidine
2′-O-ribosylguanosine (phosphate)


4-thio-pseudoisocytidine
Wybutosine


4-thio-1-methyl-pseudoisocytidine
Peroxywybutosine


4-thio-1-methyl-1-deaza-pseudoisocytidine
Hydroxywybutosine


1-methyl-1-deaza-pseudoisocytidine
undermodified hydroxywybutosine


zebularine
methylwyosine


5-aza-zebularine
queuosine


5-methyl-zebularine
epoxyqueuosine


5-aza-2-thio-zebularine
galactosyl-queuosine


2-thio-zebularine
mannosyl-queuosine


2-methoxy-cytidine
7-cyano-7-deazaguanosine


2-methoxy-5-methyl-cytidine
7-aminomethyl-7-deazaguanosine


4-methoxy-pseudoisocytidine
archaeosine


4-methoxy-1-methyl-pseudoisocytidine
5,2′-O-dimethyluridine


2-aminopurine
4-thiouridine


2,6-diaminopurine
5-methyl-2-thiouridine


7-deaza-adenine
2-thio-2′-O-methyluridine


7-deaza-8-aza-adenine
3-(3-amino-3-carboxypropyl)uridine


7-deaza-2-aminopurine
5-methoxyuridine


7-deaza-8-aza-2-aminopurine
uridine 5-oxyacetic acid


7-deaza-2,6- diaminopurine
uridine 5-oxyacetic acid methyl ester


7-deaza-8-aza-2,6-diarninopurine
5-(carboxyhydroxymethyl)uridine)


1-methyladenosine
5-(carboxyhydroxymethyl)uridine methyl ester


N6-isopentenyladenosine
5-methoxycarbonylmethyluridine


N6-(cis-hydroxyisopentenyl)adenosine
5-methoxycarbonylmethyl-2′-O-methyluridine


2-methylthio-N6-(cis-hydroxyisopentenyl)
5-methoxycarbonylmethyl-2-thiouridine


adenosine
5-aminomethyl-2-thiouridine


N6-glycinylcarbamoyladenosine
5-methylaminomethyluridine


N6-threonylcarbamoyladenosine
5-methylaminomethyl-2-thiouridine


2-methylthio-N6-threonyl
5-methylaminomethyl-2-selenouridine


carbamoyladenosine
5-carbamoylmethyluridine


N6,N6-dimethyladenosine
5-carbamoylmethyl-2′-O-methyluridine


7-methyladenine
5-carboxymethylaminomethyluridine


2-methylthio-adenine
5-carboxymethylaminomethyl-2′-O-


2-methoxy-adenine
methyluridine


inosine
5-carboxymethylaminomethyl-2-thiouridine


1-methyl-inosine
N4,2′-O-dimethylcytidine


wyosine
5-carboxymethyluridine


wybutosine
N6,2′-O-dimethyladenosine


7-deaza-guanosine
N,N6,O-2′-trimethyladenosine


7-deaza-8-aza-guanosine
N2,7-dimethylguanosine


6-thio-guanosine
N2,N2,7-trimethylguanosine


6-thio-7-deaza-guanosine
3,2′-O-dimethyluridine


6-thio-7-deaza-8-aza-guanosine
5-methyldihydrouridine


7-methyl-guanosine
5-formyl-2′-O-methylcytidine


6-thio-7-methyl-guanosine
1,2′-O-dimethylguanosine


7-methylinosine
4-demethylwyosine


6-methoxy-guanosine
Isowyosine


1-methylguanosine
N6-acetyladenosine


N2-methylguanosine



N2,N2-dimethylguanosine



8-oxo-guanosine



7-methyl-8-oxo-guanosine



1-methyl-6-thio-guanosine
















TABLE 14





Backbone modifications















2′-O-Methyl backbone


Peptide Nucleic Acid (PNA) backbone


phosphorothioate backbone


morpholino backbone


carbamate backbone


siloxane backbone


sulfide backbone


sulfoxide backbone


sulfone backbone


formacetyl backbone


thioformacetyl backbone


methyleneformacetyl backbone


riboacetyl backbone


alkene containing backbone


sulfamate backbone


sulfonate backbone


sulfonamide backbone


methyleneimino backbone


methylenehydrazino backbone


amide backbone
















TABLE 15





Modified caps















m7GpppA


m7GpppC


m2,7GpppG


m2,2,7GpppG


m7Gpppm7G


m7,2′OmeGpppG


m72′dGpppG


m7,3′OmeGpppG


m7,3′dGpppG


GppppG


m7GppppG


m7GppppA


m7GppppC


m2,7GppppG


m2,2,7GppppG


m7Gppppm7G


m7,2′OmeGppppG


m72′dGppppG


m7,3′OmeGppppG


m7,3′dGppppG









The nucleotides comprising the template of the gene modifying system can be natural or modified bases, or a combination thereof. For example, the template may contain pseudouridine, dihydrouridine, inosine, 7-methylguanosine, or other modified bases. In some embodiments, the template may contain locked nucleic acid nucleotides. In some embodiments, the modified bases used in the template do not inhibit the reverse transcription of the template. In some embodiments, the modified bases used in the template may improve reverse transcription, e.g., specificity or fidelity.
In some embodiments, an RNA component of the system (e.g., a template RNA or a gRNA) comprises one or more nucleotide modifications. In some embodiments, the modification pattern of a gRNA can significantly affect in vivo activity compared to unmodified or end-modified guides (e.g., as shown in FIG. 1D from Finn et al. Cell Rep 22(9):2227-2235 (2018); incorporated herein by reference in its entirety). Without wishing to be bound by theory, this process may be due, at least in part, to a stabilization of the RNA conferred by the modifications. Non-limiting examples of such modifications may include 2′-O-methyl (2′-O-Me), 2′-0-(2-methoxyethyl) (2′-0-MOE), 2′-fluoro (2′-F), phosphorothioate (PS) bond between nucleotides, G-C substitutions, and inverted abasic linkages between nucleotides and equivalents thereof.
In some embodiments, the template RNA (e.g., at the portion thereof that binds a target site) or the guide RNA comprises a 5′ terminus region. In some embodiments, the template RNA or the guide RNA does not comprise a 5′ terminus region. In some embodiments, the 5′ terminus region comprises a gRNA spacer region, e.g., as described with respect to sgRNA in Briner AE et al, Molecular Cell 56: 333-339 (2014) (incorporated herein by reference in its entirety; applicable herein, e.g., to all guide RNAs). In some embodiments, the 5′ terminus region comprises a 5′ end modification. In some embodiments, a 5′ terminus region with or without a spacer region may be associated with a crRNA, trRNA, sgRNA and/or dgRNA. The gRNA spacer region can, in some instances, comprise a guide region, guide domain, or targeting domain.
In some embodiments, the template RNAs (e.g., at the portion thereof that binds a target site) or guide RNAs described herein comprises any of the sequences shown in Table 4 of WO2018107028A1, incorporated herein by reference in its entirety. In some embodiments, where a sequence shows a guide and/or spacer region, the composition may comprise this region or not. In some embodiments, a guide RNA comprises one or more of the modifications of any of the sequences shown in Table 4 of WO2018107028A1, e.g., as identified therein by a SEQ ID NO. In embodiments, the nucleotides may be the same or different, and/or the modification pattern shown may be the same or similar to a modification pattern of a guide sequence as shown in Table 4 of WO2018107028A1. In some embodiments, a modification pattern includes the relative position and identity of modifications of the gRNA or a region of the gRNA (e.g. 5′ terminus region, lower stem region, bulge region, upper stem region, nexus region, hairpin 1 region, hairpin 2 region, 3′ terminus region). In some embodiments, the modification pattern contains at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the modifications of any one of the sequences shown in the sequence column of Table 4 of WO2018107028A1, and/or over one or more regions of the sequence. In some embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the modification pattern of any one of the sequences shown in the sequence column of Table 4 of WO2018107028A1. In some embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over one or more regions of the sequence shown in Table 4 of WO2018107028A1, e.g., in a 5′ terminus region, lower stem region, bulge region, upper stem region, nexus region, hairpin 1 region, hairpin 2 region, and/or 3′ terminus region. In some embodiments, the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the modification pattern of a sequence over the 5′ terminus region. In some embodiments, the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the lower stem. In some embodiments, the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the bulge. In some embodiments, the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the upper stem. In some embodiments, the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the nexus. In some embodiments, the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the hairpin 1. In some embodiments, the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the hairpin 2. In some embodiments, the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the 3′ terminus. In some embodiments, the modification pattern differs from the modification pattern of a sequence of Table 4 of WO2018107028A1, or a region (e.g. 5′ terminus, lower stem, bulge, upper stem, nexus, hairpin 1, hairpin 2, 3′ terminus) of such a sequence, e.g., at 0, 1, 2, 3, 4, 5, 6, or more nucleotides. In some embodiments, the gRNA comprises modifications that differ from the modifications of a sequence of Table 4 of WO2018107028A1, e.g., at 0, 1, 2, 3, 4, 5, 6, or more nucleotides. In some embodiments, the gRNA comprises modifications that differ from modifications of a region (e.g. 5′ terminus, lower stem, bulge, upper stem, nexus, hairpin 1, hairpin 2, 3′ terminus) of a sequence of Table 4 of WO2018107028A1, e.g., at 0, 1, 2, 3, 4, 5, 6, or more nucleotides.
In some embodiments, the template RNAs (e.g., at the portion thereof that binds a target site) or the gRNA comprises a 2′-O-methyl (2′-O-Me) modified nucleotide. In some embodiments, the gRNA comprises a 2′-O-(2-methoxy ethyl) (2′-O-moe) modified nucleotide. In some embodiments, the gRNA comprises a 2′-fluoro (2′-F) modified nucleotide. In some embodiments, the gRNA comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, the gRNA comprises a 5′ end modification, a 3′ end modification, or 5′ and 3′ end modifications. In some embodiments, the 5′ end modification comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, the 5′ end modification comprises a 2′-O-methyl (2′-O-Me), 2′-O-(2-methoxy ethyl) (2′-O-MOE), and/or 2′-fluoro (2′-F) modified nucleotide. In some embodiments, the 5′ end modification comprises at least one phosphorothioate (PS) bond and one or more of a 2′-O-methyl (2′-O-Me), 2′-O-(2-methoxyethyl) (2′-O-MOE), and/or 2′-fluoro (2′-F) modified nucleotide. The end modification may comprise a phosphorothioate (PS), 2′-O-methyl (2′-O-Me), 2′-O-(2-methoxyethyl) (2′-O-MOE), and/or 2′-fluoro (2′-F) modification. Equivalent end modifications are also encompassed by embodiments described herein. In some embodiments, the template RNA or gRNA comprises an end modification in combination with a modification of one or more regions of the template RNA or gRNA. Additional exemplary modifications and methods for protecting RNA, e.g., gRNA, and formulae thereof, are described in WO2018126176A1, which is incorporated herein by reference in its entirety.
In some embodiments, a template RNA described herein comprises three phosphorothioate linkages at the 5′ end and three phosphorothioate linkages at the 3′ end. In some embodiments, a template RNA described herein comprises three 2′-O-methyl ribonucleotides at the 5′ end and three 2′-O-methyl ribonucleotides at the 3′ end. In some embodiments, the 5′ most three nucleotides of the template RNA are 2′-O-methyl ribonucleotides, the 5′ most three internucleotide linkages of the template RNA are phosphorothioate linkages, the 3′ most three nucleotides of the template RNA are 2′-O-methyl ribonucleotides, and the 3′ most three internucleotide linkages of the template RNA are phosphorothioate linkages. In some embodiments, the template RNA comprises alternating blocks of ribonucleotides and 2′-O-methyl ribonucleotides, for instance, blocks of between 12 and 28 nucleotides in length. In some embodiments, the central portion of the template RNA comprises the alternating blocks and the 5′ and 3′ ends each comprise three 2′-O-methyl ribonucleotides and three phosphorothioate linkages.
In some embodiments, structure-guided and systematic approaches are used to introduce modifications (e.g., 2′-OMe-RNA, 2′-F-RNA, and PS modifications) to a template RNA or guide RNA, for example, as described in Mir et al. Nat Commun 9:2641 (2018) (incorporated by reference herein in its entirety). In some embodiments, the incorporation of 2′-F-RNAs increases thermal and nuclease stability of RNA:RNA or RNA:DNA duplexes, e.g., while minimally interfering with C3′-endo sugar puckering. In some embodiments, 2′-F may be better tolerated than 2′-OMe at positions where the 2′-OH is important for RNA:DNA duplex stability. In some embodiments, a crRNA comprises one or more modifications that do not reduce Cas9 activity, e.g., C10, C20, or C21 (fully modified), e.g., as described in Supplementary Table 1 of Mir et al. Nat Commun 9:2641 (2018), incorporated herein by reference in its entirety. In some embodiments, a tracrRNA comprises one or more modifications that do not reduce Cas9 activity, e.g., T2, T6, T7, or T8 (fully modified) of Supplementary Table 1 of Mir et al. Nat Commun 9:2641 (2018). In some embodiments, a crRNA comprises one or more modifications (e.g., as described herein) may be paired with a tracrRNA comprising one or more modifications, e.g., C20 and T2. In some embodiments, a gRNA comprises a chimera, e.g., of a crRNA and a tracrRNA (e.g., Jinek et al. Science 337(6096):816-821 (2012)). In embodiments, modifications from the crRNA and tracrRNA are mapped onto the single-guide chimera, e.g., to produce a modified gRNA with enhanced stability.
In some embodiments, gRNA molecules may be modified by the addition or subtraction of the naturally occurring structural components, e.g., hairpins. In some embodiments, a gRNA may comprise a gRNA with one or more 3′ hairpin elements deleted, e.g., as described in WO2018106727, incorporated herein by reference in its entirety. In some embodiments, a gRNA may contain an added hairpin structure, e.g., an added hairpin structure in the spacer region, which was shown to increase specificity of a CRISPR-Cas system in the teachings of Kocak et al. Nat Biotechnol 37(6):657-666 (2019). Additional modifications, including examples of shortened gRNA and specific modifications improving in vivo activity, can be found in US20190316121, incorporated herein by reference in its entirety.
In some embodiments, structure-guided and systematic approaches (e.g., as described in Mir et al. Nat Commun 9:2641 (2018); incorporated herein by reference in its entirety) are employed to find modifications for the template RNA. In embodiments, the modifications are identified with the inclusion or exclusion of a guide region of the template RNA. In some embodiments, a structure of polypeptide bound to template RNA is used to determine non-protein-contacted nucleotides of the RNA that may then be selected for modifications, e.g., with lower risk of disrupting the association of the RNA with the polypeptide. Secondary structures in a template RNA can also be predicted in silico by software tools, e.g., the RNAstructure tool available at rna.urmc.rochester.edu/RNAstructureWeb (Bellaousov et al. Nucleic Acids Res 41:W471-W474 (2013); incorporated by reference herein in its entirety), e.g., to determine secondary structures for selecting modifications, e.g., hairpins, stems, and/or bulges.
Production of Compositions and Systems
As will be appreciated by one of skill, methods of designing and constructing nucleic acid constructs and proteins or polypeptides (such as the systems, constructs and polypeptides described herein) are routine in the art. Generally, recombinant methods may be used. See, in general, Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013). Methods of designing, preparing, evaluating, purifying and manipulating nucleic acid compositions are described in Green and Sambrook (Eds.), Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).
The disclosure provides, in part, a nucleic acid, e.g., vector, encoding a gene modifying polypeptide described herein, a template nucleic acid described herein, or both. In some embodiments, a vector comprises a selective marker, e.g., an antibiotic resistance marker. In some embodiments, the antibiotic resistance marker is a kanamycin resistance marker. In some embodiments, the antibiotic resistance marker does not confer resistance to beta-lactam antibiotics. In some embodiments, the vector does not comprise an ampicillin resistance marker. In some embodiments, the vector comprises a kanamycin resistance marker and does not comprise an ampicillin resistance marker. In some embodiments, a vector encoding a gene modifying polypeptide is integrated into a target cell genome (e.g., upon administration to a target cell, tissue, organ, or subject). In some embodiments, a vector encoding a gene modifying polypeptide is not integrated into a target cell genome (e.g., upon administration to a target cell, tissue, organ, or subject). In some embodiments, a vector encoding a template nucleic acid (e.g., template RNA) is not integrated into a target cell genome (e.g., upon administration to a target cell, tissue, organ, or subject). In some embodiments, if a vector is integrated into a target site in a target cell genome, the selective marker is not integrated into the genome. In some embodiments, if a vector is integrated into a target site in a target cell genome, genes or sequences involved in vector maintenance (e.g., plasmid maintenance genes) are not integrated into the genome. In some embodiments, if a vector is integrated into a target site in a target cell genome, transfer regulating sequences (e.g., inverted terminal repeats, e.g., from an AAV) are not integrated into the genome. In some embodiments, administration of a vector (e.g., encoding a gene modifying polypeptide described herein, a template nucleic acid described herein, or both) to a target cell, tissue, organ, or subject results in integration of a portion of the vector into one or more target sites in the genome(s) of said target cell, tissue, organ, or subject. In some embodiments, less than 99, 95, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1% of target sites (e.g., no target sites) comprising integrated material comprise a selective marker (e.g., an antibiotic resistance gene), a transfer regulating sequence (e.g., an inverted terminal repeat, e.g., from an AAV), or both from the vector.
Exemplary methods for producing a therapeutic pharmaceutical protein or polypeptide described herein involve expression in mammalian cells, although recombinant proteins can also be produced using insect cells, yeast, bacteria, or other cells under control of appropriate promoters. Mammalian expression vectors may comprise non-transcribed elements such as an origin of replication, a suitable promoter, and other 5′ or 3′ flanking non-transcribed sequences, and 5′ or 3′ non-translated sequences such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, splice, and polyadenylation sites may be used to provide other genetic elements required for expression of a heterologous DNA sequence. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).
Various mammalian cell culture systems can be employed to express and manufacture recombinant protein. Examples of mammalian expression systems include CHO, COS, HEK293, HeLA, and BHK cell lines. Processes of host cell culture for production of protein therapeutics are described in Zhou and Kantardjieff (Eds.), Mammalian Cell Cultures for Biologics Manufacturing (Advances in Biochemical Engineering/Biotechnology), Springer (2014). Compositions described herein may include a vector, such as a viral vector, e.g., a lentiviral vector, encoding a recombinant protein. In some embodiments, a vector, e.g., a viral vector, may comprise a nucleic acid encoding a recombinant protein.
Purification of protein therapeutics is described in Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and in Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010).
The disclosure also provides compositions and methods for the production of template nucleic acid molecules (e.g., template RNAs) with specificity for a gene modifying polypeptide and/or a genomic target site. In an aspect, the method comprises production of RNA segments including an upstream homology segment, a heterologous object sequence segment, a gene modifying polypeptide binding motif, and a gRNA segment.
Therapeutic Applications
In some embodiments, a gene modifying system as described herein can be used to modify a cell (e.g., an animal cell, plant cell, or fungal cell). In some embodiments, a gene modifying system as described herein can be used to modify a mammalian cell (e.g., a human cell). In some embodiments, a gene modifying system as described herein can be used to modify a cell from a livestock animal (e.g., a cow, horse, sheep, goat, pig, llama, alpaca, camel, yak, chicken, duck, goose, or ostrich). In some embodiments, a gene modifying system as described herein can be used as a laboratory tool or a research tool, or used in a laboratory method or research method, e.g., to modify an animal cell, e.g., a mammalian cell (e.g., a human cell), a plant cell, or a fungal cell.
By integrating coding genes into a RNA sequence template, the gene modifying system can address therapeutic needs, for example, by providing expression of a therapeutic transgene in individuals with loss-of-function mutations, by replacing gain-of-function mutations with normal transgenes, by providing regulatory sequences to eliminate gain-of-function mutation expression, and/or by controlling the expression of operably linked genes, transgenes and systems thereof. In certain embodiments, the RNA sequence template encodes a promotor region specific to the therapeutic needs of the host cell, for example a tissue specific promotor or enhancer. In still other embodiments, a promotor can be operably linked to a coding sequence.
Accordingly, provided herein are methods for treating phenylketonuria (PKU) or hyperphenylalaninemia (e.g., mild or severe hyperphenylalaninemia) in a subject in need thereof. In some embodiments, treatment results in amelioration of one or more symptoms associated with PKU or hyperphenylalaninemia.
In some embodiments, a system herein is used to treat a subject having a mutation in R408 (e.g., R408W), R261 (e.g., R261Q), R243 (e.g., R243Q), and/or IVS10-11G (e.g., IVS10-11G>A).
In some embodiments, treatment with a system disclosed herein results in correction of the R408W, R261Q, R243Q, and/or IVS10-11G>A mutation in between about 5-50% (e.g., about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, or about 10%) of cells. In some embodiments, treatment with a system disclosed herein results in correction of the R408W, R261Q, R243Q, and/or IVS10-11G>A mutation in between about 5-50% (e.g., about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, or about 10%) of DNA from the treated cells.
In some embodiments, treatment with a gene modifying system described herein results in one or more of:

    
    
        (a) an increase in phenylalanine hydroxylase (PAH) activity, efficiency, and/or function;
        (b) a decrease in the concentration of phenylalanine in the blood and/or cerebrospinal fluid;
        (c) increase in the concentration of tyrosine in the blood
        (d) a restoration of normal synthesis of dopamine, norepinephrine, and/or melanin;
        (e) a reduction in ureagenesis; and/or
        (f) an improvement in protein retention and/or Phe utilization

as compared to a subject having PKU that has not been treated with a gene modifying system described herein.

Administration and Delivery

    
    


The compositions and systems described herein may be used in vitro or in vivo. In some embodiments the system or components of the system are delivered to cells (e.g., mammalian cells, e.g., human cells), e.g., in vitro or in vivo. In some embodiments, the cells are eukaryotic cells, e.g., cells of a multicellular organism, e.g., an animal, e.g., a mammal (e.g., human, swine, bovine), a bird (e.g., poultry, such as chicken, turkey, or duck), or a fish. In some embodiments, the cells are non-human animal cells (e.g., a laboratory animal, a livestock animal, or a companion animal). In some embodiments, the cell is a stem cell (e.g., a hematopoietic stem cell), a fibroblast, or a T cell. In some embodiments, the cell is an immune cell, e.g., a T cell (e.g., a Treg, CD4, CD8, γδ, or memory T cell), B cell (e.g., memory B cell or plasma cell), or NK cell. In some embodiments, the cell is a non-dividing cell, e.g., a non-dividing fibroblast or non-dividing T cell. In some embodiments, the cell is an HSC and p53 is not upregulated or is upregulated by less than 10%, 5%, 2%, or 1%, e.g., as determined according to the method described in Example 30 of PCT/US2019/048607. The skilled artisan will understand that the components of the gene modifying system may be delivered in the form of polypeptide, nucleic acid (e.g., DNA, RNA), and combinations thereof.
In one embodiment the system and/or components of the system are delivered as nucleic acid. For example, the gene modifying polypeptide may be delivered in the form of a DNA or RNA encoding the polypeptide, and the template RNA may be delivered in the form of RNA or its complementary DNA to be transcribed into RNA. In some embodiments the system or components of the system are delivered on 1, 2, 3, 4, or more distinct nucleic acid molecules. In some embodiments the system or components of the system are delivered as a combination of DNA and RNA. In some embodiments the system or components of the system are delivered as a combination of DNA and protein. In some embodiments the system or components of the system are delivered as a combination of RNA and protein. In some embodiments the gene modifying polypeptide is delivered as a protein.
In some embodiments the system or components of the system are delivered to cells, e.g. mammalian cells or human cells, using a vector. The vector may be, e.g., a plasmid or a virus. In some embodiments, delivery is in vivo, in vitro, ex vivo, or in situ. In some embodiments the virus is an adeno associated virus (AAV), a lentivirus, or an adenovirus. In some embodiments the system or components of the system are delivered to cells with a viral-like particle or a virosome. In some embodiments the delivery uses more than one virus, viral-like particle or virosome.
In one embodiment, the compositions and systems described herein can be formulated in liposomes or other similar vesicles. Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes may be anionic, neutral or cationic. Liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB) (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review).
Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparation of multilamellar vesicle lipids are known in the art (see for example U.S. Pat. No. 6,693,086, the teachings of which relating to multilamellar vesicle lipid preparation are incorporated herein by reference). Although vesicle formation can be spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review). Extruded lipids can be prepared by extruding through filters of decreasing size, as described in Templeton et al., Nature Biotech, 15:647-652, 1997, the teachings of which relating to extruded lipid preparation are incorporated herein by reference.
A variety of nanoparticles can be used for delivery, such as a liposome, a lipid nanoparticle, a cationic lipid nanoparticle, an ionizable lipid nanoparticle, a polymeric nanoparticle, a gold nanoparticle, a dendrimer, a cyclodextrin nanoparticle, a micelle, or a combination of the foregoing.
Lipid nanoparticles are an example of a carrier that provides a biocompatible and biodegradable delivery system for the pharmaceutical compositions described herein. Nanostructured lipid carriers (NLCs) are modified solid lipid nanoparticles (SLNs) that retain the characteristics of the SLN, improve drug stability and loading capacity, and prevent drug leakage. Polymer nanoparticles (PNPs) are an important component of drug delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), a type of carrier that combines liposomes and polymers, may also be employed. These nanoparticles possess the complementary advantages of PNPs and liposomes. A PLN is composed of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell offers good biocompatibility. As such, the two components increase the drug encapsulation efficiency rate, facilitate surface modification, and prevent leakage of water-soluble drugs. For a review, see, e.g., Li et al. 2017, Nanomaterials 7, 122; doi:10.3390/nano7060122.
Exosomes can also be used as drug delivery vehicles for the compositions and systems described herein. For a review, see Ha et al. July 2016. Acta Pharmaceutica Sinica B. Volume 6, Issue 4, Pages 287-296; doi.org/10.1016/j.apsb.2016.02.001.
Fusosomes interact and fuse with target cells, and thus can be used as delivery vehicles for a variety of molecules. They generally consist of a bilayer of amphipathic lipids enclosing a lumen or cavity and a fusogen that interacts with the amphipathic lipid bilayer. The fusogen component has been shown to be engineerable in order to confer target cell specificity for the fusion and payload delivery, allowing the creation of delivery vehicles with programmable cell specificity (see for example Patent Application WO2020014209, the teachings of which relating to fusosome design, preparation, and usage are incorporated herein by reference).
In some embodiments, the protein component(s) of the gene modifying system may be pre-associated with the template nucleic acid (e.g., template RNA). For example, in some embodiments, the gene modifying polypeptide may be first combined with the template nucleic acid (e.g., template RNA) to form a ribonucleoprotein (RNP) complex. In some embodiments, the RNP may be delivered to cells via, e.g., transfection, nucleofection, virus, vesicle, LNP, exosome, fusosome.
A gene modifying system can be introduced into cells, tissues and multicellular organisms. In some embodiments the system or components of the system are delivered to the cells via mechanical means or physical means.
Formulation of protein therapeutics is described in Meyer (Ed.), Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).
Tissue Specific Activity/Administration
In some embodiments, a system described herein can make use of one or more feature (e.g., a promoter or microRNA binding site) to limit activity in off-target cells or tissues.
In some embodiments, a nucleic acid described herein (e.g., a template RNA or a DNA encoding a template RNA) comprises a promoter sequence, e.g., a tissue specific promoter sequence. In some embodiments, the tissue-specific promoter is used to increase the target-cell specificity of a gene modifying system. For instance, the promoter can be chosen on the basis that it is active in a target cell type but not active in (or active at a lower level in) a non-target cell type. Thus, even if the promoter integrated into the genome of a non-target cell, it would not drive expression (or only drive low level expression) of an integrated gene. A system having a tissue-specific promoter sequence in the template RNA may also be used in combination with a microRNA binding site, e.g., in the template RNA or a nucleic acid encoding a gene modifying protein, e.g., as described herein. A system having a tissue-specific promoter sequence in the template RNA may also be used in combination with a DNA encoding a gene modifying polypeptide, driven by a tissue-specific promoter, e.g., to achieve higher levels of gene modifying protein in target cells than in non-target cells. In some embodiments, e.g., for liver indications, a tissue-specific promoter is selected from Table 3 of WO2020014209, incorporated herein by reference.
In some embodiments, a nucleic acid described herein (e.g., a template RNA or a DNA encoding a template RNA) comprises a microRNA binding site. In some embodiments, the microRNA binding site is used to increase the target-cell specificity of a gene modifying system. For instance, the microRNA binding site can be chosen on the basis that is recognized by a miRNA that is present in a non-target cell type, but that is not present (or is present at a reduced level relative to the non-target cell) in a target cell type. Thus, when the template RNA is present in a non-target cell, it would be bound by the miRNA, and when the template RNA is present in a target cell, it would not be bound by the miRNA (or bound but at reduced levels relative to the non-target cell). While not wishing to be bound by theory, binding of the miRNA to the template RNA may interfere with its activity, e.g., may interfere with insertion of the heterologous object sequence into the genome. Accordingly, the system would edit the genome of target cells more efficiently than it edits the genome of non-target cells, e.g., the heterologous object sequence would be inserted into the genome of target cells more efficiently than into the genome of non-target cells, or an insertion or deletion is produced more efficiently in target cells than in non-target cells. A system having a microRNA binding site in the template RNA (or DNA encoding it) may also be used in combination with a nucleic acid encoding a gene modifying polypeptide, wherein expression of the gene modifying polypeptide is regulated by a second microRNA binding site, e.g., as described herein. In some embodiments, e.g., for liver indications, a miRNA is selected from Table 4 of WO2020014209, incorporated herein by reference.
In some embodiments, the template RNA comprises a microRNA sequence, an siRNA sequence, a guide RNA sequence, or a piwi RNA sequence.
Promoters
In some embodiments, one or more promoter or enhancer elements are operably linked to a nucleic acid encoding a gene modifying protein or a template nucleic acid, e.g., that controls expression of the heterologous object sequence. In certain embodiments, the one or more promoter or enhancer elements comprise cell-type or tissue specific elements. In some embodiments, the promoter or enhancer is the same or derived from the promoter or enhancer that naturally controls expression of the heterologous object sequence. For example, the ornithine transcarbomylase promoter and enhancer may be used to control expression of the ornithine transcarbomylase gene in a system or method provided by the invention for correcting ornithine transcarbomylase deficiencies. In some embodiments, the promoter is a promoter of Table 16 or 17 or a functional fragment or variant thereof.
Exemplary tissue specific promoters that are commercially available can be found, for example, at a uniform resource locator (e.g., invivogen.com/tissue-specific-promoters). In some embodiments, a promoter is a native promoter or a minimal promoter, e.g., which consists of a single fragment from the 5′ region of a given gene. In some embodiments, a native promoter comprises a core promoter and its natural 5′ UTR. In some embodiments, the 5′ UTR comprises an intron. In other embodiments, these include composite promoters, which combine promoter elements of different origins or were generated by assembling a distal enhancer with a minimal promoter of the same origin.
Exemplary cell or tissue specific promoters are provided in the tables, below, and exemplary nucleic acid sequences encoding them are known in the art and can be readily accessed using a variety of resources, such as the NCBI database, including RefSeq, as well as the Eukaryotic Promoter Database (//epd.epfl.ch//index.php).







TABLE 16







Exemplary cell or tissue-specific promoters










Promoter
Target cells







B29 Promoter
B cells



CD14 Promoter
Monocytic Cells



CD43 Promoter
Leukocytes and platelets



CD45 Promoter
Hematopoeitic cells



CD68 promoter
macrophages



Desmin promoter
muscle cells



Elastase-1
pancreatic acinar cells



promoter




Endoglin promoter
endothelial cells



fibronectin
differentiating cells, healing



promoter
tissue



Flt-1 promoter
endothelial cells



GFAP promoter
Astrocytes



GPIIB promoter
megakaryocytes



ICAM-2 Promoter
Endothelial cells



INF-Beta promoter
Hematopoeitic cells



Mb promoter
muscle cells



Nphs1 promoter
podocytes



OG-2 promoter
Osteoblasts, Odonblasts



SP-B promoter
Lung



Syn1 promoter
Neurons



WASP promoter
Hematopoeitic cells



SV40/bAlb
Liver



promoter




SV40/bAlb
Liver



promoter




SV40/Cd3
Leukocytes and platelets



promoter




SV40/CD45
hematopoeitic cells



promoter




NSE/RU5′
Mature Neurons



promoter

















TABLE 17







Additional exemplary cell or tissue-specific promoters









Promoter
Gene Description
Gene Specificity





APOA2
Apolipoprotein A-II
Hepatocytes (from hepatocyte




progenitors)


SERPINA
Serpin peptidase inhibitor, clade A
Hepatocytes


1 (hAAT)
(alpha-1
(from definitive endoderm



antiproteinase, antitrypsin), member 1
stage)



(also named alpha 1 anti-tryps in)



CYP3A
Cytochrome P450, family 3,
Mature Hepatocytes



subfamily A, polypeptide



MIR122
MicroRNA 122
Hepatocytes




(from early stage embryonic




liver cells)




and endoderm







Pancreatic specific promoters









INS
Insulin
Pancreatic beta cells




(from definitive endoderm stage)


IRS2
Insulin receptor substrate 2
Pancreatic beta cells


Pdx1
Pancreatic and duodenal
Pancreas



homeobox 1
(from definitive endoderm stage)


Alx3
Aristaless-like homeobox 3
Pancreatic beta cells




(from definitive endoderm stage)


Ppy
Pancreatic polypeptide
PP pancreatic cells




(gamma cells)







Cardiac specific promoters









Myh6
Myosin, heavy chain 6, cardiac
Late differentiation marker of cardiac


(aMHC)
muscle, alpha
muscle cells (atrial specificity)


MYL2
Myosin, light chain 2, regulatory,
Late differentiation marker of cardiac


(MLC-2v)
cardiac, slow
muscle cells (ventricular specificity)


ITNN13
Troponin I type 3 (cardiac)
Cardiomyocytes


(cTnl)

(from immature state)


ITNN13
Troponin I type 3 (cardiac)
Cardiomyocytes


(cTnl)

(from immature state)


NPPA
Natriuretic peptide precursor A (also
Atrial specificity in adult cells


(ANF)
named Atrial Natriuretic Factor)



Slc8a1
Solute carrier family 8
Cardiomyocytes from early


(Ncx1)
(sodium/calcium exchanger), member
developmental stages



1








CNS specific promoters









SYN1
Synapsin I
Neurons


(hSyn)




GFAP
Glial fibrillary acidic protein
Astrocytes


INA
Internexin neuronal intermediate
Neuroprogenitors



filament protein, alpha (a-internexin)



NES
Nestin
Neuroprogenitors and ectoderm


MOBP
Myelin-associated oligodendrocyte
Oligodendrocytes



basic protein



MBP
Myelin basic protein
Oligodendrocytes


TH
Tyrosine hydroxylase
Dopaminergic neurons


FOXA2
Forkhead box A2
Dopaminergic neurons (also used as a


(HNF3

marker of endoderm)


beta)









Skin specific promoters









FLG
Filaggrin
Keratinocytes from granular layer


K14
Keratin 14
Keratinocytes from granular




and basal layers


TGM3
Transglutaminase 3
Keratinocytes from granular layer







Immune cell specific promoters









ITGAM
Integrin, alpha M (complement
Monocytes, macrophages, granulocytes,


(CD11B)
component 3 receptor 3 subunit)
natural killer cells







Urogential cell specific promoters









Pbsn
Probasin
Prostatic epithelium


Upk2
Uroplakin 2
Bladder


Sbp
Spermine binding protein
Prostate


Fer114
Fer-1-like 4
Bladder







Endothelial cell specific promoters









ENG
Endoglin
Endothelial cells







Pluripotent and embryonic cell specific promoters









Oct4
POU class 5 homeobox 1
Pluripotent cells


(POU5F1)

(germ cells, ES cells, iPS cells)


NANOG
Nanog homeobox
Pluripotent cells




(ES cells, iPS cells)


Synthetic
Synthetic promoter based on a Oct-4
Pluripotent cells (ES cells, iPS cells)


Oct4
core enhancer element



T
Brachyury
Mesoderm


brachyury




NES
Nestin
Neuroprogenitors and Ectoderm


SOX17
SRY (sex determining region Y)-box
Endoderm



17



FOXA2
Forkhead box A2
Endoderm (also used as a marker of


(HNFJ

dopaminergic neurons)


beta)




MIR122
MicroRNA 122
Endoderm and hepatocytes




(from early stage embryonic liver cells~









Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544; incorporated herein by reference in its entirety).
In some embodiments, a nucleic acid encoding a gene modifying protein or template nucleic acid is operably linked to a control element, e.g., a transcriptional control element, such as a promoter. The transcriptional control element may, in some embodiment, be functional in either a eukaryotic cell, e.g., a mammalian cell; or a prokaryotic cell (e.g., bacterial or archaeal cell). In some embodiments, a nucleotide sequence encoding a polypeptide is operably linked to multiple control elements, e.g., that allow expression of the nucleotide sequence encoding the polypeptide in both prokaryotic and eukaryotic cells.
For illustration purposes, examples of spatially restricted promoters include, but are not limited to, neuron-specific promoters, adipocyte-specific promoters, cardiomyocyte-specific promoters, smooth muscle-specific promoters, photoreceptor-specific promoters, etc. Neuron-specific spatially restricted promoters include, but are not limited to, a neuron-specific enolase (NSE) promoter (see, EMBL HSENO2, X51956); an aromatic amino acid decarboxylase (AADC) promoter, a neurofilament promoter (see, e.g., GenBank HUMNFL, L04147); a synapsin promoter (see, e.g., GenBank HUMSYNIB, M55301); a thy-1 promoter (see, e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn, et al. (2010) Nat. Med. 16(10):1161-1166); a serotonin receptor promoter (see, e.g., GenBank S62283); a tyrosine hydroxylase promoter (TH) (see, e.g., Oh et al. (2009) Gene Ther 16:437; Sasaoka et at (1992) Mol. Brain Res. 16:274; Boundy et al. (1998) J. Neurosci. 18:9989; and Kaneda et al. (1991) Neuron 6:583-594); a GnRH promoter (see, e.g., Radovick et al. (1991) Proc. Natl. Acad. Sci. USA 88:3402-3406); an L7 promoter (see, e.g., Oberdick et al. (1990) Science 248:223-226); a DNMT promoter (see, e.g., Bartge et al. (1988) Proc. Natl. Acad. Sci. USA 85:3648-3652); Ern enkephalin promoter (see, e.g., Comb et al. (1988) EMBO J. 117:3793-3805); a myelin basic protein (MBP) promoter; a Ca2+-calmodulin-dependent protein kinase II-alpha (CamKIIα) promoter (see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93:13250; and Casanova et al. (2001) Genesis 31:37); a CMV enhancer/platelet-derived growth factor-β promoter (see, e.g., Liu et al. (2004) Gene Therapy 11:52-60); and the like.
Adipocyte-specific spatially restricted promoters include, but are not limited to, the aP2 gene promoter/enhancer, e.g., a region from −5.4 kb to +21 bp of a human aP2 gene (see, e.g., Tozzo et al. (1997) Endocrinol. 138:1604; Ross et al. (1990) Proc. Natl. Acad. Sci. USA 87:9590; and Pavjani et al. (2005) Nat. Med. 11:797); a glucose transporter-4 (GLUT4) promoter (see, e.g., Knight et al. (2003) Proc. Natl. Acad. Sci. USA 100:14725); a fatty acid translocase (FAT/CD36) promoter (see, e.g., Kuriki et al. (2002) Biol. Pharm. Bull. 25:1476; and Sato et al. (2002) J. Biol. Chem. 277:15703); a stearoyl-CoA desaturase-1 (SCD1) promoter (Libor et al. (1999) J. Biol. Chem. 274:20603); a leptin promoter (see, e.g., Mason et al. (1998) Endocrinol. 139:1013; and Chen et al. (1999) Biochem. Biophys. Res. Comm. 262:187); an adiponectin promoter (see, e.g., Kita et al. (2005) Biochem. Biophys. Res. Comm. 331:484; and Chakrabarti (2010) Endocrinol. 151:2408); an adipsin promoter (see, e.g., Platt et al. (1989) Proc. Natl. Acad. Sci. USA 86:7490); a resistin promoter (see, e.g., Seo et al. (2003) Molec. Endocrinol. 17:1522); and the like.
Cardiomyocyte-specific spatially restricted promoters include, but are not limited to, control sequences derived from the following genes: myosin light chain-2, α-myosin heavy chain, AE3, cardiac troponin C, cardiac actin, and the like. Franz et al. (1997) Cardiovasc. Res. 35:560-566; Robbins et al. (1995) Ann. N.Y. Acad. Sci. 752:492-50:5; Linn et al. (1995) Circ. Res. 76:584-591; Parmacek et al. (1994) Mol. Cell. Biol. 14:1870-1885; Hunter et al. (1993) Hypertension 22:608-617; and Sartoreili et al. (1992) Proc. Natl. Acad. Sci. USA 89:4047-4051.
Smooth muscle-specific spatially restricted promoters include, but are not limited to, an SM22α promoter (see, e.g., Akyürek et al. (2000) Mol. Med. 6:983; and U.S. Pat. No. 7,169,874); a smoothelin promoter (see, e.g., WO2001/018048); an α-smooth muscle actin promoter; and the like. For example, a 0.4 kb region of the SM22α promoter, within which lie two CArG elements, has been shown to mediate vascular smooth muscle cell-specific expression (see, e.g., Kim, et al. (1997) Mol. Cell. Biol. 17, 2266-2278; Li, et al., (1996) J. Cell Biol. 132, 849-859; and Moessler, et al. (1996) Development 122, 2415-2425).
Photoreceptor-specific spatially restricted promoters include, but are not limited to, a rhodopsin promoter; a rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Via. Sci. 44:4076); a beta phosphodiesterase gene promoter (Nicoud et at (2007) J. Gene Med. 9:1015); a retinitis pigmentosa gene promoter (Nicoud et al. (2007) supra); an interphotoreceptor retinoid-binding protein (IMP) gene enhancer (Nicoud et al. (2007) supra); an IRBP gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225); and the like.
In some embodiments, a gene modifying system, e.g., DNA encoding a gene modifying polypeptide, DNA encoding a template RNA, or DNA or RNA encoding a heterologous object sequence, is designed such that one or more elements is operably linked to a tissue-specific promoter, e.g., a promoter that is active in T-cells. In further embodiments, the T-cell active promoter is inactive in other cell types, e.g., B-cells, NK cells. In some embodiments, the T-cell active promoter is derived from a promoter for a gene encoding a component of the T-cell receptor, e.g., TRAC, TRBC, TRGC, TRDC. In some embodiments, the T-cell active promoter is derived from a promoter for a gene encoding a component of a T-cell-specific cluster of differentiation protein, e.g., CD3, e.g., CD3D, CD3E, CD3G, CD3Z. In some embodiments, T-cell-specific promoters in gene modifying systems are discovered by comparing publicly available gene expression data across cell types and selecting promoters from the genes with enhanced expression in T-cells. In some embodiments, promoters may be selecting depending on the desired expression breadth, e.g., promoters that are active in T-cells only, promoters that are active in NK cells only, promoters that are active in both T-cells and NK cells.
Cell-specific promoters known in the art may be used to direct expression of a gene modifying protein, e.g., as described herein. Nonlimiting exemplary mammalian cell-specific promoters have been characterized and used in mice expressing Cre recombinase in a cell-specific manner. Certain nonlimiting exemplary mammalian cell-specific promoters are listed in Table 1 of U.S. Pat. No. 9,845,481, incorporated herein by reference.
In some embodiments, a vector as described herein comprises an expression cassette. Typically, an expression cassette comprises the nucleic acid molecule of the instant invention operatively linked to a promoter sequence. For example, a promoter is operatively linked with a coding sequence when it is capable of affecting the expression of that coding sequence (e.g., the coding sequence is under the transcriptional control of the promoter). Encoding sequences can be operatively linked to regulatory sequences in sense or antisense orientation. In certain embodiments, the promoter is a heterologous promoter. In certain embodiments, an expression cassette may comprise additional elements, for example, an intron, an enhancer, a polyadenylation site, a woodchuck response element (WRE), and/or other elements known to affect expression levels of the encoding sequence. A promoter typically controls the expression of a coding sequence or functional RNA. In certain embodiments, a promoter sequence comprises proximal and more distal upstream elements and can further comprise an enhancer element. An enhancer can typically stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. In certain embodiments, the promoter is derived in its entirety from a native gene. In certain embodiments, the promoter is composed of different elements derived from different naturally occurring promoters. In certain embodiments, the promoter comprises a synthetic nucleotide sequence. It will be understood by those skilled in the art that different promoters will direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions or to the presence or the absence of a drug or transcriptional co-factor. Ubiquitous, cell-type-specific, tissue-specific, developmental stage-specific, and conditional promoters, for example, drug-responsive promoters (e.g., tetracycline-responsive promoters) are well known to those of skill in the art. Exemplary promoters include, but are not limited to, the phosphoglycerate kinase (PKG) promoter, CAG (composite of the CMV enhancer the chicken beta actin promoter (CBA) and the rabbit beta globin intron), NSF (neuronal specific enolase), synapsin or NeuN promoters, the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP), a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), SFFV promoter, rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. Other promoters can be of human origin or from other species, including from mice. Common promoters include, e.g., the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, [beta]-actin, rat insulin promoter, the phosphoglycerate kinase promoter, the human alpha-1 antitrypsin (hAAT) promoter, the transthyretin promoter, the TBG promoter and other liver-specific promoters, the desmin promoter and similar muscle-specific promoters, the EF1-alpha promoter, hybrid promoters with multi-tissue specificity, promoters specific for neurons like synapsin and glyceraldehyde-3-phosphate dehydrogenase promoter, all of which are promoters well known and readily available to those of skill in the art, can be used to obtain high-level expression of the coding sequence of interest. In addition, sequences derived from non-viral genes, such as the murine metallothionein gene, will also find use herein. Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, CA). Additional exemplary promoter sequences are described, for example, in WO2018213786A1 (incorporated by reference herein in its entirety).
In some embodiments, the apolipoprotein E enhancer (ApoE) or a functional fragment thereof is used, e.g., to drive expression in the liver. In some embodiments, two copies of the ApoE enhancer or a functional fragment thereof are used. In some embodiments, the ApoE enhancer or functional fragment thereof is used in combination with a promoter, e.g., the human alpha-1 antitrypsin (hAAT) promoter.
In some embodiments, the regulatory sequences impart tissue-specific gene expression capabilities. In some cases, the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner. Various tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: a liver-specific thyroxin binding globulin (TBG) promoter, a insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, a α-myosin heavy chain (α-MHC) promoter, or a cardiac Troponin (cTnT) promoter. Other exemplary promoters include Beta-actin promoter, hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 241185-96 (1997)); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998); immunoglobulin heavy chain promoter; T cell receptor α-chain promoter, neuronal such as neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), and others. Additional exemplary promoter sequences are described, for example, in U.S. Pat. No. 10,300,146 (incorporated herein by reference in its entirety). In some embodiments, a tissue-specific regulatory element, e.g., a tissue-specific promoter, is selected from one known to be operably linked to a gene that is highly expressed in a given tissue, e.g., as measured by RNA-seq or protein expression data, or a combination thereof. Methods for analyzing tissue specificity by expression are taught in Fagerberg et al. Mol Cell Proteomics 13(2):397-406 (2014), which is incorporated herein by reference in its entirety.
In some embodiments, a vector described herein is a multicistronic expression construct. Multicistronic expression constructs include, for example, constructs harboring a first expression cassette, e.g. comprising a first promoter and a first encoding nucleic acid sequence, and a second expression cassette, e.g. comprising a second promoter and a second encoding nucleic acid sequence. Such multicistronic expression constructs may, in some instances, be particularly useful in the delivery of non-translated gene products, such as hairpin RNAs, together with a polypeptide, for example, a gene modifying polypeptide and gene modifying template. In some embodiments, multicistronic expression constructs may exhibit reduced expression levels of one or more of the included transgenes, for example, because of promoter interference or the presence of incompatible nucleic acid elements in close proximity. If a multicistronic expression construct is part of a viral vector, the presence of a self-complementary nucleic acid sequence may, in some instances, interfere with the formation of structures necessary for viral reproduction or packaging.
In some embodiments, the sequence encodes an RNA with a hairpin. In some embodiments, the hairpin RNA is a guide RNA, a template RNA, a shRNA, or a microRNA. In some embodiments, the first promoter is an RNA polymerase I promoter. In some embodiments, the first promoter is an RNA polymerase II promoter. In some embodiments, the second promoter is an RNA polymerase III promoter. In some embodiments, the second promoter is a U6 or H1 promoter.
Without wishing to be bound by theory, multicistronic expression constructs may not achieve optimal expression levels as compared to expression systems containing only one cistron. One of the suggested causes of lower expression levels achieved with multicistronic expression constructs comprising two or more promoter elements is the phenomenon of promoter interference (see, e.g., Curtin J A, Dane A P, Swanson A, Alexander I E, Ginn S L. Bidirectional promoter interference between two widely used internal heterologous promoters in a late-generation lentiviral construct. Gene Ther. 2008 March; 15(5):384-90; and Martin-Duque P, Jezzard S, Kaftansis L, Vassaux G. Direct comparison of the insulating properties of two genetic elements in an adenoviral vector containing two different expression cassettes. Hum Gene Ther. 2004 October; 15(10):995-1002; both references incorporated herein by reference for disclosure of promoter interference phenomenon. In some embodiments, the problem of promoter interference may be overcome, e.g., by producing multicistronic expression constructs comprising only one promoter driving transcription of multiple encoding nucleic acid sequences separated by internal ribosomal entry sites, or by separating cistrons comprising their own promoter with transcriptional insulator elements. In some embodiments, single-promoter driven expression of multiple cistrons may result in uneven expression levels of the cistrons. In some embodiments, a promoter cannot efficiently be isolated and isolation elements may not be compatible with some gene transfer vectors, for example, some retroviral vectors.
MicroRNAs
MicroRNAs (miRNAs) and other small interfering nucleic acids generally regulate gene expression via target RNA transcript cleavage/degradation or translational repression of the target messenger RNA (mRNA). miRNAs may, in some instances, be natively expressed, typically as final 19-25 non-translated RNA products. miRNAs generally exhibit their activity through sequence-specific interactions with the 3′ untranslated regions (UTR) of target mRNAs. These endogenously expressed miRNAs may form hairpin precursors that are subsequently processed into an miRNA duplex, and further into a mature single stranded miRNA molecule This mature miRNA generally guides a muitiprotein complex, miRISC, which identifies target 3′ UTR regions of target mRNAs based upon their complementarity to the mature miRNA. Useful transgene products may include, for example, miRNAs or miRNA binding sites that regulate the expression of a linked polypeptide. A non-limiting list of miRNA genes; the products of these genes and their homologues are useful as transgenes or as targets for small interfering nucleic acids (e.g., miRNA sponges, antisense oligonucleotides), e.g., in methods such as those listed in U.S. Ser. No. 10/300,146, 22:25-25:48, are herein incorporated by reference. In some embodiments, one or more binding sites for one or more of the foregoing miRNAs are incorporated in a transgene, e.g., a transgene delivered by a rAAV vector, e.g., to inhibit the expression of the transgene in one or more tissues of an animal harboring the transgene. In some embodiments, a binding site may be selected to control the expression of a transgene in a tissue specific manner. For example, binding sites for the liver-specific miR-122 may be incorporated into a transgene to inhibit expression of that transgene in the liver. Additional exemplary miRNA sequences are described, for example, in U.S. Pat. No. 10,300,146 (incorporated herein by reference in its entirety).
An miR inhibitor or miRNA inhibitor is generally an agent that blocks miRNA expression and/or processing. Examples of such agents include, but are not limited to, microRNA antagonists, microRNA specific antisense, microRNA sponges, and microRNA oligonucleotides (double-stranded, hairpin, short oligonucleotides) that inhibit miRNA, interaction with a Drosha complex. MicroRNA inhibitors, e.g., miRNA sponges, can be expressed in cells from transgenes (e.g., as described in Ebert, M. S. Nature Methods, Epub Aug. 12, 2007; incorporated by reference herein in its entirety). In some embodiments, microRNA sponges, or other miR inhibitors, are used with the AAVs. microRNA sponges generally specifically inhibit miRNAs through a complementary heptameric seed sequence. In some embodiments, an entire family of miRNAs can be silenced using a single sponge sequence. Other methods for silencing miRNA function (derepression of miRNA targets) in cells will be apparent to one of ordinary skill in the art.
In some embodiments, a gene modifying system, template RNA, or polypeptide described herein is administered to or is active in (e.g., is more active in) a target tissue, e.g., a first tissue. In some embodiments, the gene modifying system, template RNA, or polypeptide is not administered to or is less active in (e.g., not active in) a non-target tissue. In some embodiments, a gene modifying system, template RNA, or polypeptide described herein is useful for modifying DNA in a target tissue, e.g., a first tissue, (e.g., and not modifying DNA in a non-target tissue).
In some embodiments, a gene modifying system comprises (a) a polypeptide described herein or a nucleic acid encoding the same, (b) a template nucleic acid (e.g., template RNA) described herein, and (c) one or more first tissue-specific expression-control sequences specific to the target tissue, wherein the one or more first tissue-specific expression-control sequences specific to the target tissue are in operative association with (a), (b), or (a) and (b), wherein, when associated with (a), (a) comprises a nucleic acid encoding the polypeptide.
In some embodiments, the nucleic acid in (b) comprises RNA.
In some embodiments, the nucleic acid in (b) comprises DNA.
In some embodiments, the nucleic acid in (b): (i) is single-stranded or comprises a single-stranded segment, e.g., is single-stranded DNA or comprises a single-stranded segment and one or more double stranded segments; (ii) has inverted terminal repeats; or (iii) both (i) and (ii).
In some embodiments, the nucleic acid in (b) is double-stranded or comprises a double-stranded segment.
In some embodiments, (a) comprises a nucleic acid encoding the polypeptide.
In some embodiments, the nucleic acid in (a) comprises RNA.
In some embodiments, the nucleic acid in (a) comprises DNA.
In some embodiments, the nucleic acid in (a): (i) is single-stranded or comprises a single-stranded segment, e.g., is single-stranded DNA or comprises a single-stranded segment and one or more double stranded segments; (ii) has inverted terminal repeats; or (iii) both (i) and (ii).
In some embodiments, the nucleic acid in (a) is double-stranded or comprises a double-stranded segment.
In some embodiments, the nucleic acid in (a), (b), or (a) and (b) is linear.
In some embodiments, the nucleic acid in (a), (b), or (a) and (b) is circular, e.g., a plasmid or minicircle.
In some embodiments, the heterologous object sequence is in operative association with a first promoter.
In some embodiments, the one or more first tissue-specific expression-control sequences comprises a tissue specific promoter.
In some embodiments, the tissue-specific promoter comprises a first promoter in operative association with: (i) the heterologous object sequence, (ii) a nucleic acid encoding the retroviral RT, or (iii) (i) and (ii).
In some embodiments, the one or more first tissue-specific expression-control sequences comprises a tissue-specific microRNA recognition sequence in operative association with: (i) the heterologous object sequence, (ii) a nucleic acid encoding the retroviral RT domain, or (iii) (i) and (ii).
In some embodiments, a system comprises a tissue-specific promoter, and the system further comprises one or more tissue-specific microRNA recognition sequences, wherein: (i) the tissue specific promoter is in operative association with: (I) the heterologous object sequence, (II) a nucleic acid encoding the retroviral RT domain, or (III) (I) and (II); and/or (ii) the one or more tissue-specific microRNA recognition sequences are in operative association with: (I) the heterologous object sequence, (II) a nucleic acid encoding the retroviral RT, or (III) (I) and (II).
In some embodiments, wherein (a) comprises a nucleic acid encoding the polypeptide, the nucleic acid comprises a promoter in operative association with the nucleic acid encoding the polypeptide.
In some embodiments, the nucleic acid encoding the polypeptide comprises one or more second tissue-specific expression-control sequences specific to the target tissue in operative association with the polypeptide coding sequence.
In some embodiments, the one or more second tissue-specific expression-control sequences comprises a tissue specific promoter.
In some embodiments, the tissue-specific promoter is the promoter in operative association with the nucleic acid encoding the polypeptide.
In some embodiments, the one or more second tissue-specific expression-control sequences comprises a tissue-specific microRNA recognition sequence.
In some embodiments, the promoter in operative association with the nucleic acid encoding the polypeptide is a tissue-specific promoter, the system further comprising one or more tissue-specific microRNA recognition sequences.
In some embodiments, a nucleic acid component of a system provided by the invention is a sequence (e.g., encoding the polypeptide or comprising a heterologous object sequence) flanked by untranslated regions (UTRs) that modify protein expression levels. Various 5′ and 3′ UTRs can affect protein expression. For example, in some embodiments, the coding sequence may be preceded by a 5′ UTR that modifies RNA stability or protein translation. In some embodiments, the sequence may be followed by a 3′ UTR that modifies RNA stability or translation. In some embodiments, the sequence may be preceded by a 5′ UTR and followed by a 3′ UTR that modify RNA stability or translation. In some embodiments, the 5′ and/or 3′ UTR may be selected from the 5′ and 3′ UTRs of complement factor 3 (C3) (CACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAG CACC; SEQ ID NO: 11,004) or orosomucoid 1 (ORM1) (CAGGACACAGCCTTGGATCAGGACAGAGACTTGGGGGCCATCCTGCCCCTCCAACC CGACATGTGTACCTCAGCTTTTTCCCTCACTTGCATCAATAAAGCTTCTGTGTTTGGA ACAGCTAA; SEQ ID NO: 11,005) (Asrani et al. RNA Biology 2018). In certain embodiments, the 5′ UTR is the 5′ UTR from C3 and the 3′ UTR is the 3′ UTR from ORM1. In certain embodiments, a 5′ UTR and 3′ UTR for protein expression, e.g., mRNA (or DNA encoding the RNA) for a gene modifying polypeptide or heterologous object sequence, comprise optimized expression sequences. In some embodiments, the 5′ UTR comprises GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 11,006) and/or the 3′ UTR comprising UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA (SEQ ID NO: 11,007), e.g., as described in Richner et al. Cell 168(6): P1114-1125 (2017), the sequences of which are incorporated herein by reference. In some embodiments, a 5′ and/or 3′ UTR may be selected to enhance protein expression. In some embodiments, a 5′ and/or 3′ UTR may be selected to modify protein expression such that overproduction inhibition is minimized. In some embodiments, UTRs are around a coding sequence, e.g., outside the coding sequence and in other embodiments proximal to the coding sequence. In some embodiments, additional regulatory elements (e.g., miRNA binding sites, cis-regulatory sites) are included in the UTRs.
In some embodiments, an open reading frame of a gene modifying system, e.g., an ORF of an mRNA (or DNA encoding an mRNA) encoding a gene modifying polypeptide or one or more ORFs of an mRNA (or DNA encoding an mRNA) of a heterologous object sequence, is flanked by a 5′ and/or 3′ untranslated region (UTR) that enhances the expression thereof. In some embodiments, the 5′ UTR of an mRNA component (or transcript produced from a DNA component) of the system comprises the sequence 5′-GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC-3′; SEQ ID NO: 11,008). In some embodiments, the 3′ UTR of an mRNA component (or transcript produced from a DNA component) of the system comprises the sequence 5′-UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA-3′ (SEQ ID NO: 11,009). This combination of 5′ UTR and 3′ UTR has been shown to result in desirable expression of an operably linked ORF by Richner et al. Cell 168(6): P1114-1125 (2017), the teachings and sequences of which are incorporated herein by reference. In some embodiments, a system described herein comprises a DNA encoding a transcript, wherein the DNA comprises the corresponding 5′ UTR and 3′ UTR sequences, with T substituting for U in the above-listed sequence). In some embodiments, a DNA vector used to produce an RNA component of the system further comprises a promoter upstream of the 5′ UTR for initiating in vitro transcription, e.g, a T7, T3, or SP6 promoter. The 5′ UTR above begins with GGG, which is a suitable start for optimizing transcription using T7 RNA polymerase. For tuning transcription levels and altering the transcription start site nucleotides to fit alternative 5′ UTRs, the teachings of Davidson et al. Pac Symp Biocomput 433-443 (2010) describe T7 promoter variants, and the methods of discovery thereof, that fulfill both of these traits.
Viral Vectors and Components Thereof
Viruses are a useful source of delivery vehicles for the systems described herein, in addition to a source of relevant enzymes or domains as described herein, e.g., as sources of polymerases and polymerase functions used herein, e.g., DNA-dependent DNA polymerase, RNA-dependent RNA polymerase, RNA-dependent DNA polymerase, DNA-dependent RNA polymerase, reverse transcriptase. Some enzymes, e.g., reverse transcriptases, may have multiple activities, e.g., be capable of both RNA-dependent DNA polymerization and DNA-dependent DNA polymerization, e.g., first and second strand synthesis. In some embodiments, the virus used as a gene modifying delivery system or a source of components thereof may be selected from a group as described by Baltimore Bacteriol Rev 35(3):235-241 (1971).
In some embodiments, the virus is selected from a Group I virus, e.g., is a DNA virus and packages dsDNA into virions. In some embodiments, the Group I virus is selected from, e.g., Adenoviruses, Herpesviruses, Poxviruses.
In some embodiments, the virus is selected from a Group II virus, e.g., is a DNA virus and packages ssDNA into virions. In some embodiments, the Group II virus is selected from, e.g., Parvoviruses. In some embodiments, the parvovirus is a dependoparvovirus, e.g., an adeno-associated virus (AAV).
In some embodiments, the virus is selected from a Group III virus, e.g., is an RNA virus and packages dsRNA into virions. In some embodiments, the Group III virus is selected from, e.g., Reoviruses. In some embodiments, one or both strands of the dsRNA contained in such virions is a coding molecule able to serve directly as mRNA upon transduction into a host cell, e.g., can be directly translated into protein upon transduction into a host cell without requiring any intervening nucleic acid replication or polymerization steps.
In some embodiments, the virus is selected from a Group IV virus, e.g., is an RNA virus and packages ssRNA(+) into virions. In some embodiments, the Group IV virus is selected from, e.g., Coronaviruses, Picornaviruses, Togaviruses. In some embodiments, the ssRNA(+) contained in such virions is a coding molecule able to serve directly as mRNA upon transduction into a host cell, e.g., can be directly translated into protein upon transduction into a host cell without requiring any intervening nucleic acid replication or polymerization steps.
In some embodiments, the virus is selected from a Group V virus, e.g., is an RNA virus and packages ssRNA(−) into virions. In some embodiments, the Group V virus is selected from, e.g., Orthomyxoviruses, Rhabdoviruses. In some embodiments, an RNA virus with an ssRNA(−) genome also carries an enzyme inside the virion that is transduced to host cells with the viral genome, e.g., an RNA-dependent RNA polymerase, capable of copying the ssRNA(−) into ssRNA(+) that can be translated directly by the host.
In some embodiments, the virus is selected from a Group VI virus, e.g., is a retrovirus and packages ssRNA(+) into virions. In some embodiments, the Group VI virus is selected from, e.g., retroviruses. In some embodiments, the retrovirus is a lentivirus, e.g., HIV-1, HIV-2, SIV, BIV. In some embodiments, the retrovirus is a spumavirus, e.g., a foamy virus, e.g., HFV, SFV, BFV. In some embodiments, the ssRNA(+) contained in such virions is a coding molecule able to serve directly as mRNA upon transduction into a host cell, e.g., can be directly translated into protein upon transduction into a host cell without requiring any intervening nucleic acid replication or polymerization steps. In some embodiments, the ssRNA(+) is first reverse transcribed and copied to generate a dsDNA genome intermediate from which mRNA can be transcribed in the host cell. In some embodiments, an RNA virus with an ssRNA(+) genome also carries an enzyme inside the virion that is transduced to host cells with the viral genome, e.g., an RNA-dependent DNA polymerase, capable of copying the ssRNA(+) into dsDNA that can be transcribed into mRNA and translated by the host. In some embodiments, the reverse transcriptase from a Group VI retrovirus is incorporated as the reverse transcriptase domain of a gene modifying polypeptide.
In some embodiments, the virus is selected from a Group VII virus, e.g., is a retrovirus and packages dsRNA into virions. In some embodiments, the Group VII virus is selected from, e.g., Hepadnaviruses. In some embodiments, one or both strands of the dsRNA contained in such virions is a coding molecule able to serve directly as mRNA upon transduction into a host cell, e.g., can be directly translated into protein upon transduction into a host cell without requiring any intervening nucleic acid replication or polymerization steps. In some embodiments, one or both strands of the dsRNA contained in such virions is first reverse transcribed and copied to generate a dsDNA genome intermediate from which mRNA can be transcribed in the host cell. In some embodiments, an RNA virus with a dsRNA genome also carries an enzyme inside the virion that is transduced to host cells with the viral genome, e.g., an RNA-dependent DNA polymerase, capable of copying the dsRNA into dsDNA that can be transcribed into mRNA and translated by the host. In some embodiments, the reverse transcriptase from a Group VII retrovirus is incorporated as the reverse transcriptase domain of a gene modifying polypeptide.
In some embodiments, virions used to deliver nucleic acid in this invention may also carry enzymes involved in the process of gene modification. For example, a retroviral virion may contain a reverse transcriptase domain that is delivered into a host cell along with the nucleic acid. In some embodiments, an RNA template may be associated with a gene modifying polypeptide within a virion, such that both are co-delivered to a target cell upon transduction of the nucleic acid from the viral particle. In some embodiments, the nucleic acid in a virion may comprise DNA, e.g., linear ssDNA, linear dsDNA, circular ssDNA, circular dsDNA, minicircle DNA, dbDNA, ceDNA. In some embodiments, the nucleic acid in a virion may comprise RNA, e.g., linear ssRNA, linear dsRNA, circular ssRNA, circular dsRNA. In some embodiments, a viral genome may circularize upon transduction into a host cell, e.g., a linear ssRNA molecule may undergo a covalent linkage to form a circular ssRNA, a linear dsRNA molecule may undergo a covalent linkage to form a circular dsRNA or one or more circular ssRNA. In some embodiments, a viral genome may replicate by rolling circle replication in a host cell. In some embodiments, a viral genome may comprise a single nucleic acid molecule, e.g., comprise a non-segmented genome. In some embodiments, a viral genome may comprise two or more nucleic acid molecules, e.g., comprise a segmented genome. In some embodiments, a nucleic acid in a virion may be associated with one or proteins. In some embodiments, one or more proteins in a virion may be delivered to a host cell upon transduction. In some embodiments, a natural virus may be adapted for nucleic acid delivery by the addition of virion packaging signals to the target nucleic acid, wherein a host cell is used to package the target nucleic acid containing the packaging signals.
In some embodiments, a virion used as a delivery vehicle may comprise a commensal human virus. In some embodiments, a virion used as a delivery vehicle may comprise an anellovirus, the use of which is described in WO2018232017A1, which is incorporated herein by reference in its entirety.
AAV Administration
In some embodiments, an adeno-associated virus (AAV) is used in conjunction with the system, template nucleic acid, and/or polypeptide described herein. In some embodiments, an AAV is used to deliver, administer, or package the system, template nucleic acid, and/or polypeptide described herein. In some embodiments, the AAV is a recombinant AAV (rAAV).
In some embodiments, a system comprises (a) a polypeptide described herein or a nucleic acid encoding the same, (b) a template nucleic acid (e.g., template RNA) described herein, and (c) one or more first tissue-specific expression-control sequences specific to the target tissue, wherein the one or more first tissue-specific expression-control sequences specific to the target tissue are in operative association with (a), (b), or (a) and (b), wherein, when associated with (a), (a) comprises a nucleic acid encoding the polypeptide.
In some embodiments, a system described herein further comprises a first recombinant adeno-associated virus (rAAV) capsid protein; wherein the at least one of (a) or (b) is associated with the first rAAV capsid protein, wherein at least one of (a) or (b) is flanked by AAV inverted terminal repeats (ITRs).
In some embodiments, (a) and (b) are associated with the first rAAV capsid protein.
In some embodiments, (a) and (b) are on a single nucleic acid.
In some embodiments, the system further comprises a second rAAV capsid protein, wherein at least one of (a) or (b) is associated with the second rAAV capsid protein, and wherein the at least one of (a) or (b) associated with the second rAAV capsid protein is different from the at least one of (a) or (b) is associated with the first rAAV capsid protein.
In some embodiments, the at least one of (a) or (b) is associated with the first or second rAAV capsid protein is dispersed in the interior of the first or second rAAV capsid protein, which first or second rAAV capsid protein is in the form of an AAV capsid particle.
In some embodiments, the system further comprises a nanoparticle, wherein the nanoparticle is associated with at least one of (a) or (b).
In some embodiments, (a) and (b), respectively are associated with: a) a first rAAV capsid protein and a second rAAV capsid protein; b) a nanoparticle and a first rAAV capsid protein; c) a first rAAV capsid protein; d) a first adenovirus capsid protein; e) a first nanoparticle and a second nanoparticle; or f) a first nanoparticle.
Viral vectors are useful for delivering all or part of a system provided by the invention, e.g., for use in methods provided by the invention. Systems derived from different viruses have been employed for the delivery of polypeptides or nucleic acids; for example: integrase-deficient lentivirus, adenovirus, adeno-associated virus (AAV), herpes simplex virus, and baculovirus (reviewed in Hodge et al. Hum Gene Ther 2017; Narayanavari et al. Crit Rev Biochem Mol Biol 2017; Boehme et al. Curr Gene Ther 2015).
Adenoviruses are common viruses that have been used as gene delivery vehicles given well-defined biology, genetic stability, high transduction efficiency, and ease of large-scale production (see, for example, review by Lee et al. Genes & Diseases 2017). They possess linear dsDNA genomes and come in a variety of serotypes that differ in tissue and cell tropisms. In order to prevent replication of infectious virus in recipient cells, adenovirus genomes used for packaging are deleted of some or all endogenous viral proteins, which are provided in trans in viral production cells. This renders the genomes helper-dependent, meaning they can only be replicated and packaged into viral particles in the presence of the missing components provided by so-called helper functions. A helper-dependent adenovirus system with all viral ORFs removed may be compatible with packaging foreign DNA of up to −37 kb (Parks et al. J Virol 1997). In some embodiments, an adenoviral vector is used to deliver DNA corresponding to the polypeptide or template component of the gene modifying system, or both are contained on separate or the same adenoviral vector. In some embodiments, the adenovirus is a helper-dependent adenovirus (HD-AdV) that is incapable of self-packaging. In some embodiments, the adenovirus is a high-capacity adenovirus (HC-AdV) that has had all or a substantial portion of endogenous viral ORFs deleted, while retaining the necessary sequence components for packaging into adenoviral particles. For this type of vector, the only adenoviral sequences required for genome packaging are noncoding sequences: the inverted terminal repeats (ITRs) at both ends and the packaging signal at the 5′-end (Jager et al. Nat Protoc 2009). In some embodiments, the adenoviral genome also comprises stuffer DNA to meet a minimal genome size for optimal production and stability (see, for example, Hausl et al. Mol Ther 2010). In some embodiments, an adenovirus is used to deliver a gene modifying system to the liver.
In some embodiments, an adenovirus is used to deliver a gene modifying system to HSCs, e.g., HDAd5/35++. HDAd5/35++ is an adenovirus with modified serotype 35 fibers that de-target the vector from the liver (Wang et al. Blood Adv 2019). In some embodiments, the adenovirus that delivers a gene modifying system to HSCs utilizes a receptor that is expressed specifically on primitive HSCs, e.g., CD46.
Adeno-associated viruses (AAV) belong to the parvoviridae family and more specifically constitute the dependoparvovirus genus. The AAV genome is composed of a linear single-stranded DNA molecule which contains approximately 4.7 kilobases (kb) and consists of two major open reading frames (ORFs) encoding the non-structural Rep (replication) and structural Cap (capsid) proteins. A second ORF within the cap gene was identified that encodes the assembly-activating protein (AAP). The DNAs flanking the AAV coding regions are two cis-acting inverted terminal repeat (ITR) sequences, approximately 145 nucleotides in length, with interrupted palindromic sequences that can be folded into energetically stable hairpin structures that function as primers of DNA replication. In addition to their role in DNA replication, the ITR sequences have been shown to be involved in viral DNA integration into the cellular genome, rescue from the host genome or plasmid, and encapsidation of viral nucleic acid into mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. 158:97-129). In some embodiments, one or more gene modifying nucleic acid components is flanked by ITRs derived from AAV for viral packaging. See, e.g., WO2019113310.
In some embodiments, one or more components of the gene modifying system are carried via at least one AAV vector. In some embodiments, the at least one AAV vector is selected for tropism to a particular cell, tissue, organism. In some embodiments, the AAV vector is pseudotyped, e.g., AAV2/8, wherein AAV2 describes the design of the construct but the capsid protein is replaced by that from AAV8. It is understood that any of the described vectors could be pseudotype derivatives, wherein the capsid protein used to package the AAV genome is derived from that of a different AAV serotype. Without wishing to be limited in vector choice, a list of exemplary AAV serotypes can be found in Table 18. In some embodiments, an AAV to be employed for gene modifying may be evolved for novel cell or tissue tropism as has been demonstrated in the literature (e.g., Davidsson et al. Proc Natl Acad Sci USA 2019).
In some embodiments, the AAV delivery vector is a vector which has two AAV inverted terminal repeats (ITRs) and a nucleotide sequence of interest (for example, a sequence coding for a gene modifying polypeptide or a DNA template, or both), each of said ITRs having an interrupted (or noncontiguous) palindromic sequence, i.e., a sequence composed of three segments: a first segment and a last segment that are identical when read 5′→3′ but hybridize when placed against each other, and a segment that is different that separates the identical segments. See, for example, WO2012123430.
Conventionally, AAV virions with capsids are produced by introducing a plasmid or plasmids encoding the rAAV or scAAV genome, Rep proteins, and Cap proteins (Grimm et al, 1998). Upon introduction of these helper plasmids in trans, the AAV genome is “rescued” (i.e., released and subsequently recovered) from the host genome, and is further encapsidated to produce infectious AAV. In some embodiments, one or more gene modifying nucleic acids are packaged into AAV particles by introducing the ITR-flanked nucleic acids into a packaging cell in conjunction with the helper functions.
In some embodiments, the AAV genome is a so called self-complementary genome (referred to as scAAV), such that the sequence located between the ITRs contains both the desired nucleic acid sequence (e.g., DNA encoding the gene modifying polypeptide or template, or both) in addition to the reverse complement of the desired nucleic acid sequence, such that these two components can fold over and self-hybridize. In some embodiments, the self-complementary modules are separated by an intervening sequence that permits the DNA to fold back on itself, e.g., forms a stem-loop. An scAAV has the advantage of being poised for transcription upon entering the nucleus, rather than being first dependent on ITR priming and second-strand synthesis to form dsDNA. In some embodiments, one or more gene modifying components is designed as an scAAV, wherein the sequence between the AAV ITRs contains two reverse complementing modules that can self-hybridize to create dsDNA.
In some embodiments, nucleic acid (e.g., encoding a polypeptide, or a template, or both) delivered to cells is closed-ended, linear duplex DNA (CELiD DNA or ceDNA). In some embodiments, ceDNA is derived from the replicative form of the AAV genome (Li et al. PLoS One 2013). In some embodiments, the nucleic acid (e.g., encoding a polypeptide, or a template DNA, or both) is flanked by ITRs, e.g., AAV ITRs, wherein at least one of the ITRs comprises a terminal resolution site and a replication protein binding site (sometimes referred to as a replicative protein binding site). In some embodiments, the ITRs are derived from an adeno-associated virus, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or a combination thereof. In some embodiments, the ITRs are symmetric. In some embodiments, the ITRs are asymmetric. In some embodiments, at least one Rep protein is provided to enable replication of the construct. In some embodiments, the at least one Rep protein is derived from an adeno-associated virus, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or a combination thereof. In some embodiments, ceDNA is generated by providing a production cell with (i) DNA flanked by ITRs, e.g., AAV ITRs, and (ii) components required for ITR-dependent replication, e.g., AAV proteins Rep78 and Rep52 (or nucleic acid encoding the proteins). In some embodiments, ceDNA is free of any capsid protein, e.g., is not packaged into an infectious AAV particle. In some embodiments, ceDNA is formulated into LNPs (see, for example, WO2019051289A1).
In some embodiments, the ceDNA vector consists of two self-complementary sequences, e.g., asymmetrical or symmetrical or substantially symmetrical ITRs as defined herein, flanking said expression cassette, wherein the ceDNA vector is not associated with a capsid protein. In some embodiments, the ceDNA vector comprises two self-complementary sequences found in an AAV genome, where at least one ITR comprises an operative Rep-binding element (RBE) (also sometimes referred to herein as “RBS”) and a terminal resolution site (trs) of AAV or a functional variant of the RBE. See, for example, WO2019113310.
In some embodiments, the AAV genome comprises two genes that encode four replication proteins and three capsid proteins, respectively. In some embodiments, the genes are flanked on either side by 145-bp inverted terminal repeats (ITRs). In some embodiments, the virion comprises up to three capsid proteins (Vp1, Vp2, and/or Vp3), e.g., produced in a 1:1:10 ratio. In some embodiments, the capsid proteins are produced from the same open reading frame and/or from differential splicing (Vp1) and alternative translational start sites (Vp2 and Vp3, respectively). Generally, Vp3 is the most abundant subunit in the virion and participates in receptor recognition at the cell surface defining the tropism of the virus. In some embodiments, Vp1 comprises a phospholipase domain, e.g., which functions in viral infectivity, in the N-terminus of Vp1.
In some embodiments, packaging capacity of the viral vectors limits the size of the gene modifying system that can be packaged into the vector. For example, the packaging capacity of the AAVs can be about 4.5 kb (e.g., about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0 kb), e.g., including one or two inverted terminal repeats (ITRs), e.g., 145 base ITRs.
In some embodiments, recombinant AAV (rAAV) comprises cis-acting 145-bp ITRs flanking vector transgene cassettes, e.g., providing up to 4.5 kb for packaging of foreign DNA. Subsequent to infection, rAAV can, in some instances, express a fusion protein of the invention and persist without integration into the host genome by existing episomally in circular head-to-tail concatemers. rAAV can be used, for example, in vitro and in vivo. In some embodiments, AAV-mediated gene delivery requires that the length of the coding sequence of the gene is equal or greater in size than the wild-type AAV genome.
AAV delivery of genes that exceed this size and/or the use of large physiological regulatory elements can be accomplished, for example, by dividing the protein(s) to be delivered into two or more fragments. In some embodiments, the N-terminal fragment is fused to an intein-N sequence. In some embodiments, the C-terminal fragment is fused to an intein-C sequence. In embodiments, the fragments are packaged into two or more AAV vectors.
In some embodiments, dual AAV vectors are generated by splitting a large transgene expression cassette in two separate halves (5′ and 3′ ends, or head and tail), e.g., wherein each half of the cassette is packaged in a single AAV vector (of <5 kb). The re-assembly of the full-length transgene expression cassette can, in some embodiments, then be achieved upon co-infection of the same cell by both dual AAV vectors. In some embodiments, co-infection is followed by one or more of: (1) homologous recombination (HR) between 5′ and 3′ genomes (dual AAV overlapping vectors); (2) ITR-mediated tail-to-head concatemerization of 5′ and 3′ genomes (dual AAV trans-splicing vectors); and/or (3) a combination of these two mechanisms (dual AAV hybrid vectors). In some embodiments, the use of dual AAV vectors in vivo results in the expression of full-length proteins. In some embodiments, the use of the dual AAV vector platform represents an efficient and viable gene transfer strategy for transgenes of greater than about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 kb in size. In some embodiments, AAV vectors can also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides. In some embodiments, AAV vectors can be used for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest.94:1351 (1994); each of which is incorporated herein by reference in their entirety). The construction of recombinant AAV vectors is described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989) (incorporated by reference herein in their entirety).
In some embodiments, a gene modifying polypeptide described herein (e.g., with or without one or more guide nucleic acids) can be delivered using AAV, lentivirus, adenovirus or other plasmid or viral vector types, in particular, using formulations and doses from, for example, U.S. Pat. No. 8,454,972 (formulations, doses for adenovirus), U.S. Pat. No. 8,404,658 (formulations, doses for AAV) and U.S. Pat. No. 5,846,946 (formulations, doses for DNA plasmids) and from clinical trials and publications regarding the clinical trials involving lentivirus, AAV and adenovirus. For example, for AAV, the route of administration, formulation and dose can be as described in U.S. Pat. No. 8,454,972 and as in clinical trials involving AAV. For adenovirus, the route of administration, formulation and dose can be as described in U.S. Pat. No. 8,404,658 and as in clinical trials involving adenovirus. For plasmid delivery, the route of administration, formulation and dose can be as described in U.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids. Doses can be based on or extrapolated to an average 70 kg individual (e.g. a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed. In some embodiments, the viral vectors can be injected into the tissue of interest. For cell-type specific gene modifying, the expression of the gene modifying polypeptide and optional guide nucleic acid can, in some embodiments, be driven by a cell-type specific promoter.
In some embodiments, AAV allows for low toxicity, for example, due to the purification method not requiring ultracentrifugation of cell particles that can activate the immune response. In some embodiments, AAV allows low probability of causing insertional mutagenesis, for example, because it does not substantially integrate into the host genome.
In some embodiments, AAV has a packaging limit of about 4.4, 4.5, 4.6, 4.7, or 4.75 kb. In some embodiments, a gene modifying polypeptide-encoding sequence, promoter, and transcription terminator can fit into a single viral vector. SpCas9 (4.1 kb) may, in some instances, be difficult to package into AAV. Therefore, in some embodiments, a gene modifying polypeptide coding sequence is used that is shorter in length than other gene modifying polypeptide coding sequences or base editors. In some embodiments, the gene modifying polypeptide encoding sequences are less than about 4.5 kb, 4.4 kb, 4.3 kb, 4.2 kb, 4.1 kb, 4 kb, 3.9 kb, 3.8 kb, 3.7 kb, 3.6 kb, 3.5 kb, 3.4 kb, 3.3 kb, 3.2 kb, 3.1 kb, 3 kb, 2.9 kb, 2.8 kb, 2.7 kb, 2.6 kb, 2.5 kb, 2 kb, or 1.5 kb.
An AAV can be AAV1, AAV2, AAV5 or any combination thereof. In some embodiments, the type of AAV is selected with respect to the cells to be targeted; e.g., AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereof can be selected for targeting brain or neuronal cells; or AAV4 can be selected for targeting cardiac tissue. In some embodiments, AAV8 is selected for delivery to the liver. Exemplary AAV serotypes as to these cells are described, for example, in Grimm, D. et al, J. Virol.82: 5887-5911 (2008) (incorporated herein by reference in its entirety). In some embodiments, AAV refers all serotypes, subtypes, and naturally-occurring AAV as well as recombinant AAV. AAV may be used to refer to the virus itself or a derivative thereof. In some embodiments, AAV includes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2/8, AAVrh10, AAVLK03, AV10, AAV11, AAV 12, rh10, and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. Additional exemplary AAV serotypes are listed in Table 18.







TABLE 18







Exemplary AAV serotypes.









Target Tissue
Vehicle
Reference





Liver
AAV (AAV81, AAVrh.81,
1. Wang et al., Mol. Ther. 18,



AAVhu.371, AAV2/8,
118-25 (2010)



AAV2/rh102, AAV9, AAV2,
2. Ginn et al., JHEP Reports,



NP403, NP592,3, AAV3B5,
100065 (2019)



AAV-DJ4, AAV-LK014,
3. Paulk et al., Mol. Ther. 26,



AAV-LK024, AAV-LK034,
289-303 (2018).



AAV-LK194, AAV57
4. L. Lisowski et al., Nature.



Adenovirus (Ad5, HC-AdV6)
506, 382-6 (2014).




5. L. Wang et al., Mol. Ther.




23, 1877-87 (2015).




6. Hausl Mol Ther (2010)




7. Davidoff et al., Mol. Ther.




11, 875-88 (2005)


Lung
AAV (AAV4, AAV5,
1. Duncan et al., Mol Ther



AAV61, AAV9, H222)
Methods Clin Dev (2018)



Adenovirus (Ad5, Ad3,
2. Cooney et al., Am J Respir



Ad21, Ad14)3
Cell Mol Biol (2019)




3. Li et al., Mol Ther Methods




Clin Dev (2019)


Skin
AAV (AAV61, AAV-LK192)
1. Petek et al., Mol. Ther.




(2010)




2. L. Lisowski et al., Nature.




506, 382-6 (2014).


HSCs
Adenovirus (HDAd5/35++)
Wang et al. Blood Adv (2019)









In some embodiments, a pharmaceutical composition (e.g., comprising an AAV as described herein) has less than 10% empty capsids, less than 8% empty capsids, less than 7% empty capsids, less than 5% empty capsids, less than 3% empty capsids, or less than 1% empty capsids. In some embodiments, the pharmaceutical composition has less than about 5% empty capsids. In some embodiments, the number of empty capsids is below the limit of detection. In some embodiments, it is advantageous for the pharmaceutical composition to have low amounts of empty capsids, e.g., because empty capsids may generate an adverse response (e.g., immune response, inflammatory response, liver response, and/or cardiac response), e.g., with little or no substantial therapeutic benefit.
In some embodiments, the residual host cell protein (rHCP) in the pharmaceutical composition is less than or equal to 100 ng/ml rHCP per 1×1013 vg/ml, e.g., less than or equal to 40 ng/ml rHCP per 1×1013 vg/ml or 1-50 ng/ml rHCP per 1×1013 vg/ml. In some embodiments, the pharmaceutical composition comprises less than 10 ng rHCP per 1.0×1013 vg, or less than 5 ng rHCP per 1.0×1013 vg, less than 4 ng rHCP per 1.0×1013 vg, or less than 3 ng rHCP per 1.0×1013 vg, or any concentration in between. In some embodiments, the residual host cell DNA (hcDNA) in the pharmaceutical composition is less than or equal to 5×106 pg/ml hcDNA per 1×1013 vg/ml, less than or equal to 1.2×106 pg/ml hcDNA per 1×1013 vg/ml, or 1×105 pg/ml hcDNA per 1×1013 vg/ml. In some embodiments, the residual host cell DNA in said pharmaceutical composition is less than 5.0×105 pg per 1×1013 vg, less than 2.0×105 pg per 1.0×1013 vg, less than 1.1×105 pg per 1.0×1013 vg, less than 1.0×105 pg hcDNA per 1.0×1013 vg, less than 0.9×105 pg hcDNA per 1.0×1013 vg, less than 0.8×105 pg hcDNA per 1.0×1013 vg, or any concentration in between.
In some embodiments, the residual plasmid DNA in the pharmaceutical composition is less than or equal to 1.7×105 pg/ml per 1.0×1013 vg/ml, or 1×105 pg/ml per 1×1.0×1013 vg/ml, or 1.7×106 pg/ml per 1.0×1013 vg/ml. In some embodiments, the residual DNA plasmid in the pharmaceutical composition is less than 10.0×105 pg by 1.0×1013 vg, less than 8.0×105 pg by 1.0×1013 vg or less than 6.8×105 pg by 1.0×1013 vg. In embodiments, the pharmaceutical composition comprises less than 0.5 ng per 1.0×1013 vg, less than 0.3 ng per 1.0×1013 vg, less than 0.22 ng per 1.0×1013 vg or less than 0.2 ng per 1.0×1013 vg or any intermediate concentration of bovine serum albumin (BSA). In embodiments, the benzonase in the pharmaceutical composition is less than 0.2 ng by 1.0×1013 vg, less than 0.1 ng by 1.0×1013 vg, less than 0.09 ng by 1.0×1013 vg, less than 0.08 ng by 1.0×1013 vg or any intermediate concentration. In embodiments, Poloxamer 188 in the pharmaceutical composition is about 10 to 150 ppm, about 15 to 100 ppm or about 20 to 80 ppm. In embodiments, the cesium in the pharmaceutical composition is less than 50 pg/g (ppm), less than 30 pg/g (ppm) or less than 20 pg/g (ppm) or any intermediate concentration.
In embodiments, the pharmaceutical composition comprises total impurities, e.g., as determined by SDS-PAGE, of less than 10%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or any percentage in between. In embodiments, the total purity, e.g., as determined by SDS-PAGE, is greater than 90%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or any percentage in between. In embodiments, no single unnamed related impurity, e.g., as measured by SDS-PAGE, is greater than 5%, greater than 4%, greater than 3% or greater than 2%, or any percentage in between. In embodiments, the pharmaceutical composition comprises a percentage of filled capsids relative to total capsids (e.g., peak 1+peak 2 as measured by analytical ultracentrifugation) of greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 91.9%, greater than 92%, greater than 93%, or any percentage in between. In embodiments of the pharmaceutical composition, the percentage of filled capsids measured in peak 1 by analytical ultracentrifugation is 20-80%, 25-75%, 30-75%, 35-75%, or 37.4-70.3%. In embodiments of the pharmaceutical composition, the percentage of filled capsids measured in peak 2 by analytical ultracentrifugation is 20-80%, 20-70%, 22-65%, 24-62%, or 24.9-60.1%.
In one embodiment, the pharmaceutical composition comprises a genomic titer of 1.0 to 5.0×1013 vg/mL, 1.2 to 3.0×1013 vg/mL or 1.7 to 2.3×1013 vg/ml. In one embodiment, the pharmaceutical composition exhibits a biological load of less than 5 CFU/mL, less than 4 CFU/mL, less than 3 CFU/mL, less than 2 CFU/mL or less than 1 CFU/mL or any intermediate contraction. In embodiments, the amount of endotoxin according to USP, for example, USP <85> (incorporated by reference in its entirety) is less than 1.0 EU/mL, less than 0.8 EU/mL or less than 0.75 EU/mL. In embodiments, the osmolarity of a pharmaceutical composition according to USP, for example, USP <785> (incorporated by reference in its entirety) is 350 to 450 mOsm/kg, 370 to 440 mOsm/kg or 390 to 430 mOsm/kg. In embodiments, the pharmaceutical composition contains less than 1200 particles that are greater than 25 μm per container, less than 1000 particles that are greater than 25 μm per container, less than 500 particles that are greater than 25 μm per container or any intermediate value. In embodiments, the pharmaceutical composition contains less than 10,000 particles that are greater than 10 μm per container, less than 8000 particles that are greater than 10 μm per container or less than 600 particles that are greater than 10 pm per container.
In one embodiment, the pharmaceutical composition has a genomic titer of 0.5 to 5.0×1013 vg/mL, 1.0 to 4.0×1013 vg/mL, 1.5 to 3.0×1013 vg/ml or 1.7 to 2.3×1013 vg/ml. In one embodiment, the pharmaceutical composition described herein comprises one or more of the following: less than about 0.09 ng benzonase per 1.0×1013 vg, less than about 30 pg/g (ppm) of cesium, about 20 to 80 ppm Poloxamer 188, less than about 0.22 ng BSA per 1.0×1013 vg, less than about 6.8×105 pg of residual DNA plasmid per 1.0×1013 vg, less than about 1.1×105 pg of residual hcDNA per 1.0×1013 vg, less than about 4 ng of rHCP per 1.0×1013 vg, pH 7.7 to 8.3, about 390 to 430 mOsm/kg, less than about 600 particles that are >25 μm in size per container, less than about 6000 particles that are >10 μm in size per container, about 1.7×1013-2.3×1013 vg/mL genomic titer, infectious titer of about 3.9×108 to 8.4×1010 IU per 1.0×1013 vg, total protein of about 100-300 μg per 1.0×1013 vg, mean survival of >24 days in A7SMA mice with about 7.5×1013 vg/kg dose of viral vector, about 70 to 130% relative potency based on an in vitro cell based assay and/or less than about 5% empty capsid. In various embodiments, the pharmaceutical compositions described herein comprise any of the viral particles discussed here, retain a potency of between ±20%, between ±15%, between ±10% or within ±5% of a reference standard. In some embodiments, potency is measured using a suitable in vitro cell assay or in vivo animal model.
Additional methods of preparation, characterization, and dosing AAV particles are taught in WO2019094253, which is incorporated herein by reference in its entirety.
Additional rAAV constructs that can be employed consonant with the invention include those described in Wang et al 2019, available at: //doi.org/10.1038/s41573-019-0012-9, including Table 1 thereof, which is incorporated by reference in its entirety.
Lipid Nanoparticles
The methods and systems provided herein may employ any suitable carrier or delivery modality, including, in certain embodiments, lipid nanoparticles (LNPs). Lipid nanoparticles, in some embodiments, comprise one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or zwitterionic lipids); one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of WO2019217941; incorporated herein by reference in its entirety); one or more sterols (e.g., cholesterol); and, optionally, one or more targeting molecules (e.g., conjugated receptors, receptor ligands, antibodies); or combinations of the foregoing.
Lipids that can be used in nanoparticle formations (e.g., lipid nanoparticles) include, for example those described in Table 4 of WO2019217941, which is incorporated by reference—e.g., a lipid-containing nanoparticle can comprise one or more of the lipids in Table 4 of WO2019217941. Lipid nanoparticles can include additional elements, such as polymers, such as the polymers described in Table 5 of WO2019217941, incorporated by reference.
In some embodiments, conjugated lipids, when present, can include one or more of PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2′,3′-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypoly ethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those described in Table 2 of WO2019051289 (incorporated by reference), and combinations of the foregoing.
In some embodiments, sterols that can be incorporated into lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those in WO2009/127060 or US2010/0130588, which are incorporated by reference. Additional exemplary sterols include phytosterols, including those described in Eygeris et al (2020), dx.doi.org/10.1021/acs.nanolett.0c01386, incorporated herein by reference.
In some embodiments, the lipid particle comprises an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits aggregation of particles, and a sterol. The amounts of these components can be varied independently and to achieve desired properties. For example, in some embodiments, the lipid nanoparticle comprises an ionizable lipid is in an amount from about 20 mol % to about 90 mol % of the total lipids (in other embodiments it may be 20-70% (mol), 30-60% (mol) or 40-50% (mol); about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle), a non-cationic lipid in an amount from about 5 mol % to about 30 mol % of the total lipids, a conjugated lipid in an amount from about 0.5 mol % to about 20 mol % of the total lipids, and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipids. The ratio of total lipid to nucleic acid (e.g., encoding the gene modifying polypeptide or template nucleic acid) can be varied as desired. For example, the total lipid to nucleic acid (mass or weight) ratio can be from about 10:1 to about 30:1.
In some embodiments, an ionizable lipid may be a cationic lipid, an ionizable cationic lipid, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that can be readily protonated. In some embodiments, the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. In some embodiments, the lipid particle comprises a cationic lipid in formulation with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyn lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol and polymer conjugated lipids. In some embodiments, the cationic lipid may be an ionizable cationic lipid. An exemplary cationic lipid as disclosed herein may have an effective pKa over 6.0. In embodiments, a lipid nanoparticle may comprise a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa), than the first cationic lipid. A lipid nanoparticle may comprise between 40 and 60 mol percent of a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid, and a therapeutic agent, e.g., a nucleic acid (e.g., RNA) described herein (e.g., a template nucleic acid or a nucleic acid encoding a gene modifying polypeptide), encapsulated within or associated with the lipid nanoparticle. In some embodiments, the nucleic acid is co-formulated with the cationic lipid. The nucleic acid may be adsorbed to the surface of an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the nucleic acid may be encapsulated in an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the lipid nanoparticle may comprise a targeting moiety, e.g., coated with a targeting agent. In embodiments, the LNP formulation is biodegradable. In some embodiments, a lipid nanoparticle comprising one or more lipid described herein, e.g., Formula (i), (ii), (ii), (vii) and/or (ix) encapsulates at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or 100% of an RNA molecule, e.g., template RNA and/or a mRNA encoding the gene modifying polypeptide.
In some embodiments, the lipid to nucleic acid ratio (mass/mass ratio; w/w ratio) can be in the range of from about 1:1 to about 25:1, from about 10:1 to about 14:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of lipids and nucleic acid can be adjusted to provide a desired N/P ratio, for example, N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher. Generally, the lipid nanoparticle formulation's overall lipid content can range from about 5 mg/ml to about 30 mg/mL.
Exemplary ionizable lipids that can be used in lipid nanoparticle formulations include, without limitation, those listed in Table 1 of WO2019051289, incorporated herein by reference. Additional exemplary lipids include, without limitation, one or more of the following formulae: X of US2016/0311759; I of US20150376115 or in US2016/0376224; I, II or III of US20160151284; I, IA, II, or IIA of US20170210967; I-c of US20150140070; A of US2013/0178541; I of US2013/0303587 or US2013/0123338; I of US2015/0141678; II, III, IV, or V of US2015/0239926; I of US2017/0119904; I or II of WO2017/117528; A of US2012/0149894; A of US2015/0057373; A of WO2013/116126; A of US2013/0090372; A of US2013/0274523; A of US2013/0274504; A of US2013/0053572; A of WO2013/016058; A of WO2012/162210; I of US2008/042973; I, II, III, or IV of US2012/01287670; I or II of US2014/0200257; I, II, or III of US2015/0203446; I or III of US2015/0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of US2014/0308304; of US2013/0338210; I, II, III, or IV of WO2009/132131; A of US2012/01011478; I or XXXV of US2012/0027796; XIV or XVII of US2012/0058144; of US2013/0323269; I of US2011/0117125; I, II, or III of US2011/0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US2012/0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US2011/0076335; I or II of US2006/008378; I of US2013/0123338; I or X-A-Y-Z of US2015/0064242; XVI, XVII, or XVIII of US2013/0022649; I, II, or III of US2013/0116307; I, II, or III of US2013/0116307; I or II of US2010/0062967; I-X of US2013/0189351; I of US2014/0039032; V of US2018/0028664; I of US2016/0317458; I of US2013/0195920; 5, 6, or 10 of U.S. Pat. No. 10,221,127; 111-3 of WO2018/081480; I-5 or I-8 of WO2020/081938; 18 or 25 of U.S. Pat. No. 9,867,888; A of US2019/0136231; II of WO2020/219876; 1 of US2012/0027803; OF-02 of US2019/0240349; 23 of U.S. Pat. No. 10,086,013; cKK-E12/A6 of Miao et al (2020); C12-200 of WO2010/053572; 7C1 of Dahlman et al (2017); 304-O13 or 503-O13 of Whitehead et al; TS-P4C2 of U.S. Pat. No. 9,708,628; I of WO2020/106946; I of WO2020/106946.
In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,3 1Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is the lipid ATX-002, e.g., as described in Example 10 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is (13Z,16Z)-A,A-dimethyl-3-nonyldocosa-13,16-dien-1-amine (Compound 32), e.g., as described in Example 11 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Compound 6 or Compound 22, e.g., as described in Example 12 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102); e.g., as described in Example 1 of U.S. Pat. No. 9,867,888 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (LP01) e.g., as synthesized in Example 13 of WO2015/095340 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Di((Z)-non-2-en-1-yl) 9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g. as synthesized in Example 7, 8, or 9 of US2012/0027803 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is 1,1′-((2-(4-(2-((2-(Bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-01) (C12-200), e.g., as synthesized in Examples 14 and 16 of WO2010/053572 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is; Imidazole cholesterol ester (ICE) lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-4-yl)propanoate, e.g., Structure (I) from WO2020/106946 (incorporated by reference herein in its entirety).
Some non-limiting examples of lipid compounds that may be used (e.g., in combination with other lipid components) to form lipid nanoparticles for the delivery of compositions described herein, e.g., nucleic acid (e.g., RNA) described herein (e.g., a template nucleic acid or a nucleic acid encoding a gene modifying polypeptide) includes,




In some embodiments an LNP comprising Formula (i) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.




In some embodiments an LNP comprising Formula (ii) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.




In some embodiments an LNP comprising Formula (iii) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.




In some embodiments an LNP comprising Formula (v) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.




In some embodiments an LNP comprising Formula (vi) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.




In some embodiments an LNP comprising Formula (viii) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.




In some embodiments an LNP comprising Formula (ix) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.




wherein

    
    
        X1 is O, NR1, or a direct bond, X2 is C2-5 alkylene, X3 is C(=0) or a direct bond, R1 is H or Me, R3 is Ci-3 alkyl, R2 is Ci-3 alkyl, or R2 taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X2 form a 4-, 5-, or 6-membered ring, or X1 is NR1, R1 and R2 taken together with the nitrogen atoms to which they are attached form a 5- or 6-membered ring, or R2 taken together with R3 and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y1 is C2-12 alkylene. Y2 is selected from
    
    






    
    
        n is 0 to 3, R4 is Ci-15 alkyl, Z1 is Ci-6 alkylene or a direct bond,
        Z2 is
    
    






    
    
        (in either orientation) or absent, provided that if Z1 is a direct bond, Z2 is absent;
        R5 is C5-9 alkyl or C6-10 alkoxy, R6 is C5-9 alkyl or C6-10 alkoxy, W is methylene or a direct bond, and R7 is H or Me, or a salt thereof, provided that if R3 and R2 are C2 alkyls, X1 is O, X2 is linear C3 alkylene, X3 is C(=0), Y1 is linear Ce alkylene, (Y2)n-R4 is
    
    






R4 is linear C5 alkyl, Z1 is C2 alkylene, Z2 is absent, W is methylene, and R7 is H, then R5 and R6 are not Cx alkoxy.

In some embodiments an LNP comprising Formula (xii) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.




In some embodiments an LNP comprising Formula (xi) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.




In some embodiments an LNP comprises a compound of Formula (xiii) and a compound of Formula (xiv).




In some embodiments an LNP comprising Formula (xv) is used to deliver a gene modifying composition described herein to the liver and/or hepatocyte cells.




In some embodiments an LNP comprising a formulation of Formula (xvi) is used to deliver a gene modifying composition described herein to the lung endothelial cells.




In some embodiments, a lipid compound used to form lipid nanoparticles for the delivery of compositions described herein, e.g., nucleic acid (e.g., RNA) described herein (e.g., a template nucleic acid or a nucleic acid encoding a gene modifying polypeptide) is made by one of the following reactions:




Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, di stearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), di stearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl. Additional exemplary lipids, in certain embodiments, include, without limitation, those described in Kim et al. (2020) dx.doi.org/10.1021/acs.nanolett.0c01386, incorporated herein by reference. Such lipids include, in some embodiments, plant lipids found to improve liver transfection with mRNA (e.g., DGTS). In some embodiments, the non-cationic lipid may have the following structure,




Other examples of non-cationic lipids suitable for use in the lipid nanopartieles include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like. Other non-cationic lipids are described in WO2017/099823 or US patent publication US2018/0028664, the contents of which is incorporated herein by reference in their entirety.
In some embodiments, the non-cationic lipid is oleic acid or a compound of Formula I, II, or IV of US2018/0028664, incorporated herein by reference in its entirety. The non-cationic lipid can comprise, for example, 0-30% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle. In embodiments, the molar ratio of ionizable lipid to the neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).
In some embodiments, the lipid nanoparticles do not comprise any phospholipids.
In some aspects, the lipid nanoparticle can further comprise a component, such as a sterol, to provide membrane integrity. One exemplary sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-choiestanol, 53-coprostanol, choiesteryl-(2-hydroxy)-ethyl ether, choiesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue, e.g., choiesteryl-(4′-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in PCT publication WO2009/127060 and US patent publication US2010/0130588, each of which is incorporated herein by reference in its entirety.
In some embodiments, the component providing membrane integrity, such as a sterol, can comprise 0-50% (mol) (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%) of the total lipid present in the lipid nanoparticle. In some embodiments, such a component is 20-50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle.
In some embodiments, the lipid nanoparticle can comprise a polyethylene glycol (PEG) or a conjugated lipid molecule. Generally, these are used to inhibit aggregation of lipid nanoparticles and/or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid.
Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), 1,2-dimyristoyl-sn-glycerol, methoxypoly ethylene glycol (DMG-PEG-2K), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2′,3′-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or a mixture thereof. Additional exemplary PEG-lipid conjugates are described, for example, in U.S. Pat. Nos. 5,885,613, 6,287,591, US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2010/0130588, US2016/0376224, US2017/0119904, and US/099823, the contents of all of which are incorporated herein by reference in their entirety. In some embodiments, a PEG-lipid is a compound of Formula III, III-a-I, III-b-1, III-b-2, or V of US2018/0028664, the content of which is incorporated herein by reference in its entirety. In some embodiments, a PEG-lipid is of Formula II of US20150376115 or US2016/0376224, the content of both of which is incorporated herein by reference in its entirety. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-di sterylglycamide, PEG-cholesterol (1-[8′-(Cholest-5-en-3[beta]-oxy)carboxamido-3′,6′-dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-Ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises a structure selected from:




In some embodiments, lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates can be used in place of or in addition to the PEG-lipid.
Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids are described in the PCT and LIS patent applications listed in Table 2 of WO2019051289A9 and in WO2020106946A1, the contents of all of which are incorporated herein by reference in their entirety.
In some embodiments an LNP comprises a compound of Formula (xix), a compound of Formula (xxi) and a compound of Formula (xxv). In some embodiments an LNP comprising a formulation of Formula (xix), Formula (xxi) and Formula (xxv) is used to deliver a gene modifying composition described herein to the lung or pulmonary cells.
In some embodiments, a lipid nanoparticle may comprise one or more cationic lipids selected from Formula (i), Formula (ii), Formula (iii), Formula (vii), and Formula (ix). In some embodiments, the LNP may further comprise one or more neutral lipid, e.g., DSPC, DPPC, DMPC, DOPC, POPC, DOPE, SM, a steroid, e.g., cholesterol, and/or one or more polymer conjugated lipid, e.g., a pegylated lipid, e.g., PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer or a PEG dialkyoxypropylcarbamate.
In some embodiments, the PEG or the conjugated lipid can comprise 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG or the conjugated lipid content is 0.5-10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle. Molar ratios of the ionizable lipid, non-cationic-lipid, sterol, and PEG/conjugated lipid can be varied as needed. For example, the lipid particle can comprise 30-70% ionizable lipid by mole or by total weight of the composition, 0-60% cholesterol by mole or by total weight of the composition, 0-30% non-cationic-lipid by mole or by total weight of the composition and 1-10% conjugated lipid by mole or by total weight of the composition. Preferably, the composition comprises 30-40% ionizable lipid by mole or by total weight of the composition, 40-50% cholesterol by mole or by total weight of the composition, and 10-20% non-cationic-lipid by mole or by total weight of the composition. In some other embodiments, the composition is 50-75% ionizable lipid by mole or by total weight of the composition, 20-40% cholesterol by mole or by total weight of the composition, and 5 to 10% non-cationic-lipid, by mole or by total weight of the composition and 1-10% conjugated lipid by mole or by total weight of the composition. The composition may contain 60-70% ionizable lipid by mole or by total weight of the composition, 25-35% cholesterol by mole or by total weight of the composition, and 5-10% non-cationic-lipid by mole or by total weight of the composition. The composition may also contain up to 90% ionizable lipid by mole or by total weight of the composition and 2 to 15% non-cationic lipid by mole or by total weight of the composition. The formulation may also be a lipid nanoparticle formulation, for example comprising 8-30% ionizable lipid by mole or by total weight of the composition, 5-30% non-cationic lipid by mole or by total weight of the composition, and 0-20% cholesterol by mole or by total weight of the composition; 4-25% ionizable lipid by mole or by total weight of the composition, 4-25% non-cationic lipid by mole or by total weight of the composition, 2 to 25% cholesterol by mole or by total weight of the composition, 10 to 35% conjugate lipid by mole or by total weight of the composition, and 5% cholesterol by mole or by total weight of the composition; or 2-30% ionizable lipid by mole or by total weight of the composition, 2-30% non-cationic lipid by mole or by total weight of the composition, 1 to 15% cholesterol by mole or by total weight of the composition, 2 to 35% conjugate lipid by mole or by total weight of the composition, and 1-20% cholesterol by mole or by total weight of the composition; or even up to 90% ionizable lipid by mole or by total weight of the composition and 2-10% non-cationic lipids by mole or by total weight of the composition, or even 100% cationic lipid by mole or by total weight of the composition. In some embodiments, the lipid particle formulation comprises ionizable lipid, phospholipid, cholesterol and a PEG-ylated lipid in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation comprises ionizable lipid, cholesterol and a PEG-ylated lipid in a molar ratio of 60:38.5:1.5.
In some embodiments, the lipid particle comprises ionizable lipid, non-cationic lipid (e.g. phospholipid), a sterol (e.g., cholesterol) and a PEG-ylated lipid, where the molar ratio of lipids ranges from 20 to 70 mole percent for the ionizable lipid, with a target of 40-60, the mole percent of non-cationic lipid ranges from 0 to 30, with a target of 0 to 15, the mole percent of sterol ranges from 20 to 70, with a target of 30 to 50, and the mole percent of PEG-ylated lipid ranges from 1 to 6, with a target of 2 to 5.
In some embodiments, the lipid particle comprises ionizable lipid/non-cationic-lipid/sterol/conjugated lipid at a molar ratio of 50:10:38.5:1.5.
In an aspect, the disclosure provides a lipid nanoparticle formulation comprising phospholipids, lecithin, phosphatidylcholine and phosphatidylethanolamine.
In some embodiments, one or more additional compounds can also be included. Those compounds can be administered separately or the additional compounds can be included in the lipid nanoparticles of the invention. In other words, the lipid nanoparticles can contain other compounds in addition to the nucleic acid or at least a second nucleic acid, different than the first. Without limitations, other additional compounds can be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, an extract made from biological materials, or any combinations thereof.
In some embodiments, a lipid nanoparticle (or a formulation comprising lipid nanoparticles) lacks reactive impurities (e.g., aldehydes or ketones), or comprises less than a preselected level of reactive impurities (e.g., aldehydes or ketones). While not wishing to be bound by theory, in some embodiments, a lipid reagent is used to make a lipid nanoparticle formulation, and the lipid reagent may comprise a contaminating reactive impurity (e.g., an aldehyde or ketone). A lipid regent may be selected for manufacturing based on having less than a preselected level of reactive impurities (e.g., aldehydes or ketones). Without wishing to be bound by theory, in some embodiments, aldehydes can cause modification and damage of RNA, e.g., cross-linking between bases and/or covalently conjugating lipid to RNA (e.g., forming lipid-RNA adducts). This may, in some instances, lead to failure of a reverse transcriptase reaction and/or incorporation of inappropriate bases, e.g., at the site(s) of lesion(s), e.g., a mutation in a newly synthesized target DNA.
In some embodiments, a lipid nanoparticle formulation is produced using a lipid reagent comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content. In some embodiments, a lipid nanoparticle formulation is produced using a lipid reagent comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, a lipid nanoparticle formulation is produced using a lipid reagent comprising: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, the lipid nanoparticle formulation is produced using a plurality of lipid reagents, and each lipid reagent of the plurality independently meets one or more criterion described in this paragraph. In some embodiments, each lipid reagent of the plurality meets the same criterion, e.g., a criterion of this paragraph.
In some embodiments, the lipid nanoparticle formulation comprises less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content. In some embodiments, the lipid nanoparticle formulation comprises less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, the lipid nanoparticle formulation comprises: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
In some embodiments, one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content. In some embodiments, one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.
In some embodiments, total aldehyde content and/or quantity of any single reactive impurity (e.g., aldehyde) species is determined by liquid chromatography (LC), e.g., coupled with tandem mass spectrometry (MS/MS), e.g., according to the method described in Example 40 of PCT/US21/20948. In some embodiments, reactive impurity (e.g., aldehyde) content and/or quantity of reactive impurity (e.g., aldehyde) species is determined by detecting one or more chemical modifications of a nucleic acid molecule (e.g., an RNA molecule, e.g., as described herein) associated with the presence of reactive impurities (e.g., aldehydes), e.g., in the lipid reagents. In some embodiments, reactive impurity (e.g., aldehyde) content and/or quantity of reactive impurity (e.g., aldehyde) species is determined by detecting one or more chemical modifications of a nucleotide or nucleoside (e.g., a ribonucleotide or ribonucleoside, e.g., comprised in or isolated from a template nucleic acid, e.g., as described herein) associated with the presence of reactive impurities (e.g., aldehydes), e.g., in the lipid reagents, e.g., according to the method described in Example 41 of PCT/US21/20948. In embodiments, chemical modifications of a nucleic acid molecule, nucleotide, or nucleoside are detected by determining the presence of one or more modified nucleotides or nucleosides, e.g., using LC-MS/MS analysis, e.g., according to the method described in Example 41 of PCT/US21/20948.
In some embodiments, a nucleic acid (e.g., RNA) described herein (e.g., a template nucleic acid or a nucleic acid encoding a gene modifying polypeptide) does not comprise an aldehyde modification, or comprises less than a preselected amount of aldehyde modifications. In some embodiments, on average, a nucleic acid has less than 50, 20, 10, 5, 2, or 1 aldehyde modifications per 1000 nucleotides, e.g., wherein a single cross-linking of two nucleotides is a single aldehyde modification. In some embodiments, the aldehyde modification is an RNA adduct (e.g., a lipid-RNA adduct). In some embodiments, the aldehyde-modified nucleotide is cross-linking between bases. In some embodiments, a nucleic acid (e.g., RNA) described herein comprises less than 50, 20, 10, 5, 2, or 1 cross-links between nucleotide.
In some embodiments, LNPs are directed to specific tissues by the addition of targeting domains. For example, biological ligands may be displayed on the surface of LNPs to enhance interaction with cells displaying cognate receptors, thus driving association with and cargo delivery to tissues wherein cells express the receptor. In some embodiments, the biological ligand may be a ligand that drives delivery to the liver, e.g., LNPs that display GalNAc result in delivery of nucleic acid cargo to hepatocytes that display asialoglycoprotein receptor (ASGPR). The work of Akinc et al. Mol Ther 18(7):1357-1364 (2010) teaches the conjugation of a trivalent GalNAc ligand to a PEG-lipid (GalNAc-PEG-DSG) to yield LNPs dependent on ASGPR for observable LNP cargo effect (see, e.g., FIG. 6 therein). Other ligand-displaying LNP formulations, e.g., incorporating folate, transferrin, or antibodies, are discussed in WO2017223135, which is incorporated herein by reference in its entirety, in addition to the references used therein, namely Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control Release. 20:63-68; Peer et al., Proc Natl Acad Sci USA. 2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., Nat Biotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630; and Peer and Lieberman, Gene Ther. 2011 18:1127-1133.
In some embodiments, LNPs are selected for tissue-specific activity by the addition of a Selective ORgan Targeting (SORT) molecule to a formulation comprising traditional components, such as ionizable cationic lipids, amphipathic phospholipids, cholesterol and poly(ethylene glycol) (PEG) lipids. The teachings of Cheng et al. Nat Nanotechnol 15(4):313-320 (2020) demonstrate that the addition of a supplemental “SORT” component precisely alters the in vivo RNA delivery profile and mediates tissue-specific (e.g., lungs, liver, spleen) gene delivery and editing as a function of the percentage and biophysical property of the SORT molecule.
In some embodiments, the LNPs comprise biodegradable, ionizable lipids. In some embodiments, the LNPs comprise (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate) or another ionizable lipid. See, e.g, lipids of WO2019/067992, WO/2017/173054, WO2015/095340, and WO2014/136086, as well as references provided therein. In some embodiments, the term cationic and ionizable in the context of LNP lipids is interchangeable, e.g., wherein ionizable lipids are cationic depending on the pH.
In some embodiments, an LNP described herein comprises a lipid described in Table 19.







TABLE 19







Exemplary Lipids












Molecular



LIPID ID
Chemical Name
Weight
Structure





LIPIDV- 003
(9Z,12Z)-3-((4,4- bis(octyloxy) butanoyl)oxy)-2- ((((3- (diethylamino) propoxy)carbonyl) oxy)methyl) propyl octadeca-9, 12-dienoate
852.29






 


LIPIDV- 004
Heptadecan- 9-yl 8-((2- hydroxyethyl) (8-(nonyloxy)-8- oxooctyl) amino)octanoate
710.18






 


LIPIDV- 005

919.56













In some embodiments, multiple components of a gene modifying system may be prepared as a single LNP formulation, e.g., an LNP formulation comprises mRNA encoding for the gene modifying polypeptide and an RNA template. Ratios of nucleic acid components may be varied in order to maximize the properties of a therapeutic. In some embodiments, the ratio of RNA template to mRNA encoding a gene modifying polypeptide is about 1:1 to 100:1, e.g., about 1:1 to 20:1, about 20:1 to 40:1, about 40:1 to 60:1, about 60:1 to 80:1, or about 80:1 to 100:1, by molar ratio. In other embodiments, a system of multiple nucleic acids may be prepared by separate formulations, e.g., one LNP formulation comprising a template RNA and a second LNP formulation comprising an mRNA encoding a gene modifying polypeptide. In some embodiments, the system may comprise more than two nucleic acid components formulated into LNPs. In some embodiments, the system may comprise a protein, e.g., a gene modifying polypeptide, and a template RNA formulated into at least one LNP formulation.
In some embodiments, the average LNP diameter of the LNP formulation may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the LNP formulation may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of the LNP formulation may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation may be from about 70 nm to about 100 nm. In a particular embodiment, the average LNP diameter of the LNP formulation may be about 80 nm. In some embodiments, the average LNP diameter of the LNP formulation may be about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation ranges from about 1 mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from about 20 mm to about 80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from about 35 mm to about 50 mm, or from about 38 mm to about 42 mm.
An LNP may, in some instances, be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of an LNP, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. An LNP may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of an LNP may be from about 0.10 to about 0.20.
The zeta potential of an LNP may be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential may describe the surface charge of an LNP. Lipid nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of an LNP may be from about −10 mV to about +20 mV, from about −10 mV to about +15 mV, from about −10 mV to about +10 mV, from about −10 mV to about +5 mV, from about −10 mV to about 0 mV, from about −10 mV to about −5 mV, from about −5 mV to about +20 mV, from about −5 mV to about +15 mV, from about −5 mV to about +10 mV, from about −5 mV to about +5 mV, from about −5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.
The efficiency of encapsulation of a protein and/or nucleic acid, e.g., gene modifying polypeptide or mRNA encoding the polypeptide, describes the amount of protein and/or nucleic acid that is encapsulated or otherwise associated with an LNP after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing the lipid nanoparticle before and after breaking up the lipid nanoparticle with one or more organic solvents or detergents. An anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence may be used to measure the amount of free protein and/or nucleic acid (e.g., RNA) in a solution. For the lipid nanoparticles described herein, the encapsulation efficiency of a protein and/or nucleic acid may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.
An LNP may optionally comprise one or more coatings. In some embodiments, an LNP may be formulated in a capsule, film, or table having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness or density.
Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by WO2020061457, which is incorporated herein by reference in its entirety.
In some embodiments, in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Minis Bio). In certain embodiments, LNPs are formulated using the GenVoy_ILM ionizable lipid mix (Precision NanoSystems). In certain embodiments, LNPs are formulated using 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012), incorporated herein by reference in its entirety.
LNP formulations optimized for the delivery of CRISPR-Cas systems, e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA, are described in WO2019067992 and WO2019067910, both incorporated by reference.
Additional specific LNP formulations useful for delivery of nucleic acids are described in U.S. Pat. Nos. 8,158,601 and 8,168,775, both incorporated by reference, which include formulations used in patisiran, sold under the name ONPATTRO.
Exemplary dosing of gene modifying LNP may include about 0.1, 0.25, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, or 100 mg/kg (RNA). Exemplary dosing of AAV comprising a nucleic acid encoding one or more components of the system may include an MOI of about 1011, 1012, 1013, and 1014 vg/kg.
Kits, Articles of Manufacture, and Pharmaceutical Compositions
In an aspect the disclosure provides a kit comprising a gene modifying polypeptide or a gene modifying system, e.g., as described herein. In some embodiments, the kit comprises a gene modifying polypeptide (or a nucleic acid encoding the polypeptide) and a template RNA (or DNA encoding the template RNA). In some embodiments, the kit further comprises a reagent for introducing the system into a cell, e.g., transfection reagent, LNP, and the like. In some embodiments, the kit is suitable for any of the methods described herein. In some embodiments, the kit comprises one or more elements, compositions (e.g., pharmaceutical compositions), gene modifying polypeptides, and/or gene modifying systems, or a functional fragment or component thereof, e.g., disposed in an article of manufacture. In some embodiments, the kit comprises instructions for use thereof.
In an aspect, the disclosure provides an article of manufacture, e.g., in which a kit as described herein, or a component thereof, is disposed.
In an aspect, the disclosure provides a pharmaceutical composition comprising a gene modifying polypeptide or a gene modifying system, e.g., as described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises a template RNA and/or an RNA encoding the polypeptide. In embodiments, the pharmaceutical composition has one or more (e.g., 1, 2, 3, or 4) of the following characteristics:

    
    
        (a) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) DNA template relative to the template RNA and/or the RNA encoding the polypeptide, e.g., on a molar basis;
        (b) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) uncapped RNA relative to the template RNA and/or the RNA encoding the polypeptide, e.g., on a molar basis;
        (c) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) partial length RNAs relative to the template RNA and/or the RNA encoding the polypeptide, e.g., on a molar basis;
        (d) substantially lacks unreacted cap dinucleotides.

Chemistry, Manufacturing, and Controls (CMC)

    
    


Purification of protein therapeutics is described, for example, in Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and in Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010).
In some embodiments, a gene modifying system, polypeptide, and/or template nucleic acid (e.g., template RNA) conforms to certain quality standards. In some embodiments, a gene modifying system, polypeptide, and/or template nucleic acid (e.g., template RNA) produced by a method described herein conforms to certain quality standards. Accordingly, the disclosure is directed, in some aspects, to methods of manufacturing a gene modifying system, polypeptide, and/or template nucleic acid (e.g., template RNA) that conforms to certain quality standards, e.g., in which said quality standards are assayed. The disclosure is also directed, in some aspects, to methods of assaying said quality standards in a gene modifying system, polypeptide, and/or template nucleic acid (e.g., template RNA). In some embodiments, quality standards include, but are not limited to, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) of the following:

    
    
        (i) the length of the template RNA, e.g., whether the template RNA has a length that is above a reference length or within a reference length range, e.g., whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the template RNA present is greater than 100, 125, 150, 175, or 200 nucleotides long;
        (ii) the presence, absence, and/or length of a polyA tail on the template RNA, e.g., whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the template RNA present contains a polyA tail (e.g., a polyA tail that is at least 5, 10, 20, 30, 50, 70, 100 nucleotides in length (SEQ ID NO: 37640));
        (iii) the presence, absence, and/or type of a 5′ cap on the template RNA, e.g., whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the template RNA present contains a 5′ cap, e.g., whether that cap is a 7-methylguanosine cap, e.g., a O-Me-m7G cap;
        (iv) the presence, absence, and/or type of one or more modified nucleotides (e.g., selected from pseudouridine, dihydrouridine, inosine, 7-methylguanosine, 1-N-methylpseudouridine (1-5-methoxyuridine (5-MO-U), 5-methylcytidine (5mC), or a locked nucleotide) in the template RNA, e.g., whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the template RNA present contains one or more modified nucleotides;
        (v) the stability of the template RNA (e.g., over time and/or under a pre-selected condition), e.g., whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the template RNA remains intact (e.g., greater than 100, 125, 150, 175, or 200 nucleotides long) after a stability test;
        (vi) the potency of the template RNA in a system for modifying DNA, e.g., whether at least 1% of target sites are modified after a system comprising the template RNA is assayed for potency;
        (vii) the length of the polypeptide, first polypeptide, or second polypeptide, e.g., whether the polypeptide, first polypeptide, or second polypeptide has a length that is above a reference length or within a reference length range, e.g., whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the polypeptide, first polypeptide, or second polypeptide present is greater than 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids long (and optionally, no larger than 2500, 2000, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, or 600 amino acids long);
        (viii) the presence, absence, and/or type of post-translational modification on the polypeptide, first polypeptide, or second polypeptide, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the polypeptide, first polypeptide, or second polypeptide contains phosphorylation, methylation, acetylation, myristoylation, palmitoylation, isoprenylation, glipyatyon, or lipoylation, or any combination thereof;
        (ix) the presence, absence, and/or type of one or more artificial, synthetic, or non-canonical amino acids (e.g., selected from ornithine, (3-alanine, GABA, 6-Aminolevulinic acid, PABA, a D-amino acid (e.g., D-alanine or D-glutamate), aminoisobutyric acid, dehydroalanine, cystathionine, lanthionine, Djenkolic acid, Diaminopimelic acid, Homoalanine, Norvaline, Norleucine, Homonorleucine, homoserine, O-methyl-homoserine and O-ethyl-homoserine, ethionine, selenocysteine, selenohomocysteine, selenomethionine, selenoethionine, tellurocysteine, or telluromethionine) in the polypeptide, first polypeptide, or second polypeptide, e.g., whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the polypeptide, first polypeptide, or second polypeptide present contains one or more artificial, synthetic, or non-canonical amino acids;
        (x) the stability of the polypeptide, first polypeptide, or second polypeptide (e.g., over time and/or under a pre-selected condition), e.g., whether at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the polypeptide, first polypeptide, or second polypeptide remains intact (e.g., greater than 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids long (and optionally, no larger than 2500, 2000, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, or 600 amino acids long)) after a stability test;
        (xi) the potency of the polypeptide, first polypeptide, or second polypeptide in a system for modifying DNA, e.g., whether at least 1% of target sites are modified after a system comprising the polypeptide, first polypeptide, or second polypeptide is assayed for potency; or
        (xii) the presence, absence, and/or level of one or more of a pyrogen, virus, fungus, bacterial pathogen, or host cell protein, e.g., whether the system is free or substantially free of pyrogen, virus, fungus, bacterial pathogen, or host cell protein contamination.
    
    


In some embodiments, a system or pharmaceutical composition described herein is endotoxin free.
In some embodiments, the presence, absence, and/or level of one or more of a pyrogen, virus, fungus, bacterial pathogen, and/or host cell protein is determined. In embodiments, whether the system is free or substantially free of pyrogen, virus, fungus, bacterial pathogen, and/or host cell protein contamination is determined.
In some embodiments, a pharmaceutical composition or system as described herein has one or more (e.g., 1, 2, 3, or 4) of the following characteristics:

    
    
        (a) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) DNA template relative to the template RNA and/or the RNA encoding the polypeptide, e.g., on a molar basis;
        (b) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) uncapped RNA relative to the template RNA and/or the RNA encoding the polypeptide, e.g., on a molar basis;
        (c) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) partial length RNAs relative to the template RNA and/or the RNA encoding the polypeptide, e.g., on a molar basis;
        (d) substantially lacks unreacted cap dinucleotides.
    
    


EXAMPLES
Example 1: Screening Configurations of Template RNAs that Correct the R408W Mutation in a Genomic Landing Pad in Human Cells
This example describes the use of gene modifying system containing a gene modifying polypeptide and template RNAs comprising varied lengths of heterologous object sequences and PBS sequences to quantify the activity of template RNAs for correction of the R408W mutation. In this example, a template RNA contains:

    
    
        (1) a gRNA spacer;
        (2) a gRNA scaffold;
        (3) a heterologous object sequence; and
        (4) a primer binding site (PBS) sequence.
    
    


One or more template RNAs described in Tables 1A-4D can be tested as described in this example. The heterologous object sequences and PBS sequences were designed to correct the PAH mutation in a landing pad by replacing a “T” nucleotide with a “C” nucleotide at the mutation site via gene editing, to reverse a R408W mutation in the corresponding protein.
A cell line is created to have a “landing pad” or a stable integration that mimics a region of the PAH gene that contains the R408W mutation site and flanking sequences. The DNA for the landing pad is chemically synthesized and cloned into the pLenti-N-tGFP vector. The cloned landing pad sequence in the lentiviral expression vector is confirmed and the sequence is verified by Sanger sequencing of the landing pad. The sequence verified plasmids (9 μg) along with the lentiviral packaging mix (9 μg, Biosettia) are transfected using Lipofectamine2000™ according to the manufacturer instructions into a packaging cell line, LentiX-293T (Takara Bio). The transfected cells are incubated at 37° C., 5% CO2 for 48 hours (including one medium change at 24 hrs) and the viral particle containing medium is collected from the cell culture dish. The collected medium is filtered through a 0.2 μm filter to remove cell debris and is prepared for transduction of HEK293T cells. The virus-containing medium is diluted in DMEM and mixed with polybrene to prepare a dilution series for transduction of HEK293T cells where the final concentration of polybrene is 8 μg/ml. The HEK293T cells are grown in virus containing medium for 48 hours and then split with fresh medium. The split cells are grown to confluence and transduction efficiency of the different dilutions of virus is measured by GFP expression via flow cytometry and ddPCR detection of the genomic integrated lentivirus that contained GFP and the PAH landing pads.
A gene modifying system comprising a (i) compatible gene modifying polypeptide described herein, e.g., having: an NLS of Table 11, a compatible Cas9 domain having a sequence of Table 8 (e.g., SpyCas9-SpRY), a linker of Table 10, an RT sequence of Table 6 (e.g., MLVMS_P03355_PLV919), and a second NLS of Table 11 and (ii) a template RNA of any of Tables 1A-4D (e.g., a template RNA of ID #1) is transfected into the HEK293T landing pad cell line. The gene modifying polypeptide and the template RNAs are delivered by nucleofection in RNA format. Specifically, 1 μg of gene modifying polypeptide mRNA is combined with 10 μM template RNAs. The mRNA and template RNAs are added to 25 μL SF buffer containing 250,000 HEK293T landing pad cells and cells are nucleofected using program DS-150. After nucleofection, are were grown at 37° C., 5% CO2 for 3 days prior to cell lysis and genomic DNA extraction. To analyze gene editing activity, primers flanking the PAH mutation site are used to amplify across the locus. Amplicons are analyzed via short read sequencing using an Illumina MiSeq. In some embodiments, the assay will indicate that at least 5%, 10%, 20%, 30%, 40%, or 50% of copies of the PAH gene in the sample are converted to the desired wild-type sequence.
Example 2: Gene Modifying Polypeptide Selection by Pooled Screening in HEK293T & U2OS Cells
This example describes the use of an RNA gene modifying system for the targeted editing of a coding sequence in the human genome. More specifically, this example describes the infection of HEK293T and U2OS cells with a library of gene modifying candidates, followed by transfection of a template guide RNA (tgRNA) for in vitro gene modifying in the cells, e.g., as a means of evaluating a new gene modifying polypeptide for editing activity in human cells by a pooled screening approach.
The gene modifying polypeptide library candidates assayed herein each comprise: 1) a S. pyogenes (Spy) Cas9 nickase containing an N863A mutation that inactivates one endonuclease active site; 2) one of the 122 peptide linkers depicted at Table 10; and 3) a reverse transcriptase (RT) domain from Table 6 of retroviral origin. The particular retroviral RT domains utilized were selected if they were expected to function as a monomer. For each selected RT domain, the wild-type sequences were tested, as well as versions with point mutations installed in the primary wild-type sequence. In particular, 143 RT domains were tested, either wild type or containing various mutations. In total, 17,446 Cas-linker-RT gene modifying polypeptides were tested.
The system described here is a two-component system comprising: 1) an expression plasmid encoding a human codon-optimized gene modifying polypeptide library candidate within a lentiviral cassette, and 2) a tgRNA expression plasmid expressing a non-coding tgRNA sequence that is recognized by Cas and localizes it to the genomic locus of interest, and that also templates reverse transcription of the desired edit into the genome by the RT domain, driven by a U6 promoter. The lentiviral cassette comprises: (i) a CMV promoter for expression in mammalian cells; (ii) a gene modifying polypeptide library candidate as shown; (iii) a self-cleaving T2A polypeptide; (iv) a puromycin resistance gene enabling selection in mammalian cells; and (v) a polyA tail termination signal.
To prepare a pool of cells expressing gene modifying polypeptide library candidates, HEK293T or U2OS cells were transduced with pooled lentiviral preparations of the gene modifying candidate plasmid library. HEK293 Lenti-X cells were seeded in 15 cm plates (12×106 cells) prior to lentiviral plasmid transfection. Lentiviral plasmid transfection using the Lentiviral Packaging Mix (Biosettia, 27 ug) and the plasmid DNA for the gene modifying candidate library (27 ug) was performed the following day using Lipofectamine 2000 and Opti-MEM media according to the manufacturer's protocol. Extracellular DNA was removed by a full media change the next day and virus-containing media was harvested 48 hours after. Lentiviral media was concentrated using Lenti-X Concentrator (TaKaRa Biosciences) and 5 mL lentiviral aliquots were made and stored at −80° C. Lentiviral titering was performed by enumerating colony forming units post Puromycin selection. HEK293T or U2OS cells carrying a BFP-expressing genomic landing pad were seeded at 6×107 cells in culture plates and transduced at a 0.3 multiplicity of infection (MOI) to minimize multiple infections per cell. Puromycin (2.5 ug/mL) was added 48 hours post infection to allow for selection of infected cells. Cells were kept under puromycin selection for at least 7 days and then scaled up for tgRNA electroporation.
To determine the genome-editing capacity of the gene modifying library candidates in the assay, infected BFP-expressing HEK293T or U2OS cells were then transfected by electroporation of 250,000 cells/well with 200 ng of a tgRNA (either g4 or g10) plasmid, designed to convert BFP to GFP, at sufficient cell count for >1000× coverage per library candidate.
The g4 tgRNA (5′ to 3′) is as follows: 20 nucleotide spacer region (GCCGAAGCACTGCACGCCGT; SEQ ID NO: 11,011), a scaffold region (GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC; SEQ ID NO: 11,012), the template region encoding the single base pair substitution to change BFP to GFP (bold) and a PAM inactivation that introduces a synonymous point mutation in the SpyCas9 PAM (NGG to NCG) that prevents re-engagement of the gene modifying polypeptide upon completion of a functional gene modifying reaction (underline) (ACCCTGACGTACG; SEQ ID NO: 11,013), and the 13 nucleotide PBS (GCGTGCAGTGCTT; SEQ ID NO: 11,014).
Similarly, the g10 tgRNA (5′ to 3′) is as follows: 20 nucleotide spacer region (AGAAGTCGTGCTGCTTCATG; SEQ ID NO: 11,015), a scaffold region (GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGC; SEQ ID NO: 11,016), the template region encoding the single base pair substitution to change BFP to GFP (bold) and a PAM inactivation that introduces a synonymous point mutation in the SpyCas9 PAM (NGG to NGA) that prevents re-engagement of the gene modifying polypeptide upon completion of a functional gene modifying reaction (underline) (ACCCTGACCTACGGCGTGCAGTGCTTCGGCCGCTACCCCGATCACAT; SEQ ID NO: 11,017), and 13 nucleotide PBS (GAAGCAGCACGAC; SEQ ID NO: 11,018).
To assess the genome-editing capacity of the various constructs in the assay, cells were sorted by Fluorescence-Activated Cell Sorting (FACS) for GFP expression 6-7 days post-electroporation. Cells were sorted and harvested as distinct populations of unedited (BFP+) cells, edited (GFP+) cells and imperfect edit (BFP−, GFP−) cells. A sample of unsorted cells was also harvested as the input population to determine enrichment during analysis.
To determine which gene modifying library candidates have genome-editing capacity in this assay, genomic DNA (gDNA) was harvested from sorted and unsorted cell populations, and analyzed by sequencing the gene modifying library candidates in each population. Briefly, gene modifying sequences were amplified from the genome using primers specific to the lentiviral cassette, amplified in a second round of PCR to dilute genomic DNA, and then sequenced using Oxford Nanopore Sequencing Technology according to the manufacturer's protocol.
After quality control of sequencing reads, reads of at least 1500 and no more than 3200 nucleotides were mapped to the gene modifying polypeptide library sequences and those containing a minimum of an 80% match to a library sequence were considered to be successfully aligned to a given candidate. To identify gene modifying candidates capable of performing gene editing in the assay, the read count of each library candidate in the edited population was compared to its read count in the initial, unsorted population. For purposes of this pooled screen, gene modifying candidates with genome-editing capacity were selected as those candidates that were enriched in the converted (GFP+) population relative to unsorted (input) cells and wherein the enrichment was determined to be at or above the enrichment level of a reference (Element ID No: 17380).
A large number of gene modifying polypeptide candidates were determined to be enriched in the GFP+ cell populations. For example, of the 17,446 candidates tested, over 3,300 exhibited enrichment in GFP+ sorted populations (relative to unsorted) that was at least equivalent to that of the reference under similar experimental conditions (HEK293T using g4 tgRNA; HEK293T cells using g10 tgRNA; or U2OS cells using g4 tgRNA), shown in Table D. Although the 17,446 candidates were also tested in U2OS cells using g10 tgRNA, the pooled screen did not yield candidates that were enriched in the converted (GFP+) population relative to unsorted (input) cells under that experimental condition; further investigation is required to explain these results.







TABLE D







Combinations of linker and RT sequences


screened. The amino acid sequence of


each RT in this table is provided in


Table 6.









Linker amino acid
SEQ ID



sequence
NO:
RT domain name





EAAAKGSS
12,001
PERV_Q4VFZ2_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
12,002
MLVMS_P03355_PLV919


AK




 


PAPEAAAK
12,003
MLVFF_P26809_3mutA


 


EAAAKPAPGGG
12,004
MLVFF_P26809_3mutA


 


GSSGSSGSSGSSGSSGSS
12,005
PERV_Q4VFZ2_3mut


 


PAPGGGEAAAK
12,006
MLVAV_P03356_3mutA


 


AEAAAKEAAAKEAAAKEA
12,007
MLVMS_P03355_PLV919


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GSSEAAAK
12,008
MLVFF_P26809_3mutA


 


EAAAKPAPGGS
12,009
MLVFF_P26809_3mutA


 


GGSGGSGGSGGSGGSGGS
12,010
MLVFF_P26809_3mutA


 


AEAAAKEAAAKEAAAKEA
12,011
XMRV6_A1Z651_3mutA


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


AEAAAKEAAAKEAAAKEA
12,012
PERV_Q4VFZ2_3mutA_WS


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


EAAAKEAAAKEAAAK
12,013
MLVFF_P26809_3mutA


 


PAPEAAAKGSS
12,014
MLVFF_P26809_3mutA


 


AEAAAKEAAAKEAAAKEA
12,015
PERV_Q4VFZ2_3mutA_WS


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


EAAAKEAAAKEAAAK
12,016
PERV_Q4VFZ2_3mutA_WS


 


AEAAAKEAAAKEAAAKEA
12,017
AVIRE_P03360_3mutA


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


PAPAPAPAPAP
12,018
MLVCB_P08361_3mutA


 


PAPAPAPAPAP
12,019
MLVFF_P26809_3mutA


 


EAAAKGGSPAP
12,020
PERV_Q4VFZ2_3mutA_WS


 


PAP

MLVMS_P03355_PLV919


 


PAPGGGGSS
12,022
WMSV_P03359_3mutA


 


SGSETPGTSESATPES
12,023
MLVFF_P26809_3mutA


 


PAPEAAAKGSS
12,024
XMRV6_A1Z651_3mutA


 


EAAAKGGSGGG
12,025
MLVMS_P03355_PLV919


 


GGGGSGGGGS
12,026
MLVFF_P26809_3mutA


 


GGGPAPGSS
12,027
MLVAV_P03356_3mutA


 


GGSGGSGGSGGSGGSGGS
12,028
XMRV6_A1Z651_3mut


 


GGGGSGGGGSGGGGSGGG
12,029
MLVCB_P08361_3mutA


GGGGGSGGGGS




 


GSSPAP
12,030
AVIRE_P03360_3mutA


 


EAAAKGSSPAP
12,031
MLVFF_P26809_3mutA


 


GSSGGGEAAAK
12,032
MLVFF_P26809_3mutA


 


GGSGGSGGSGGSGGSGGS
12,033
MLVMS_P03355_3mutA_WS


 


PAPAPAPAP
12,034
MLVFF_P26809_3mutA


 


EAAAKEAAAKEAAAKEAA
12,035
XMRV6_A1Z651_3mutA


AK




 


EAAAKGGSPAP
12,036
MLVMS_P03355_3mutA_WS


 


PAPGGSEAAAK
12,037
AVIRE_P03360_3mutA


 


GGGGSGGGGSGGGGSGGG
12,038
AVIRE_P03360_3mutA


GSGGGGSGGGGS




 


EAAAKGGGGSEAAAK
12,039
MLVCB_P08361_3mutA


 


AEAAAKEAAAKEAAAKEA
12,040
WMSV_P03359_3mutA


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GSS

MLVMS_P03355_PLV919


 


GSSGSSGSSGSS
12,042
MLVMS_P03355_PLV919


 


GSSPAPEAAAK
12,043
XMRV6_A1Z651_3mutA


 


GGSPAPEAAAK
12,044
MLVFF_P26809_3mutA


 


GGGEAAAKGGS
12,045
MLVFF_P26809_3mutA


 


EAAAKEAAAKEAAAKEAA
12,046
PERV_Q4VFZ2_3mutA_WS


AKEAAAK




 


GGGGGGGG
12,047
PERV_Q4VFZ2_3mut


 


GGGPAP
12,048
MLVCB_P08361_3mutA


 


PAPAPAPAPAPAP
12,049
MLVCB_P08361_3mutA


 


GGSGGSGGSGGSGGSGGS
12,050
MLVCB_P08361_3mutA


 


PAP

MLVMS_P03355_3mutA_WS


 


GGSGGSGGSGGSGGSGGS
12,052
PERV_Q4VFZ2_3mutA_WS


 


PAPAPAPAPAPAP
12,053
MLVMS_P03355_PLV919


 


EAAAKPAPGSS
12,054
MLVMS_P03355_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
12,055
MLVMS_P03355_3mutA_WS


AK




 


EAAAKGGS
12,056
MLVMS_P03355_3mutA_WS


 


GGGGSEAAAKGGGGS
12,057
MLVFF_P26809_3mutA


 


EAAAKPAPGSS
12,058
MLVFF_P26809_3mutA


 


GGGGSGGGGGGGGSGGGG
12,059
MLVMS_P03355_PLV919


S




 


EAAAKGGGGGS
12,060
MLVMS_P03355_PLV919


 


GGSPAP
12,061
XMRV6_A1Z651_3mutA


 


EAAAKGGGPAP
12,062
MLVMS_P03355_PLV919


 


EAAAKEAAAKEAAAKEAA
12,063
MLVFF_P26809_3mutA


AKEAAAK




 


PAP

MLVCB_P08361_3mutA


 


EAAAK
12,065
XMRV6_A1Z651_3mutA


 


GGSGSSPAP
12,066
PERV_Q4VFZ2_3mutA_WS


 


GSSGSSGSSGSSGSSGSS
12,067
MLVMS_P03355_PLV919


 


GSSEAAAKGGG
12,068
MLVAV_P03356_3mutA


 


GGGEAAAKGGS
12,069
XMRV6_A1Z651_3mutA


 


EAAAKGGGGSEAAAK
12,070
MLVAV_P03356_3mutA


 


GGGGSGGGGSGGGGS
12,071
MLVFF_P26809_3mutA


 


GGGGGGGGSGGGGSGGGG
12,072
AVIRE_P03360_3mutA


S




 


SGSETPGTSESATPES
12,073
AVIRE_P03360_3mutA


 


GGGEAAAKPAP
12,074
MLVFF_P26809_3mutA


 


EAAAKGSSGGG
12,075
MLVMS_P03355_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
12,076
WMSV_P03359_3mut


AKEAAAK




 


GGSGGSGGSGGS
12,077
XMRV6_A1Z651_3mutA


 


GGSEAAAKPAP
12,078
MLVFF_P26809_3mutA


 


EAAAKGSSGGG
12,079
XMRV6_A1Z651_3mutA


 


GGGGS
12,080
MLVFF_P26809_3mutA


 


GGGEAAAKGSS
12,081
MLVMS_P03355_PLV919


 


PAPAPAPAPAPAP
12,082
MLVAV_P03356_3mutA


 


GGGGSGGGGSGGGGSGGG
12,083
MLVCB_P08361_3mutA


GS




 


GGGEAAAKGSS
12,084
MLVCB_P08361_3mutA


 


PAPGGSGSS
12,085
MLVFF_P26809_3mutA


 


GSAGSAAGSGEF
12,086
MLVCB_P08361_3mutA


 


PAPGGSEAAAK
12,087
MLVMS_P03355_3mutA_WS


 


GGSGSS
12,088
XMRV6_A1Z651_3mutA


 


PAPGGGGSS
12,089
MLVMS_P03355_PLV919


 


GSSGSSGSS
12,090
XMRV6_A1Z651_3mut


 


AEAAAKEAAAKEAAAKEA
12,091
MLVMS_P03355_3mutA_WS


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


EAAAK
12,092
MLVMS_P03355_PLV919


 


GSSGSSGSSGSS
12,093
MLVFF_P26809_3mutA


 


PAPGGGGSS
12,094
MLVCB_P08361_3mutA


 


GGGEAAAKGGS
12,095
MLVCB_P08361_3mutA


 


PAPGGGEAAAK
12,096
MLVMS_P03355_PLV919


 


GGGGGSPAP
12,097
XMRV6_A1Z651_3mutA


 


EAAAKGGS
12,098
XMRV6_A1Z651_3mutA


 


EAAAKGSSPAP
12,099
XMRV6_A1Z651_3mut


 


PAPEAAAK
12,100
MLVAV_P03356_3mutA


 


GGSGGSGGSGGS
12,101
MLVMS_P03355_3mutA_WS


 


GGGPAPGGS
12,102
MLVMS_P03355_PLV919


 


GSSGSSGSSGSS
12,103
PERV_Q4VFZ2_3mutA_WS


 


EAAAKPAPGGS
12,104
MLVCB_P08361_3mutA


 


GSSGSS
12,105
MLVFF_P26809_3mutA


 


EAAAKEAAAKEAAAKEAA
12,106
MLVCB_P08361_3mutA


AK




 


EAAAKEAAAKEAAAKEAA
12,107
FLV_P10273_3mutA


AK




 


GSS

MLVFF_P26809_3mutA


 


EAAAKEAAAK
12,109
MLVMS_P03355_3mutA_WS


 


PAPEAAAKGGG
12,110
MLVAV_P03356_3mutA


 


GGSGSSEAAAK
12,111
MLVFF_P26809_3mutA


 


EAAAKEAAAKEAAAKEAA
12,112
PERV_Q4VFZ2


AKEAAAK




 


GSSEAAAKPAP
12,113
AVIRE_P03360_3mutA


 


EAAAKEAAAKEAAAKEAA
12,114
MLVCB_P08361_3mutA


AKEAAAK




 


EAAAKGGG
12,115
MLVFF_P26809_3mutA


 


GSSPAPGGG
12,116
MLVCB_P08361_3mutA


 


GGGPAPGSS
12,117
MLVMS_P03355_PLV919


 


GGGGGS
12,118
MLVMS_P03355_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
12,119
PERV_Q4VFZ2_3mut


AKEAAAKEAAAK




 


GGGGSGGGGSGGGGSGGG
12,120
WMSV_P03359_3mutA


GSGGGGS




 


EAAAKEAAAKEAAAK
12,121
PERV_Q4VFZ2_3mut


 


PAPAPAPAP
12,122
MLVCB_P08361_3mutA


 


GSSGSSGSSGSSGSS
12,123
PERV_Q4VFZ2_3mut


 


GGGGSSEAAAK
12,124
MLVMS_P03355_3mutA_WS


 


GGSGGSGGSGGS
12,125
MLVCB_P08361_3mutA


 


PAPEAAAKGGS
12,126
MLVCB_P08361_3mutA


 


EAAAKEAAAKEAAAKEAA
12,127
MLVCB_P08361_3mutA


AKEAAAKEAAAK




 


EAAAKGGGGSEAAAK
12,128
MLVMS_P03355_PLV919


 


EAAAKGGGGSEAAAK
12,129
MLVMS_P03355_3mutA_WS


 


EAAAKGGGPAP
12,130
XMRV6_A1Z651_3mut


 


EAAAKEAAAKEAAAKEAA
12,131
MLVMS_P03355_3mutA_WS


AKEAAAK




 


AEAAAKEAAAKEAAAKEA
12,132
FLV_P10273_3mutA


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GGSEAAAKGGG
12,133
MLVMS_P03355_3mutA_WS


 


GGGGSGGGGSGGGGSGGG
12,134
KORV_Q9TTC1-Pro_3mutA


GSGGGGSGGGGS




 


GGGPAPGGS
12,135
MLVCB_P08361_3mutA


 


PAPAPAPAPAPAP
12,136
XMRV6_A1Z651_3mutA


 


GGSGSSGGG
12,137
XMRV6_A1Z651_3mutA


 


GGSGSSGGG
12,138
MLVCB_P08361_3mutA


 


GGGEAAAKGGS
12,139
MLVMS_P03355_3mutA_WS


 


EAAAK
12,140
MLVCB_P08361_3mutA


 


GGSPAPGSS
12,141
MLVMS_P03355_3mutA_WS


 


GGGGSSEAAAK
12,142
PERV_Q4VFZ2_3mut


 


PAPAPAPAPAP
12,143
MLVBM_Q7SVK7_3mut


 


EAAAKEAAAKEAAAKEAA
12,144
MLVAV_P03356_3mutA


AK




 


GGGGGSGSS
12,145
MLVCB_P08361_3mutA


 


EAAAKGSSPAP
12,146
MLVMS_P03355_3mutA_WS


 


PAPAPAPAPAPAP
12,147
MLVMS_P03355_3mutA_WS


 


GSSGGGGGS
12,148
MLVMS_P03355_3mutA_WS


 


PAPGSSGGG
12,149
MLVMS_P03355_PLV919


 


GGSGGGPAP
12,150
MLVCB_P08361_3mutA


 


GGGGGGG
12,151
MLVCB_P08361_3mutA


 


GSSGSSGSSGSSGSSGSS
12,152
MLVCB_P08361_3mutA


 


GGGPAPGGS
12,153
MLVFF_P26809_3mutA


 


EAAAKGGSGGG
12,154
PERV_Q4VFZ2_3mut


 


EAAAKGGGGSS
12,155
MLVMS_P03355_3mutA_WS


 


GSSGSSGSSGSSGSSGSS
12,156
MLVMS_P03355_3mut


 


GGGGSGGGGSGGGGSGGG
12,157
MLVBM_Q7SVK7_3mutA_WS


GS




 


PAPAPAPAPAP
12,158
MLVMS_P03355_PLV919


 


GGGEAAAKGGS
12,159
MLVMS_P03355_PLV919


 


AEAAAKEAAAKEAAAKEA
12,160
MLVMS_P03355_3mut


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GSAGSAAGSGEF
12,161
MLVMS_P03355_3mutA_WS


 


GSSGSSGSSGSSGSS
12,162
MLVFF_P26809_3mutA


 


EAAAKGGSGSS
12,163
MLVFF_P26809_3mutA


 


PAPGGG
12,164
MLVFF_P26809_3mutA


 


GGGPAPGSS
12,165
XMRV6_A1Z651_3mutA


 


PAPEAAAKGGS
12,166
AVIRE_P03360_3mutA


 


PAPGGGEAAAK
12,167
MLVFF_P26809_3mut


 


GGGGSSEAAAK
12,168
MLVCB_P08361_3mutA


 


EAAAK
12,169
MLVMS_P03355_PLV919


 


GGGGSGGGGSGGGGSGGG
12,170
BAEVM_P10272_3mutA


GSGGGGSGGGGS




 


GGSGGGEAAAK
12,171
MLVMS_P03355_PLV919


 


AEAAAKEAAAKEAAAKEA
12,172
MLVFF_P26809_3mutA


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GSSPAPGGS
12,173
XMRV6_A1Z651_3mutA


 


GGSGGGPAP
12,174
MLVMS_P03355_PLV919


 


EAAAK
12,175
AVIRE_P03360_3mutA


 


GSS

XMRV6_A1Z651_3mutA


 


GGSGGSGGS
12,177
MLVFF_P26809_3mutA


 


EAAAKEAAAKEAAAKEAA
12,178
AVIRE_P03360_3mut


AK




 


PAPEAAAKGGG
12,179
PERV_Q4VFZ2_3mutA_WS


 


GGGGGSEAAAK
12,180
BAEVM_P10272_3mutA


 


GGSGSSGGG
12,181
MLVMS_P03355_3mutA_WS


 


GGGGGGG
12,182
MLVMS_P03355_3mutA_WS


 


GSSEAAAKPAP
12,183
PERV_Q4VFZ2_3mut


 


GGGGGSEAAAK
12,184
WMSV_P03359_3mut


 


GGGGSGGGGSGGGGSGGG
12,185
MLVFF_P26809_3mut


GSGGGGS




 


GGGEAAAKGGS
12,186
AVIRE_P03360_3mutA


 


GGSPAPGGG
12,187
AVIRE_P03360_3mutA


 


GSAGSAAGSGEF
12,188
MLVAV_P03356_3mutA


 


EAAAK
12,189
MLVAV_P03356_3mutA


 


EAAAKPAPGSS
12,190
WMSV_P03359_3mutA


 


EAAAKEAAAKEAAAKEAA
12,191
PERV_Q4VFZ2_3mutA_WS


AKEAAAKEAAAK




 


GGSEAAAKPAP
12,192
MLVCB_P08361_3mutA


 


PAPAPAPAPAPAP
12,193
MLVBM_Q7SVK7_3mutA_WS


 


GGSPAPGGG
12,194
MLVMS_P03355_3mutA_WS


 


GGSEAAAKGGG
12,195
MLVMS_P03355_3mut


 


GGSGGSGGSGGS
12,196
MLVFF_P26809_3mutA


 


EAAAKEAAAKEAAAKEAA
12,197
MLVFF_P26809_3mutA


AKEAAAKEAAAK




 


GGG

AVIRE_P03360_3mutA


 


AEAAAKEAAAKEAAAKEA
12,199
PERV_Q4VFZ2_3mut


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GGSGGSGGSGGS
12,200
MLVMS_P03355_3mutA_WS


 


GGGEAAAK
12,201
MLVCB_P08361_3mutA


 


GSSGSSGSSGSSGSSGSS
12,202
MLVMS_P03355_3mutA_WS


 


GSSGGGPAP
12,203
MLVMS_P03355_3mutA_WS


 


GSSEAAAKPAP
12,204
MLVFF_P26809_3mutA


 


EAAAKEAAAK
12,205
MLVMS_P03355_PLV919


 


GGGGSGGGGSGGGGSGGG
12,206
MLVCB_P08361_3mut


GSGGGGSGGGGS




 


GGGGGG
12,207
MLVMS_P03355_3mutA_WS


 


GGSGSSGGG
12,208
MLVFF_P26809_3mutA


 


GSSGGGEAAAK
12,209
PERV_Q4VFZ2_3mutA_WS


 


PAPAPAPAPAP
12,210
PERV_Q4VFZ2_3mut


 


EAAAKEAAAKEAAAKEAA
12,211
SFV3L_P27401_2mut


AKEAAAKEAAAK




 


EAAAKGGSGGG
12,212
BAEVM_P10272_3mutA


 


GGGGSSPAP
12,213
PERV_Q4VFZ2_3mutA_WS


 


GGGEAAAKPAP
12,214
MLVMS_P03355_PLV919


 


GGSGGGPAP
12,215
BAEVM_P10272_3mutA


 


PAPGSSGGS
12,216
MLVMS_P03355_PLV919


 


GGSGGGPAP
12,217
MLVMS_P03355_3mutA_WS


 


EAAAKGGSPAP
12,218
PERV_Q4VFZ2_3mutA_WS


 


EAAAKGGSGGG
12,219
MLVMS_P03355_3mutA_WS


 


PAPGSSGGG
12,220
MLVFF_P26809_3mutA


 


GSSEAAAKGGS
12,221
MLVFF_P26809_3mutA


 


PAPGSSEAAAK
12,222
MLVFF_P26809_3mutA


 


EAAAKGSSPAP
12,223
KORV_Q9TTC1-Pro_3mutA


 


EAAAKEAAAKEAAAKEAA
12,224
MLVBM_Q7SVK7_3mutA_WS


AKEAAAK




 


PAPGSSEAAAK
12,225
MLVMS_P03355_PLV919


 


EAAAKGSSGGG
12,226
MLVMS_P03355_3mutA_WS


 


EAAAKGGGGGS
12,227
AVIRE_P03360_3mutA


 


EAAAKEAAAKEAAAK
12,228
MLVMS_P03355_PLV919


 


PAPAPAPAPAPAP
12,229
MLVFF_P26809_3mutA


 


GGGGSGGGGSGGGGS
12,230
MLVCB_P08361_3mutA


 


PAPGGSEAAAK
12,231
MLVCB_P08361_3mutA


 


PAPGSSEAAAK
12,232
MLVBM_Q7SVK7_3mutA_WS


 


PAPEAAAKGSS
12,233
AVIRE_P03360_3mutA


 


GGSPAPGSS
12,234
WMSV_P03359_3mutA


 


PAPGGSGGG
12,235
MLVMS_P03355_PLV919


 


EAAAKGGSGSS
12,236
MLVMS_P03355_3mutA_WS


 


GGSGGG
12,237
MLVFF_P26809_3mutA


 


GGSEAAAKGSS
12,238
KORV_Q9TTC1_3mutA


 


AEAAAKEAAAKEAAAKEA
12,239
MLVCB_P08361_3mutA


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


PAPAPAPAPAPAP
12,240
PERV_Q4VFZ2_3mutA_WS


 


PAPEAAAK
12,241
MLVMS_P03355_3mutA_WS


 


GGSEAAAKGGG
12,242
MLVMS_P03355_PLV919


 


GSSPAP
12,243
MLVMS_P03355_3mutA_WS


 


GGGGSS
12,244
MLVMS_P03355_PLV919


 


GGGEAAAKPAP
12,245
AVIRE_P03360_3mutA


 


EAAAKPAPGGS
12,246
MLVAV_P03356_3mutA


 


EAAAKGGGPAP
12,247
MLVAV_P03356_3mutA


 


PAPGGSEAAAK
12,248
BAEVM_P10272_3mutA


 


PAPGGSGSS
12,249
MLVMS_P03355_3mutA_WS


 


PAPGGSGSS
12,250
AVIRE_P03360_3mutA


 


GGSGGGPAP
12,251
MLVMS_P03355_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
12,252
BAEVM_P10272_3mutA


AK




 


GGGGSGGGGSGGGGSGGG
12,253
MLVMS_P03355_PLV919


GSGGGGS




 


GGGGSSPAP
12,254
MLVCB_P08361_3mutA


 


GSSGGGPAP
12,255
MLVFF_P26809_3mutA


 


GGGGSSGGS
12,256
MLVMS_P03355_PLV919


 


GGSGGG
12,257
MLVCB_P08361_3mutA


 


GSSGGGGGS
12,258
MLVMS_P03355_PLV919


 


SGGSSGGSSGSETPGTSE
12,259
XMRV6_A1Z651_3mutA


SATPESSGGSSGGSS




 


GGGGGSGSS
12,260
KORV_Q9TTC1_3mut


 


GGGEAAAKGGS
12,261
BAEVM_P10272_3mutA


 


GGSGGG
12,262
BAEVM_P10272_3mutA


 


PAPAPAP
12,263
KORV_Q9TTC1-Pro_3mut


 


AEAAAKEAAAKEAAAKEA
12,264
SFV3L_P27401_2mutA


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


AEAAAKEAAAKEAAAKEA
12,265
MLVBM_Q7SVK7_3mutA_WS


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GSSGSSGSSGSSGSS
12,266
MLVMS_P03355_3mutA_WS


 


GSSGGGEAAAK
12,267
MLVMS_P03355_3mutA_WS


 


GSSGGSEAAAK
12,268
MLVFF_P26809_3mutA


 


PAP

MLVMS_P03355_PLV919


 


EAAAKGGGGSEAAAK
12,270
MLVBM_Q7SVK7_3mutA_WS


 


PAPAP
12,271
AVIRE_P03360_3mutA


 


PAP

MLVFF_P26809_3mutA


 


GSSGGG
12,273
MLVMS_P03355_3mut


 


GSSPAPGGS
12,274
MLVFF_P26809_3mutA


 


PAPAPAPAP
12,275
XMRV6_A1Z651_3mutA


 


EAAAKGSSGGS
12,276
PERV_Q4VFZ2_3mut


 


PAPEAAAKGGG
12,277
KORV_Q9TTC1-Pro_3mutA


 


PAPGGS
12,278
MLVCB_P08361_3mutA


 


EAAAKGGG
12,279
MLVCB_P08361_3mutA


 


GSSEAAAKPAP
12,280
MLVMS_P03355_PLV919


 


PAPGGS
12,281
MLVFF_P26809_3mutA


 


EAAAKGGS
12,282
MLVCB_P08361_3mutA


 


EAAAKEAAAKEAAAKEAA
12,283
FLV_P10273_3mutA


AKEAAAKEAAAK




 


PAPGGSEAAAK
12,284
MLVAV_P03356_3mutA


 


GSS

MLVCB_P08361_3mutA


 


GSSGSSGSSGSS
12,286
AVIRE_P03360_3mutA


 


GSSGSSGSS
12,287
MLVFF_P26809_3mutA


 


GSSGGG
12,288
MLVMS_P03355_PLV919


 


EAAAK
12,289
MLVFF_P26809_3mutA


 


GGSPAPEAAAK
12,290
MLVCB_P08361_3mutA


 


GGSGSS
12,291
MLVCB_P08361_3mutA


 


GSSPAPGGG
12,292
MLVMS_P03355_PLV919


 


EAAAKEAAAKEAAAKEAA
12,293
MLVAV_P03356_3mutA


AKEAAAK




 


EAAAKGSSPAP
12,294
FLV_P10273_3mutA


 


GGGGSS
12,295
XMRV6_A1Z651_3mutA


 


GGSPAPGSS
12,296
MLVMS_P03355_PLV919


 


EAAAKEAAAKEAAAKEAA
12,297
MLVMS_P03355_3mutA_WS


AKEAAAK




 


PAPEAAAKGGG
12,298
FLV_P10273_3mutA


 


EAAAKPAPGGS
12,299
XMRV6_A1Z651_3mut


 


PAPAP
12,300
BAEVM_P10272_3mutA


 


EAAAKEAAAKEAAAKEAA
12,301
MLVMS_P03355_PLV919


AK




 


GSSPAPGGG
12,302
MLVMS_P03355_PLV919


 


EAAAKGGGPAP
12,303
KORV_Q9TTC1_3mutA


 


PAPEAAAK
12,304
MLVMS_P03355_PLV919


 


PAPGGGEAAAK
12,305
PERV_Q4VFZ2_3mutA_WS


 


EAAAKGSSGGS
12,306
MLVMS_P03355_3mutA_WS


 


EAAAKEAAAKEAAAK
12,307
MLVMS_P03355_PLV919


 


GSSEAAAK
12,308
MLVMS_P03355_3mutA_WS


 


GSSGSSGSSGSS
12,309
MLVMS_P03355_3mutA_WS


 


GGGGSGGGGSGGGGSGGG
12,310
MLVMS_P03355_3mutA_WS


GS




 


EAAAKGGGGSEAAAK
12,311
MLVMS_P03355_3mut


 


GGS

MLVCB_P08361_3mutA


 


GGGGSGGGGSGGGGSGGG
12,313
XMRV6_A1Z651_3mutA


GSGGGGSGGGGS




 


GGSGSSPAP
12,314
MLVCB_P08361_3mutA


 


GGGGSGGGGSGGGGS
12,315
XMRV6_A1Z651_3mutA


 


PAPAPAPAPAP
12,316
BAEVM_P10272_3mutA


 


PAPAPAPAPAP
12,317
MLVMS_P03355_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
12,318
MLVBM_Q7SVK7_3mut


AK




 


GGGGSGGGGSGGGGSGGG
12,319
BAEVM_P10272_3mutA


GSGGGGS




 


GGSGGSGGS
12,320
MLVMS_P03355_3mutA_WS


 


EAAAKPAPGSS
12,321
MLVMS_P03355_PLV919


 


GSS

MLVMS_P03355_3mutA_WS


 


PAPEAAAKGGS
12,323
MLVMS_P03355_3mutA_WS


 


GGGPAPGGS
12,324
MLVMS_P03355_3mutA_WS


 


EAAAKGGGGSS
12,325
MLVAV_P03356_3mutA


 


GSSGSSGSSGSSGSS
12,326
MLVFF_P26809_3mut


 


SGSETPGTSESATPES
12,327
PERV_Q4VFZ2_3mut


 


GGSEAAAKGGG
12,328
MLVMS_P03355_3mut


 


GSSGSSGSSGSSGSSGSS
12,329
AVIRE_P03360_3mutA


 


PAPAPAPAPAPAP
12,330
AVIRE_P03360_3mut


 


GGSGGS
12,331
XMRV6_A1Z651_3mutA


 


PAPGSSEAAAK
12,332
MLVCB_P08361_3mut


 


GGSPAPEAAAK
12,333
PERV_Q4VFZ2_3mut


 


EAAAKGGGGGS
12,334
MLVCB_P08361_3mutA


 


GGSGGSGGSGGS
12,335
MLVMS_P03355_PLV919


 


GGGGSSEAAAK
12,336
MLVMS_P03355_PLV919


 


GSSEAAAKGGG
12,337
MLVFF_P26809_3mutA


 


PAPGGS
12,338
MLVMS_P03355_3mutA_WS


 


EAAAKGGSGGG
12,339
MLVCB_P08361_3mutA


 


EAAAKGGG
12,340
PERV_Q4VFZ2_3mut


 


PAPGGS
12,341
XMRV6_A1Z651_3mutA


 


GSSPAPGGG
12,342
XMRV6_A1Z651_3mutA


 


PAPEAAAKGGG
12,343
MLVMS_P03355_3mutA_WS


 


GSSEAAAKGGG
12,344
PERV_Q4VFZ2_3mutA_WS


 


PAPGGSEAAAK
12,345
XMRV6_A1Z651_3mutA


 


GGGGGS
12,346
MLVMS_P03355_3mutA_WS


 


GGSPAPEAAAK
12,347
MLVMS_P03355_3mutA_WS


 


GGGPAP
12,348
MLVFF_P26809_3mutA


 


PAPGSSGGG
12,349
XMRV6_A1Z651_3mutA


 


PAPGSSGGG
12,350
MLVBM_Q7SVK7_3mutA_WS


 


GGGEAAAKGSS
12,351
MLVMS_P03355_3mutA_WS


 


GSSEAAAKGGS
12,352
MLVCB_P08361_3mutA


 


PAPGGSGSS
12,353
MLVCB_P08361_3mutA


 


EAAAKGGGGSEAAAK
12,354
BAEVM_P10272_3mutA


 


PAPAPAP
12,355
PERV_Q4VFZ2_3mutA_WS


 


GGGGGG
12,356
MLVAV_P03356_3mutA


 


GSSPAPEAAAK
12,357
MLVCB_P08361_3mutA


 


GGSGGSGGS
12,358
MLVMS_P03355_3mutA_WS


 


GSSGSSGSSGSSGSS
12,359
XMRV6_A1Z651_3mut


 


GGGPAPGGS
12,360
XMRV6_A1Z651_3mutA


 


GGGPAPEAAAK
12,361
BAEVM_P10272_3mutA


 


GGSGGG
12,362
AVIRE_P03360_3mutA


 


SGSETPGTSESATPES
12,363
PERV_Q4VFZ2_3mutA_WS


 


EAAAKGSSPAP
12,364
MLVMS_P03355_PLV919


 


GSSEAAAK
12,365
XMRV6_A1Z651_3mut


 


GSSGGSGGG
12,366
MLVFF_P26809_3mutA


 


EAAAKEAAAKEAAAKEAA
12,367
WMSV_P03359_3mutA


AKEAAAK




 


GGGGSEAAAKGGGGS
12,368
MLVMS_P03355_PLV919


 


PAPGGGGSS
12,369
MLVMS_P03355_3mutA_WS


 


SGSETPGTSESATPES
12,370
MLVMS_P03355_3mutA_WS


 


GGSPAPEAAAK
12,371
KORV_Q9TTC1-Pro_3mutA


 


GSSEAAAKGGG
12,372
MLVMS_P03355_3mutA_WS


 


GSSEAAAK
12,373
WMSV_P03359_3mutA


 


GGGGSEAAAKGGGGS
12,374
AVIRE_P03360_3mutA


 


GSS

WMSV_P03359_3mutA


 


PAPGGSEAAAK
12,376
MLVFF_P26809_3mutA


 


GGGGS
12,377
MLVMS_P03355_3mutA_WS


 


GGGPAP
12,378
MLVMS_P03355_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
12,379
MLVMS_P03355_3mutA_WS


AKEAAAKEAAAK




 


EAAAKPAPGSS
12,380
PERV_Q4VFZ2_3mut


 


EAAAKPAPGSS
12,381
MLVCB_P08361_3mutA


 


GGGGGG
12,382
WMSV_P03359_3mutA


 


EAAAKPAPGGS
12,383
MLVMS_P03355_PLV919


 


PAPGGGEAAAK
12,384
PERV_Q4VFZ2_3mut


 


EAAAKEAAAKEAAAKEAA
12,385
AVIRE_P03360_3mutA


AKEAAAK




 


GSSEAAAKPAP
12,386
XMRV6_A1Z651_3mutA


 


PAPGGSEAAAK
12,387
MLVBM_Q7SVK7_3mutA_WS


 


PAPGSS
12,388
MLVCB_P08361_3mutA


 


EAAAKGGG
12,389
MLVMS_P03355_3mutA_WS


 


EAAAKPAP
12,390
MLVCB_P08361_3mutA


 


PAPEAAAKGGS
12,391
MLVBM_Q7SVK7_3mutA_WS


 


GGSPAPGGG
12,392
MLVCB_P08361_3mutA


 


PAPGGSGSS
12,393
WMSV_P03359_3mutA


 


EAAAKEAAAKEAAAKEAA
12,394
MLVMS_P03355_PLV919


AKEAAAKEAAAK




 


GGSGGGPAP
12,395
MLVMS_P03355_PLV919


 


AEAAAKEAAAKEAAAKEA
12,396
MLVMS_P03355


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


PAPEAAAKGSS
12,397
MLVCB_P08361_3mutA


 


EAAAKGSS
12,398
MLVMS_P03355_3mutA_WS


 


GGSGGS
12,399
MLVMS_P03355_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
12,400
BAEVM_P10272_3mutA


AKEAAAK




 


GGGGSEAAAKGGGGS
12,401
FLV_P10273_3mutA


 


GGSEAAAKGGG
12,402
MLVCB_P08361_3mutA


 


GSSGSSGSSGSSGSS
12,403
BAEVM_P10272_3mutA


 


GGGGSGGGGSGGGGSGGG
12,404
MLVFF_P26809_3mutA


GSGGGGSGGGGS




 


EAAAKGGG
12,405
PERV_Q4VFZ2_3mut


 


GGGGGSEAAAK
12,406
MLVCB_P08361_3mutA


 


EAAAKPAPGGS
12,407
MLVMS_P03355_3mutA_WS


 


GGGGGSGSS
12,408
XMRV6_A1Z651_3mutA


 


PAPGSSEAAAK
12,409
MLVMS_P03355_3mutA_WS


 


GSSEAAAKPAP
12,410
MLVCB_P08361_3mutA


 


EAAAKGSSPAP
12,411
MLVAV_P03356_3mutA


 


GGGPAPGGS
12,412
WMSV_P03359_3mutA


 


GGSPAP
12,413
MLVMS_P03355_3mutA_WS


 


GGSEAAAKGGG
12,414
MLVMS_P03355_3mutA_WS


 


GGGGGGGG
12,415
MLVFF_P26809_3mutA


 


GGGGGGGGSGGGGSGGGG
12,416
MLVMS_P03355_3mutA_WS


SGGGGSGGGGS




 


GGGGSGGGGSGGGGSGGG
12,417
MLVBM_Q7SVK7_3mutA_WS


GSGGGGSGGGGS




 


GSSPAPGGG
12,418
MLVAV_P03356_3mutA


 


GGGGGG
12,419
AVIRE_P03360_3mutA


 


GSSGGS
12,420
MLVMS_P03355_3mutA_WS


 


GGSPAPGSS
12,421
MLVFF_P26809_3mutA


 


PAPEAAAKGGG
12,422
PERV_Q4VFZ2_3mut


 


EAAAKGGGPAP
12,423
MLVFF_P26809_3mutA


 


GGGEAAAKGGS
12,424
MLVMS_P03355_PLV919


 


GGSGSSPAP
12,425
MLVFF_P26809_3mutA


 


SGSETPGTSESATPES
12,426
WMSV_P03359_3mutA


 


PAPGGSEAAAK
12,427
MLVBM_Q7SVK7_3mutA_WS


 


GGSGGG
12,428
MLVMS_P03355_PLV919


 


GGGGSSPAP
12,429
PERV_Q4VFZ2_3mut


 


GGGEAAAKGSS
12,430
MLVAV_P03356_3mutA


 


PAPAPAPAPAPAP
12,431
MLVMS_P03355_3mutA_WS


 


EAAAKGGGGSEAAAK
12,432
PERV_Q4VFZ2


 


EAAAKEAAAKEAAAKEAA
12,433
MLVMS_P03355_PLV919


AKEAAAK




 


GGGGGSEAAAK
12,434
PERV_Q4VFZ2_3mut


 


PAPGSSEAAAK
12,435
MLVCB_P08361_3mutA


 


GSAGSAAGSGEF
12,436
PERV_Q4VFZ2_3mutA_WS


 


EAAAKGGGGSEAAAK
12,437
MLVFF_P26809_3mutA


 


GGSPAPGGG
12,438
PERV_Q4VFZ2_3mutA_WS


 


GSSEAAAKGGG
12,439
AVIRE_P03360_3mutA


 


GGGEAAAKPAP
12,440
MLVMS_P03355_3mutA_WS


 


GGGPAP
12,441
AVIRE_P03360_3mutA


 


GGSEAAAK
12,442
MLVCB_P08361_3mutA


 


SGGSSGGSSGSETPGTSE
12,443
PERV_Q4VFZ2_3mut


SATPESSGGSSGGSS




 


EAAAKPAPGGS
12,444
MLVBM_Q7SVK7_3mutA_WS


 


AEAAAKEAAAKEAAAKEA
12,445
XMRV6_A1Z651_3mut


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GGGGGGGG
12,446
MLVCB_P08361_3mutA


 


PAPGSS
12,447
PERV_Q4VFZ2_3mut


 


EAAAK
12,448
PERV_Q4VFZ2_3mut


 


GSAGSAAGSGEF
12,449
MLVMS_P03355_3mutA_WS


 


PAPGGGEAAAK
12,450
PERV_Q4VFZ2_3mut


 


EAAAKGSSGGS
12,451
MLVFF_P26809_3mut


 


GGGGSEAAAKGGGGS
12,452
BAEVM_P10272_3mutA


 


GGGGSGGGGSGGGGS
12,453
MLVMS_P03355_PLV919


 


EAAAKGGGGSEAAAK
12,454
BAEVM_P10272_3mut


 


PAPGGGEAAAK
12,455
MLVMS_P03355_3mutA_WS


 


GGSEAAAKPAP
12,456
MLVMS_P03355_3mutA_WS


 


PAPAP
12,457
MLVCB_P08361_3mutA


 


PAPAP
12,458
MLVFF_P26809_3mutA


 


GGSPAP
12,459
AVIRE_P03360_3mutA


 


EAAAKGSSGGS
12,460
MLVCB_P08361_3mutA


 


PAPGSSGGS
12,461
AVIRE_P03360_3mutA


 


EAAAKGGGGSEAAAK
12,462
XMRV6_A1Z651_3mutA


 


PAPAPAP
12,463
BAEVM_P10272_3mutA


 


GGSGGSGGSGGSGGSGGS
12,464
MLVMS_P03355_PLV919


 


GGGGGSGSS
12,465
MLVMS_P03355_PLV919


 


PAPGSSEAAAK
12,466
XMRV6_A1Z651_3mut


 


GGSEAAAKPAP
12,467
XMRV6_A1Z651_3mutA


 


EAAAKEAAAKEAAAKEAA
12,468
XMRV6_A1Z651_3mut


AK




 


AEAAAKEAAAKEAAAKEA
12,469
WMSV_P03359_3mut


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GGSGGGEAAAK
12,470
XMRV6_A1Z651_3mutA


 


GGGEAAAK
12,471
XMRV6_A1Z651_3mutA


 


GGGGSGGGGSGGGGS
12,472
MLVMS_P03355_3mutA_WS


 


GGSGGSGGSGGSGGS
12,473
MLVFF_P26809_3mutA


 


GSSGGGGGS
12,474
MLVMS_P03355_3mut


 


PAPGGSEAAAK
12,475
MLVMS_P03355_3mutA_WS


 


GSSGGSPAP
12,476
MLVMS_P03355_3mutA_WS


 


SGSETPGTSESATPES
12,477
XMRV6_A1Z651_3mutA


 


GGGGGGGGS
12,478
MLVMS_P03355_PLV919


 


PAPAPAPAPAP
12,479
MLVMS_P03355_3mut


 


GSSGSS
12,480
XMRV6_A1Z651_3mutA


 


GSSEAAAKPAP
12,481
PERV_Q4VFZ2_3mut


 


GGSGSSGGG
12,482
MLVMS_P03355_3mutA_WS


 


EAAAKEAAAK
12,483
MLVCB_P08361_3mutA


 


GSSGSSGSSGSS
12,484
MLVMS_P03355_3mutA_WS


 


GSSPAPGGG
12,485
PERV_Q4VFZ2_3mutA_WS


 


EAAAKEAAAKEAAAK
12,486
MLVMS_P03355_3mutA_WS


 


AEAAAKEAAAKEAAAKEA
12,487
SFV1_P23074_2mutA


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GGGGSGGGGSGGGGSGGG
12,488
MLVMS_P03355_PLV919


GSGGGGSGGGGS




 


GSAGSAAGSGEF
12,489
MLVMS_P03355_PLV919


 


PAPGSSEAAAK
12,490
MLVMS_P03355_3mutA_WS


 


GGSEAAAK
12,491
MLVMS_P03355_3mutA_WS


 


GSSGSSGSSGSSGSS
12,492
PERV_Q4VFZ2_3mutA_WS


 


GGSEAAAKPAP
12,493
PERV_Q4VFZ2_3mutA_WS


 


GGSGGSGGS
12,494
MLVCB_P08361_3mutA


 


EAAAKGGSGSS
12,495
MLVCB_P08361_3mutA


 


GGGGSGGGGSGGGGSGGG
12,496
FLV_P10273_3mutA


GSGGGGS




 


EAAAKEAAAKEAAAKEAA
12,497
MLVBM_Q7SVK7_3mutA_WS


AK




 


GGSGSSPAP
12,498
BAEVM_P10272_3mutA


 


EAAAKEAAAKEAAAKEAA
12,499
XMRV6_A1Z651_3mutA


AKEAAAK




 


GGGGSGGGGSGGGGSGGG
12,500
MLVBM_Q7SVK7_3mutA_WS


GSGGGGS




 


GGSGSS
12,501
WMSV_P03359_3mutA


 


PAPEAAAK
12,502
MLVCB_P08361_3mutA


 


EAAAKPAP
12,503
BAEVM_P10272_3mutA


 


GSSPAP
12,504
PERV_Q4VFZ2_3mutA_WS


 


GGGPAP
12,505
PERV_Q4VFZ2_3mutA_WS


 


EAAAKGGSGSS
12,506
MLVMS_P03355_3mutA_WS


 


EAAAKGGGGSEAAAK
12,507
AVIRE_P03360_3mutA


 


GGSGGG
12,508
KORV_Q9TTC1-Pro_3mutA


 


GSSPAP
12,509
MLVFF_P26809_3mutA


 


GGSGSSEAAAK
12,510
BAEVM_P10272_3mutA


 


PAPGSSGGS
12,511
BAEVM_P10272_3mutA


 


GGGGGG
12,512
MLVFF_P26809_3mutA


 


PAPGGSEAAAK
12,513
MLVMS_P03355_PLV919


 


PAPGGS
12,514
MLVMS_P03355_PLV919


 


GGSGGSGGSGGS
12,515
BAEVM_P10272_3mutA


 


GSSPAP
12,516
MLVCB_P08361_3mutA


 


PAPAPAPAP
12,517
MLVMS_P03355_3mutA_WS


 


GGGGGG
12,518
MLVCB_P08361_3mutA


 


GSSGSSGSSGSSGSSGSS
12,519
KORV_Q9TTC1-Pro_3mutA


 


GSSEAAAKGGS
12,520
BAEVM_P10272_3mutA


 


GGSEAAAK
12,521
FLV_P10273_3mutA


 


GGSGGSGGSGGSGGS
12,522
KORV_Q9TTC1-Pro_3mutA


 


GSSPAPEAAAK
12,523
PERV_Q4VFZ2_3mut


 


GSSGSSGSSGSSGSS
12,524
XMRV6_A1Z651_3mutA


 


EAAAKPAPGGS
12,525
MLVMS_P03355_3mut


 


SGGSSGGSSGSETPGTSE
12,526
FLV_P10273_3mut


SATPESSGGSSGGSS




 


GGSPAPEAAAK
12,527
XMRV6_A1Z651_3mut


 


EAAAKGGSGGG
12,528
MLVFF_P26809_3mutA


 


EAAAKEAAAKEAAAKEAA
12,529
MLVFF_P26809_3mutA


AK




 


GSSPAP
12,530
WMSV_P03359_3mutA


 


PAPAPAPAP
12,531
MLVAV_P03356_3mutA


 


PAPGGSEAAAK
12,532
KORV_Q9TTC1_3mut


 


GGSGSSEAAAK
12,533
MLVBM_Q7SVK7_3mutA_WS


 


GSSGGG
12,534
MLVCB_P08361_3mutA


 


GGGEAAAKGSS
12,535
PERV_Q4VFZ2_3mut


 


PAPGGSGGG
12,536
MLVFF_P26809_3mutA


 


AEAAAKEAAAKEAAAKEA
12,537
FFV_O93209


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


PAPGGGGSS
12,538
MLVMS_P03355_3mutA_WS


 


EAAAKGGS
12,539
MLVAV_P03356_3mutA


 


EAAAKEAAAKEAAAKEAA
12,540
MLVBM_Q7SVK7_3mutA_WS


AKEAAAKEAAAK




 


GGSGGSGGS
12,541
WMSV_P03359_3mutA


 


PAPAP
12,542
MLVMS_P03355_3mutA_WS


 


GSSGGGEAAAK
12,543
MLVAV_P03356_3mutA


 


GGGGSSEAAAK
12,544
MLVFF_P26809_3mutA


 


EAAAKGSSGGS
12,545
MLVMS_P03355_PLV919


 


EAAAKGGGGSEAAAK
12,546
MLVMS_P03355_3mutA_WS


 


GGGGGGGG
12,547
MLVMS_P03355_PLV919


 


GSSGSSGSS
12,548
MLVMS_P03355_PLV919


 


GGGEAAAKPAP
12,549
PERV_Q4VFZ2_3mutA_WS


 


GGGGGSGSS
12,550
MLVMS_P03355_3mutA_WS


 


GGGGGGG
12,551
MLVMS_P03355_PLV919


 


GGS

MLVMS_P03355_PLV919


 


GSSGGG
12,553
MLVMS_P03355_3mutA_WS


 


EAAAKGGSGSS
12,554
PERV_Q4VFZ2_3mutA_WS


 


PAPGSSEAAAK
12,555
MLVMS_P03355_PLV919


 


GSSEAAAKPAP
12,556
MLVMS_P03355_PLV919


 


GGSPAPGSS
12,557
BAEVM_P10272_3mutA


 


GSAGSAAGSGEF
12,558
MLVCB_P08361_3mut


 


GGSPAPGGG
12,559
PERV_Q4VFZ2_3mut


 


GGGGSGGGGSGGGGSGGG
12,560
MLVMS_P03355_3mut


GS




 


GSSGSSGSS
12,561
PERV_Q4VFZ2_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
12,562
PERV_Q4VFZ2_3mut


AKEAAAKEAAAK




 


GGGGSEAAAKGGGGS
12,563
MLVCB_P08361_3mutA


 


GGSEAAAKGSS
12,564
MLVAV_P03356_3mutA


 


EAAAKGGGGSEAAAK
12,565
MLVCB_P08361_3mut


 


EAAAKEAAAKEAAAKEAA
12,566
XMRV6_A1Z651_3mutA


AKEAAAKEAAAK




 


PAPGGGEAAAK
12,567
MLVMS_P03355_3mutA_WS


 


GSSGGGEAAAK
12,568
PERV_Q4VFZ2_3mutA_WS


 


GSSGSS
12,569
MLVCB_P08361_3mut


 


PAPAPAPAPAPAP
12,570
PERV_Q4VFZ2_3mut


 


GGSPAPGGG
12,571
MLVFF_P26809_3mutA


 


GGSGGSGGSGGSGGS
12,572
MLVCB_P08361_3mutA


 


EAAAKEAAAK
12,573
MLVFF_P26809_3mutA


 


AEAAAKEAAAKEAAAKEA
12,574
GALV_P21414_3mut


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


PAPAPAPAPAPAP
12,575
WMSV_P03359_3mutA


 


GGGEAAAKGGS
12,576
KORV_Q9TTC1_3mutA


 


EAAAKGGGPAP
12,577
KORV_Q9TTC1_3mut


 


PAPEAAAKGSS
12,578
MLVBM_Q7SVK7_3mutA_WS


 


PAPEAAAKGSS
12,579
FLV_P10273_3mutA


 


PAPGGSEAAAK
12,580
MLVMS_P03355_3mut


 


GSSPAPGGG
12,581
BAEVM_P10272_3mutA


 


GGGEAAAKPAP
12,582
KORV_Q9TTC1-Pro_3mutA


 


GGGGSGGGGS
12,583
MLVMS_P03355_PLV919


 


GGGEAAAKGSS
12,584
MLVFF_P26809_3mutA


 


PAPGGGGSS
12,585
MLVBM_Q7SVK7_3mutA_WS


 


GSSEAAAK
12,586
BAEVM_P10272_3mutA


 


GGGGGGGG
12,587
MLVMS_P03355_PLV919


 


PAPGSSGGS
12,588
MLVAV_P03356_3mutA


 


GGGGSGGGGSGGGGSGGG
12,589
BAEVM_P10272_3mutA


GS




 


PAP

MLVMS_P03355_3mut


 


EAAAKGSSPAP
12,591
XMRV6_A1Z651_3mutA


 


PAPEAAAKGGS
12,592
MLVFF_P26809_3mutA


 


GSSGGGEAAAK
12,593
BAEVM_P10272_3mutA


 


PAPAPAP
12,594
MLVMS_P03355_3mutA_WS


 


GGSEAAAKGGG
12,595
MLVMS_P03355_PLV919


 


GSSEAAAK
12,596
PERV_Q4VFZ2_3mut


 


GGGG
12,597
MLVMS_P03355_3mutA_WS


 


GGGGGS
12,598
MLVMS_P03355_3mut


 


GGGGSSEAAAK
12,599
PERV_Q4VFZ2_3mut


 


EAAAKEAAAKEAAAKEAA
12,600
SFV3L_P27401-Pro_2mutA


AKEAAAKEAAAK




 


GGSEAAAKGSS
12,601
MLVMS_P03355_3mutA_WS


 


PAPGSSGGS
12,602
XMRV6_A1Z651_3mutA


 


GGSPAP
12,603
MLVMS_P03355_3mutA_WS


 


GGGGSSEAAAK
12,604
BAEVM_P10272_3mut


 


GGSGGSGGSGGS
12,605
AVIRE_P03360_3mutA


 


PAPGSSGGS
12,606
MLVFF_P26809_3mutA


 


GSSPAPGGG
12,607
MLVMS_P03355_3mutA_WS


 


GGGGGGG
12,608
MLVMS_P03355_3mutA_WS


 


EAAAKGGGGGS
12,609
MLVMS_P03355_3mutA_WS


 


EAAAKGGSGGG
12,610
MLVMS_P03355_PLV919


 


GGGGSSEAAAK
12,611
XMRV6_A1Z651_3mutA


 


GGGGSEAAAKGGGGS
12,612
MLVBM_Q7SVK7_3mutA_WS


 


GSSGSS
12,613
MLVMS_P03355_PLV919


 


GGSGGG
12,614
MLVMS_P03355_PLV919


 


PAPEAAAKGGG
12,615
AVIRE_P03360_3mutA


 


AEAAAKEAAAKEAAAKEA
12,616
FOAMV_P14350-Pro_2mutA


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GGGGGSGSS
12,617
PERV_Q4VFZ2_3mut


 


GSSGSSGSSGSSGSS
12,618
KORV_Q9TTC1-Pro_3mut


 


GGGGSEAAAKGGGGS
12,619
MLVMS_P03355_3mutA_WS


 


GGGGGSPAP
12,620
FLV_P10273_3mut


 


GGGEAAAK
12,621
MLVMS_P03355_3mutA_WS


 


GGSGGSGGSGGS
12,622
FLV_P10273_3mutA


 


GGG

MLVMS_P03355_PLV919


 


GGSPAPEAAAK
12,624
BAEVM_P10272_3mutA


 


EAAAKEAAAK
12,625
FLV_P10273_3mutA


 


GGGEAAAKPAP
12,626
BAEVM_P10272_3mutA


 


GGGEAAAKGGS
12,627
PERV_Q4VFZ2_3mut


 


GGSGGSGGS
12,628
PERV_Q4VFZ2_3mut


 


EAAAKGGGPAP
12,629
XMRV6_A1Z651_3mutA


 


EAAAK
12,630
MLVBM_Q7SVK7_3mutA_WS


 


PAPEAAAKGGG
12,631
PERV_Q4VFZ2_3mut


 


EAAAKGSS
12,632
MLVCB_P08361_3mutA


 


GGSEAAAKGGG
12,633
MLVBM_Q7SVK7_3mutA_WS


 


GGGGSGGGGSGGGGSGGG
12,634
XMRV6_A1Z651_3mutA


GS




 


GGGGSGGGGGGGGSGGGG
12,635
BAEVM_P10272_3mut


SGGGGS




 


GGGGSSPAP
12,636
PERV_Q4VFZ2_3mutA_WS


 


GGSGGSGGSGGSGGSGGS
12,637
PERV_Q4VFZ2_3mut


 


GGGEAAAKPAP
12,638
PERV_Q4VFZ2_3mut


 


EAAAKEAAAK
12,639
BAEVM_P10272_3mutA


 


GGSGSSEAAAK
12,640
XMRV6_A1Z651_3mutA


 


PAPEAAAKGSS
12,641
WMSV_P03359_3mutA


 


PAPAPAPAPAP
12,642
XMRV6_A1Z651_3mutA


 


GSSGGGEAAAK
12,643
MLVMS_P03355_PLV919


 


GSSPAPGGG
12,644
MLVFF_P26809_3mutA


 


GGSPAPEAAAK
12,645
MLVFF_P26809_3mut


 


PAPGGSEAAAK
12,646
PERV_Q4VFZ2_3mut


 


GGGGSS
12,647
MLVFF_P26809_3mutA


 


GGSGSSGGG
12,648
BAEVM_P10272_3mutA


 


GSSGGGEAAAK
12,649
MLVMS_P03355_3mutA_WS


 


EAAAKGGS
12,650
MLVBM_Q7SVK7_3mutA_WS


 


GGGPAPGGS
12,651
MLVMS_P03355_PLV919


 


EAAAKEAAAK
12,652
MLVMS_P03355_PLV919


 


GSSGSSGSS
12,653
MLVMS_P03355_PLV919


 


GGGEAAAKPAP
12,654
MLVAV_P03356_3mutA


 


SGSETPGTSESATPES
12,655
FLV_P10273_3mutA


 


PAPAPAPAPAP
12,656
KORV_Q9TTC1-Pro_3mut


 


AEAAAKEAAAKEAAAKEA
12,657
BAEVM_P10272_3mutA


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


PAPGSSGGG
12,658
MLVMS_P03355_3mutA_WS


 


GSSGGGEAAAK
12,659
XMRV6_A1Z651_3mutA


 


GGGGSGGGGSGGGGSGGG
12,660
XMRV6_A1Z651_3mutA


GSGGGGS




 


GGGGSSPAP
12,661
MLVFF_P26809_3mutA


 


GGSGGGPAP
12,662
PERV_Q4VFZ2_3mutA_WS


 


GSS

PERV_Q4VFZ2_3mut


 


EAAAKGSSPAP
12,664
MLVMS_P03355_3mut


 


EAAAKGGG
12,665
XMRV6_A1Z651_3mutA


 


GSSGSSGSSGSS
12,666
WMSV_P03359_3mutA


 


PAPEAAAKGSS
12,667
MLVMS_P03355_PLV919


 


GSSEAAAK
12,668
AVIRE_P03360_3mutA


 


EAAAKGGSGSS
12,669
AVIRE_P03360_3mutA


 


GSSEAAAK
12,670
MLVMS_P03355_3mut


 


GGSGSSEAAAK
12,671
MLVMS_P03355_PLV919


 


GGSEAAAKGGG
12,672
MLVFF_P26809_3mutA


 


GGGGSGGGGSGGGGSGGG
12,673
MLVAV_P03356_3mutA


GS




 


PAPAPAPAPAPAP
12,674
MLVFF_P26809_3mut


 


EAAAKPAPGSS
12,675
KORV_Q9TTC1-Pro_3mut


 


PAPGSSEAAAK
12,676
MLVAV_P03356_3mutA


 


GGGGSSPAP
12,677
WMSV_P03359_3mutA


 


EAAAKGGGGGS
12,678
MLVMS_P03355_3mutA_WS


 


GGGEAAAKGGS
12,679
MLVMS_P03355_3mut


 


GGSGSSGGG
12,680
MLVMS_P03355_3mut


 


GGGPAPGGS
12,681
MLVAV_P03356_3mutA


 


PAPGGGGGS
12,682
MLVMS_P03355_PLV919


 


GGGPAPGSS
12,683
PERV_Q4VFZ2_3mut


 


GGGGGGG
12,684
MLVFF_P26809_3mutA


 


GGSGGGGSS
12,685
MLVCB_P08361_3mutA


 


GGGGGG
12,686
FLV_P10273_3mutA


 


GGSEAAAKGSS
12,687
PERV_Q4VFZ2_3mut


 


GGSPAPGGG
12,688
BAEVM_P10272_3mutA


 


GGSPAPGSS
12,689
AVIRE_P03360_3mutA


 


GGSGGSGGSGGS
12,690
KORV_Q9TTC1_3mut


 


EAAAKEAAAKEAAAKEAA
12,691
MLVBM_Q7SVK7_3mut


AKEAAAK




 


PAPGSSGGS
12,692
XMRV6_A1Z651_3mut


 


EAAAKGGGGSS
12,693
PERV_Q4VFZ2_3mutA_WS


 


GGSGGSGGSGGSGGS
12,694
PERV_Q4VFZ2_3mutA_WS


 


PAPGGSGGG
12,695
MLVMS_P03355_PLV919


 


PAPGSSGGG
12,696
PERV_Q4VFZ2_3mutA_WS


 


GSSGSS
12,697
BAEVM_P10272_3mutA


 


EAAAKGSS
12,698
MLVFF_P26809_3mutA


 


GGGPAP
12,699
MLVMS_P03355_PLV919


 


EAAAKGGGGGS
12,700
MLVFF_P26809_3mutA


 


EAAAKGGSPAP
12,701
MLVBM_Q7SVK7_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
12,702
WMSV_P03359_3mutA


AKEAAAKEAAAK




 


GSSPAPGGG
12,703
MLVBM_Q7SVK7_3mutA_WS


 


GGGEAAAKGSS
12,704
AVIRE_P03360_3mutA


 


GGGGSSEAAAK
12,705
AVIRE_P03360_3mutA


 


GGGGGGGG
12,706
PERV_Q4VFZ2_3mutA_WS


 


PAPGSSEAAAK
12,707
BAEVM_P10272_3mutA


 


EAAAKGSS
12,708
MLVFF_P26809_3mut


 


GSSEAAAKGGG
12,709
MLVCB_P08361_3mutA


 


GGSEAAAK
12,710
MLVBM_Q7SVK7_3mutA_WS


 


GSSEAAAKGGG
12,711
PERV_Q4VFZ2_3mutA_WS


 


PAPGGSGGG
12,712
WMSV_P03359_3mutA


 


GSSGGSGGG
12,713
MLVCB_P08361_3mutA


 


EAAAKGSSGGG
12,714
FLV_P10273_3mutA


 


GSSEAAAK
12,715
MLVCB_P08361_3mutA


 


GSSGGGEAAAK
12,716
MLVMS_P03355_3mut


 


GGGGSGGGGS
12,717
MLVCB_P08361_3mutA


 


EAAAKGGGGSEAAAK
12,718
MLVBM_Q7SVK7_3mutA_WS


 


EAAAKGGG
12,719
PERV_Q4VFZ2_3mutA_WS


 


EAAAKGGSPAP
12,720
MLVMS_P03355_PLV919


 


GGGPAPGGS
12,721
AVIRE_P03360_3mutA


 


GSSEAAAK
12,722
MLVBM_Q7SVK7_3mutA_WS


 


GSSGGGEAAAK
12,723
PERV_Q4VFZ2_3mut


 


SGSETPGTSESATPES
12,724
MLVMS_P03355_PLV919


 


GGSGSSPAP
12,725
MLVMS_P03355_3mut


 


GGGGGG
12,726
MLVBM_Q7SVK7_3mutA_WS


 


GGSPAPGGG
12,727
XMRV6_A1Z651_3mutA


 


GGSGSS
12,728
PERV_Q4VFZ2_3mutA_WS


 


PAP

MLVBM_Q7SVK7_3mutA_WS


 


EAAAKPAPGSS
12,730
MLVMS_P03355_PLV919


 


EAAAKGGG
12,731
MLVMS_P03355_3mut


 


GSSEAAAKPAP
12,732
PERV_Q4VFZ2_3mutA_WS


 


GGGGSS
12,733
MLVMS_P03355_3mutA_WS


 


GGSGSSEAAAK
12,734
PERV_Q4VFZ2_3mut


 


GGGGSS
12,735
BAEVM_P10272_3mutA


 


PAPAP
12,736
MLVFF_P26809_3mut


 


PAPEAAAKGGG
12,737
BAEVM_P10272_3mutA


 


EAAAKGGS
12,738
MLVMS_P03355_PLV919


 


PAPAPAPAPAPAP
12,739
PERV_Q4VFZ2_3mutA_WS


 


GGGGGSEAAAK
12,740
MLVMS_P03355_3mut


 


PAPGGS
12,741
PERV_Q4VFZ2_3mut


 


GGGGSS
12,742
MLVCB_P08361_3mutA


 


GGGGS
12,743
MLVAV_P03356_3mutA


 


GSSPAPEAAAK
12,744
MLVMS_P03355_PLV919


 


GGGGSSGGS
12,745
MLVFF_P26809_3mutA


 


PAPEAAAKGSS
12,746
MLVMS_P03355_PLV919


 


GGSGSSEAAAK
12,747
MLVMS_P03355_3mutA_WS


 


EAAAKGGG
12,748
MLVAV_P03356_3mutA


 


PAPGSSEAAAK
12,749
FLV_P10273_3mutA


 


EAAAKGSSGGG
12,750
MLVCB_P08361_3mutA


 


PAPEAAAK
12,751
KORV_Q9TTC1-Pro_3mutA


 


GGSPAPEAAAK
12,752
KORV_Q9TTC1-Pro_3mut


 


GGSGGSGGSGGSGGSGGS
12,753
MLVAV_P03356_3mutA


 


GSSEAAAKPAP
12,754
MLVBM_Q7SVK7_3mutA_WS


 


AEAAAKEAAAKEAAAKEA
12,755
KORV_Q9TTC1-Pro_3mutA


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GSSGGGEAAAK
12,756
XMRV6_A1Z651_3mut


 


PAPGGSGGG
12,757
AVIRE_P03360_3mutA


 


PAPGGSEAAAK
12,758
PERV_Q4VFZ2_3mutA_WS


 


GGGGS
12,759
MLVMS_P03355_3mutA_WS


 


GGGGSGGGGSGGGGS
12,760
MLVBM_Q7SVK7_3mutA_WS


 


PAPAPAPAPAP
12,761
PERV_Q4VFZ2_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
12,762
MLVMS_P03355_3mut


AKEAAAK




 


GSSGGSEAAAK
12,763
MLVMS_P03355_3mutA_WS


 


GGSGGSGGSGGS
12,764
WMSV_P03359_3mutA


 


EAAAKGSSGGG
12,765
WMSV_P03359_3mutA


 


EAAAKGGG
12,766
PERV_Q4VFZ2_3mutA_WS


 


SGSETPGTSESATPES
12,767
PERV_Q4VFZ2_3mut


 


PAPGSSGGS
12,768
MLVMS_P03355_3mutA_WS


 


PAPEAAAKGSS
12,769
PERV_Q4VFZ2_3mut


 


PAPEAAAK
12,770
AVIRE_P03360_3mutA


 


GSSEAAAKGGG
12,771
BAEVM_P10272_3mutA


 


GSSPAP
12,772
MLVAV_P03356_3mutA


 


EAAAKEAAAKEAAAKEAA
12,773
MLVFF_P26809_3mut


AK




 


PAPGGSGSS
12,774
MLVAV_P03356_3mutA


 


GGGGSGGGGSGGGGS
12,775
PERV_Q4VFZ2_3mutA_WS


 


GSSGGSEAAAK
12,776
MLVCB_P08361_3mutA


 


EAAAKGGS
12,777
KORV_Q9TTC1-Pro_3mutA


 


EAAAKGGS
12,778
MLVFF_P26809_3mutA


 


GGSPAP
12,779
MLVMS_P03355_PLV919


 


GGSGSS
12,780
MLVMS_P03355_PLV919


 


SGSETPGTSESATPES
12,781
WMSV_P03359_3mut


 


GGGGGGG
12,782
WMSV_P03359_3mut


 


GGSPAPGSS
12,783
MLVCB_P08361_3mutA


 


GGGGSSGGS
12,784
WMSV_P03359_3mut


 


PAPGGS
12,785
MLVMS_P03355_PLV919


 


PAPGSSGGS
12,786
MLVCB_P08361_3mutA


 


EAAAKEAAAKEAAAKEAA
12,787
MLVFF_P26809_3mut


AKEAAAK




 


SGGSSGGSSGSETPGTSE
12,788
PERV_Q4VFZ2_3mut


SATPESSGGSSGGSS




 


GGSGGSGGSGGSGGS
12,789
BAEVM_P10272_3mutA


 


GSSEAAAK
12,790
PERV_Q4VFZ2_3mut


 


EAAAKEAAAKEAAAKEAA
12,791
KORV_Q9TTC1-Pro_3mutA


AK




 


GGSGGSGGSGGSGGS
12,792
MLVMS_P03355_3mut


 


PAPAPAPAPAPAP
12,793
MLVMS_P03355_3mut


 


GGSPAPEAAAK
12,794
MLVMS_P03355_PLV919


 


EAAAK
12,795
WMSV_P03359_3mutA


 


EAAAKGSSGGS
12,796
MLVBM_Q7SVK7_3mutA_WS


 


GGSGGGGSS
12,797
MLVMS_P03355_3mutA_WS


 


GGGEAAAKPAP
12,798
MLVMS_P03355_3mut


 


EAAAKGGSGGG
12,799
XMRV6_A1Z651_3mutA


 


GGGGGSEAAAK
12,800
KORV_Q9TTC1-Pro_3mutA


 


GGGGGG
12,801
BAEVM_P10272_3mutA


 


GGGGGG
12,802
MLVMS_P03355_3mut


 


GGGGGGG
12,803
MLVBM_Q7SVK7_3mutA_WS


 


AEAAAKEAAAKEAAAKEA
12,804
AVIRE_P03360


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


PAPGSSGGS
12,805
PERV_Q4VFZ2_3mut


 


GGGGGS
12,806
XMRV6_A1Z651_3mut


 


EAAAKPAP
12,807
XMRV6_A1Z651_3mutA


 


GGG

MLVMS_P03355_3mutA_WS


 


AEAAAKEAAAKEAAAKEA
12,809
FLV_P10273_3mut


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


EAAAKGSSPAP
12,810
MLVMS_P03355_3mut


 


SGSETPGTSESATPES
12,811
BAEVM_P10272_3mutA


 


GGSPAPEAAAK
12,812
MLVMS_P03355_3mut


 


GSSGSSGSSGSS
12,813
MLVAV_P03356_3mutA


 


AEAAAKEAAAKEAAAKEA
12,814
MLVMS_P03355_3mut


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GGSPAP
12,815
MLVCB_P08361_3mutA


 


GGGGGSEAAAK
12,816
MLVMS_P03355_3mutA_WS


 


GGGGG
12,817
MLVFF_P26809_3mutA


 


GSSEAAAK
12,818
MLVAV_P03356_3mutA


 


GGS

BAEVM_P10272_3mut


 


EAAAKGGSPAP
12,820
MLVCB_P08361_3mutA


 


PAPAPAPAP
12,821
FLV_P10273_3mutA


 


PAPGGGEAAAK
12,822
MLVCB_P08361_3mutA


 


GGGGSSEAAAK
12,823
MLVMS_P03355_3mutA_WS


 


GGGGG
12,824
PERV_Q4VFZ2_3mutA_WS


 


GGSGGSGGSGGSGGSGGS
12,825
PERV_Q4VFZ2_3mut


 


GGGGG
12,826
MLVMS_P03355_3mut


 


PAPEAAAKGGG
12,827
MLVBM_Q7SVK7_3mutA_WS


 


GSSGGGPAP
12,828
XMRV6_A1Z651_3mutA


 


GSSGSSGSSGSSGSSGSS
12,829
PERV_Q4VFZ2_3mutA_WS


 


EAAAKGGSPAP
12,830
PERV_Q4VFZ2_3mut


 


GSSGGSEAAAK
12,831
MLVMS_P03355_PLV919


 


GSS

PERV_Q4VFZ2_3mut


 


EAAAKGGS
12,833
WMSV_P03359_3mutA


 


GGGGGSPAP
12,834
PERV_Q4VFZ2_3mutA_WS


 


EAAAKGSS
12,835
MLVMS_P03355_PLV919


 


EAAAKGGGGSS
12,836
KORV_Q9TTC1-Pro_3mutA


 


PAPGSSGGG
12,837
PERV_Q4VFZ2_3mut


 


GGGGSSEAAAK
12,838
MLVFF_P26809_3mut


 


PAPAPAP
12,839
MLVMS_P03355_3mut


 


GSSGGSEAAAK
12,840
XMRV6_A1Z651_3mut


 


PAPEAAAKGSS
12,841
MLVMS_P03355_3mutA_WS


 


GGSGGSGGSGGSGGS
12,842
MLVMS_P03355_3mutA_WS


 


GGSGSSPAP
12,843
XMRV6_A1Z651_3mutA


 


GGGGSSPAP
12,844
MLVMS_P03355_PLV919


 


GGGGS
12,845
MLVCB_P08361_3mutA


 


EAAAKEAAAKEAAAKEAA
12,846
PERV_Q4VFZ2_3mutA_WS


AK




 


EAAAKEAAAK
12,847
KORV_Q9TTC1_3mutA


 


PAPGGGEAAAK
12,848
BAEVM_P10272_3mutA


 


GSSGGSEAAAK
12,849
XMRV6_A1Z651_3mutA


 


EAAAKEAAAKEAAAKEAA
12,850
FLV_P10273_3mut


AKEAAAKEAAAK




 


GSSEAAAKPAP
12,851
MLVMS_P03355_3mutA_WS


 


EAAAKPAPGSS
12,852
PERV_Q4VFZ2_3mutA_WS


 


GSSGGSPAP
12,853
XMRV6_A1Z651_3mutA


 


GSSEAAAKGGG
12,854
PERV_Q4VFZ2_3mut


 


GGGEAAAKGGS
12,855
WMSV_P03359_3mutA


 


GSSEAAAKGGG
12,856
MLVFF_P26809_3mut


 


PAPAPAP
12,857
KORV_Q9TTC1-Pro_3mutA


 


EAAAKGGSPAP
12,858
MLVMS_P03355_3mutA_WS


 


PAPGGSEAAAK
12,859
PERV_Q4VFZ2_3mut


 


GGGGS
12,860
MLVBM_Q7SVK7_3mutA_WS


 


EAAAKGSSGGG
12,861
KORV_Q9TTC1_3mut


 


EAAAKGGGPAP
12,862
MLVCB_P08361_3mutA


 


EAAAKGSS
12,863
BAEVM_P10272_3mutA


 


GGSPAPGGG
12,864
MLVBM_Q7SVK7_3mutA_WS


 


GGGGSEAAAKGGGGS
12,865
MLVMS_P03355_3mutA_WS


 


GGGEAAAKGGS
12,866
PERV_Q4VFZ2_3mutA_WS


 


EAAAKGGGGSS
12,867
MLVMS_P03355_3mutA_WS


 


EAAAKGGGPAP
12,868
MLVFF_P26809_3mut


 


GSSPAP
12,869
PERV_Q4VFZ2_3mutA_WS


 


EAAAKGGS
12,870
MLVMS_P03355_3mut


 


GGGGSS
12,871
KORV_Q9TTC1-Pro_3mutA


 


EAAAKGSSPAP
12,872
MLVMS_P03355_3mutA_WS


 


GGGPAP
12,873
PERV_Q4VFZ2_3mut


 


EAAAKGSSGGS
12,874
XMRV6_A1Z651_3mutA


 


PAPGGG
12,875
MLVAV_P03356_3mutA


 


GSSPAPEAAAK
12,876
BAEVM_P10272_3mutA


 


GGGPAP
12,877
MLVBM_Q7SVK7_3mutA_WS


 


GSSGGGGGS
12,878
AVIRE_P03360_3mutA


 


SGSETPGTSESATPES
12,879
MLVMS_P03355_PLV919


 


GGGPAP
12,880
MLVFF_P26809_3mut


 


EAAAKGGGGSS
12,881
XMRV6_A1Z651_3mutA


 


GGGGSSPAP
12,882
XMRV6_A1Z651_3mut


 


GGGGSEAAAKGGGGS
12,883
MLVMS_P03355_3mut


 


GSSPAP
12,884
MLVBM_Q7SVK7_3mutA_WS


 


GGSGSSEAAAK
12,885
FLV_P10273_3mutA


 


SGSETPGTSESATPES
12,886
MLVBM_Q7SVK7_3mutA_WS


 


PAPGGG
12,887
AVIRE_P03360_3mutA


 


GGGEAAAKPAP
12,888
MLVMS_P03355_3mutA_WS


 


EAAAKGGSGSS
12,889
PERV_Q4VFZ2_3mut


 


GGSPAPGGG
12,890
MLVAV_P03356_3mutA


 


PAPGGSGSS
12,891
BAEVM_P10272_3mutA


 


GSSGGSPAP
12,892
MLVFF_P26809_3mutA


 


EAAAKGSSGGG
12,893
PERV_Q4VFZ2_3mut


 


GGGGSGGGGS
12,894
PERV_Q4VFZ2_3mutA_WS


 


GSSGGGGGS
12,895
BAEVM_P10272_3mutA


 


GGGGSSGGS
12,896
MLVBM_Q7SVK7_3mutA_WS


 


EAAAKGGS
12,897
PERV_Q4VFZ2_3mutA_WS


 


GSSGSSGSSGSS
12,898
MLVMS_P03355_3mut


 


GGS

MLVMS_P03355_3mutA_WS


 


GSSGGSEAAAK
12,900
MLVBM_Q7SVK7_3mutA_WS


 


SGGSSGGSSGSETPGTSE
12,901
XMRV6_A1Z651


SATPESSGGSSGGSS




 


GGGGG
12,902
FLV_P10273_3mutA


 


PAPEAAAKGSS
12,903
PERV_Q4VFZ2_3mut


 


GGGGGG
12,904
WMSV_P03359_3mut


 


EAAAKGGG
12,905
BAEVM_P10272_3mutA


 


GGGGSS
12,906
MLVMS_P03355_3mutA_WS


 


GSSGGGEAAAK
12,907
KORV_Q9TTC1_3mut


 


GGSGSS
12,908
AVIRE_P03360_3mutA


 


EAAAKPAP
12,909
MLVMS_P03355_3mut


 


EAAAKEAAAKEAAAK
12,910
FLV_P10273_3mutA


 


GGGG
12,911
XMRV6_A1Z651_3mutA


 


GSSPAPGGS
12,912
BAEVM_P10272_3mutA


 


GSSGGGGGS
12,913
MLVFF_P26809_3mutA


 


GGGGSSGGS
12,914
MLVAV_P03356_3mutA


 


GGS

PERV_Q4VFZ2_3mut


 


GGGGG
12,916
WMSV_P03359_3mutA


 


GSSGSSGSSGSSGSSGSS
12,917
FLV_P10273_3mutA


 


PAPGGGGSS
12,918
MLVAV_P03356_3mutA


 


GGGGGGGG
12,919
BAEVM_P10272_3mutA


 


SGSETPGTSESATPES
12,920
MLVCB_P08361_3mutA


 


PAPGGG
12,921
BAEVM_P10272_3mutA


 


GSSGSSGSS
12,922
MLVCB_P08361_3mutA


 


GGSGSS
12,923
MLVMS_P03355_3mutA_WS


 


EAAAKGGGGSEAAAK
12,924
WMSV_P03359_3mutA


 


GGGGGGGG
12,925
FLV_P10273_3mutA


 


GSSGSS
12,926
MLVMS_P03355_3mutA_WS


 


PAPEAAAKGGS
12,927
XMRV6_A1Z651_3mutA


 


EAAAKEAAAK
12,928
MLVMS_P03355_3mut


 


GGGGSGGGGSGGGGS
12,929
BAEVM_P10272_3mutA


 


EAAAKGSSPAP
12,930
MLVMS_P03355_PLV919


 


GGGGSSEAAAK
12,931
MLVMS_P03355_3mut


 


GGGGSSEAAAK
12,932
BAEVM_P10272_3mutA


 


PAPGGSGSS
12,933
PERV_Q4VFZ2_3mut


 


GGSGGGEAAAK
12,934
MLVFF_P26809_3mut


 


PAPEAAAKGGS
12,935
PERV_Q4VFZ2_3mut


 


GGGPAPGSS
12,936
AVIRE_P03360_3mut


 


PAPGGSGGG
12,937
PERV_Q4VFZ2_3mutA_WS


 


GGGGGGGG
12,938
PERV_Q4VFZ2_3mutA_WS


 


GSSEAAAK
12,939
MLVMS_P03355_3mutA_WS


 


GGGGSGGGGSGGGGS
12,940
PERV_Q4VFZ2_3mutA_WS


 


EAAAKGGS
12,941
MLVMS_P03355_3mut


 


GGGGGSGSS
12,942
MLVCB_P08361_3mut


 


GGGPAP
12,943
KORV_Q9TTC1-Pro_3mutA


 


EAAAKPAPGGG
12,944
MLVCB_P08361_3mut


 


GSSGGSPAP
12,945
MLVCB_P08361_3mutA


 


SGGSSGGSSGSETPGTSE
12,946
MLVMS_P03355_3mut


SATPESSGGSSGGSS




 


PAPAPAPAP
12,947
MLVMS_P03355_3mut


 


GSSGGS
12,948
XMRV6_A1Z651_3mutA


 


GSSEAAAKGGG
12,949
MLVMS_P03355_3mut


 


GGSGSSPAP
12,950
MLVMS_P03355_3mutA_WS


 


GSSEAAAKGGS
12,951
MLVMS_P03355_PLV919


 


EAAAKEAAAKEAAAKEAA
12,952
BAEVM_P10272_3mut


AKEAAAK




 


PAPGGGGSS
12,953
KORV_Q9TTC1_3mutA


 


EAAAKGSS
12,954
MLVMS_P03355_3mutA_WS


 


AEAAAKEAAAKEAAAKEA
12,955
FFV_O93209_2mut


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GGSGGSGGSGGSGGSGGS
12,956
BAEVM_P10272_3mutA


 


GGGGGG
12,957
MLVMS_P03355_PLV919


 


PAPEAAAK
12,958
BAEVM_P10272_3mutA


 


GGSGSSEAAAK
12,959
MLVAV_P03356_3mutA


 


GGG

MLVCB_P08361_3mutA


 


GGGGG
12,961
MLVCB_P08361_3mutA


 


GGSGGSGGSGGS
12,962
KORV_Q9TTC1-Pro_3mutA


 


GSSGSSGSSGSSGSSGSS
12,963
XMRV6_A1Z651_3mutA


 


GSSEAAAKPAP
12,964
FLV_P10273_3mutA


 


GGGEAAAKPAP
12,965
MLVCB_P08361_3mutA


 


GSSGSSGSS
12,966
MLVMS_P03355_3mutA_WS


 


PAPAPAPAP
12,967
MLVMS_P03355_PLV919


 


EAAAKGGG
12,968
MLVMS_P03355_PLV919


 


PAPAPAPAPAPAP
12,969
FLV_P10273_3mutA


 


EAAAKGGSGSS
12,970
MLVMS_P03355_3mut


 


GGGGGG
12,971
PERV_Q4VFZ2_3mutA_WS


 


PAPGGG
12,972
MLVCB_P08361_3mutA


 


GGGGGSGSS
12,973
KORV_Q9TTC1_3mutA


 


GGGGSGGGGSGGGGSGGG
12,974
XMRV6_A1Z651_3mut


GS




 


GGSGGSGGS
12,975
KORV_Q9TTC1-Pro_3mutA


 


EAAAKPAPGGG
12,976
MLVMS_P03355_3mutA_WS


 


AEAAAKEAAAKEAAAKEA
12,977
XMRV6_A1Z651


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GGGGSGGGGSGGGGSGGG
12,978
FLV_P10273_3mutA


GSGGGGSGGGGS




 


EAAAKGGGGSEAAAK
12,979
PERV_Q4VFZ2_3mutA_WS


 


GGGPAPGSS
12,980
AVIRE_P03360_3mutA


 


GGGGG
12,981
MLVMS_P03355_3mutA_WS


 


GGGGSGGGGSGGGGSGGG
12,982
MLVMS_P03355_3mut


GSGGGGSGGGGS




 


GGGGSGGGGS
12,983
MLVMS_P03355_3mutA_WS


 


EAAAKGGSPAP
12,984
XMRV6_A1Z651_3mutA


 


EAAAKGSSPAP
12,985
AVIRE_P03360_3mutA


 


PAPGGSGSS
12,986
KORV_Q9TTC1-Pro_3mutA


 


GSS

MLVBM_Q7SVK7_3mutA_WS


 


GSS

WMSV_P03359_3mut


 


GGGPAPGSS
12,989
MLVFF_P26809_3mutA


 


EAAAKPAP
12,990
MLVMS_P03355_3mut


 


GSSPAPEAAAK
12,991
FLV_P10273_3mutA


 


GGSPAPGSS
12,992
MLVBM_Q7SVK7_3mutA_WS


 


GGGGGSEAAAK
12,993
XMRV6_A1Z651_3mut


 


PAPEAAAKGGG
12,994
WMSV_P03359_3mutA


 


PAPGGG
12,995
PERV_Q4VFZ2_3mut


 


GGSPAPEAAAK
12,996
WMSV_P03359_3mutA


 


GGSGGGGSS
12,997
PERV_Q4VFZ2_3mut


 


EAAAKGGGGSS
12,998
PERV_Q4VFZ2_3mut


 


EAAAKGGSPAP
12,999
AVIRE_P03360_3mut


 


GGSGGGGSS
13,000
WMSV_P03359_3mutA


 


PAPGSSEAAAK
13,001
MLVFF_P26809_3mut


 


GSSEAAAK
13,002
MLVMS_P03355_PLV919


 


GSAGSAAGSGEF
13,003
AVIRE_P03360_3mutA


 


EAAAKGGSGSS
13,004
MLVMS_P03355_3mut


 


GGSEAAAKPAP
13,005
MLVMS_P03355_PLV919


 


GGGGSGGGGSGGGGSGGG
13,006
MLVFF_P26809_3mutA


GSGGGGS




 


PAPGSSEAAAK
13,007
PERV_Q4VFZ2_3mutA_WS


 


GGGGSSPAP
13,008
MLVMS_P03355_3mutA_WS


 


PAPAPAP
13,009
MLVCB_P08361_3mutA


 


EAAAKPAPGGG
13,010
MLVBM_Q7SVK7_3mutA_WS


 


GGGPAPGSS
13,011
BAEVM_P10272_3mutA


 


PAP

MLVMS_P03355_3mutA_WS


 


PAPGGSGGG
13,013
MLVMS_P03355_3mutA_WS


 


GGSGGSGGSGGSGGS
13,014
MLVBM_Q7SVK7_3mutA_WS


 


PAPAPAPAP
13,015
XMRV6_A1Z651_3mut


 


GSSPAPGGG
13,016
MLVMS_P03355_3mutA_WS


 


GSSPAPGGG
13,017
MLVMS_P03355_3mut


 


PAPGGG
13,018
MLVMS_P03355_PLV919


 


GGGEAAAKGSS
13,019
WMSV_P03359_3mut


 


EAAAKGSS
13,020
KORV_Q9TTC1-Pro_3mutA


 


EAAAKGGS
13,021
PERV_Q4VFZ2_3mut


 


EAAAKEAAAKEAAAKEAA
13,022
PERV_Q4VFZ2_3mut


AKEAAAK




 


PAPEAAAKGGG
13,023
MLVMS_P03355_PLV919


 


EAAAKGSSGGG
13,024
MLVFF_P26809_3mut


 


AEAAAKEAAAKEAAAKEA
13,025
PERV_Q4VFZ2


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


EAAAKEAAAKEAAAKEAA
13,026
MLVAV_P03356_3mutA


AKEAAAKEAAAK




 


GSSGGSGGG
13,027
MLVFF_P26809_3mut


 


GSSGSSGSSGSS
13,028
PERV_Q4VFZ2_3mutA_WS


 


GGSPAPGGG
13,029
MLVMS_P03355_PLV919


 


GSS

BAEVM_P10272_3mut


 


GGGPAPGSS
13,031
MLVMS_P03355_3mutA_WS


 


GGGGSS
13,032
KORV_Q9TTC1_3mutA


 


GSSGGSGGG
13,033
BAEVM_P10272_3mutA


 


EAAAKEAAAKEAAAK
13,034
MLVCB_P08361_3mutA


 


SGGSSGGSSGSETPGTSE
13,035
FLV_P10273_3mutA


SATPESSGGSSGGSS




 


PAPGGGGGS
13,036
PERV_Q4VFZ2_3mut


 


PAPAPAPAPAP
13,037
KORV_Q9TTC1-Pro_3mutA


 


EAAAK
13,038
MLVMS_P03355_3mutA_WS


 


GGG

MLVCB_P08361_3mut


 


GGSEAAAKGGG
13,040
BAEVM_P10272_3mutA


 


GGGGGSGSS
13,041
MLVAV_P03356_3mutA


 


EAAAKGSSPAP
13,042
MLVBM_Q7SVK7_3mutA_WS


 


GGSGGSGGS
13,043
XMRV6_A1Z651_3mut


 


EAAAKPAPGGG
13,044
KORV_Q9TTC1-Pro_3mutA


 


GGGPAPEAAAK
13,045
FLV_P10273_3mutA


 


GGSPAPEAAAK
13,046
MLVMS_P03355_3mutA_WS


 


GGSGGSGGSGGSGGS
13,047
MLVFF_P26809_3mut


 


EAAAKGGSGSS
13,048
MLVMS_P03355_PLV919


 


GGGEAAAKGGS
13,049
MLVBM_Q7SVK7_3mutA_WS


 


PAPAPAPAP
13,050
BAEVM_P10272_3mutA


 


EAAAKEAAAKEAAAKEAA
13,051
MLVMS_P03355_3mut


AK




 


EAAAKPAP
13,052
XMRV6_A1Z651_3mut


 


EAAAKEAAAK
13,053
MLVBM_Q7SVK7_3mutA_WS


 


EAAAKGGG
13,054
BAEVM_P10272_3mut


 


EAAAKGSS
13,055
MLVAV_P03356_3mutA


 


EAAAKEAAAKEAAAKEAA
13,056
MLVFF_P26809_3mut


AKEAAAKEAAAK




 


GGGPAPGSS
13,057
PERV_Q4VFZ2_3mutA_WS


 


GGGG
13,058
PERV_Q4VFZ2_3mut


 


EAAAKGGSGSS
13,059
MLVMS_P03355_PLV919


 


GGGGSGGGGSGGGGS
13,060
MLVMS_P03355_3mutA_WS


 


EAAAK
13,061
MLVMS_P03355_3mutA_WS


 


GGGGSS
13,062
PERV_Q4VFZ2


 


PAPEAAAKGGS
13,063
MLVCB_P08361_3mut


 


GSS

MLVMS_P03355_3mut


 


GSAGSAAGSGEF
13,065
MLVFF_P26809_3mutA


 


EAAAKEAAAKEAAAKEAA
13,066
KORV_Q9TTC1-Pro_3mut


AKEAAAKEAAAK




 


GGGGSGGGGS
13,067
AVIRE_P03360_3mutA


 


EAAAK
13,068
MLVMS_P03355_3mut


 


GGGPAPGGS
13,069
PERV_Q4VFZ2_3mut


 


GGGGSGGGGSGGGGS
13,070
MLVMS_P03355_PLV919


 


PAPGGG
13,071
MLVMS_P03355_3mutA_WS


 


GGGEAAAKPAP
13,072
PERV_Q4VFZ2_3mutA_WS


 


EAAAKPAPGSS
13,073
KORV_Q9TTC1-Pro_3mutA


 


PAPGSS
13,074
KORV_Q9TTC1_3mutA


 


GSAGSAAGSGEF
13,075
PERV_Q4VFZ2_3mut


 


PAPGGGGSS
13,076
KORV_Q9TTC1-Pro_3mutA


 


GSSGGGEAAAK
13,077
MLVCB_P08361_3mutA


 


GSS

AVIRE_P03360_3mutA


 


GSSGSSGSSGSS
13,079
XMRV6_A1Z651_3mutA


 


PAPEAAAKGGG
13,080
MLVMS_P03355_PLV919


 


GGGPAPEAAAK
13,081
MLVCB_P08361_3mutA


 


PAPGGGGGS
13,082
MLVCB_P08361_3mutA


 


EAAAKEAAAKEAAAKEAA
13,083
PERV_Q4VFZ2_3mutA_WS


AK




 


GGGGGSPAP
13,084
MLVFF_P26809_3mutA


 


GSSGSSGSSGSSGSS
13,085
PERV_Q4VFZ2


 


GSSPAPEAAAK
13,086
MLVMS_P03355_PLV919


 


GSSGSSGSSGSSGSSGSS
13,087
MLVBM_Q7SVK7_3mutA_WS


 


GSSGSSGSSGSSGSSGSS
13,088
MLVMS_P03355_3mutA_WS


 


GGSPAPEAAAK
13,089
MLVAV_P03356_3mutA


 


GSSGGG
13,090
BAEVM_P10272_3mut


 


EAAAKGSSGGS
13,091
KORV_Q9TTC1-Pro_3mutA


 


GGSGSSEAAAK
13,092
MLVMS_P03355_3mutA_WS


 


GGGPAPEAAAK
13,093
MLVFF_P26809_3mutA


 


GGGPAPGGS
13,094
MLVMS_P03355_3mutA_WS


 


GGGGG
13,095
MLVMS_P03355_PLV919


 


GGGEAAAKPAP
13,096
MLVBM_Q7SVK7_3mutA_WS


 


GGGGGGGGS
13,097
WMSV_P03359_3mut


 


GGGPAPEAAAK
13,098
PERV_Q4VFZ2_3mut


 


GGSGSSEAAAK
13,099
MLVMS_P03355_PLV919


 


EAAAKGGGPAP
13,100
MLVMS_P03355_3mutA_WS


 


GSSGSSGSSGSSGSS
13,101
KORV_Q9TTC1-Pro_3mutA


 


PAPAP
13,102
WMSV_P03359_3mutA


 


GGSPAPGSS
13,103
MLVAV_P03356_3mutA


 


GGSGGGPAP
13,104
MLVMS_P03355_3mut


 


GGSPAP
13,105
MLVMS_P03355_PLV919


 


EAAAKGGSPAP
13,106
PERV_Q4VFZ2_3mut


 


GSSPAPGGG
13,107
KORV_Q9TTC1-Pro_3mutA


 


GSAGSAAGSGEF
13,108
MLVMS_P03355_3mut


 


GGSPAP
13,109
PERV_Q4VFZ2_3mut


 


GSSGSS
13,110
KORV_Q9TTC1-Pro_3mut


 


GGGPAPGSS
13,111
MLVMS_P03355_3mutA_WS


 


AEAAAKEAAAKEAAAKEA
13,112
FOAMV_P14350


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


PAPGSSGGG
13,113
MLVMS_P03355_PLV919


 


GGSEAAAKPAP
13,114
BAEVM_P10272_3mutA


 


GGGGGS
13,115
MLVCB_P08361_3mutA


 


PAPEAAAKGGS
13,116
MLVMS_P03355_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
13,117
BAEVM_P10272_3mutA


AKEAAAKEAAAK




 


GGSEAAAK
13,118
BAEVM_P10272_3mutA


 


GSSPAPEAAAK
13,119
MLVMS_P03355_3mutA_WS


 


PAPGGG
13,120
WMSV_P03359_3mut


 


EAAAKPAP
13,121
PERV_Q4VFZ2_3mut


 


GSSGSSGSSGSSGSS
13,122
WMSV_P03359_3mut


 


PAPGGG
13,123
MLVBM_Q7SVK7_3mutA_WS


 


GGSGGGEAAAK
13,124
BAEVM_P10272_3mutA


 


PAPGGS
13,125
MLVMS_P03355_3mut


 


GGSGGSGGSGGS
13,126
MLVBM_Q7SVK7_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
13,127
PERV_Q4VFZ2_3mut


AK




 


GGSEAAAKGGG
13,128
WMSV_P03359_3mutA


 


GGGPAP
13,129
BAEVM_P10272_3mutA


 


GGGGSGGGGSGGGGSGGG
13,130
XMRV6_A1Z651_3mut


GSGGGGSGGGGS




 


GGSPAPGSS
13,131
KORV_Q9TTC1_3mut


 


GGGPAPGSS
13,132
MLVMS_P03355_3mut


 


GGGGSSGGS
13,133
BAEVM_P10272_3mutA


 


GGGEAAAKGSS
13,134
KORV_Q9TTC1-Pro_3mutA


 


PAPAP
13,135
MLVBM_Q7SVK7_3mutA_WS


 


GGSPAPGGG
13,136
PERV_Q4VFZ2_3mut


 


PAPGSS
13,137
PERV_Q4VFZ2_3mutA_WS


 


GSSGGSPAP
13,138
MLVBM_Q7SVK7_3mutA_WS


 


EAAAKGGGGSEAAAK
13,139
PERV_Q4VFZ2_3mut


 


GSSEAAAKGGS
13,140
KORV_Q9TTC1-Pro_3mut


 


PAPAPAPAP
13,141
KORV_Q9TTC1-Pro_3mutA


 


GGSEAAAKPAP
13,142
WMSV_P03359_3mutA


 


PAPGGS
13,143
FLV_P10273_3mutA


 


EAAAKGGGPAP
13,144
PERV_Q4VFZ2_3mut


 


GGSGSSGGG
13,145
AVIRE_P03360_3mutA


 


EAAAKGGSGSS
13,146
BAEVM_P10272_3mutA


 


SGGSSGGSSGSETPGTSE
13,147
MLVCB_P08361_3mutA


SATPESSGGSSGGSS




 


GSSEAAAKGGS
13,148
XMRV6_A1Z651_3mutA


 


GGGGG
13,149
BAEVM_P10272_3mutA


 


GGGGSGGGGSGGGGSGGG
13,150
SFV3L_P27401_2mutA


GSGGGGSGGGGS




 


GGGEAAAKGSS
13,151
MLVMS_P03355_PLV919


 


EAAAKGGGGSEAAAK
13,152
KORV_Q9TTC1_3mutA


 


EAAAKGGG
13,153
AVIRE_P03360_3mut


 


GGSGGG
13,154
MLVMS_P03355_3mutA_WS


 


GGSGSSGGG
13,155
MLVMS_P03355_PLV919


 


GGGGSGGGGSGGGGGGGG
13,156
KORV_Q9TTC1_3mut


SGGGGSGGGGS




 


GGGGSEAAAKGGGGS
13,157
KORV_Q9TTC1_3mutA


 


PAPAPAPAPAP
13,158
FLV_P10273_3mutA


 


GGS

MLVBM_Q7SVK7_3mutA_WS


 


GGGGGSEAAAK
13,160
MLVBM_Q7SVK7_3mutA_WS


 


GSSGSSGSSGSSGSS
13,161
MLVMS_P03355_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
13,162
MLVMS_P03355_3mut


AKEAAAK




 


GGSGSSGGG
13,163
PERV_Q4VFZ2_3mut


 


PAP

MLVFF_P26809_3mut


 


GSSPAPEAAAK
13,165
MLVAV_P03356_3mutA


 


EAAAKGGGGSS
13,166
MLVMS_P03355_3mut


 


GGGEAAAKGGS
13,167
XMRV6_A1Z651_3mut


 


GGSGGGPAP
13,168
MLVBM_Q7SVK7_3mutA_WS


 


GSAGSAAGSGEF
13,169
BAEVM_P10272_3mutA


 


GSSEAAAK
13,170
MLVCB_P08361_3mut


 


PAPGSS
13,171
MLVMS_P03355_3mut


 


EAAAKEAAAKEAAAK
13,172
MLVAV_P03356_3mutA


 


GSAGSAAGSGEF
13,173
XMRV6_A1Z651_3mutA


 


GSSGSSGSSGSS
13,174
BAEVM_P10272_3mutA


 


AEAAAKEAAAKEAAAKEA
13,175
KORV_Q9TTC1-Pro_3mut


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GGGGSSEAAAK
13,176
WMSV_P03359_3mut


 


GSSGGGEAAAK
13,177
MLVBM_Q7SVK7_3mutA_WS


 


EAAAKPAP
13,178
MLVFF_P26809_3mutA


 


GGSPAPGGG
13,179
KORV_Q9TTC1_3mutA


 


PAPEAAAK
13,180
FLV_P10273_3mutA


 


GSSGSSGSS
13,181
MLVBM_Q7SVK7_3mutA_WS


 


GSSGGGEAAAK
13,182
FLV_P10273_3mutA


 


GGSPAP
13,183
MLVBM_Q7SVK7_3mutA_WS


 


GSAGSAAGSGEF
13,184
KORV_Q9TTC1-Pro_3mutA


 


PAPGGSEAAAK
13,185
MLVMS_P03355_PLV919


 


GGSPAPEAAAK
13,186
MLVBM_Q7SVK7_3mutA_WS


 


GGGGGSPAP
13,187
MLVBM_Q7SVK7_3mutA_WS


 


EAAAKGSSPAP
13,188
WMSV_P03359_3mut


 


EAAAKGGGPAP
13,189
MLVBM_Q7SVK7_3mutA_WS


 


PAPGSS
13,190
KORV_Q9TTC1-Pro_3mutA


 


GGSGSSGGG
13,191
BAEVM_P10272_3mut


 


SGGSSGGSSGSETPGTSE
13,192
FFV_O93209-Pro_2mut


SATPESSGGSSGGSS




 


GGSGGSGGSGGSGGSGGS
13,193
WMSV_P03359_3mutA


 


GGSGGSGGS
13,194
PERV_Q4VFZ2_3mutA_WS


 


GGGGG
13,195
PERV_Q4VFZ2_3mutA_WS


 


GGGPAP
13,196
FLV_P10273_3mutA


 


PAPGGSGGG
13,197
XMRV6_A1Z651_3mutA


 


GGGGSEAAAKGGGGS
13,198
XMRV6_A1Z651_3mut


 


EAAAKGSSGGG
13,199
KORV_Q9TTC1-Pro_3mutA


 


GSSGGSEAAAK
13,200
WMSV_P03359_3mut


 


EAAAKGGSGSS
13,201
PERV_Q4VFZ2_3mut


 


PAPAPAPAPAP
13,202
PERV_Q4VFZ2_3mut


 


GGGGSGGGGSGGGGSGGG
13,203
MLVMS_P03355_3mutA_WS


GSGGGGSGGGGS




 


GGGGGGG
13,204
KORV_Q9TTC1_3mutA


 


EAAAK
13,205
KORV_Q9TTC1-Pro_3mutA


 


GGGEAAAKGGS
13,206
KORV_Q9TTC1-Pro_3mutA


 


GGGEAAAKGGS
13,207
PERV_Q4VFZ2_3mutA_WS


 


GGGGGSPAP
13,208
XMRV6_A1Z651_3mut


 


GGGGSGGGGSGGGGSGGG
13,209
MLVFF_P26809_3mut


GS




 


GGGGGGG
13,210
MLVFF_P26809_3mut


 


PAPAPAPAPAPAP
13,211
AVIRE_P03360_3mutA


 


GSSPAPGGG
13,212
FLV_P10273_3mutA


 


GGGGGSPAP
13,213
MLVMS_P03355_3mutA_WS


 


GGGGSGGGGSGGGGS
13,214
MLVMS_P03355_3mut


 


GGGGSGGGGSGGGGS
13,215
KORV_Q9TTC1_3mut


 


GSSEAAAKGGS
13,216
MLVAV_P03356_3mutA


 


GSSGSSGSSGSSGSS
13,217
MLVMS_P03355_3mut


 


EAAAKGGGGGS
13,218
PERV_Q4VFZ2_3mutA_WS


 


GSSGGGGGS
13,219
PERV_Q4VFZ2_3mut


 


GGGEAAAKPAP
13,220
MLVMS_P03355_3mut


 


GSSGGSPAP
13,221
PERV_Q4VFZ2_3mutA_WS


 


GSSGGGPAP
13,222
BAEVM_P10272_3mutA


 


GGGGGSGSS
13,223
MLVMS_P03355_PLV919


 


AEAAAKEAAAKEAAAKEA
13,224
BAEVM_P10272_3mut


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


PAPEAAAK
13,225
MLVMS_P03355_3mut


 


GGGGSGGGGSGGGGS
13,226
FLV_P10273_3mutA


 


GGSGSSGGG
13,227
WMSV_P03359_3mutA


 


EAAAKGGS
13,228
PERV_Q4VFZ2_3mut


 


EAAAKGSSPAP
13,229
MLVCB_P08361_3mut


 


EAAAKGGSGSS
13,230
WMSV_P03359_3mutA


 


GSSGSS
13,231
PERV_Q4VFZ2_3mutA_WS


 


PAPAPAPAP
13,232
MLVMS_P03355_PLV919


 


GGSGGG
13,233
PERV_Q4VFZ2_3mutA_WS


 


GSS

MLVBM_Q7SVK7_3mutA_WS


 


PAP

KORV_Q9TTC1-Pro_3mutA


 


GGSGSSEAAAK
13,236
MLVFF_P26809_3mut


 


PAPEAAAKGSS
13,237
KORV_Q9TTC1-Pro_3mutA


 


GGSGGS
13,238
MLVCB_P08361_3mutA


 


GGGGGGG
13,239
PERV_Q4VFZ2_3mutA_WS


 


GGSPAPEAAAK
13,240
MLVBM_Q7SVK7_3mut


 


EAAAKEAAAKEAAAKEAA
13,241
KORV_Q9TTC1_3mutA


AKEAAAKEAAAK




 


GGSPAP
13,242
MLVMS_P03355_3mut


 


GGSEAAAKGGG
13,243
PERV_Q4VFZ2_3mut


 


GGGGSGGGGS
13,244
FLV_P10273_3mutA


 


GGGEAAAK
13,245
BAEVM_P10272_3mutA


 


GGGGSGGGGSGGGGSGGG
13,246
SFV3L_P27401_2mut


GSGGGGSGGGGS




 


GGSEAAAKPAP
13,247
KORV_Q9TTC1-Pro_3mutA


 


GSSGGGEAAAK
13,248
MLVMS_P03355_PLV919


 


GGGGGSEAAAK
13,249
MLVMS_P03355_PLV919


 


EAAAKGGSGGG
13,250
MLVMS_P03355_3mutA_WS


 


GGGGSSPAP
13,251
MLVAV_P03356_3mutA


 


EAAAKEAAAK
13,252
MLVMS_P03355_3mutA_WS


 


AEAAAKEAAAKEAAAKEA
13,253
SFV3L_P27401_2mut


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GSSGSSGSSGSSGSS
13,254
MLVMS_P03355_PLV919


 


GSSGGG
13,255
KORV_Q9TTC1-Pro_3mutA


 


GSSGGS
13,256
MLVFF_P26809_3mutA


 


GGGGSGGGGS
13,257
XMRV6_A1Z651_3mutA


 


PAPGSS
13,258
MLVBM_Q7SVK7_3mutA_WS


 


GGGPAPEAAAK
13,259
XMRV6_A1Z651_3mutA


 


EAAAKGGS
13,260
MLVFF_P26809_3mut


 


GSS

KORV_Q9TTC1_3mutA


 


GGGG
13,262
PERV_Q4VFZ2_3mut


 


GGGGGSEAAAK
13,263
AVIRE_P03360_3mutA


 


GSSGSSGSSGSSGSS
13,264
MLVMS_P03355_PLV919


 


PAPGGSGGG
13,265
PERV_Q4VFZ2_3mut


 


GGGPAP
13,266
PERV_Q4VFZ2_3mut


 


GGGPAPEAAAK
13,267
AVIRE_P03360_3mutA


 


GGGEAAAK
13,268
MLVCB_P08361_3mut


 


GGG

MLVFF_P26809_3mutA


 


EAAAKPAPGSS
13,270
XMRV6_A1Z651_3mutA


 


GGSGSSEAAAK
13,271
PERV_Q4VFZ2_3mutA_WS


 


EAAAKGSS
13,272
MLVMS_P03355_3mut


 


GGSGSSEAAAK
13,273
BAEVM_P10272_3mut


 


GGSGGG
13,274
MLVBM_Q7SVK7_3mutA_WS


 


GGGPAP
13,275
MLVMS_P03355_PLV919


 


GGSPAPGGG
13,276
PERV_Q4VFZ2_3mutA_WS


 


GGGGGSEAAAK
13,277
MLVFF_P26809_3mutA


 


EAAAKGSSGGS
13,278
MLVBM_Q7SVK7_3mut


 


PAPAP
13,279
XMRV6_A1Z651_3mut


 


GSSPAPGGS
13,280
MLVBM_Q7SVK7_3mutA_WS


 


GSSEAAAKGGG
13,281
WMSV_P03359_3mutA


 


EAAAKGGGGGS
13,282
PERV_Q4VFZ2_3mut


 


GSSGSSGSSGSSGSS
13,283
MLVCB_P08361_3mutA


 


EAAAKGGGGSS
13,284
PERV_Q4VFZ2_3mut


 


EAAAKGSS
13,285
PERV_Q4VFZ2_3mut


 


EAAAKEAAAKEAAAKEAA
13,286
AVIRE_P03360_3mutA


AKEAAAKEAAAK




 


EAAAKGGS
13,287
MLVCB_P08361_3mut


 


GSSGGSEAAAK
13,288
MLVAV_P03356_3mutA


 


EAAAKPAPGGS
13,289
PERV_Q4VFZ2_3mut


 


GGSGGS
13,290
MLVAV_P03356_3mutA


 


EAAAKGSSGGG
13,291
AVIRE_P03360_3mutA


 


GGSGGSGGSGGS
13,292
PERV_Q4VFZ2_3mut


 


GGGGGGGG
13,293
KORV_Q9TTC1_3mutA


 


GGSGSSEAAAK
13,294
MLVCB_P08361_3mutA


 


EAAAKGGG
13,295
MLVBM_Q7SVK7_3mutA_WS


 


GGGGSGGGGSGGGGS
13,296
MLVCB_P08361_3mut


 


GGSGGSGGSGGS
13,297
PERV_Q4VFZ2_3mutA_WS


 


PAPAPAPAPAP
13,298
WMSV_P03359_3mut


 


EAAAKEAAAKEAAAKEAA
13,299
PERV_Q4VFZ2_3mut


AK




 


GGSGGSGGS
13,300
XMRV6_A1Z651_3mutA


 


PAPGGGGSS
13,301
BAEVM_P10272_3mutA


 


GSSEAAAKGGS
13,302
MLVCB_P08361_3mut


 


GSSGGGPAP
13,303
MLVCB_P08361_3mutA


 


GGSGSS
13,304
MLVBM_Q7SVK7_3mutA_WS


 


GGGGGSEAAAK
13,305
MLVAV_P03356_3mutA


 


GSSEAAAK
13,306
PERV_Q4VFZ2_3mutA_WS


 


GGGGGSGSS
13,307
MLVBM_Q7SVK7_3mutA_WS


 


EAAAKGGSGSS
13,308
MLVFF_P26809_3mut


 


PAP

FLV_P10273_3mutA


 


GGGGG
13,310
MLVMS_P03355_3mutA_WS


 


EAAAK
13,311
PERV_Q4VFZ2_3mut


 


GSS

FLV_P10273_3mutA


 


PAPAPAPAPAPAP
13,313
KORV_Q9TTC1-Pro_3mutA


 


EAAAKEAAAKEAAAKEAA
13,314
MLVCB_P08361_3mut


AK




 


EAAAKGGGGSEAAAK
13,315
XMRV6_A1Z651_3mut


 


PAPGGSGGG
13,316
MLVBM_Q7SVK7_3mutA_WS


 


GGSGGGPAP
13,317
WMSV_P03359_3mutA


 


GGGGSSEAAAK
13,318
MLVBM_Q7SVK7_3mutA_WS


 


PAPGGGGSS
13,319
MLVCB_P08361_3mut


 


GGSGGSGGSGGS
13,320
PERV_Q4VFZ2_3mutA_WS


 


PAPGGSGGG
13,321
MLVMS_P03355_3mutA_WS


 


GSSPAPGGS
13,322
MLVCB_P08361_3mutA


 


GSSGSSGSS
13,323
MLVFF_P26809_3mut


 


PAPGGGGGS
13,324
MLVBM_Q7SVK7_3mutA_WS


 


GSSPAP
13,325
PERV_Q4VFZ2_3mut


 


GGSGGG
13,326
KORV_Q9TTC1-Pro_3mut


 


EAAAKGGGGSEAAAK
13,327
PERV_Q4VFZ2_3mutA_WS


 


GGSPAPEAAAK
13,328
PERV_Q4VFZ2_3mutA_WS


 


EAAAKPAP
13,329
BAEVM_P10272_3mut


 


GGGGSGGGGSGGGGGGGG
13,330
MLVMS_P03355_3mut


SGGGGSGGGGS




 


EAAAKGGGGSS
13,331
MLVFF_P26809_3mut


 


EAAAKEAAAK
13,332
MLVCB_P08361_3mut


 


GSSEAAAKGGS
13,333
PERV_Q4VFZ2_3mut


 


GGSPAP
13,334
KORV_Q9TTC1-Pro_3mutA


 


EAAAKEAAAKEAAAKEAA
13,335
MLVMS_P03355_3mutA_WS


AK




 


GSSGSSGSSGSSGSS
13,336
BAEVM_P10272_3mut


 


PAPEAAAK
13,337
MLVMS_P03355_3mut


 


GSSGGSPAP
13,338
PERV_Q4VFZ2


 


GGGPAPGGS
13,339
BAEVM_P10272_3mutA


 


EAAAKPAPGGS
13,340
MLVMS_P03355_PLV919


 


GGGGSGGGGS
13,341
PERV_Q4VFZ2


 


GGGEAAAK
13,342
KORV_Q9TTC1-Pro_3mut


 


EAAAKGGGGGS
13,343
FLV_P10273_3mutA


 


GGSPAPGSS
13,344
MLVMS_P03355_3mut


 


GSSPAPEAAAK
13,345
MLVMS_P03355_3mutA_WS


 


GSAGSAAGSGEF
13,346
MLVBM_Q7SVK7_3mutA_WS


 


EAAAK
13,347
BAEVM_P10272_3mutA


 


EAAAKGGGGSS
13,348
BAEVM_P10272_3mutA


 


GGG

WMSV_P03359_3mut


 


GGSGSSPAP
13,350
BAEVM_P10272_3mut


 


GGSEAAAKPAP
13,351
MLVBM_Q7SVK7_3mutA_WS


 


EAAAKGGSGSS
13,352
MLVCB_P08361_3mut


 


PAPGSS
13,353
MLVAV_P03356_3mutA


 


PAPEAAAKGGG
13,354
MLVCB_P08361_3mutA


 


AEAAAKEAAAKEAAAKEA
13,355
FOAMV_P14350-Pro_2mut


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GSSGSSGSS
13,356
PERV_Q4VFZ2_3mut


 


PAPGGG
13,357
MLVMS_P03355_3mut


 


PAPGGS
13,358
PERV_Q4VFZ2_3mut


 


GSSGGG
13,359
MLVMS_P03355_PLV919


 


GSSGSSGSSGSSGSSGSS
13,360
WMSV_P03359_3mut


 


PAP

AVIRE_P03360_3mutA


 


EAAAKGSSPAP
13,362
MLVBM_Q7SVK7_3mutA_WS


 


GSSGSSGSSGSS
13,363
MLVMS_P03355_PLV919


 


GGGGSGGGGSGGGGSGGG
13,364
AVIRE_P03360


GSGGGGS




 


GGGGS
13,365
PERV_Q4VFZ2_3mut


 


EAAAKGSSGGG
13,366
MLVBM_Q7SVK7_3mutA_WS


 


GGGGGG
13,367
KORV_Q9TTC1-Pro_3mut


 


GGSGSSEAAAK
13,368
PERV_Q4VFZ2_3mut


 


GSSPAPEAAAK
13,369
MLVBM_Q7SVK7_3mutA_WS


 


GGGGSGGGGS
13,370
MLVBM_Q7SVK7_3mutA_WS


 


GSSGGGGGS
13,371
MLVAV_P03356_3mutA


 


GSAGSAAGSGEF
13,372
WMSV_P03359_3mutA


 


GGGEAAAKGSS
13,373
BAEVM_P10272_3mutA


 


AEAAAKEAAAKEAAAKEA
13,374
FFV_O93209-Pro_2mut


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


PAPGGSGGG
13,375
MLVCB_P08361_3mutA


 


EAAAKEAAAKEAAAKEAA
13,376
SFV3L_P27401_2mut


AKEAAAK




 


GGSGSSPAP
13,377
MLVMS_P03355_PLV919


 


GGGGGG
13,378
PERV_Q4VFZ2_3mut


 


EAAAKEAAAKEAAAKEAA
13,379
PERV_Q4VFZ2_3mut


AKEAAAK




 


EAAAKGSSPAP
13,380
MLVFF_P26809_3mut


 


GGGPAPGGS
13,381
MLVBM_Q7SVK7_3mutA_WS


 


AEAAAKEAAAKEAAAKEA
13,382
SFV3L_P27401


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


PAP

PERV_Q4VFZ2_3mut


 


EAAAKGGS
13,384
MLVMS_P03355_PLV919


 


GSSGGSEAAAK
13,385
WMSV_P03359_3mutA


 


GGSGSSEAAAK
13,386
KORV_Q9TTC1-Pro_3mutA


 


EAAAKEAAAKEAAAK
13,387
PERV_Q4VFZ2


 


GGSGGGEAAAK
13,388
MLVMS_P03355_3mutA_WS


 


GGGGSGGGGSGGGGSGGG
13,389
BAEVM_P10272_3mut


GS




 


EAAAKGSS
13,390
XMRV6_A1Z651_3mutA


 


GSSGGGGGS
13,391
WMSV_P03359_3mutA


 


GSSGSSGSSGSSGSSGSS
13,392
MLVFF_P26809_3mutA


 


GGSGSS
13,393
MLVAV_P03356_3mutA


 


EAAAKGGGGSEAAAK
13,394
MLVMS_P03355_PLV919


 


EAAAKGGGPAP
13,395
PERV_Q4VFZ2


 


GGSEAAAKGGG
13,396
MLVAV_P03356_3mutA


 


EAAAKEAAAKEAAAKEAA
13,397
MLVBM_Q7SVK7_3mut


AKEAAAKEAAAK




 


EAAAKEAAAKEAAAKEAA
13,398
KORV_Q9TTC1-Pro_3mutA


AKEAAAKEAAAK




 


GSSPAPEAAAK
13,399
MLVFF_P26809_3mutA


 


GGGGSEAAAKGGGGS
13,400
PERV_Q4VFZ2_3mut


 


GSSGSSGSSGSS
13,401
PERV_Q4VFZ2_3mut


 


GGSEAAAK
13,402
MLVFF_P26809_3mutA


 


GGGGGGGG
13,403
MLVMS_P03355_3mut


 


GSSGGG
13,404
XMRV6_A1Z651_3mutA


 


EAAAKGGS
13,405
BAEVM_P10272_3mutA


 


GGGGS
13,406
BAEVM_P10272_3mutA


 


GGSEAAAKGGG
13,407
KORV_Q9TTC1-Pro_3mutA


 


GGSGSSGGG
13,408
KORV_Q9TTC1_3mutA


 


GGSGSSEAAAK
13,409
WMSV_P03359_3mut


 


EAAAKGGSGSS
13,410
MLVBM_Q7SVK7_3mutA_WS


 


GGS

BAEVM_P10272_3mutA


 


GGGPAPGSS
13,412
WMSV_P03359_3mutA


 


GSSGSSGSSGSSGSS
13,413
AVIRE_P03360_3mut


 


GGGEAAAKPAP
13,414
XMRV6_A1Z651_3mut


 


GSSGGG
13,415
MLVFF_P26809_3mutA


 


GGSPAPGSS
13,416
PERV_Q4VFZ2_3mut


 


PAPGGS
13,417
MLVCB_P08361_3mut


 


PAPAPAPAPAP
13,418
KORV_Q9TTC1_3mutA


 


GSSGGS
13,419
MLVCB_P08361_3mutA


 


GSSGGSEAAAK
13,420
PERV_Q4VFZ2_3mut


 


EAAAKGSSGGS
13,421
MLVMS_P03355_PLV919


 


EAAAKGGG
13,422
WMSV_P03359_3mut


 


PAPGGGGGS
13,423
BAEVM_P10272_3mutA


 


GGGGSEAAAKGGGGS
13,424
WMSV_P03359_3mutA


 


EAAAKEAAAKEAAAKEAA
13,425
MLVMS_P03355_3mutA_WS


AKEAAAKEAAAK




 


GGS

KORV_Q9TTC1-Pro_3mutA


 


GSSGGSPAP
13,427
BAEVM_P10272_3mutA


 


GGG

MLVMS_P03355_PLV919


 


PAPGSS
13,429
KORV_Q9TTC1-Pro_3mut


 


GGSEAAAKGGG
13,430
FLV_P10273_3mutA


 


GGSEAAAKPAP
13,431
PERV_Q4VFZ2_3mutA_WS


 


GGGGSSPAP
13,432
XMRV6_A1Z651_3mutA


 


EAAAKEAAAKEAAAKEAA
13,433
PERV_Q4VFZ2_3mutA_WS


AKEAAAK




 


GGGG
13,434
PERV_Q4VFZ2_3mutA_WS


 


GGSEAAAKPAP
13,435
MLVMS_P03355_3mut


 


PAPGSSGGG
13,436
MLVMS_P03355_3mutA_WS


 


PAPEAAAKGGS
13,437
AVIRE_P03360_3mut


 


GGGGSSPAP
13,438
MLVMS_P03355_3mutA_WS


 


GGGGSGGGGSGGGGSGGG
13,439
PERV_Q4VFZ2_3mut


GS




 


GGGEAAAK
13,440
MLVMS_P03355_3mut


 


GGGGSS
13,441
MLVFF_P26809_3mut


 


GGSPAPGSS
13,442
XMRV6_A1Z651_3mut


 


GGGGS
13,443
KORV_Q9TTC1-Pro_3mutA


 


EAAAKGSSGGS
13,444
FLV_P10273_3mutA


 


GSS

MLVMS_P03355_PLV919


 


GGGG
13,446
MLVMS_P03355_PLV919


 


GSSGGS
13,447
MLVMS_P03355_PLV919


 


GGSGGSGGSGGS
13,448
MLVMS_P03355_3mut


 


PAPEAAAKGGS
13,449
MLVMS_P03355_3mut


 


EAAAKGSSGGG
13,450
BAEVM_P10272_3mutA


 


GSSEAAAK
13,451
KORV_Q9TTC1-Pro_3mutA


 


GSAGSAAGSGEF
13,452
KORV_Q9TTC1_3mutA


 


GGGGGSEAAAK
13,453
MLVCB_P08361_3mut


 


GGGG
13,454
WMSV_P03359_3mut


 


GGGGSSEAAAK
13,455
MLVMS_P03355_PLV919


 


PAPGGG
13,456
WMSV_P03359_3mutA


 


EAAAKGGSGGG
13,457
MLVAV_P03356_3mutA


 


GGGPAPGGS
13,458
MLVMS_P03355_3mut


 


EAAAKPAP
13,459
PERV_Q4VFZ2_3mutA_WS


 


GSSGSSGSS
13,460
KORV_Q9TTC1-Pro_3mutA


 


GSSPAPGGS
13,461
XMRV6_A1Z651_3mut


 


GGGGGSPAP
13,462
BAEVM_P10272_3mutA


 


GGSGSSGGG
13,463
PERV_Q4VFZ2_3mutA_WS


 


GGGEAAAKGSS
13,464
AVIRE_P03360_3mut


 


GSSEAAAK
13,465
FLV_P10273_3mutA


 


EAAAK
13,466
MLVMS_P03355_3mut


 


EAAAKGGSGSS
13,467
WMSV_P03359_3mut


 


GSSEAAAKGGG
13,468
PERV_Q4VFZ2_3mut


 


PAPGSSGGG
13,469
BAEVM_P10272_3mutA


 


EAAAKGGGGGS
13,470
MLVMS_P03355_3mut


 


GGSEAAAKPAP
13,471
AVIRE_P03360_3mut


 


GGGPAPGGS
13,472
XMRV6_A1Z651_3mut


 


GGGGS
13,473
KORV_Q9TTC1_3mutA


 


GGSGGSGGSGGSGGS
13,474
XMRV6_A1Z651_3mut


 


GGGPAP
13,475
KORV_Q9TTC1-Pro_3mut


 


EAAAKPAP
13,476
MLVBM_Q7SVK7_3mutA_WS


 


GGSEAAAK
13,477
MLVMS_P03355_PLV919


 


GSSEAAAKPAP
13,478
KORV_Q9TTC1-Pro_3mutA


 


GGSGSS
13,479
MLVMS_P03355_3mut


 


EAAAKPAPGGG
13,480
PERV_Q4VFZ2_3mut


 


GGSPAPEAAAK
13,481
KORV_Q9TTC1_3mutA


 


GGSEAAAKGGG
13,482
AVIRE_P03360_3mutA


 


GGGGSEAAAKGGGGS
13,483
MLVMS_P03355_PLV919


 


GSSGGGEAAAK
13,484
KORV_Q9TTC1-Pro_3mutA


 


EAAAKGGGPAP
13,485
WMSV_P03359_3mut


 


GSSPAP
13,486
XMRV6_A1Z651_3mutA


 


AEAAAKEAAAKEAAAKEA
13,487
SFV3L_P27401-Pro


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GGSEAAAKGSS
13,488
MLVMS_P03355_PLV919


 


GSSGGSEAAAK
13,489
KORV_Q9TTC1-Pro_3mutA


 


GGSEAAAKGSS
13,490
KORV_Q9TTC1-Pro_3mutA


 


EAAAKGGG
13,491
AVIRE_P03360_3mutA


 


GSSGGSEAAAK
13,492
BAEVM_P10272_3mutA


 


GGGGSEAAAKGGGGS
13,493
KORV_Q9TTC1-Pro_3mut


 


PAPGSSEAAAK
13,494
MLVMS_P03355_3mut


 


PAPEAAAK
13,495
WMSV_P03359_3mut


 


PAPGGSGSS
13,496
PERV_Q4VFZ2_3mutA_WS


 


PAPGSS
13,497
BAEVM_P10272_3mut


 


PAPGGGGGS
13,498
MLVMS_P03355_3mut


 


EAAAKPAPGSS
13,499
MLVBM_Q7SVK7_3mutA_WS


 


GSSPAPGGS
13,500
MLVMS_P03355_PLV919


 


GGSGSSEAAAK
13,501
MLVMS_P03355_3mut


 


GGGGGG
13,502
KORV_Q9TTC1-Pro_3mutA


 


EAAAKEAAAKEAAAKEAA
13,503
MLVBM_Q7SVK7_3mut


AK




 


GGSPAPGSS
13,504
MLVMS_P03355_PLV919


 


PAPAPAPAPAP
13,505
MLVCB_P08361_3mut


 


GGSGSSPAP
13,506
WMSV_P03359_3mutA


 


EAAAKGGSGGG
13,507
PERV_Q4VFZ2_3mutA_WS


 


GSSGSSGSSGSSGSS
13,508
PERV_Q4VFZ2_3mut


 


AEAAAKEAAAKEAAAKEA
13,509
KORV_Q9TTC1_3mutA


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GSSGGGEAAAK
13,510
WMSV_P03359_3mutA


 


GSSGGSEAAAK
13,511
FLV_P10273_3mutA


 


GGGGGGGG
13,512
PERV_Q4VFZ2_3mut


 


PAPGGSEAAAK
13,513
FLV_P10273_3mutA


 


GGGGSSPAP
13,514
BAEVM_P10272_3mutA


 


PAPAPAPAP
13,515
WMSV_P03359_3mut


 


GGSEAAAKPAP
13,516
PERV_Q4VFZ2_3mut


 


PAPGGSGGG
13,517
BAEVM_P10272_3mutA


 


EAAAKEAAAKEAAAKEAA
13,518
MLVMS_P03355_3mut


AKEAAAKEAAAK




 


GGGGSGGGGSGGGGS
13,519
PERV_Q4VFZ2_3mut


 


GGSGGGPAP
13,520
PERV_Q4VFZ2_3mut


 


GGGPAPEAAAK
13,521
MLVFF_P26809_3mut


 


GGGGGSGSS
13,522
MLVMS_P03355_3mutA_WS


 


GSS

MLVCB_P08361_3mut


 


GGGGGSPAP
13,524
MLVMS_P03355_PLV919


 


GGSPAP
13,525
MLVAV_P03356_3mutA


 


GGGPAPGGS
13,526
KORV_Q9TTC1-Pro_3mutA


 


PAPGSSGGG
13,527
FLV_P10273_3mutA


 


PAPGSSGGG
13,528
WMSV_P03359_3mutA


 


PAPGGS
13,529
MLVBM_Q7SVK7_3mutA_WS


 


GGGEAAAKGSS
13,530
PERV_Q4VFZ2_3mutA_WS


 


GGSEAAAKGSS
13,531
MLVBM_Q7SVK7_3mutA_WS


 


PAPGGSEAAAK
13,532
MLVCB_P08361_3mut


 


GGSEAAAKGGG
13,533
XMRV6_A1Z651_3mutA


 


GGSGGGGSS
13,534
WMSV_P03359_3mut


 


GGGEAAAKPAP
13,535
KORV_Q9TTC1_3mutA


 


EAAAKGSS
13,536
KORV_Q9TTC1-Pro_3mut


 


PAPEAAAKGSS
13,537
MLVFF_P26809_3mut


 


GSAGSAAGSGEF
13,538
PERV_Q4VFZ2_3mut


 


EAAAKGGGGGS
13,539
WMSV_P03359_3mut


 


EAAAKGSSPAP
13,540
WMSV_P03359_3mutA


 


GGGGSEAAAKGGGGS
13,541
XMRV6_A1Z651_3mutA


 


GSSEAAAKPAP
13,542
SFV3L_P27401-Pro_2mutA


 


GGGGGG
13,543
PERV_Q4VFZ2_3mutA_WS


 


PAPGGS
13,544
BAEVM_P10272_3mut


 


PAP

AVIRE_P03360_3mut


 


PAPAPAP
13,546
MLVBM_Q7SVK7_3mutA_WS


 


GGGG
13,547
PERV_Q4VFZ2_3mutA_WS


 


GSSGGSEAAAK
13,548
MLVBM_Q7SVK7_3mut


 


GGSGGGGSS
13,549
MLVFF_P26809_3mut


 


GGGGSSGGS
13,550
AVIRE_P03360_3mutA


 


GSSPAPGGG
13,551
PERV_Q4VFZ2_3mutA_WS


 


GGSEAAAKPAP
13,552
MLVMS_P03355_PLV919


 


PAP

KORV_Q9TTC1-Pro_3mut


 


GSSGGS
13,554
PERV_Q4VFZ2_3mut


 


GGGGG
13,555
PERV_Q4VFZ2_3mut


 


GSSGGGPAP
13,556
FLV_P10273_3mutA


 


GSSEAAAKGGG
13,557
KORV_Q9TTC1-Pro_3mut


 


EAAAKEAAAKEAAAKEAA
13,558
MLVCB_P08361_3mut


AKEAAAKEAAAK




 


GGSEAAAKPAP
13,559
MLVCB_P08361_3mut


 


PAPAPAPAPAPAP
13,560
BAEVM_P10272_3mutA


 


GGGGSEAAAKGGGGS
13,561
MLVMS_P03355_3mut


 


EAAAKPAPGSS
13,562
MLVMS_P03355_3mut


 


GSSGSSGSSGSSGSS
13,563
MLVBM_Q7SVK7_3mutA_WS


 


PAPEAAAKGSS
13,564
MLVAV_P03356_3mut


 


AEAAAKEAAAKEAAAKEA
13,565
AVIRE_P03360_3mut


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


AEAAAKEAAAKEAAAKEA
13,566
PERV_Q4VFZ2_3mut


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GGSEAAAKGGG
13,567
PERV_Q4VFZ2_3mutA_WS


 


GGSGGGGSS
13,568
MLVFF_P26809_3mutA


 


PAPEAAAKGSS
13,569
MLVCB_P08361_3mut


 


GGG

PERV_Q4VFZ2_3mutA_WS


 


GGSGGGEAAAK
13,571
MLVMS_P03355_3mut


 


EAAAKGGGGSS
13,572
WMSV_P03359_3mut


 


GSSPAPGGG
13,573
WMSV_P03359_3mutA


 


EAAAKGSSGGG
13,574
PERV_Q4VFZ2_3mut


 


GGSGGGEAAAK
13,575
PERV_Q4VFZ2_3mutA_WS


 


GGSGGSGGSGGSGGS
13,576
PERV_Q4VFZ2_3mutA_WS


 


EAAAKPAPGGS
13,577
PERV_Q4VFZ2_3mutA_WS


 


GGGGGSEAAAK
13,578
PERV_Q4VFZ2_3mutA_WS


 


GSSPAP
13,579
MLVFF_P26809_3mut


 


GGGEAAAKPAP
13,580
AVIRE_P03360_3mut


 


GSSGGSEAAAK
13,581
MLVMS_P03355_PLV919


 


EAAAKPAPGGS
13,582
WMSV_P03359_3mutA


 


PAPGGG
13,583
KORV_Q9TTC1_3mutA


 


EAAAKGSSPAP
13,584
KORV_Q9TTC1-Pro_3mut


 


GSSPAPEAAAK
13,585
MLVFF_P26809_3mut


 


GGSGGGEAAAK
13,586
MLVFF_P26809_3mutA


 


GSSGSSGSS
13,587
WMSV_P03359_3mutA


 


EAAAKGGS
13,588
BAEVM_P10272_3mut


 


EAAAKPAPGGS
13,589
KORV_Q9TTC1_3mutA


 


EAAAKPAPGGS
13,590
BAEVM_P10272_3mutA


 


GSSGGGGGS
13,591
PERV_Q4VFZ2_3mut


 


PAPGGGGSS
13,592
PERV_Q4VFZ2_3mut


 


GSSGSSGSS
13,593
WMSV_P03359_3mut


 


EAAAKEAAAKEAAAKEAA
13,594
WMSV_P03359_3mut


AK




 


GGS

AVIRE_P03360_3mut


 


EAAAKPAPGSS
13,596
MLVFF_P26809_3mut


 


EAAAKGGG
13,597
KORV_Q9TTC1_3mut


 


PAPGSSEAAAK
13,598
MLVMS_P03355_3mut


 


PAPGSSGGS
13,599
MLVMS_P03355_PLV919


 


GSSPAPEAAAK
13,600
MLVMS_P03355_3mut


 


GSSGSSGSSGSSGSSGSS
13,601
WMSV_P03359_3mutA


 


GGGGS
13,602
BAEVM_P10272_3mut


 


GSSPAP
13,603
MLVMS_P03355_3mut


 


EAAAKGGGGSEAAAK
13,604
KORV_Q9TTC1-Pro_3mutA


 


EAAAKEAAAK
13,605
WMSV_P03359_3mutA


 


GGGGSSGGS
13,606
MLVCB_P08361_3mutA


 


PAPGGSEAAAK
13,607
BAEVM_P10272_3mut


 


EAAAKGGSPAP
13,608
MLVFF_P26809_3mut


 


GSSGGSGGG
13,609
MLVBM_Q7SVK7_3mutA_WS


 


GSSGGS
13,610
PERV_Q4VFZ2_3mut


 


PAPGGSGSS
13,611
PERV_Q4VFZ2_3mutA_WS


 


EAAAKGGSGSS
13,612
KORV_Q9TTC1-Pro_3mutA


 


PAPAP
13,613
MLVCB_P08361_3mut


 


EAAAKGSSPAP
13,614
PERV_Q4VFZ2_3mutA_WS


 


EAAAKPAPGGG
13,615
MLVMS_P03355_PLV919


 


GGGGSGGGGSGGGGGGGG
13,616
MLVBM_Q7SVK7_3mut


SGGGGSGGGGS




 


EAAAKGGGGSS
13,617
MLVMS_P03355_PLV919


 


PAPEAAAK
13,618
PERV_Q4VFZ2_3mut


 


EAAAKPAPGSS
13,619
BAEVM_P10272_3mutA


 


GGSPAP
13,620
PERV_Q4VFZ2_3mutA_WS


 


GGSGGS
13,621
BAEVM_P10272_3mutA


 


PAPEAAAKGSS
13,622
KORV_Q9TTC1_3mut


 


PAPGSS
13,623
MLVMS_P03355_PLV919


 


PAPAPAPAPAP
13,624
MLVAV_P03356_3mutA


 


GGG

XMRV6_A1Z651_3mutA


 


GGGPAP
13,626
PERV_Q4VFZ2_3mutA_WS


 


GSSPAPEAAAK
13,627
KORV_Q9TTC1_3mutA


 


PAP

BAEVM_P10272_3mutA


 


GGSPAP
13,629
BAEVM_P10272_3mutA


 


PAPEAAAKGGS
13,630
MLVMS_P03355_PLV919


 


PAPGSSGGS
13,631
PERV_Q4VFZ2_3mutA_WS


 


PAPAPAPAPAPAP
13,632
PERV_Q4VFZ2_3mut


 


EAAAKEAAAKEAAAK
13,633
MLVCB_P08361_3mut


 


GGSGGSGGSGGSGGS
13,634
MLVMS_P03355_PLV919


 


EAAAKPAPGGS
13,635
MLVMS_P03355_3mut


 


GGSGGS
13,636
MLVMS_P03355_PLV919


 


EAAAKPAP
13,637
MLVMS_P03355_3mutA_WS


 


GGSEAAAK
13,638
XMRV6_A1Z651_3mutA


 


GGSGGG
13,639
KORV_Q9TTC1_3mut


 


GGSGGGEAAAK
13,640
PERV_Q4VFZ2_3mut


 


PAPEAAAKGGG
13,641
AVIRE_P03360


 


PAPAP
13,642
PERV_Q4VFZ2_3mut


 


GSS

KORV_Q9TTC1-Pro_3mutA


 


EAAAKGSSGGG
13,644
MLVAV_P03356_3mutA


 


GGSPAPGSS
13,645
MLVBM_Q7SVK7_3mutA_WS


 


PAPEAAAK
13,646
MLVAV_P03356_3mut


 


EAAAKGGSPAP
13,647
BAEVM_P10272_3mutA


 


PAPAPAPAP
13,648
WMSV_P03359_3mutA


 


PAPGGSEAAAK
13,649
MLVMS_P03355_3mut


 


GGSGGSGGSGGS
13,650
WMSV_P03359_3mut


 


GGGGGSGSS
13,651
XMRV6_A1Z651_3mut


 


PAPGGSGGG
13,652
KORV_Q9TTC1_3mutA


 


GGS

MLVMS_P03355_3mut


 


EAAAK
13,654
WMSV_P03359_3mut


 


GGGEAAAKGSS
13,655
MLVBM_Q7SVK7_3mutA_WS


 


GGSPAPGSS
13,656
MLVCB_P08361_3mut


 


GGSEAAAKPAP
13,657
PERV_Q4VFZ2_3mut


 


GGGGSGGGGSGGGGSGGG
13,658
MLVCB_P08361_3mutA


GSGGGGS




 


GGSGSS
13,659
BAEVM_P10272_3mutA


 


GGGEAAAKGSS
13,660
WMSV_P03359_3mutA


 


EAAAKGGSPAP
13,661
WMSV_P03359_3mut


 


GSSPAPEAAAK
13,662
MLVMS_P03355_3mut


 


GGSGGSGGSGGS
13,663
MLVMS_P03355_PLV919


 


GSSPAPEAAAK
13,664
WMSV_P03359_3mut


 


GSSGSSGSSGSS
13,665
PERV_Q4VFZ2


 


GGSGSSEAAAK
13,666
WMSV_P03359_3mutA


 


GGSGGG
13,667
MLVFF_P26809_3mut


 


GGSPAPGGG
13,668
MLVFF_P26809_3mut


 


GGSGGSGGS
13,669
BAEVM_P10272_3mutA


 


GGGGSSEAAAK
13,670
MLVBM_Q7SVK7_3mut


 


GGSPAPGSS
13,671
MLVMS_P03355_3mut


 


EAAAKPAPGSS
13,672
AVIRE_P03360_3mut


 


GGGGSSGGS
13,673
FLV_P10273_3mutA


 


GGSPAPEAAAK
13,674
PERV_Q4VFZ2_3mut


 


GGSEAAAK
13,675
MLVMS_P03355_3mutA_WS


 


GSSGSSGSSGSS
13,676
MLVCB_P08361_3mutA


 


EAAAKEAAAKEAAAKEAA
13,677
MLVMS_P03355_PLV919


AKEAAAK




 


GGGGG
13,678
PERV_Q4VFZ2_3mut


 


GGSEAAAKGSS
13,679
MLVCB_P08361_3mutA


 


GSSGGG
13,680
MLVBM_Q7SVK7_3mutA_WS


 


PAPGSSGGG
13,681
KORV_Q9TTC1-Pro_3mutA


 


GGSGGS
13,682
BAEVM_P10272_3mut


 


EAAAKGGGGGS
13,683
MLVBM_Q7SVK7_3mutA_WS


 


GGSGSSPAP
13,684
MLVCB_P08361_3mut


 


PAPGSSGGG
13,685
KORV_Q9TTC1


 


PAPGGSGGG
13,686
MLVMS_P03355_3mut


 


GGGG
13,687
WMSV_P03359_3mutA


 


EAAAKGGSPAP
13,688
MLVCB_P08361_3mut


 


GSSGSS
13,689
FLV_P10273_3mutA


 


GGSEAAAKPAP
13,690
SFV3L_P27401_2mut


 


EAAAKGSSGGS
13,691
MLVAV_P03356_3mutA


 


AEAAAKEAAAKEAAAKEA
13,692
MLVAV_P03356_3mutA


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


EAAAKGGSGSS
13,693
PERV_Q4VFZ2_3mutA_WS


 


GGGGG
13,694
MLVCB_P08361_3mut


 


GGGEAAAK
13,695
BAEVM_P10272_3mut


 


GGSGGSGGSGGS
13,696
MLVCB_P08361_3mut


 


EAAAKEAAAKEAAAKEAA
13,697
PERV_Q4VFZ2


AKEAAAKEAAAK




 


PAPAPAPAPAP
13,698
MLVMS_P03355_3mutA_WS


 


EAAAKEAAAK
13,699
XMRV6_A1Z651_3mut


 


GSSGGSEAAAK
13,700
PERV_Q4VFZ2_3mutA_WS


 


PAPGGSEAAAK
13,701
KORV_Q9TTC1-Pro_3mutA


 


EAAAKGGGPAP
13,702
MLVBM_Q7SVK7_3mutA_WS


 


PAPGGSGSS
13,703
PERV_Q4VFZ2


 


SGSETPGTSESATPES
13,704
MLVMS_P03355_3mut


 


GGSGGS
13,705
MLVMS_P03355_PLV919


 


EAAAKGGS
13,706
FLV_P10273_3mut


 


GGSPAPGSS
13,707
MLVMS_P03355_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
13,708
FFV_O93209_2mut


AK




 


GSSGGSGGG
13,709
MLVMS_P03355_3mutA_WS


 


PAPGSSEAAAK
13,710
WMSV_P03359_3mut


 


PAPAPAPAPAPAP
13,711
KORV_Q9TTC1_3mutA


 


GGGGSS
13,712
BAEVM_P10272_3mut


 


GGGGSEAAAKGGGGS
13,713
AVIRE_P03360_3mut


 


GSSPAPEAAAK
13,714
KORV_Q9TTC1-Pro_3mutA


 


PAPEAAAKGGG
13,715
MLVBM_Q7SVK7_3mut


 


EAAAKEAAAK
13,716
WMSV_P03359_3mut


 


EAAAK
13,717
SFV3L_P27401-Pro_2mutA


 


GSSGGSGGG
13,718
XMRV6_A1Z651_3mutA


 


GGGEAAAKPAP
13,719
WMSV_P03359_3mutA


 


GGSGGS
13,720
MLVFF_P26809_3mutA


 


EAAAKEAAAKEAAAKEAA
13,721
FOAMV_P14350_2mutA


AKEAAAKEAAAK




 


GGGGG
13,722
MLVAV_P03356_3mutA


 


GSSGGSEAAAK
13,723
BAEVM_P10272_3mut


 


SGGSSGGSSGSETPGTSE
13,724
SFV1_P23074


SATPESSGGSSGGSS




 


GGSGGGPAP
13,725
MLVCB_P08361_3mut


 


GGSGSS
13,726
PERV_Q4VFZ2_3mut


 


SGSETPGTSESATPES
13,727
MLVFF_P26809_3mut


 


EAAAKGGSPAP
13,728
MLVMS_P03355_3mut


 


PAPAP
13,729
PERV_Q4VFZ2_3mut


 


AEAAAKEAAAKEAAAKEA
13,730
MLVBM_Q7SVK7_3mut


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GGGGGS
13,731
BAEVM_P10272_3mutA


 


EAAAKEAAAK
13,732
AVIRE_P03360_3mut


 


GSSGGSEAAAK
13,733
PERV_Q4VFZ2_3mut


 


GGGEAAAK
13,734
WMSV_P03359_3mut


 


GSSGGGEAAAK
13,735
AVIRE_P03360_3mutA


 


GGG

XMRV6_A1Z651_3mut


 


GGGGSEAAAKGGGGS
13,737
BAEVM_P10272_3mut


 


GGGG
13,738
MLVMS_P03355_3mut


 


GGSGGS
13,739
MLVMS_P03355_3mutA_WS


 


GGSGGGGSS
13,740
MLVBM_Q7SVK7_3mutA_WS


 


GSSPAPGGS
13,741
PERV_Q4VFZ2_3mut


 


GSSPAPEAAAK
13,742
PERV_Q4VFZ2_3mutA_WS


 


EAAAKGGS
13,743
WMSV_P03359_3mut


 


GGSGGSGGSGGS
13,744
PERV_Q4VFZ2_3mut


 


GGGGSSEAAAK
13,745
KORV_Q9TTC1-Pro_3mut


 


PAPAPAPAPAPAP
13,746
MLVAV_P03356_3mut


 


EAAAKGSSGGG
13,747
MLVMS_P03355_PLV919


 


GGGGG
13,748
MLVBM_Q7SVK7_3mutA_WS


 


AEAAAKEAAAKEAAAKEA
13,749
FFV_O93209_2mutA


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


SGGSSGGSSGSETPGTSE
13,750
KORV_Q9TTC1-Pro_3mut


SATPESSGGSSGGSS




 


GGSPAPGGG
13,751
MLVMS_P03355_3mutA_WS


 


GGGEAAAKGGS
13,752
MLVMS_P03355_3mut


 


GGGEAAAK
13,753
PERV_Q4VFZ2_3mut


 


PAPEAAAKGGG
13,754
MLVMS_P03355_3mut


 


GSSGSSGSSGSSGSSGSS
13,755
BAEVM_P10272_3mutA


 


AEAAAKEAAAKEAAAKEA
13,756
GALV_P21414_3mutA


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


EAAAKGGSPAP
13,757
FFV_O93209-Pro


 


EAAAKEAAAK
13,758
MLVFF_P26809_3mut


 


GGGGSGGGGSGGGGSGGG
13,759
PERV_Q4VFZ2_3mutA_WS


GSGGGGSGGGGS




 


GGSGGSGGSGGS
13,760
MLVAV_P03356_3mutA


 


EAAAKEAAAKEAAAKEAA
13,761
SFV3L_P27401_2mutA


AKEAAAK




 


GSSGSSGSSGSSGSSGSS
13,762
BAEVM_P10272_3mut


 


GGGGS
13,763
MLVMS_P03355_PLV919


 


AEAAAKEAAAKEAAAKEA
13,764
SFV1_P23074


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GGGGSGGGGS
13,765
KORV_Q9TTC1-Pro_3mutA


 


GGGGSGGGGS
13,766
MLVMS_P03355_3mut


 


GGSGSS
13,767
KORV_Q9TTC1_3mutA


 


GSSPAPGGG
13,768
PERV_Q4VFZ2_3mut


 


GSSGGSPAP
13,769
PERV_Q4VFZ2_3mutA_WS


 


PAPGGS
13,770
PERV_Q4VFZ2_3mutA_WS


 


GGSPAPEAAAK
13,771
FOAMV_P14350_2mutA


 


GGGPAPGGS
13,772
SFV3L_P27401_2mut


 


PAPGSSGGG
13,773
MLVCB_P08361_3mut


 


GSSGGGEAAAK
13,774
AVIRE_P03360_3mut


 


GSSGGG
13,775
XMRV6_A1Z651_3mut


 


GSSGSS
13,776
PERV_Q4VFZ2_3mut


 


GSSGGG
13,777
MLVAV_P03356_3mutA


 


PAPGGGGGS
13,778
PERV_Q4VFZ2_3mut


 


GSSEAAAK
13,779
MLVMS_P03355_3mut


 


PAPGGG
13,780
FLV_P10273_3mutA


 


GGGGSGGGGS
13,781
PERV_Q4VFZ2_3mut


 


GSSGGS
13,782
MLVMS_P03355_PLV919


 


GGGGSGGGGS
13,783
SFV3L_P27401_2mut


 


EAAAKGGSGSS
13,784
FLV_P10273_3mutA


 


GSSEAAAKGGS
13,785
MLVMS_P03355_3mutA_WS


 


PAPGSSEAAAK
13,786
SFV3L_P27401_2mutA


 


GGGGSGGGGS
13,787
SFV3L_P27401-Pro_2mutA


 


PAPGSSEAAAK
13,788
PERV_Q4VFZ2_3mut


 


PAPGSSEAAAK
13,789
PERV_Q4VFZ2


 


GGSPAPGGG
13,790
AVIRE_P03360_3mut


 


GGGGGS
13,791
PERV_Q4VFZ2_3mutA_WS


 


GGGGSSGGS
13,792
PERV_Q4VFZ2_3mut


 


PAPAPAPAP
13,793
AVIRE_P03360_3mutA


 


GGSGGS
13,794
WMSV_P03359_3mutA


 


GGGPAPGGS
13,795
PERV_Q4VFZ2_3mut


 


GGSGGSGGSGGSGGS
13,796
MLVMS_P03355_PLV919


 


GGSGGG
13,797
PERV_Q4VFZ2_3mut


 


EAAAKEAAAK
13,798
SFV3L_P27401_2mut


 


PAPGSS
13,799
XMRV6_A1Z651_3mut


 


GSSEAAAK
13,800
MLVFF_P26809_3mut


 


GGSPAPGGG
13,801
MLVMS_P03355_3mut


 


EAAAKGGG
13,802
WMSV_P03359_3mutA


 


GSSEAAAKGGS
13,803
PERV_Q4VFZ2_3mutA_WS


 


GSSGGSPAP
13,804
FFV_O93209


 


GGGGGS
13,805
KORV_Q9TTC1-Pro_3mut


 


GSSGGG
13,806
MLVCB_P08361_3mut


 


GSSGSS
13,807
MLVCB_P08361_3mutA


 


GGSEAAAKPAP
13,808
BAEVM_P10272_3mut


 


EAAAKGGGGSS
13,809
MLVCB_P08361_3mut


 


EAAAKPAPGGS
13,810
KORV_Q9TTC1-Pro_3mutA


 


GSSGSSGSSGSSGSS
13,811
MLVAV_P03356_3mutA


 


GGGGSEAAAKGGGGS
13,812
PERV_Q4VFZ2_3mutA_WS


 


GGSGSS
13,813
KORV_Q9TTC1-Pro_3mut


 


GSS

SFV3L_P27401-Pro_2mutA


 


PAPAP
13,815
BAEVM_P10272_3mut


 


EAAAKPAP
13,816
BAEVM_P10272


 


EAAAKEAAAKEAAAKEAA
13,817
KORV_Q9TTC1-Pro_3mut


AKEAAAK




 


GGGGGGG
13,818
PERV_Q4VFZ2_3mutA_WS


 


GGGGS
13,819
MLVMS_P03355_3mut


 


GSSGGG
13,820
FLV_P10273_3mutA


 


PAPAPAPAPAP
13,821
FLV_P10273_3mut


 


EAAAKEAAAKEAAAK
13,822
WMSV_P03359_3mutA


 


GSSGGS
13,823
MLVBM_Q7SVK7_3mutA_WS


 


EAAAKPAPGGG
13,824
MLVMS_P03355_3mut


 


GSSPAPGGS
13,825
WMSV_P03359_3mut


 


PAPGSSGGG
13,826
PERV_Q4VFZ2_3mutA_WS


 


GSSGGG
13,827
AVIRE_P03360_3mutA


 


PAPGGSGSS
13,828
MLVFF_P26809_3mut


 


PAPGSS
13,829
PERV_Q4VFZ2_3mut


 


GGGGGSGSS
13,830
WMSV_P03359_3mutA


 


EAAAKGGGGSS
13,831
MLVBM_Q7SVK7_3mutA_WS


 


GGGGGGG
13,832
BAEVM_P10272_3mut


 


PAPEAAAKGSS
13,833
MLVMS_P03355_3mut


 


GGSGGGEAAAK
13,834
MLVMS_P03355_PLV919


 


EAAAKGGGGGS
13,835
MLVCB_P08361_3mut


 


PAPGGS
13,836
KORV_Q9TTC1-Pro_3mut


 


GGGG
13,837
FLV_P10273_3mutA


 


EAAAKGGSGSS
13,838
MLVBM_Q7SVK7_3mutA_WS


 


GGGGSSGGS
13,839
MLVMS_P03355_3mutA_WS


 


GGGGGGGG
13,840
WMSV_P03359_3mut


 


GGSGSSGGG
13,841
MLVMS_P03355_PLV919


 


GSSEAAAKGGS
13,842
KORV_Q9TTC1-Pro_3mutA


 


EAAAKPAPGSS
13,843
MLVCB_P08361_3mut


 


GGSPAPGSS
13,844
KORV_Q9TTC1_3mutA


 


PAPGSSGGG
13,845
BAEVM_P10272_3mut


 


EAAAKPAPGSS
13,846
WMSV_P03359_3mut


 


GGSPAPEAAAK
13,847
XMRV6_A1Z651_3mutA


 


GSSPAP
13,848
FLV_P10273_3mutA


 


GSS

BAEVM_P10272_3mutA


 


EAAAKPAPGGS
13,850
FLV_P10273_3mutA


 


GGSGSSPAP
13,851
FLV_P10273_3mutA


 


PAPGSSGGS
13,852
MLVMS_P03355_3mut


 


GSAGSAAGSGEF
13,853
PERV_Q4VFZ2_3mutA_WS


 


GSSGGSEAAAK
13,854
KORV_Q9TTC1_3mutA


 


GSSGGS
13,855
MLVMS_P03355_3mutA_WS


 


EAAAKGGGGSEAAAK
13,856
SFV3L_P27401_2mut


 


GSSGGS
13,857
PERV_Q4VFZ2_3mutA_WS


 


GGSPAPEAAAK
13,858
FLV_P10273_3mut


 


GGSEAAAKGSS
13,859
PERV_Q4VFZ2_3mutA_WS


 


GSSPAPEAAAK
13,860
PERV_Q4VFZ2_3mutA_WS


 


GGSGSSGGG
13,861
PERV_Q4VFZ2_3mut


 


GGGG
13,862
AVIRE_P03360_3mutA


 


GGSEAAAKPAP
13,863
WMSV_P03359_3mut


 


GSSGGSPAP
13,864
MLVAV_P03356_3mutA


 


GSSGGSEAAAK
13,865
MLVMS_P03355_3mut


 


PAPEAAAKGGS
13,866
KORV_Q9TTC1-Pro_3mut


 


GGSPAP
13,867
PERV_Q4VFZ2_3mutA_WS


 


GGSEAAAK
13,868
MLVAV_P03356_3mutA


 


EAAAKGGGGSEAAAK
13,869
KORV_Q9TTC1-Pro_3mut


 


SGGSSGGSSGSETPGTSE
13,870
MLVMS_P03355_PLV919


SATPESSGGSSGGSS




 


GSSEAAAK
13,871
KORV_Q9TTC1_3mutA


 


GGG

AVIRE_P03360


 


GGSEAAAKGSS
13,873
MLVBM_Q7SVK7_3mut


 


GGSEAAAKGSS
13,874
MLVMS_P03355_3mut


 


GGSPAPEAAAK
13,875
MLVCB_P08361_3mut


 


GGSGGGEAAAK
13,876
MLVCB_P08361_3mut


 


GGSEAAAKPAP
13,877
MLVMS_P03355_3mutA_WS


 


EAAAKGGSGSS
13,878
KORV_Q9TTC1-Pro_3mut


 


GGGEAAAKGGS
13,879
MLVCB_P08361_3mut


 


EAAAKGGGGSEAAAK
13,880
FLV_P10273_3mutA


 


GGSPAP
13,881
MLVFF_P26809_3mut


 


GGGGSSGGS
13,882
XMRV6_A1Z651_3mutA


 


PAP

MLVCB_P08361_3mut


 


GGS

SFV3L_P27401-Pro_2mutA


 


GGGGSGGGGS
13,885
MLVMS_P03355_3mut


 


GGGEAAAKGGS
13,886
MLVAV_P03356_3mutA


 


GSSGSSGSSGSSGSSGSS
13,887
MLVMS_P03355_PLV919


 


PAPGSS
13,888
MLVCB_P08361_3mut


 


GGSGGSGGS
13,889
MLVMS_P03355_PLV919


 


PAPGGSGGG
13,890
FLV_P10273_3mutA


 


GGGGSGGGGSGGGGS
13,891
FLV_P10273_3mut


 


GGSGSSGGG
13,892
KORV_Q9TTC1-Pro_3mutA


 


GGSGGSGGS
13,893
GALV_P21414_3mutA


 


GGGEAAAKGGS
13,894
WMSV_P03359_3mut


 


SGSETPGTSESATPES
13,895
KORV_Q9TTC1_3mutA


 


EAAAKGGGGGS
13,896
KORV_Q9TTC1-Pro_3mut


 


EAAAKGSSPAP
13,897
BAEVM_P10272_3mut


 


GGGG
13,898
MLVCB_P08361_3mut


 


GGGGSGGGGSGGGGSGGG
13,899
MLVBM_Q7SVK7_3mut


GSGGGGS




 


GSSGGSGGG
13,900
MLVMS_P03355_PLV919


 


GGSGSS
13,901
MLVFF_P26809_3mut


 


EAAAKGGS
13,902
AVIRE_P03360_3mutA


 


GSSEAAAKGGS
13,903
MLVBM_Q7SVK7_3mutA_WS


 


EAAAKPAPGGG
13,904
WMSV_P03359_3mut


 


PAPGSSGGG
13,905
MLVCB_P08361_3mutA


 


GGGGSSEAAAK
13,906
KORV_Q9TTC1-Pro_3mutA


 


GSSEAAAKPAP
13,907
BAEVM_P10272_3mutA


 


PAPGGGEAAAK
13,908
MLVBM_Q7SVK7_3mutA_WS


 


GGSGGGEAAAK
13,909
MLVCB_P08361_3mutA


 


GGGGSGGGGSGGGGSGGG
13,910
FFV_O93209


GSGGGGSGGGGS




 


EAAAKGGGGGS
13,911
GALV_P21414_3mutA


 


GGSPAPGGG
13,912
MLVMS_P03355_3mut


 


GSSGSSGSS
13,913
FLV_P10273_3mutA


 


EAAAK
13,914
MLVBM_Q7SVK7_3mut


 


GGGGSSGGS
13,915
MLVMS_P03355_3mut


 


GGSGSSPAP
13,916
PERV_Q4VFZ2_3mut


 


EAAAKEAAAKEAAAKEAA
13,917
BAEVM_P10272_3mut


AK




 


GGGPAPGSS
13,918
MLVMS_P03355_3mut


 


GSSPAPGGS
13,919
PERV_Q4VFZ2_3mutA_WS


 


PAPAP
13,920
FLV_P10273_3mutA


 


PAPAPAPAP
13,921
PERV_Q4VFZ2_3mut


 


GGGGGSEAAAK
13,922
GALV_P21414_3mutA


 


GGGGGSGSS
13,923
BAEVM_P10272_3mutA


 


GGGEAAAKGSS
13,924
KORV_Q9TTC1_3mutA


 


GGGGGSPAP
13,925
AVIRE_P03360_3mut


 


GGGGGSEAAAK
13,926
SFV3L_P27401_2mutA


 


GGS

KORV_Q9TTC1_3mutA


 


GGGGGGG
13,928
PERV_Q4VFZ2_3mut


 


SGSETPGTSESATPES
13,929
SFV3L_P27401_2mutA


 


EAAAKGGSGGG
13,930
MLVMS_P03355_3mut


 


GGGGS
13,931
MLVFF_P26809_3mut


 


EAAAKGSSGGG
13,932
BAEVM_P10272_3mut


 


EAAAKPAPGGS
13,933
MLVF5_P26810_3mutA


 


SGGSSGGSSGSETPGTSE
13,934
SFV3L_P27401_2mutA


SATPESSGGSSGGSS




 


GGSPAPGGG
13,935
WMSV_P03359_3mutA


 


GSAGSAAGSGEF
13,936
MLVFF_P26809_3mut


 


GGGGSSGGS
13,937
MLVMS_P03355_3mutA_WS


 


GGGGGGG
13,938
MLVCB_P08361_3mut


 


GSSEAAAK
13,939
WMSV_P03359_3mut


 


PAPGSS
13,940
FLV_P10273_3mutA


 


GSSGGG
13,941
PERV_Q4VFZ2_3mutA_WS


 


PAPGGG
13,942
MLVFF_P26809_3mut


 


GGGGGSPAP
13,943
MLVMS_P03355_3mut


 


GGSEAAAK
13,944
XMRV6_A1Z651_3mut


 


GSSGGG
13,945
PERV_Q4VFZ2_3mut


 


GGSGGSGGSGGS
13,946
MLVMS_P03355_3mut


 


PAPAP
13,947
AVIRE_P03360_3mut


 


GGSEAAAK
13,948
PERV_Q4VFZ2_3mut


 


GGGGS
13,949
MLVMS_P03355_PLV919


 


GGGG
13,950
BAEVM_P10272_3mutA


 


EAAAKGGGGSS
13,951
MLVCB_P08361_3mutA


 


EAAAKEAAAKEAAAK
13,952
GALV_P21414_3mutA


 


PAPGGGEAAAK
13,953
KORV_Q9TTC1


 


EAAAKGGSPAP
13,954
MLVMS_P03355_3mut


 


GGSGSSEAAAK
13,955
MLVMS_P03355_3mut


 


GGSPAPEAAAK
13,956
FLV_P10273_3mutA


 


GGGGGGG
13,957
PERV_Q4VFZ2_3mut


 


EAAAKEAAAKEAAAKEAA
13,958
SFV1_P23074_2mutA


AKEAAAKEAAAK




 


EAAAKGSSGGS
13,959
MLVMS_P03355_3mut


 


GSSEAAAKPAP
13,960
MLVFF_P26809_3mut


 


GGGGSS
13,961
FLV_P10273_3mutA


 


EAAAKGGSGGG
13,962
AVIRE_P03360_3mutA


 


GGSGGS
13,963
PERV_Q4VFZ2_3mutA_WS


 


GGGGGSPAP
13,964
AVIRE_P03360_3mutA


 


EAAAKEAAAKEAAAK
13,965
XMRV6_A1Z651_3mut


 


PAPEAAAKGGS
13,966
FLV_P10273_3mutA


 


GSSGGSEAAAK
13,967
MLVCB_P08361_3mut


 


EAAAKGGSGGG
13,968
MLVMS_P03355


 


GGSGGGPAP
13,969
MLVMS_P03355_3mut


 


GGS

XMRV6_A1Z651_3mut


 


GGSEAAAKPAP
13,971
MLVFF_P26809_3mut


 


EAAAKGGG
13,972
MLVMS_P03355_PLV919


 


GSSGSSGSSGSS
13,973
WMSV_P03359_3mut


 


GGSGSSPAP
13,974
PERV_Q4VFZ2_3mut


 


GGGEAAAK
13,975
MLVMS_P03355_3mutA_WS


 


GSSPAPGGS
13,976
KORV_Q9TTC1-Pro_3mutA


 


GSSEAAAKGGG
13,977
SFV3L_P27401_2mut


 


EAAAKPAPGGS
13,978
MLVCB_P08361_3mut


 


GGSGGGEAAAK
13,979
PERV_Q4VFZ2


 


GGSGSS
13,980
MLVCB_P08361_3mut


 


GGSGGGEAAAK
13,981
MLVBM_Q7SVK7_3mutA_WS


 


GGSGGSGGSGGSGGSGGS
13,982
FLV_P10273_3mut


 


PAPEAAAKGSS
13,983
MLVMS_P03355_3mut


 


EAAAKGSSGGS
13,984
WMSV_P03359_3mutA


 


GGSGSSEAAAK
13,985
MLVCB_P08361_3mut


 


GGSGSSEAAAK
13,986
KORV_Q9TTC1_3mutA


 


GSSGGSGGG
13,987
MLVMS_P03355_PLV919


 


EAAAKGGSGGG
13,988
SFV3L_P27401-Pro_2mutA


 


GGSGGS
13,989
AVIRE_P03360_3mutA


 


GSAGSAAGSGEF
13,990
MLVMS_P03355_PLV919


 


GGSGSS
13,991
GALV_P21414_3mutA


 


GGGG
13,992
MLVFF_P26809_3mutA


 


GGGGSGGGGSGGGGSGGG
13,993
WMSV_P03359_3mut


GS




 


SGSETPGTSESATPES
13,994
BAEVM_P10272_3mut


 


EAAAKEAAAKEAAAKEAA
13,995
FOAMV_P14350_2mutA


AK




 


GGGEAAAKGGS
13,996
FLV_P10273_3mutA


 


GSSGGSEAAAK
13,997
MLVFF_P26809_3mut


 


EAAAKGGGGSS
13,998
MLVAV_P03356_3mut


 


PAPGGSEAAAK
13,999
KORV_Q9TTC1-Pro_3mut


 


EAAAK
14,000
XMRV6_A1Z651_3mut


 


GSSGSSGSSGSSGSSGSS
14,001
PERV_Q4VFZ2_3mut


 


GGGG
14,002
MLVCB_P08361_3mutA


 


GSSGSS
14,003
WMSV_P03359_3mutA


 


GSSGGSPAP
14,004
AVIRE_P03360_3mut


 


GGSGGSGGS
14,005
MLVCB_P08361_3mut


 


EAAAKGGGPAP
14,006
FLV_P10273_3mutA


 


GGGGSGGGGS
14,007
MLVCB_P08361_3mut


 


GGSEAAAKGSS
14,008
PERV_Q4VFZ2_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
14,009
SFV3L_P27401_2mutA


AKEAAAKEAAAK




 


GGSGSSEAAAK
14,010
PERV_Q4VFZ2_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
14,011
SFV3L_P27401-Pro_2mutA


AK




 


GSSEAAAKGGS
14,012
FLV_P10273_3mutA


 


GGSGSS
14,013
PERV_Q4VFZ2


 


GGSGSSEAAAK
14,014
SFV3L_P27401-Pro_2mutA


 


GSSGSSGSS
14,015
XMRV6_A1Z651_3mutA


 


EAAAKGSSPAP
14,016
KORV_Q9TTC1_3mutA


 


EAAAKPAP
14,017
FLV_P10273_3mutA


 


GGSGSSEAAAK
14,018
KORV_Q9TTC1-Pro_3mut


 


GGGGSGGGGSGGGGSGGG
14,019
KORV_Q9TTC1_3mutA


GSGGGGSGGGGS




 


GGGGSGGGGSGGGGS
14,020
KORV_Q9TTC1-Pro_3mutA


 


GGGGGGG
14,021
FLV_P10273_3mut


 


EAAAKGSS
14,022
WMSV_P03359_3mut


 


EAAAKGGGPAP
14,023
MLVCB_P08361_3mut


 


GSSGSS
14,024
MLVBM_Q7SVK7_3mutA_WS


 


EAAAKGGGGGS
14,025
MLVFF_P26809_3mut


 


GGSGGGEAAAK
14,026
FLV_P10273_3mutA


 


PAPGSS
14,027
MLVFF_P26809_3mutA


 


PAPGSS
14,028
BAEVM_P10272_3mutA


 


GGSPAPGSS
14,029
AVIRE_P03360_3mut


 


GGGGSSEAAAK
14,030
MLVMS_P03355_3mut


 


GSSGGGGGS
14,031
FFV_O93209-Pro


 


EAAAKGSSPAP
14,032
PERV_Q4VFZ2_3mut


 


GSSPAPGGS
14,033
PERV_Q4VFZ2_3mut


 


GGGGGG
14,034
BAEVM_P10272_3mut


 


EAAAKGGGGSS
14,035
PERV_Q4VFZ2_3mutA_WS


 


PAPGGSEAAAK
14,036
KORV_Q9TTC1_3mutA


 


SGGSSGGSSGSETPGTSE
14,037
MLVMS_P03355_3mutA_WS


SATPESSGGSSGGSS




 


GSSGSSGSSGSS
14,038
MLVMS_P03355_3mut


 


EAAAKGSSGGG
14,039
MLVMS_P03355_PLV919


 


GGSEAAAKPAP
14,040
AVIRE_P03360_3mutA


 


GSSGSSGSSGSSGSS
14,041
WMSV_P03359_3mutA


 


GGGEAAAKPAP
14,042
FLV_P10273_3mutA


 


PAPGSSGGG
14,043
KORV_Q9TTC1_3mutA


 


GSSGSS
14,044
MLVMS_P03355_3mutA_WS


 


PAPEAAAK
14,045
BAEVM_P10272_3mut


 


GGGPAPGSS
14,046
PERV_Q4VFZ2


 


GSSGGSPAP
14,047
MLVFF_P26809_3mut


 


GGGGSS
14,048
SFV3L_P27401_2mut


 


PAPEAAAKGSS
14,049
SFV3L_P27401_2mut


 


GGSGGGPAP
14,050
XMRV6_A1Z651_3mutA


 


PAPGGS
14,051
BAEVM_P10272_3mutA


 


EAAAKGGGGGS
14,052
AVIRE_P03360_3mut


 


GSSGGSPAP
14,053
KORV_Q9TTC1-Pro_3mutA


 


GSSGGGGGS
14,054
WMSV_P03359_3mut


 


GGGEAAAKGGS
14,055
AVIRE_P03360_3mut


 


GGGEAAAKGSS
14,056
BAEVM_P10272_3mut


 


PAPEAAAKGSS
14,057
MLVAV_P03356_3mutA


 


GSSGSSGSSGSSGSS
14,058
MLVCB_P08361_3mut


 


GGSPAPGSS
14,059
FLV_P10273_3mutA


 


EAAAKGSSPAP
14,060
BAEVM_P10272_3mutA


 


GGSGGSGGSGGSGGSGGS
14,061
PERV_Q4VFZ2


 


GGGGSSEAAAK
14,062
FLV_P10273_3mutA


 


GGGGSSPAP
14,063
FFV_O93209


 


GSSGGSPAP
14,064
MLVMS_P03355_3mut


 


GGGPAPGSS
14,065
MLVMS_P03355_PLV919


 


PAPGSSGGS
14,066
PERV_Q4VFZ2_3mut


 


GGGGGSPAP
14,067
MLVFF_P26809_3mut


 


SGSETPGTSESATPES
14,068
MLVMS_P03355_3mutA_WS


 


GSSGSSGSSGSSGSS
14,069
KORV_Q9TTC1_3mutA


 


GSSPAPGGG
14,070
WMSV_P03359_3mut


 


PAPAPAPAPAPAP
14,071
SFV3L_P27401_2mutA


 


GGGPAPGGS
14,072
MLVMS_P03355_3mut


 


PAPGGSEAAAK
14,073
WMSV_P03359_3mut


 


GGGGSSEAAAK
14,074
FFV_O93209-Pro


 


GGSPAPGGG
14,075
FLV_P10273_3mutA


 


GSSPAPEAAAK
14,076
AVIRE_P03360_3mut


 


GGGEAAAK
14,077
FLV_P10273_3mutA


 


PAPEAAAKGGG
14,078
MLVCB_P08361_3mut


 


GGSPAPGGG
14,079
MLVCB_P08361_3mut


 


GGSGGGGSS
14,080
BAEVM_P10272_3mutA


 


GSSPAPEAAAK
14,081
MLVCB_P08361_3mut


 


GGSPAPGGG
14,082
KORV_Q9TTC1-Pro_3mutA


 


PAPGGSGSS
14,083
KORV_Q9TTC1_3mutA


 


GSSPAP
14,084
KORV_Q9TTC1-Pro_3mutA


 


SGSETPGTSESATPES
14,085
MLVMS_P03355


 


GSSGSSGSS
14,086
MLVAV_P03356_3mutA


 


PAPGSSGGS
14,087
PERV_Q4VFZ2_3mutA_WS


 


PAPGGS
14,088
KORV_Q9TTC1-Pro_3mutA


 


PAPEAAAKGGG
14,089
SFV3L_P27401-Pro_2mutA


 


GGSGGSGGS
14,090
BAEVM_P10272_3mut


 


PAPGGS
14,091
MLVFF_P26809_3mut


 


GSSGGSPAP
14,092
MLVMS_P03355_PLV919


 


GSSGGGGGS
14,093
FLV_P10273_3mutA


 


GGGGGSPAP
14,094
KORV_Q9TTC1-Pro_3mut


 


EAAAKPAPGSS
14,095
SFV3L_P27401-Pro_2mutA


 


EAAAKGGSPAP
14,096
KORV_Q9TTC1-Pro


 


GGGPAPEAAAK
14,097
MLVMS_P03355_PLV919


 


GGSEAAAKGSS
14,098
MLVMS_P03355


 


PAPEAAAKGSS
14,099
KORV_Q9TTC1_3mutA


 


PAPEAAAKGGS
14,100
WMSV_P03359_3mutA


 


GSSGGG
14,101
PERV_Q4VFZ2_3mutA_WS


 


EAAAKGGGGSS
14,102
MLVMS_P03355_PLV919


 


EAAAKGGSPAP
14,103
AVIRE_P03360_3mutA


 


GGGGSSGGS
14,104
MLVMS_P03355_PLV919


 


PAPEAAAKGSS
14,105
PERV_Q4VFZ2_3mutA_WS


 


EAAAKGGGGGS
14,106
BAEVM_P10272_3mut


 


GSSGGGGGS
14,107
MLVMS_P03355_3mut


 


PAPAPAPAP
14,108
KORV_Q9TTC1_3mutA


 


GGSGGSGGSGGS
14,109
MLVAV_P03356_3mut


 


PAPAPAPAP
14,110
SFV3L_P27401_2mut


 


GSSEAAAKPAP
14,111
MLVMS_P03355_3mut


 


GGSGGGEAAAK
14,112
SFV3L_P27401_2mutA


 


GSSGGSGGG
14,113
MLVMS_P03355_3mutA_WS


 


GGGGGSPAP
14,114
MLVCB_P08361_3mutA


 


GGGEAAAKGSS
14,115
XMRV6_A1Z651_3mutA


 


GGGGSSPAP
14,116
BAEVM_P10272_3mut


 


GGSGGG
14,117
PERV_Q4VFZ2_3mut


 


GGGGSS
14,118
MLVBM_Q7SVK7_3mutA_WS


 


EAAAKGSSGGS
14,119
PERV_Q4VFZ2_3mutA_WS


 


GSSGGGGGS
14,120
PERV_Q4VFZ2


 


EAAAKGSSGGS
14,121
PERV_Q4VFZ2_3mut


 


EAAAKEAAAK
14,122
MLVAV_P03356_3mut


 


GSSGGGEAAAK
14,123
MLVAV_P03356_3mut


 


GSSPAPGGG
14,124
XMRV6_A1Z651_3mut


 


GGGGSGGGGSGGGGS
14,125
PERV_Q4VFZ2_3mut


 


EAAAKEAAAKEAAAKEAA
14,126
KORV_Q9TTC1_3mutA


AK




 


EAAAKGGSGSS
14,127
MLVBM_Q7SVK7_3mut


 


PAPEAAAK
14,128
BLVJ_P03361


 


GSSGGG
14,129
FFV_O93209-Pro


 


GGSGGGEAAAK
14,130
KORV_Q9TTC1-Pro_3mutA


 


EAAAK
14,131
FLV_P10273_3mutA


 


GGGGSSPAP
14,132
MLVMS_P03355_3mut


 


GSS

SFV3L_P27401-Pro_2mut


 


PAPEAAAKGSS
14,134
BAEVM_P10272_3mut


 


GGGGGSPAP
14,135
PERV_Q4VFZ2_3mut


 


GSSGSSGSS
14,136
BAEVM_P10272_3mutA


 


GGGGSGGGGSGGGGSGGG
14,137
SFV1_P23074_2mut


GS




 


GGGGSSEAAAK
14,138
SFV3L_P27401_2mutA


 


GGGGSGGGGSGGGGSGGG
14,139
FOAMV_P14350-Pro_2mut


GS




 


PAPGSSEAAAK
14,140
MLVBM_Q7SVK7_3mutA_WS


 


GGGGGSGSS
14,141
MLVFF_P26809_3mutA


 


GGSEAAAKGGG
14,142
MLVBM_Q7SVK7_3mut


 


PAPGSSGGG
14,143
PERV_Q4VFZ2


 


GGS

PERV_Q4VFZ2_3mutA_WS


 


EAAAKGGSGSS
14,145
FLV_P10273_3mut


 


GGGEAAAK
14,146
WMSV_P03359_3mutA


 


GGSEAAAKPAP
14,147
MLVBM_Q7SVK7_3mut


 


SGSETPGTSESATPES
14,148
FOAMV_P14350-Pro_2mutA


 


EAAAKPAPGGS
14,149
AVIRE_P03360_3mut


 


EAAAKGGGGGS
14,150
KORV_Q9TTC1-Pro_3mutA


 


GGGGS
14,151
PERV_Q4VFZ2_3mut


 


GGSEAAAKGSS
14,152
MLVFF_P26809_3mutA


 


GGSEAAAKGGG
14,153
AVIRE_P03360


 


GGSGGSGGSGGSGGSGGS
14,154
SFV3L_P27401_2mut


 


GGSEAAAKGSS
14,155
SFV3L_P27401-Pro_2mutA


 


GGGEAAAKPAP
14,156
MLVCB_P08361_3mut


 


GGSEAAAK
14,157
MLVMS_P03355_PLV919


 


GGSPAPGSS
14,158
KORV_Q9TTC1-Pro_3mutA


 


GSSPAPEAAAK
14,159
WMSV_P03359_3mutA


 


GGSGSS
14,160
KORV_Q9TTC1-Pro_3mutA


 


PAPGGGGGS
14,161
AVIRE_P03360_3mut


 


PAPEAAAKGSS
14,162
FFV_O93209-Pro


 


GGSGGGEAAAK
14,163
WMSV_P03359_3mut


 


PAPGGG
14,164
MLVMS_P03355_3mut


 


EAAAKGGG
14,165
FLV_P10273_3mutA


 


GSSGSSGSSGSS
14,166
MLVCB_P08361_3mut


 


EAAAKGGSGGG
14,167
FFV_O93209


 


GSSPAPGGS
14,168
PERV_Q4VFZ2_3mutA_WS


 


GSSPAPGGS
14,169
MLVCB_P08361_3mut


 


GGGPAP
14,170
WMSV_P03359_3mutA


 


GGGPAP
14,171
KORV_Q9TTC1_3mutA


 


GGSPAPGSS
14,172
KORV_Q9TTC1-Pro_3mut


 


PAPAP
14,173
MLVMS_P03355_3mut


 


GGGGGGG
14,174
MLVMS_P03355_3mut


 


GGGGG
14,175
KORV_Q9TTC1-Pro_3mut


 


GSAGSAAGSGEF
14,176
FOAMV_P14350_2mutA


 


PAPAP
14,177
KORV_Q9TTC1-Pro_3mutA


 


GGSEAAAKGGG
14,178
SFV3L_P27401-Pro_2mutA


 


PAPAP
14,179
WMSV_P03359_3mut


 


GGGGSGGGGSGGGGS
14,180
SFV3L_P27401_2mut


 


PAPGGS
14,181
KORV_Q9TTC1_3mutA


 


GGGEAAAKPAP
14,182
FLV_P10273_3mut


 


GGGGGS
14,183
MLVAV_P03356_3mutA


 


GSSEAAAKGGG
14,184
WMSV_P03359_3mut


 


EAAAKGGGGSS
14,185
GALV_P21414_3mutA


 


GSSGGS
14,186
MLVAV_P03356_3mutA


 


GSSGGG
14,187
MLVBM_Q7SVK7_3mut


 


PAPAPAP
14,188
SFV3L_P27401-Pro_2mutA


 


GGGG
14,189
KORV_Q9TTC1_3mutA


 


EAAAKPAPGGS
14,190
MLVFF_P26809_3mut


 


GGGGSGGGGS
14,191
XMRV6_A1Z651_3mut


 


EAAAKGGG
14,192
MLVCB_P08361_3mut


 


GGGGSSPAP
14,193
KORV_Q9TTC1_3mutA


 


GSSEAAAKGGG
14,194
KORV_Q9TTC1-Pro_3mutA


 


GGGGG
14,195
BLVJ_P03361_2mutB


 


GGGEAAAKGSS
14,196
FFV_O93209-Pro


 


GSSGSSGSS
14,197
BAEVM_P10272_3mut


 


GSSGGSPAP
14,198
PERV_Q4VFZ2_3mut


 


EAAAKGGS
14,199
KORV_Q9TTC1_3mut


 


GGSPAPEAAAK
14,200
AVIRE_P03360_3mut


 


GGSEAAAK
14,201
WMSV_P03359_3mut


 


GSSGGS
14,202
KORV_Q9TTC1-Pro_3mutA


 


GGGPAPEAAAK
14,203
KORV_Q9TTC1_3mutA


 


PAPGSS
14,204
WMSV_P03359_3mutA


 


GGSEAAAKGSS
14,205
FLV_P10273_3mutA


 


EAAAKEAAAKEAAAKEAA
14,206
SFV3L_P27401


AKEAAAK




 


GSSEAAAKGGG
14,207
SFV3L_P27401-Pro_2mutA


 


GGGGSEAAAKGGGGS
14,208
KORV_Q9TTC1-Pro_3mutA


 


GGSGGSGGS
14,209
WMSV_P03359_3mut


 


GGGGGSGSS
14,210
KORV_Q9TTC1-Pro


 


GGGGSGGGGSGGGGSGGG
14,211
MLVMS_P03355_3mut


GS




 


EAAAKGGG
14,212
PERV_Q4VFZ2


 


GGSEAAAKGGG
14,213
KORV_Q9TTC1-Pro_3mut


 


GSSGGSGGG
14,214
PERV_Q4VFZ2_3mutA_WS


 


GGGGGS
14,215
PERV_Q4VFZ2_3mut


 


GSAGSAAGSGEF
14,216
PERV_Q4VFZ2


 


PAPEAAAKGSS
14,217
BAEVM_P10272_3mutA


 


GSSPAPGGG
14,218
MLVCB_P08361_3mut


 


GGGGSSPAP
14,219
KORV_Q9TTC1-Pro_3mutA


 


PAPGGSGGG
14,220
MLVFF_P26809_3mut


 


GSSPAP
14,221
KORV_Q9TTC1_3mutA


 


PAPGSS
14,222
SFV3L_P27401-Pro_2mut


 


GGSGGGGSS
14,223
MLVMS_P03355_PLV919


 


GSSGGS
14,224
WMSV_P03359_3mutA


 


EAAAKGGGGGS
14,225
PERV_Q4VFZ2


 


GGGGG
14,226
KORV_Q9TTC1_3mutA


 


EAAAKGSS
14,227
MLVMS_P03355_PLV919


 


EAAAKEAAAKEAAAKEAA
14,228
FLV_P10273_3mut


AKEAAAK




 


EAAAKEAAAKEAAAKEAA
14,229
SFV3L_P27401-Pro_2mut


AK




 


GSAGSAAGSGEF
14,230
SFV3L_P27401_2mutA


 


GGGPAPGGS
14,231
FLV_P10273_3mutA


 


GGSEAAAKGGG
14,232
MLVCB_P08361_3mut


 


PAPGGGEAAAK
14,233
BAEVM_P10272_3mut


 


EAAAKPAPGSS
14,234
FOAMV_P14350_2mut


 


GGSEAAAK
14,235
KORV_Q9TTC1_3mutA


 


GGSGSS
14,236
AVIRE_P03360


 


GGSPAPEAAAK
14,237
MLVMS_P03355_PLV919


 


GGGGS
14,238
XMRV6_A1Z651_3mut


 


GGSPAPGGG
14,239
XMRV6_A1Z651_3mut


 


EAAAKPAPGGS
14,240
PERV_Q4VFZ2


 


GSSPAP
14,241
BAEVM_P10272_3mut


 


GGSGSSGGG
14,242
FLV_P10273_3mutA


 


PAPGGG
14,243
PERV_Q4VFZ2_3mutA_WS


 


GSSGGSEAAAK
14,244
MLVBM_Q7SVK7_3mut


 


GGSEAAAK
14,245
MLVMS_P03355_3mut


 


GGGPAPGGS
14,246
MLVFF_P26809_3mut


 


GSAGSAAGSGEF
14,247
MLVBM_Q7SVK7_3mutA_WS


 


EAAAKPAPGGS
14,248
SFVCP_Q87040


 


PAPGGG
14,249
PERV_Q4VFZ2_3mutA_WS


 


GSSPAPEAAAK
14,250
MLVBM_Q7SVK7


 


PAPEAAAK
14,251
MLVBM_Q7SVK7_3mut


 


PAPGGGGGS
14,252
AVIRE_P03360_3mutA


 


GGSEAAAKPAP
14,253
MLVBM_Q7SVK7_3mut


 


EAAAKGSS
14,254
WMSV_P03359_3mutA


 


GGGEAAAK
14,255
MLVFF_P26809_3mutA


 


EAAAKEAAAKEAAAK
14,256
MLVMS_P03355_3mut


 


PAPEAAAKGGG
14,257
BAEVM_P10272_3mut


 


PAPAPAP
14,258
MLVCB_P08361_3mut


 


EAAAKPAPGGS
14,259
BAEVM_P10272_3mut


 


GGGGSGGGGS
14,260
FLV_P10273_3mut


 


GGGGSEAAAKGGGGS
14,261
KORV_Q9TTC1_3mut


 


EAAAK
14,262
FLV_P10273_3mut


 


PAPAPAP
14,263
WMSV_P03359_3mut


 


GGGGSEAAAKGGGGS
14,264
FFV_O93209-Pro


 


GGSPAPEAAAK
14,265
MLVMS_P03355_3mut


 


GGSGSSGGG
14,266
XMRV6_A1Z651_3mut


 


GGSPAPGSS
14,267
PERV_Q4VFZ2_3mut


 


SGGSSGGSSGSETPGTSE
14,268
SFV3L_P27401-Pro_2mutA


SATPESSGGSSGGSS




 


EAAAKGGGPAP
14,269
BAEVM_P10272_3mutA


 


GSSGGSEAAAK
14,270
MLVMS_P03355_3mutA_WS


 


SGSETPGTSESATPES
14,271
PERV_Q4VFZ2_3mutA_WS


 


EAAAKEAAAKEAAAKEAA
14,272
KORV_Q9TTC1-Pro_3mutA


AKEAAAK




 


GSSGSSGSS
14,273
KORV_Q9TTC1_3mutA


 


GSSPAPGGG
14,274
SFV3L_P27401-Pro_2mutA


 


GSSGGGEAAAK
14,275
KORV_Q9TTC1_3mutA


 


GGSGGGGSS
14,276
PERV_Q4VFZ2_3mutA_WS


 


GSSGGGEAAAK
14,277
MLVCB_P08361_3mut


 


GSSEAAAKGGG
14,278
MLVCB_P08361_3mut


 


GGSGGGGSS
14,279
KORV_Q9TTC1_3mutA


 


GGSGSSPAP
14,280
PERV_Q4VFZ2_3mutA_WS


 


GSSPAP
14,281
MLVMS_P03355_3mut


 


GGGGSSEAAAK
14,282
AVIRE_P03360


 


GGS

WMSV_P03359_3mut


 


EAAAKEAAAK
14,284
PERV_Q4VFZ2_3mut


 


PAPAPAPAP
14,285
MLVAV_P03356_3mut


 


GGSEAAAKGGG
14,286
KORV_Q9TTC1_3mutA


 


PAPGGG
14,287
MLVAV_P03356_3mut


 


EAAAKGSS
14,288
BAEVM_P10272_3mut


 


GGGGSGGGGS
14,289
WMSV_P03359_3mutA


 


GGSGGSGGS
14,290
SFV3L_P27401_2mut


 


EAAAK
14,291
MLVCB_P08361_3mut


 


GGGGSSGGS
14,292
WMSV_P03359_3mutA


 


GGGPAPEAAAK
14,293
MLVAV_P03356_3mutA


 


EAAAKEAAAKEAAAK
14,294
FFV_O93209


 


GSSEAAAKGGG
14,295
MLVBM_Q7SVK7_3mut


 


GGGPAPGGS
14,296
FLV_P10273_3mut


 


GGSEAAAKGGG
14,297
WMSV_P03359_3mut


 


EAAAKGGGGGS
14,298
XMRV6_A1Z651_3mutA


 


EAAAKGGSGGG
14,299
FLV_P10273_3mutA


 


GGSEAAAKGGG
14,300
SFV3L_P27401_2mutA


 


GGGGS
14,301
PERV_Q4VFZ2_3mutA_WS


 


GSSGGS
14,302
MLVMS_P03355_3mut


 


GSSGSS
14,303
MLVAV_P03356_3mutA


 


GGSPAPGGG
14,304
MLVBM_Q7SVK7_3mutA_WS


 


GSSGGGGGS
14,305
MLVF5_P26810_3mut


 


PAPAPAPAP
14,306
MLVCB_P08361_3mut


 


PAPAP
14,307
PERV_Q4VFZ2_3mutA_WS


 


PAPGSSGGS
14,308
KORV_Q9TTC1_3mut


 


PAPGSSGGG
14,309
PERV_Q4VFZ2_3mut


 


GGGEAAAK
14,310
MLVMS_P03355_PLV919


 


GGSGGSGGSGGSGGS
14,311
SFV3L_P27401-Pro_2mutA


 


GGSGGG
14,312
FLV_P10273_3mut


 


PAPEAAAKGGG
14,313
MLVFF_P26809_3mut


 


PAP

PERV_Q4VFZ2_3mutA_WS


 


PAPGGSGSS
14,315
FFV_O93209_2mut


 


EAAAKEAAAKEAAAKEAA
14,316
FFV_O93209-Pro_2mut


AKEAAAKEAAAK




 


GSSGSSGSSGSS
14,317
FFV_O93209-Pro


 


GSSGSSGSSGSSGSS
14,318
FLV_P10273_3mutA


 


GGGEAAAKPAP
14,319
PERV_Q4VFZ2


 


PAPGSSGGG
14,320
SFV3L_P27401_2mut


 


PAPGGSGSS
14,321
KORV_Q9TTC1-Pro_3mut


 


PAPAPAPAPAP
14,322
GALV_P21414_3mutA


 


GGSGGGEAAAK
14,323
PERV_Q4VFZ2_3mut


 


GSSPAP
14,324
MLVCB_P08361_3mut


 


EAAAKPAP
14,325
MLVF5_P26810_3mut


 


GGGGSGGGGSGGGGSGGG
14,326
MLVBM_Q7SVK7_3mut


GS




 


GGSGGG
14,327
WMSV_P03359_3mut


 


GGSGGSGGS
14,328
KORV_Q9TTC1_3mut


 


GGGGGGGG
14,329
MLVFF_P26809_3mut


 


GGGGSS
14,330
MLVAV_P03356_3mut


 


GSSGGGGGS
14,331
SFV3L_P27401_2mut


 


EAAAKEAAAKEAAAKEAA
14,332
GALV_P21414_3mutA


AKEAAAKEAAAK




 


GSSGSSGSS
14,333
PERV_Q4VFZ2_3mut


 


GSSPAPGGS
14,334
MLVFF_P26809_3mut


 


PAPAPAP
14,335
AVIRE_P03360_3mutA


 


EAAAKEAAAKEAAAKEAA
14,336
WMSV_P03359_3mutA


AK




 


PAPAPAPAP
14,337
SFV3L_P27401_2mutA


 


GGGGSS
14,338
MLVAV_P03356_3mutA


 


GSSGSSGSSGSSGSS
14,339
SFV3L_P27401_2mutA


 


PAPGGS
14,340
WMSV_P03359_3mutA


 


GSSEAAAKGGG
14,341
PERV_Q4VFZ2


 


GSSGGSPAP
14,342
MLVMS_P03355_PLV919


 


GSSGSSGSSGSSGSSGSS
14,343
SFV3L_P27401_2mutA


 


GGSGSSGGG
14,344
MLVCB_P08361_3mut


 


GGGPAPGSS
14,345
SFV3L_P27401-Pro_2mutA


 


GSSEAAAKGGS
14,346
WMSV_P03359_3mut


 


GSSEAAAKGGG
14,347
MLVAV_P03356_3mut


 


GGSGGGPAP
14,348
FFV_O93209-Pro


 


GSSGSS
14,349
PERV_Q4VFZ2_3mut


 


PAPGGGGGS
14,350
GALV_P21414_3mutA


 


EAAAKPAPGGS
14,351
MLVAV_P03356_3mut


 


GSSGSS
14,352
MLVMS_P03355_3mut


 


EAAAKPAPGGS
14,353
FFV_O93209-Pro


 


GGGPAPEAAAK
14,354
MLVMS_P03355_3mutA_WS


 


GSSEAAAKGGG
14,355
MLVBM_Q7SVK7_3mut


 


GGGEAAAKGGS
14,356
BAEVM_P10272_3mut


 


GSSGSS
14,357
KORV_Q9TTC1-Pro_3mutA


 


EAAAKEAAAKEAAAK
14,358
SFV1_P23074


 


PAPGSSGGS
14,359
KORV_Q9TTC1-Pro_3mut


 


PAPAPAPAPAP
14,360
MLVMS_P03355


 


GSSEAAAK
14,361
SFV3L_P27401_2mut


 


PAP

PERV_Q4VFZ2_3mut


 


GGSEAAAKGGG
14,363
MLVBM_Q7SVK7_3mut


 


GGSGGGPAP
14,364
MLVBM_Q7SVK7_3mutA_WS


 


GSSGSS
14,365
MLVMS_P03355_3mut


 


GGSEAAAK
14,366
MLVMS_P03355


 


GSSEAAAKGGS
14,367
MLVMS_P03355_PLV919


 


PAPGGGGGS
14,368
MLVFF_P26809_3mut


 


GSSGGG
14,369
PERV_Q4VFZ2_3mut


 


GSSGGS
14,370
PERV_Q4VFZ2_3mutA_WS


 


PAPGGG
14,371
BAEVM_P10272_3mut


 


PAPGSSGGG
14,372
MLVBM_Q7SVK7_3mut


 


GGSEAAAK
14,373
SFV3L_P27401_2mut


 


GSSPAPEAAAK
14,374
SFV3L_P27401-Pro_2mut


 


GSSGGSPAP
14,375
BAEVM_P10272_3mut


 


GGSPAPGSS
14,376
PERV_Q4VFZ2_3mutA_WS


 


GGSGGSGGS
14,377
PERV_Q4VFZ2


 


GGSGGGPAP
14,378
FLV_P10273_3mut


 


GGGPAPEAAAK
14,379
SFV3L_P27401_2mutA


 


GGGGS
14,380
FLV_P10273_3mutA


 


GSSGGSGGG
14,381
XMRV6_A1Z651_3mut


 


EAAAKGGGGSS
14,382
PERV_Q4VFZ2


 


GGSGSSGGG
14,383
SFV3L_P27401-Pro_2mutA


 


GGSGGSGGS
14,384
MLVFF_P26809_3mut


 


GGGPAPEAAAK
14,385
FLV_P10273_3mut


 


GSSGGGEAAAK
14,386
MLVMS_P03355_3mut


 


GGG

SFV3L_P27401_2mut


 


GSAGSAAGSGEF
14,388
WMSV_P03359_3mut


 


GSSGGGPAP
14,389
MLVMS_P03355_PLV919


 


GGGGSS
14,390
KORV_Q9TTC1-Pro_3mut


 


GGGGSSEAAAK
14,391
KORV_Q9TTC1


 


PAPGGSGGG
14,392
SFV3L_P27401_2mut


 


GSSGSSGSSGSSGSS
14,393
FFV_O93209


 


GSSGGSPAP
14,394
MLVMS_P03355_3mut


 


GGSEAAAK
14,395
KORV_Q9TTC1-Pro_3mutA


 


GGGGGGGGS
14,396
BAEVM_P10272_3mut


 


GSSEAAAKGGG
14,397
AVIRE_P03360_3mut


 


EAAAKPAPGGG
14,398
FLV_P10273_3mut


 


EAAAKGGSPAP
14,399
SFV3L_P27401-Pro_2mutA


 


GSSEAAAKPAP
14,400
MLVBM_Q7SVK7_3mut


 


GGGPAPGGS
14,401
MLVCB_P08361_3mut


 


GGG

SFV3L_P27401_2mutA


 


EAAAKGGGGSEAAAK
14,403
SFV3L_P27401_2mutA


 


GGSGSSGGG
14,404
MLVBM_Q7SVK7_3mut


 


GSAGSAAGSGEF
14,405
BAEVM_P10272_3mut


 


GGGEAAAK
14,406
FOAMV_P14350_2mutA


 


PAPEAAAKGGS
14,407
WMSV_P03359_3mut


 


PAPAPAPAPAPAP
14,408
MLVF5_P26810_3mutA


 


GGSGGGGSS
14,409
FLV_P10273_3mutA


 


PAPGSSGGS
14,410
BAEVM_P10272_3mut


 


PAPEAAAK
14,411
WMSV_P03359_3mutA


 


GSSGSSGSSGSSGSSGSS
14,412
FFV_O93209-Pro_2mut


 


GGGGGSGSS
14,413
FFV_O93209-Pro


 


GGGGGGGG
14,414
SFV3L_P27401-Pro_2mutA


 


GGGGGG
14,415
FLV_P10273_3mut


 


GSSGGSGGG
14,416
MLVAV_P03356_3mutA


 


GGGGSS
14,417
SFV3L_P27401-Pro_2mutA


 


GGSGGGPAP
14,418
FOAMV_P14350_2mut


 


GSSGSS
14,419
AVIRE_P03360_3mutA


 


EAAAKEAAAKEAAAKEAA
14,420
SFV3L_P27401-Pro_2mutA


AKEAAAK




 


EAAAKEAAAK
14,421
BAEVM_P10272_3mut


 


GSSPAPEAAAK
14,422
GALV_P21414_3mutA


 


GGSEAAAKPAP
14,423
SFV3L_P27401_2mutA


 


GGSGGGEAAAK
14,424
SFV3L_P27401-Pro_2mutA


 


EAAAKGSSPAP
14,425
FOAMV_P14350_2mut


 


GGSGSSEAAAK
14,426
SFV3L_P27401_2mut


 


GGG

PERV_Q4VFZ2


 


GGGGGSGSS
14,428
FOAMV_P14350_2mut


 


GGSGGGEAAAK
14,429
KORV_Q9TTC1-Pro_3mut


 


GSSGGSGGG
14,430
AVIRE_P03360_3mutA


 


EAAAKPAPGGG
14,431
SFV3L_P27401_2mutA


 


PAPGGSGGG
14,432
KORV_Q9TTC1-Pro_3mut


 


PAPAPAP
14,433
WMSV_P03359_3mutA


 


GSSEAAAKPAP
14,434
SFV1_P23074


 


SGGSSGGSSGSETPGTSE
14,435
SRV2_P51517


SATPESSGGSSGGSS




 


GSSGGSGGG
14,436
PERV_Q4VFZ2_3mutA_WS


 


GSSGSSGSSGSSGSSGSS
14,437
FFV_O93209


 


GSSGGGPAP
14,438
WMSV_P03359_3mut


 


PAPAPAPAPAPAP
14,439
MLVBM_Q7SVK7_3mut


 


GGGGGSPAP
14,440
KORV_Q9TTC1-Pro_3mutA


 


PAPGSS
14,441
MLVBM_Q7SVK7_3mutA_WS


 


PAPEAAAKGGS
14,442
SFV3L_P27401-Pro_2mut


 


GGGGSSPAP
14,443
MLVMS_P03355_3mut


 


GGSEAAAK
14,444
FFV_O93209-Pro


 


EAAAKPAPGGS
14,445
AVIRE_P03360_3mutA


 


PAPGSS
14,446
WMSV_P03359_3mut


 


PAPGSSGGG
14,447
SFV3L_P27401-Pro_2mutA


 


EAAAKEAAAKEAAAK
14,448
SFV3L_P27401_2mut


 


GGS

MLVRD_P11227_3mut


 


GGGGS
14,450
KORV_Q9TTC1-Pro_3mut


 


GGSGGGGSS
14,451
KORV_Q9TTC1


 


GGSGGG
14,452
MLVMS_P03355_3mutA_WS


 


GGGEAAAKPAP
14,453
BAEVM_P10272_3mut


 


EAAAKEAAAKEAAAKEAA
14,454
FLV_P10273


AKEAAAK




 


PAPGGSGGG
14,455
KORV_Q9TTC1-Pro_3mutA


 


GSSGSSGSSGSSGSSGSS
14,456
HTL1L_P0C211


 


GGGEAAAKPAP
14,457
WMSV_P03359


 


GSSGGSPAP
14,458
FFV_O93209-Pro


 


PAPAPAPAPAP
14,459
SFV3L_P27401-Pro_2mutA


 


GSSGGSEAAAK
14,460
SFV3L_P27401_2mutA


 


GGSPAPGSS
14,461
SFV3L_P27401_2mut


 


GGSGGSGGS
14,462
KORV_Q9TTC1-Pro_3mut


 


PAPEAAAKGSS
14,463
KORV_Q9TTC1-Pro_3mut


 


EAAAKGGS
14,464
KORV_Q9TTC1_3mutA


 


EAAAKGGGGSEAAAK
14,465
SFV3L_P27401-Pro_2mut


 


GGGGSSPAP
14,466
FFV_O93209-Pro


 


EAAAK
14,467
SFV3L_P27401_2mut


 


EAAAKGGGGSS
14,468
BAEVM_P10272_3mut


 


GGGGGSEAAAK
14,469
MLVBM_Q7SVK7_3mut


 


GGGG
14,470
PERV_Q4VFZ2


 


GGGGGSEAAAK
14,471
FLV_P10273_3mut


 


EAAAKGGGPAP
14,472
KORV_Q9TTC1-Pro


 


GGGGSGGGGSGGGGSGGG
14,473
FFV_O93209_2mutA


GS




 


GSSGGSGGG
14,474
PERV_Q4VFZ2_3mut


 


GGGGSGGGGSGGGGS
14,475
GALV_P21414_3mutA


 


GGSGGGEAAAK
14,476
AVIRE_P03360_3mutA


 


PAPEAAAKGGG
14,477
SFV3L_P27401_2mut


 


GGGGSGGGGS
14,478
AVIRE_P03360


 


GSSGGGEAAAK
14,479
SFV3L_P27401_2mutA


 


GGGGG
14,480
AVIRE_P03360_3mutA


 


GGSGSS
14,481
KORV_Q9TTC1_3mut


 


PAPAPAPAPAPAP
14,482
FOAMV_P14350_2mut


 


GGSEAAAKPAP
14,483
KORV_Q9TTC1-Pro_3mut


 


GGGGGG
14,484
PERV_Q4VFZ2_3mut


 


GSSGGGEAAAK
14,485
MLVBM_Q7SVK7


 


SGGSSGGSSGSETPGTSE
14,486
MLVAV_P03356


SATPESSGGSSGGSS




 


GGSPAPGSS
14,487
BAEVM_P10272_3mut


 


GGGGSSPAP
14,488
BAEVM_P10272


 


GGGGSEAAAKGGGGS
14,489
SFV3L_P27401_2mut


 


GGGGGGGG
14,490
GALV_P21414_3mutA


 


PAPAP
14,491
MLVAV_P03356_3mut


 


GGGEAAAK
14,492
PERV_Q4VFZ2_3mutA_WS


 


GSSPAPGGG
14,493
FFV_O93209_2mut


 


GGSGGSGGSGGSGGS
14,494
BAEVM_P10272


 


GGGGGS
14,495
MLVF5_P26810_3mutA


 


PAPGGGGSS
14,496
FLV_P10273_3mutA


 


GGGEAAAK
14,497
MLVBM_Q7SVK7_3mut


 


PAPEAAAKGGG
14,498
WMSV_P03359_3mut


 


GSSEAAAK
14,499
MLVBM_Q7SVK7_3mut


 


EAAAKEAAAK
14,500
AVIRE_P03360


 


EAAAKGGGGGS
14,501
MLVBM_Q7SVK7_3mut


 


GGGEAAAKGGS
14,502
SFV3L_P27401-Pro_2mutA


 


PAPAPAPAPAP
14,503
MLVF5_P26810_3mut


 


PAPGSSEAAAK
14,504
SFV3L_P27401-Pro_2mutA


 


EAAAKEAAAKEAAAK
14,505
BAEVM_P10272_3mutA


 


GGSPAPGSS
14,506
MLVMS_P03355


 


PAPGSSGGS
14,507
FLV_P10273_3mutA


 


EAAAKEAAAKEAAAKEAA
14,508
FOAMV_P14350-Pro_2mut


AK




 


EAAAKGGG
14,509
KORV_Q9TTC1_3mutA


 


EAAAKGGSGGG
14,510
MLVBM_Q7SVK7_3mut


 


GGGGGS
14,511
KORV_Q9TTC1-Pro_3mutA


 


PAPGGSGGG
14,512
WMSV_P03359_3mut


 


GGGPAPGGS
14,513
KORV_Q9TTC1_3mutA


 


GSS

FFV_O93209


 


GGSGGSGGS
14,515
PERV_Q4VFZ2_3mut


 


GGGGS
14,516
GALV_P21414_3mutA


 


GGGG
14,517
MLVF5_P26810_3mut


 


GGSEAAAKPAP
14,518
FFV_O93209-Pro_2mut


 


PAPAPAPAP
14,519
FFV_O93209-Pro


 


PAP

MLVF5_P26810_3mut


 


EAAAKEAAAKEAAAK
14,521
FFV_O93209_2mut


 


EAAAKGSS
14,522
MLVCB_P08361_3mut


 


EAAAKGGG
14,523
MLVBM_Q7SVK7_3mut


 


PAPEAAAKGGG
14,524
FFV_O93209_2mut


 


GSSGGGEAAAK
14,525
SFV1_P23074-Pro_2mut


 


PAPGGGEAAAK
14,526
GALV_P21414_3mutA


 


GGGGSGGGGSGGGGSGGG
14,527
FOAMV_P14350-Pro_2mutA


GS




 


GSSGGG
14,528
FOAMV_P14350_2mut


 


GGGGSGGGGSGGGGSGGG
14,529
SFV3L_P27401_2mutA


GS




 


GGSGSS
14,530
AVIRE_P03360_3mut


 


GGSGSSEAAAK
14,531
MMTVB_P03365_WS


 


PAPAPAP
14,532
MLVAV_P03356_3mutA


 


GSSGGSPAP
14,533
SFV3L_P27401-Pro_2mut


 


GGSPAP
14,534
AVIRE_P03360


 


GGSGGGPAP
14,535
FFV_O93209


 


GSSEAAAK
14,536
PERV_Q4VFZ2


 


GSSGGGPAP
14,537
PERV_Q4VFZ2_3mutA_WS


 


GGGGSSEAAAK
14,538
KORV_Q9TTC1_3mutA


 


GGSEAAAKPAP
14,539
SFVCP_Q87040


 


GGSGGGPAP
14,540
FOAMV_P14350_2mutA


 


GGGGSGGGGSGGGGSGGG
14,541
BLVJ_P03361_2mutB


GS




 


GGGGSSPAP
14,542
SFV3L_P27401_2mutA


 


EAAAKGGS
14,543
MLVF5_P26810_3mut


 


GGSEAAAKGSS
14,544
MLVCB_P08361_3mut


 


GGGGSSEAAAK
14,545
SFV3L_P27401_2mut


 


EAAAKGGSGGG
14,546
FOAMV_P14350_2mut


 


GGSGGS
14,547
FLV_P10273_3mut


 


EAAAKGGG
14,548
FFV_O93209-Pro


 


GSSGSSGSSGSSGSS
14,549
SFV3L_P27401


 


GSSGGGPAP
14,550
PERV_Q4VFZ2_3mutA_WS


 


PAPGGSEAAAK
14,551
SFV3L_P27401-Pro_2mutA


 


GGSPAP
14,552
KORV_Q9TTC1


 


EAAAKPAPGSS
14,553
KORV_Q9TTC1_3mutA


 


SGSETPGTSESATPES
14,554
SFV1_P23074


 


GSSPAP
14,555
SFV3L_P27401-Pro_2mutA


 


GSSPAPGGG
14,556
SFV3L_P27401_2mut


 


GGGEAAAKGSS
14,557
SFV1_P23074_2mut


 


GGGPAPGGS
14,558
BAEVM_P10272_3mut


 


EAAAKGGG
14,559
KORV_Q9TTC1-Pro_3mutA


 


GSSGGG
14,560
SFV3L_P27401-Pro_2mut


 


GGSPAPEAAAK
14,561
BAEVM_P10272_3mut


 


EAAAKGSSPAP
14,562
FFV_O93209


 


EAAAKGGGGSEAAAK
14,563
SFV3L_P27401-Pro_2mutA


 


GSSGSSGSSGSSGSS
14,564
SFV1_P23074_2mut


 


EAAAKGGSPAP
14,565
FOAMV_P14350_2mut


 


GGSGGS
14,566
KORV_Q9TTC1-Pro_3mutA


 


EAAAKGSSGGS
14,567
GALV_P21414


 


GSSGGGPAP
14,568
MLVAV_P03356


 


PAPEAAAKGGS
14,569
FOAMV_P14350_2mut


 


EAAAKPAPGGG
14,570
AVIRE_P03360_3mut


 


GGSPAP
14,571
SFV3L_P27401_2mutA


 


GGGGSGGGGS
14,572
SFV3L_P27401_2mutA


 


GGGGSS
14,573
AVIRE_P03360_3mutA


 


GGSPAPGGG
14,574
SFV3L_P27401-Pro_2mutA


 


EAAAKPAPGSS
14,575
SFV3L_P27401


 


EAAAKPAP
14,576
FOAMV_P14350-Pro_2mut


 


PAPEAAAKGSS
14,577
PERV_Q4VFZ2_3mutA_WS


 


EAAAKGGSGSS
14,578
SFV3L_P27401_2mutA


 


GGGEAAAKGSS
14,579
GALV_P21414_3mutA


 


GGGGSEAAAKGGGGS
14,580
PERV_Q4VFZ2_3mut


 


PAPGGSGSS
14,581
FFV_O93209-Pro_2mutA


 


GGSEAAAKPAP
14,582
GALV_P21414_3mutA


 


GGSGGSGGSGGSGGS
14,583
FFV_O93209-Pro


 


GSSGGSEAAAK
14,584
SFV3L_P27401-Pro_2mut


 


GGS

GALV_P21414_3mutA


 


PAPGGSEAAAK
14,586
MLVMS_P03355


 


PAPEAAAKGGS
14,587
BAEVM_P10272_3mutA


 


GGSGSSPAP
14,588
SFV3L_P27401-Pro_2mutA


 


GSSPAP
14,589
WMSV_P03359_3mut


 


GGGEAAAK
14,590
MMTVB_P03365


 


GGGGSS
14,591
PERV_Q4VFZ2_3mut


 


GGSPAPGSS
14,592
SFV3L_P27401-Pro_2mut


 


PAPGGS
14,593
MLVBM_Q7SVK7_3mut


 


EAAAKGSSPAP
14,594
MLVBM_Q7SVK7_3mut


 


GGGGSSGGS
14,595
PERV_Q4VFZ2_3mut


 


PAPAPAPAPAPAP
14,596
SFV1_P23074


 


GGSEAAAKGGG
14,597
SFV3L_P27401-Pro_2mut


 


GGSGGS
14,598
SFV1_P23074_2mut


 


GSSGGGGGS
14,599
MLVF5_P26810_3mutA


 


EAAAKGGGPAP
14,600
SFV3L_P27401


 


EAAAKEAAAKEAAAKEAA
14,601
FOAMV_P14350-Pro_2mutA


AK




 


GGGPAPGSS
14,602
SFV3L_P27401_2mutA


 


GGGGSGGGGSGGGGSGGG
14,603
SFV3L_P27401_2mut


GS




 


EAAAKEAAAKEAAAKEAA
14,604
MMTVB_P03365_WS


AK




 


PAPGSSGGS
14,605
KORV_Q9TTC1-Pro_3mutA


 


PAPGSSEAAAK
14,606
FOAMV_P14350-Pro_2mut


 


GSSPAPEAAAK
14,607
BAEVM_P10272_3mut


 


EAAAKGGGGSEAAAK
14,608
FFV_O93209-Pro


 


GGSPAP
14,609
PERV_Q4VFZ2


 


GGSGSSEAAAK
14,610
XMRV6_A1Z651_3mut


 


GGSEAAAKGGG
14,611
GALV_P21414_3mutA


 


PAPGGGGSS
14,612
AVIRE_P03360_3mutA


 


GGSGGSGGSGGS
14,613
PERV_Q4VFZ2


 


GGGGSSGGS
14,614
PERV_Q4VFZ2_3mutA_WS


 


SGGSSGGSSGSETPGTSE
14,615
BAEVM_P10272_3mutA


SATPESSGGSSGGSS




 


GGGPAP
14,616
MLVAV_P03356_3mut


 


GGGGSGGGGSGGGGSGGG
14,617
FFV_O93209_2mut


GS




 


GSSEAAAK
14,618
FFV_O93209


 


GGSPAPEAAAK
14,619
FOAMV_P14350_2mut


 


GGGGGSEAAAK
14,620
FOAMV_P14350_2mut


 


GSSPAPGGS
14,621
MLVBM_Q7SVK7_3mut


 


GSS

SFVCP_Q87040_2mut


 


EAAAKPAP
14,623
FOAMV_P14350-Pro


 


EAAAKGGG
14,624
SFV3L_P27401_2mut


 


GGGEAAAK
14,625
AVIRE_P03360_3mutA


 


PAPGSSGGG
14,626
WMSV_P03359_3mut


 


EAAAKGGSPAP
14,627
SFV3L_P27401


 


GSSGGSGGG
14,628
SFV3L_P27401-Pro_2mutA


 


GSSGGGEAAAK
14,629
GALV_P21414_3mutA


 


GGGPAPGSS
14,630
MLVBM_Q7SVK7_3mutA_WS


 


PAPGGGEAAAK
14,631
FFV_O93209-Pro_2mut


 


GSSGSSGSSGSS
14,632
SFV1_P23074_2mut


 


GGSEAAAK
14,633
PERV_Q4VFZ2_3mutA_WS


 


GGGEAAAKPAP
14,634
SFV3L_P27401_2mut


 


EAAAKGGGPAP
14,635
SFV3L_P27401_2mut


 


GGGGSSPAP
14,636
FLV_P10273_3mut


 


EAAAKPAPGSS
14,637
FFV_O93209_2mut


 


GGGGSSPAP
14,638
SFV3L_P27401_2mut


 


GSSGSS
14,639
KORV_Q9TTC1_3mutA


 


GGGGSGGGGSGGGGGGGG
14,640
BLVJ_P03361_2mut


SGGGGS




 


GGGGSSGGS
14,641
GALV_P21414_3mutA


 


EAAAKGGSGSS
14,642
FFV_O93209-Pro


 


EAAAKPAP
14,643
PERV_Q4VFZ2


 


GSSGGGEAAAK
14,644
MLVBM_Q7SVK7_3mut


 


PAPGGSGGG
14,645
BAEVM_P10272


 


EAAAKGGGPAP
14,646
MLVF5_P26810


 


GSSGSSGSS
14,647
MLVBM_Q7SVK7_3mut


 


GSSGGS
14,648
AVIRE_P03360_3mutA


 


GGSEAAAKGGG
14,649
FOAMV_P14350_2mut


 


EAAAKGGS
14,650
MLVF5_P26810_3mutA


 


GGSGSSGGG
14,651
WMSV_P03359_3mut


 


EAAAK
14,652
SFV1_P23074_2mut


 


GSSGGSPAP
14,653
SFV3L_P27401-Pro_2mutA


 


GGGGSSGGS
14,654
KORV_Q9TTC1_3mut


 


PAPGGSGGG
14,655
FFV_O93209-Pro_2mut


 


GGGPAPGGS
14,656
SFV3L_P27401_2mutA


 


GSSPAPEAAAK
14,657
FLV_P10273_3mut


 


GGSGSSPAP
14,658
SFV3L_P27401_2mut


 


GSSEAAAKGGS
14,659
SFV3L_P27401_2mut


 


PAPGGG
14,660
SFV3L_P27401_2mutA


 


SGSETPGTSESATPES
14,661
KORV_Q9TTC1-Pro_3mut


 


GGGGS
14,662
SFV1_P23074-Pro_2mutA


 


GSSGGGEAAAK
14,663
WMSV_P03359


 


EAAAKGGGGSEAAAK
14,664
MLVF5_P26810_3mutA


 


GSSEAAAKPAP
14,665
FFV_O93209


 


GGGGGG
14,666
SFV1_P23074_2mutA


 


EAAAKEAAAKEAAAK
14,667
MMTVB_P03365-Pro


 


EAAAKPAPGSS
14,668
MLVBM_Q7SVK7_3mut


 


GGSGSSEAAAK
14,669
SFV3L_P27401_2mutA


 


GGSEAAAK
14,670
MLVMS_P03355_3mut


 


GGSPAPEAAAK
14,671
SFV3L_P27401_2mut


 


GGGPAPGSS
14,672
SFV1_P23074


 


GGGGGSEAAAK
14,673
MLVBM_Q7SVK7_3mutA_WS


 


EAAAKPAPGSS
14,674
KORV_Q9TTC1-Pro


 


GSSGSSGSSGSS
14,675
SFV3L_P27401_2mut


 


EAAAKPAP
14,676
SFV3L_P27401_2mut


 


GGGEAAAK
14,677
PERV_Q4VFZ2_3mut


 


GGSGGS
14,678
SFV3L_P27401_2mutA


 


EAAAKGSSGGS
14,679
MMTVB_P03365


 


SGSETPGTSESATPES
14,680
SFV3L_P27401


 


EAAAKGSSGGG
14,681
PERV_Q4VFZ2


 


EAAAKEAAAKEAAAKEAA
14,682
MMTVB_P03365


AKEAAAKEAAAK




 


GGSGGGPAP
14,683
KORV_Q9TTC1_3mutA


 


PAPAPAPAP
14,684
SFV3L_P27401


 


GGGEAAAKGGS
14,685
SFV1_P23074_2mut


 


GSSGGSGGG
14,686
PERV_Q4VFZ2_3mut


 


PAPEAAAKGGS
14,687
FOAMV_P14350_2mutA


 


GGGEAAAKGSS
14,688
SFV3L_P27401_2mut


 


GGGGSGGGGSGGGGSGGG
14,689
MLVBM_Q7SVK7


GS




 


PAPGSSGGG
14,690
FLV_P10273


 


GGSGSSGGG
14,691
FFV_O93209


 


EAAAKPAPGSS
14,692
MLVBM_Q7SVK7


 


GSSEAAAKGGG
14,693
SFV3L_P27401_2mutA


 


GGSGGSGGSGGSGGS
14,694
MLVF5_P26810


 


GGSEAAAKPAP
14,695
SFV3L_P27401-Pro_2mutA


 


EAAAKGGSPAP
14,696
SFV3L_P27401_2mutA


 


EAAAKGGGGGS
14,697
SFV3L_P27401_2mut


 


GSSPAPEAAAK
14,698
SFV3L_P27401_2mutA


 


PAPAP
14,699
MLVBM_Q7SVK7_3mut


 


PAPGGSEAAAK
14,700
KORV_Q9TTC1-Pro


 


GGSGSS
14,701
MLVF5_P26810_3mutA


 


GGSEAAAKPAP
14,702
FFV_O93209_2mut


 


GSS

MLVMS_P03355


 


SGGSSGGSSGSETPGTSE
14,704
SFV3L_P27401-Pro


SATPESSGGSSGGSS




 


PAPGGGEAAAK
14,705
SFV3L_P27401_2mut


 


PAPGGGGGS
14,706
SFV3L_P27401-Pro_2mut


 


PAPGGSGSS
14,707
BAEVM_P10272_3mut


 


GSSGGGEAAAK
14,708
FFV_O93209


 


GGSEAAAKPAP
14,709
SFV1_P23074_2mut


 


GGGGG
14,710
FLV_P10273_3mut


 


GGGEAAAKGSS
14,711
SFV3L_P27401


 


GSSGSSGSSGSSGSS
14,712
SFV1_P23074-Pro


 


SGSETPGTSESATPES
14,713
AVIRE_P03360


 


PAPGSSGGG
14,714
MLVBM_Q7SVK7_3mut


 


GGGGSSPAP
14,715
HTL3P_Q4U0X6_2mut


 


GGGEAAAK
14,716
SFV1_P23074


 


GGSGGG
14,717
AVIRE_P03360


 


EAAAKGSSGGG
14,718
SFV3L_P27401_2mutA


 


GSSPAPEAAAK
14,719
FOAMV_P14350-Pro_2mutA


 


GGGPAPGSS
14,720
WMSV_P03359


 


EAAAKGSSGGG
14,721
MLVMS_P03355


 


GGGGGSEAAAK
14,722
MLVMS_P03355


 


EAAAKPAPGGS
14,723
SFV3L_P27401


 


EAAAKGSSPAP
14,724
SFV3L_P27401


 


GGGGGGG
14,725
FOAMV_P14350_2mutA


 


EAAAKEAAAKEAAAK
14,726
SFV3L_P27401


 


GSSPAPGGS
14,727
FFV_O93209_2mutA


 


GGGGSSEAAAK
14,728
SFV3L_P27401-Pro_2mutA


 


GGSEAAAKGSS
14,729
GALV_P21414_3mutA


 


GGSEAAAKGSS
14,730
BAEVM_P10272_3mutA


 


EAAAKPAPGGG
14,731
MLVCB_P08361


 


GSSGSSGSSGSSGSSGSS
14,732
SFV1_P23074-Pro


 


GGGGSEAAAKGGGGS
14,733
FOAMV_P14350_2mut


 


GSSPAPGGS
14,734
MLVMS_P03355_PLV919


 


GGGGGGGGS
14,735
FFV_O93209-Pro


 


GSSGGSPAP
14,736
KORV_Q9TTC1_3mutA


 


GGSGGS
14,737
GALV_P21414_3mutA


 


PAPGSSEAAAK
14,738
WMSV_P03359


 


PAPGGGGSS
14,739
MMTVB_P03365-Pro


 


GGGGSSGGS
14,740
PERV_Q4VFZ2_3mutA_WS


 


GGGGSGGGGS
14,741
FFV_O93209_2mut


 


GGGGGGGGSGGGGSGGGG
14,742
XMRV6_A1Z651


S




 


GGSGSSEAAAK
14,743
SFV1_P23074_2mut


 


GGSGGGGSS
14,744
GALV_P21414_3mutA


 


GGSEAAAKPAP
14,745
MLVBM_Q7SVK7


 


EAAAKGGSPAP
14,746
SFV1_P23074_2mutA


 


PAPAPAPAP
14,747
FFV_O93209


 


GSSGGSPAP
14,748
MMTVB_P03365-Pro


 


GGGGGSPAP
14,749
KORV_Q9TTC1_3mutA


 


EAAAKGGGPAP
14,750
PERV_Q4VFZ2


 


GSSGGSPAP
14,751
BAEVM_P10272


 


GGGGG
14,752
FFV_O93209


 


GGGGGS
14,753
FLV_P10273_3mutA


 


EAAAKEAAAKEAAAK
14,754
FOAMV_P14350


 


PAPGGG
14,755
MLVCB_P08361_3mut


 


GSSGGSEAAAK
14,756
FOAMV_P14350_2mutA


 


GGSPAPGGG
14,757
FLV_P10273_3mut


 


GSSGSSGSSGSSGSSGSS
14,758
SFV1_P23074-Pro_2mutA


 


GGSPAPEAAAK
14,759
SFV3L_P27401


 


PAPGGGGSS
14,760
HTL3P_Q4U0X6_2mutB


 


GGGGSSEAAAK
14,761
MMTVB_P03365_2mut_WS


 


PAPGGS
14,762
MLVRD_P11227_3mut


 


GGSGGSGGSGGSGGS
14,763
MMTVB_P03365


 


GSAGSAAGSGEF
14,764
AVIRE_P03360


 


GSSGGS
14,765
BAEVM_P10272_3mutA


 


GGSGGGGSS
14,766
MMTVB_P03365


 


GGSGGGGSS
14,767
WMSV_P03359


 


PAPEAAAKGSS
14,768
SFV1_P23074


 


GSSGSSGSSGSS
14,769
SFV1_P23074-Pro_2mutA


 


PAPAPAPAPAPAP
14,770
SFV3L_P27401


 


PAPGSSGGG
14,771
FLV_P10273_3mut


 


GGSGSSPAP
14,772
MLVMS_P03355


 


GGSGGGPAP
14,773
FOAMV_P14350


 


PAPGGGGGS
14,774
KORV_Q9TTC1_3mutA


 


EAAAKGSSPAP
14,775
GALV_P21414_3mutA


 


GGSGSSPAP
14,776
MLVBM_Q7SVK7_3mut


 


EAAAKGSS
14,777
SFV3L_P27401_2mut


 


GGGGGSEAAAK
14,778
WMSV_P03359


 


GGGGGGGG
14,779
SFV1_P23074-Pro


 


EAAAKEAAAK
14,780
MLVBM_Q7SVK7


 


GGGEAAAKGGS
14,781
MLVBM_Q7SVK7


 


EAAAKGGSPAP
14,782
SFV3L_P27401_2mut


 


GSSEAAAK
14,783
XMRV6_A1Z651


 


PAPGGGEAAAK
14,784
MMTVB_P03365_WS


 


GGSPAP
14,785
GALV_P21414_3mutA


 


GSSPAPGGG
14,786
MLVBM_Q7SVK7_3mutA_WS


 


GGSGSSPAP
14,787
SFV1_P23074_2mutA


 


GGS

HTL32_Q0R5R2_2mut


 


GGSGGGGSS
14,789
MMTVB_P03365-Pro


 


GGGGSGGGGSGGGGSGGG
14,790
SFVCP_Q87040_2mutA


GS




 


EAAAKGGGPAP
14,791
FOAMV_P14350_2mut


 


GSSGGGEAAAK
14,792
MMTVB_P03365


 


SGGSSGGSSGSETPGTSE
14,793
MLVBM_Q7SVK7_3mutA_WS


SATPESSGGSSGGSS




 


AEAAAKEAAAKEAAAKEA
14,794
MMTVB_P03365_WS


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


EAAAKEAAAK
14,795
FOAMV_P14350-Pro_2mut


 


GSSPAPEAAAK
14,796
FOAMV_P14350_2mutA


 


EAAAKPAPGGS
14,797
GALV_P21414_3mutA


 


GSSGGSPAP
14,798
KORV_Q9TTC1-Pro_3mut


 


GGGPAPEAAAK
14,799
MLVAV_P03356


 


GGGEAAAKPAP
14,800
SFV1_P23074-Pro_2mut


 


GGGGGSEAAAK
14,801
SFV3L_P27401_2mut


 


GGGPAPGSS
14,802
SFV3L_P27401_2mut


 


GGSEAAAKPAP
14,803
AVIRE_P03360


 


GSSGSSGSSGSSGSSGSS
14,804
SFV1_P23074-Pro_2mut


 


EAAAKGSSGGS
14,805
FOAMV_P14350_2mutA


 


GGGGGG
14,806
MLVBM_Q7SVK7_3mut


 


GSSPAPGGS
14,807
PERV_Q4VFZ2


 


GGSGSSPAP
14,808
GALV_P21414_3mutA


 


GGGPAPEAAAK
14,809
SFV3L_P27401


 


GGSGGGEAAAK
14,810
WMSV_P03359


 


GSAGSAAGSGEF
14,811
SFV1_P23074_2mut


 


GSSGGGEAAAK
14,812
MLVMS_P03355


 


GGG

MMTVB_P03365-Pro


 


PAPGSSGGS
14,814
FOAMV_P14350_2mut


 


GGGGSSPAP
14,815
FFV_O93209_2mut


 


SGGSSGGSSGSETPGTSE
14,816
MMTVB_P03365_WS


SATPESSGGSSGGSS




 


GGGGGGG
14,817
XMRV6_A1Z651


 


PAPAPAPAPAP
14,818
FOAMV_P14350


 


GGGGSGGGGSGGGGSGGG
14,819
MMTVB_P03365_2mut_WS


GS




 


GGSGGGPAP
14,820
SFV3L_P27401_2mut


 


GGGGGG
14,821
SFV1_P23074-Pro


 


EAAAKPAPGSS
14,822
SFV3L_P27401_2mut


 


GGGGSSGGS
14,823
HTL3P_Q4U0X6_2mut


 


PAPGSSEAAAK
14,824
MMTVB_P03365-Pro


 


GGGGSSPAP
14,825
FOAMV_P14350-Pro_2mut


 


PAPGSSGGS
14,826
MMTVB_P03365


 


AEAAAKEAAAKEAAAKEA
14,827
SRV2_P51517


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


PAPAPAP
14,828
MMTVB_P03365_2mut_WS


 


PAPGGGGGS
14,829
MMTVB_P03365_2mutB


 


GGGGSS
14,830
SFV1_P23074-Pro_2mutA


 


EAAAKEAAAKEAAAKEAA
14,831
SFV3L_P27401-Pro


AK




 


GGSGGSGGSGGSGGS
14,832
MMTVB_P03365-Pro


 


GGGGGGG
14,833
SFV3L_P27401_2mut


 


PAPGGGEAAAK
14,834
SFV3L_P27401


 


PAPGSS
14,835
FOAMV_P14350_2mutA


 


GGGGSGGGGS
14,836
SFVCP_Q87040_2mutA


 


GSSGGSGGG
14,837
XMRV6_A1Z651


 


GGGGSGGGGSGGGGSGGG
14,838
MLVBM_Q7SVK7


GSGGGGSGGGGS




 


GSSEAAAKGGG
14,839
FFV_O93209-Pro_2mut


 


GGSEAAAKPAP
14,840
SFV3L_P27401-Pro


 


GSSGGSGGG
14,841
SFV1_P23074_2mut


 


EAAAKGGGGSS
14,842
FOAMV_P14350_2mutA


 


GGGGGG
14,843
SFV3L_P27401_2mut


 


GGGGG
14,844
MLVBM_Q7SVK7_3mut


 


PAPEAAAKGGG
14,845
SFV3L_P27401


 


EAAAKGGSPAP
14,846
KORV_Q9TTC1_3mutA


 


GGGEAAAKPAP
14,847
SFV1_P23074_2mut


 


GSSGSSGSSGSSGSSGSS
14,848
KORV_Q9TTC1-Pro


 


EAAAKEAAAKEAAAKEAA
14,849
SFVCP_Q87040


AK




 


PAPGSSEAAAK
14,850
MLVBM_Q7SVK7


 


GSSGSSGSS
14,851
FFV_O93209-Pro_2mut


 


GSSGGGPAP
14,852
SFV3L_P27401-Pro_2mut


 


GGGPAPEAAAK
14,853
WMSV_P03359_3mut


 


GGGEAAAK
14,854
MMTVB_P03365-Pro


 


GSSGSSGSSGSS
14,855
SFV3L_P27401-Pro_2mutA


 


PAPAPAPAPAP
14,856
FFV_O93209-Pro


 


GGSPAPEAAAK
14,857
FFV_O93209-Pro_2mut


 


GSSGSSGSSGSSGSSGSS
14,858
GALV_P21414


 


EAAAKEAAAKEAAAKEAA
14,859
FOAMV_P14350


AKEAAAK




 


GGGPAPEAAAK
14,860
MMTVB_P03365-Pro


 


PAPGGSGGG
14,861
MLVF5_P26810_3mutA


 


PAPGGSGGG
14,862
FLV_P10273_3mut


 


GGGEAAAKGGS
14,863
SFV3L_P27401


 


GSAGSAAGSGEF
14,864
MLVBM_Q7SVK7_3mut


 


GSSPAPGGG
14,865
MPMV_P07572_2mutB


 


GSSGSSGSSGSSGSSGSS
14,866
FOAMV_P14350


 


GGSGGGGSS
14,867
BLVJ_P03361_2mut


 


PAPEAAAKGSS
14,868
SFV1_P23074-Pro


 


GGG

FFV_O93209


 


EAAAKGGGGSS
14,870
SFV1_P23074_2mut


 


EAAAKEAAAKEAAAKEAA
14,871
SRV2_P51517


AKEAAAKEAAAK




 


GGGGSGGGGSGGGGSGGG
14,872
MMTVB_P03365


GSGGGGSGGGGS




 


GGGEAAAKGGS
14,873
MMTVB_P03365_WS


 


GSSGSS
14,874
SFV1_P23074


 


GSSGGGGGS
14,875
SFV3L_P27401


 


GGGGSSEAAAK
14,876
SFV1_P23074


 


EAAAKGSSGGS
14,877
HTL1A_P03362_2mutB


 


GSSEAAAKGGS
14,878
GALV_P21414_3mutA


 


EAAAKGSSPAP
14,879
SFV1_P23074


 


EAAAKPAPGSS
14,880
SFV3L_P27401_2mutA


 


PAPGSSGGG
14,881
SFV3L_P27401-Pro_2mut


 


GGGGSGGGGSGGGGSGGG
14,882
SFV3L_P27401-Pro


GSGGGGSGGGGS




 


EAAAKEAAAKEAAAKEAA
14,883
MMTVB_P03365_WS


AKEAAAK




 


GGGGSSEAAAK
14,884
MLVF5_P26810_3mutA


 


EAAAKGGSPAP
14,885
GALV_P21414


 


PAPEAAAKGSS
14,886
MMTVB_P03365_WS


 


GSSGGGGGS
14,887
SFVCP_Q87040_2mut


 


GGGGSSPAP
14,888
SFV1_P23074


 


EAAAKGGGGSS
14,889
XMRV6_A1Z651


 


PAPAPAPAP
14,890
MMTVB_P03365


 


GGSEAAAKGSS
14,891
SFV3L_P27401_2mutA


 


GSSPAPGGG
14,892
MMTVB_P03365_WS


 


GGGGGG
14,893
SFV3L_P27401-Pro


 


GGSGGSGGS
14,894
FOAMV_P14350-Pro_2mut


 


PAPAPAPAPAPAP
14,895
WMSV_P03359


 


GSSPAP
14,896
MLVBM_Q7SVK7


 


GGGGGSGSS
14,897
MMTVB_P03365_2mut_WS


 


EAAAKGSSGGS
14,898
MMTVB_P03365_2mutB_WS


 


EAAAK
14,899
FFV_O93209_2mutA


 


PAPEAAAK
14,900
SFV1_P23074-Pro


 


EAAAKGGSGSS
14,901
SFV3L_P27401


 


GGSGGSGGS
14,902
FFV_O93209-Pro


 


GSSGGGEAAAK
14,903
MMTVB_P03365


 


SGGSSGGSSGSETPGTSE
14,904
MLVFF_P26809_3mutA


SATPESSGGSSGGSS




 


GGSGGSGGSGGSGGSGGS
14,905
HTL1L_P0C211_2mutB


 


GGGEAAAK
14,906
SFV3L_P27401-Pro_2mutA


 


GGGGGSGSS
14,907
MMTVB_P03365


 


GSSPAPGGS
14,908
FOAMV_P14350_2mutA


 


EAAAKGSS
14,909
MLVMS_P03355


 


GSSGGSGGG
14,910
FFV_O93209-Pro


 


GGSGGGGSS
14,911
MMTVB_P03365-Pro_2mut


 


GGSPAPGSS
14,912
FOAMV_P14350_2mut


 


GGSGGSGGSGGSGGSGGS
14,913
SFVCP_Q87040-Pro_2mut


 


GSSEAAAKGGG
14,914
FOAMV_P14350_2mutA


 


GGSGGSGGS
14,915
MMTVB_P03365-Pro


 


GSSGSSGSSGSSGSSGSS
14,916
MMTVB_P03365_2mut_WS


 


GSSGSSGSSGSSGSS
14,917
MMTVB_P03365-Pro


 


PAPEAAAK
14,918
WDSV_O92815


 


GSSGSSGSSGSSGSS
14,919
FFV_O93209-Pro_2mut


 


EAAAKGGGGSEAAAK
14,920
MMTVB_P03365-Pro


 


GGSPAPEAAAK
14,921
FOAMV_P14350


 


GSSGSS
14,922
PERV_Q4VFZ2


 


GGG

MMTVB_P03365-Pro


 


GGGGSGGGGSGGGGS
14,924
FFV_O93209_2mut


 


EAAAKEAAAKEAAAKEAA
14,925
MMTVB_P03365-Pro


AKEAAAKEAAAK




 


GGSGSSPAP
14,926
WMSV_P03359


 


GGGGGGGG
14,927
SFV3L_P27401_2mut


 


PAPGSSEAAAK
14,928
FOAMV_P14350-Pro_2mutA


 


GGGGSSPAP
14,929
FOAMV_P14350_2mut


 


GSSGGSPAP
14,930
MLVBM_Q7SVK7_3mut


 


GSSGGGGGS
14,931
GALV_P21414_3mutA


 


EAAAKEAAAKEAAAKEAA
14,932
MMTVB_P03365


AKEAAAK




 


GSSGGGGGS
14,933
SFV1_P23074_2mut


 


GGGGSEAAAKGGGGS
14,934
SFV1_P23074


 


GGGEAAAKPAP
14,935
FFV_O93209


 


PAPGGGEAAAK
14,936
SFV1_P23074


 


GGSGGGEAAAK
14,937
PERV_Q4VFZ2_3mutA_WS


 


GSSGGG
14,938
MMTVB_P03365-Pro


 


EAAAKGSSGGS
14,939
FFV_O93209_2mut


 


GGGGG
14,940
SFV1_P23074_2mut


 


GGGPAP
14,941
SFV3L_P27401


 


GSSGGSEAAAK
14,942
FFV_O93209


 


SGGSSGGSSGSETPGTSE
14,943
MMTVB_P03365-Pro


SATPESSGGSSGGSS




 


GSSGGGEAAAK
14,944
SFV1_P23074_2mutA


 


GSSGSSGSSGSSGSS
14,945
SFV3L_P27401_2mut


 


GGSEAAAKPAP
14,946
FLV_P10273


 


GGGGSGGGGS
14,947
FOAMV_P14350-Pro_2mutA


 


GSSEAAAKPAP
14,948
SFV3L_P27401


 


GGGGSEAAAKGGGGS
14,949
MMTVB_P03365-Pro


 


PAPGSSEAAAK
14,950
MLVF5_P26810_3mut


 


EAAAKGGSGGG
14,951
SFV3L_P27401


 


GGGPAPGGS
14,952
SFV3L_P27401


 


GSSEAAAKGGS
14,953
FOAMV_P14350_2mutA


 


EAAAKGGSGGG
14,954
HTL1L_P0C211


 


GSSGGSPAP
14,955
SFV3L_P27401_2mutA


 


PAPAP
14,956
FFV_O93209


 


PAPGGSGSS
14,957
MMTVB_P03365_WS


 


EAAAKGGGGGS
14,958
FOAMV_P14350_2mut


 


PAPEAAAKGGS
14,959
SFV3L_P27401_2mut


 


GSSEAAAKPAP
14,960
MMTVB_P03365-Pro


 


GGSGGS
14,961
PERV_Q4VFZ2_3mut


 


GSSEAAAKGGG
14,962
FFV_O93209-Pro_2mutA


 


EAAAK
14,963
HTL1L_P0C211


 


GSSPAP
14,964
MLVMS_P03355


 


EAAAKPAPGGG
14,965
FFV_O93209-Pro_2mut


 


GGGGSEAAAKGGGGS
14,966
SFV1_P23074-Pro_2mut


 


EAAAKGSSGGS
14,967
SFV3L_P27401


 


GSAGSAAGSGEF
14,968
FFV_O93209_2mutA


 


PAPEAAAKGGS
14,969
MMTVB_P03365_2mutB_WS


 


EAAAKEAAAKEAAAKEAA
14,970
MMTVB_P03365


AKEAAAKEAAAK




 


GGS

MMTVB_P03365


 


GGSEAAAKPAP
14,972
SFV1_P23074


 


EAAAKGSSGGG
14,973
HTLV2_P03363_2mut


 


GGSEAAAKGGG
14,974
MMTVB_P03365_WS


 


GGSGGS
14,975
FFV_O93209-Pro


 


GSSEAAAKGGS
14,976
MMTVB_P03365-Pro


 


PAPAPAPAPAP
14,977
SFV1_P23074_2mutA


 


GGSEAAAKGGG
14,978
MMTVB_P03365_2mutB_WS


 


PAPAPAPAP
14,979
MMTVB_P03365_WS


 


GGGGSGGGGSGGGGSGGG
14,980
HTL3P_Q4U0X6_2mut


GSGGGGS




 


PAPGGSEAAAK
14,981
SFV1_P23074-Pro_2mut


 


GGSGGGPAP
14,982
MMTVB_P03365


 


GSSGSSGSSGSSGSSGSS
14,983
MMTVB_P03365-Pro


 


GGSEAAAKPAP
14,984
SFV1_P23074-Pro


 


GGGEAAAKGSS
14,985
SFV3L_P27401_2mutA


 


GGGPAPGGS
14,986
AVIRE_P03360


 


PAPGGG
14,987
MLVRD_P11227


 


GGSEAAAKGSS
14,988
SFV3L_P27401_2mut


 


GGGEAAAKGSS
14,989
FOAMV_P14350_2mut


 


GGGEAAAKGSS
14,990
SFV1_P23074-Pro


 


EAAAKEAAAKEAAAKEAA
14,991
MLVAV_P03356


AK




 


EAAAKGGGPAP
14,992
JSRV_P31623_2mutB


 


EAAAKGGGGSS
14,993
FOAMV_P14350_2mut


 


EAAAKEAAAKEAAAKEAA
14,994
SRV2_P51517


AKEAAAK




 


GSSGGGGGS
14,995
FFV_O93209


 


PAPAPAP
14,996
FOAMV_P14350_2mutA


 


GGSGGSGGSGGS
14,997
FOAMV_P14350


 


GGGEAAAK
14,998
MMTVB_P03365_WS


 


GGGGGS
14,999
SFV1_P23074_2mutA


 


GGSGGS
15,000
WMSV_P03359_3mut


 


EAAAKGGS
15,001
MMTVB_P03365-Pro


 


GGGGSS
15,002
BLVJ_P03361_2mut


 


PAPAP
15,003
MMTVB_P03365-Pro_2mut


 


PAPGGG
15,004
SMRVH_P03364


 


EAAAKGGGGSS
15,005
SFV3L_P27401


 


PAPAPAPAPAP
15,006
MMTVB_P03365


 


GGGPAP
15,007
MMTVB_P03365-Pro


 


GSSGGSGGG
15,008
MMTVB_P03365


 


EAAAKGGGPAP
15,009
FOAMV_P14350_2mutA


 


GSSGSSGSSGSS
15,010
SFV1_P23074


 


GGGGSGGGGS
15,011
SFV3L_P27401


 


GSSGGSGGG
15,012
MLVF5_P26810


 


GGGEAAAKPAP
15,013
MMTVB_P03365-Pro


 


PAPEAAAK
15,014
HTLV2_P03363_2mut


 


GSSGSSGSSGSS
15,015
FOAMV_P14350_2mut


 


GSSEAAAKPAP
15,016
MMTVB_P03365-Pro


 


PAPEAAAKGGG
15,017
HTL3P_Q4U0X6_2mut


 


GGSEAAAKGSS
15,018
MMTVB_P03365-Pro


 


EAAAKPAPGGS
15,019
MMTVB_P03365_2mut_WS


 


GSSGGSEAAAK
15,020
MLVF5_P26810_3mutA


 


GGGGSGGGGSGGGGSGGG
15,021
MLVF5_P26810_3mut


GSGGGGSGGGGS




 


EAAAKGGGGSS
15,022
MMTVB_P03365-Pro


 


GGGGGSGSS
15,023
HTL1A_P03362_2mutB


 


PAPAP
15,024
FFV_O93209-Pro_2mut


 


GGGGGSPAP
15,025
HTL1C_P14078_2mut


 


GGGPAP
15,026
HTLV2_P03363_2mut


 


EAAAKGGGGSEAAAK
15,027
SFVCP_Q87040


 


GGSEAAAKGGG
15,028
FFV_O93209-Pro_2mutA


 


GSSPAPGGS
15,029
FOAMV_P14350-Pro_2mut


 


GGGGGGG
15,030
MMTVB_P03365-Pro


 


EAAAKGSS
15,031
SFV3L_P27401_2mutA


 


EAAAKGGGGSEAAAK
15,032
MMTVB_P03365-Pro


 


GGGGSEAAAKGGGGS
15,033
SFV1_P23074-Pro_2mutA


 


EAAAKGGGGSS
15,034
MMTVB_P03365


 


GGGEAAAKGGS
15,035
SFV1_P23074


 


PAPEAAAKGGG
15,036
MLVF5_P26810


 


GGGGSSGGS
15,037
MMTVB_P03365


 


GGSGSS
15,038
MMTVB_P03365


 


PAPAPAPAPAPAP
15,039
KORV_Q9TTC1


 


EAAAKGGG
15,040
SFV1_P23074-Pro_2mut


 


PAPAPAPAPAPAP
15,041
SRV2_P51517


 


GSSGSSGSSGSSGSS
15,042
FFV_O93209-Pro_2mutA


 


GGGGSS
15,043
FOAMV_P14350_2mut


 


PAPGGGEAAAK
15,044
MMTVB_P03365_WS


 


GGSGGGEAAAK
15,045
FFV_O93209-Pro_2mut


 


PAPAPAPAPAP
15,046
MMTVB_P03365_WS


 


GGGEAAAKGGS
15,047
MMTVB_P03365-Pro


 


GGGEAAAKGSS
15,048
MMTVB_P03365_2mutB


 


GSSPAPEAAAK
15,049
MMTVB_P03365_WS


 


EAAAKEAAAKEAAAKEAA
15,050
SFV1_P23074-Pro_2mutA


AKEAAAK




 


PAPGGG
15,051
SFV3L_P27401


 


GSSEAAAKGGG
15,052
MMTVB_P03365_WS


 


GGGGSSEAAAK
15,053
FOAMV_P14350_2mut


 


PAPGSSGGS
15,054
SFV1_P23074-Pro_2mut


 


GSSGSSGSSGSSGSSGSS
15,055
SFV3L_P27401


 


EAAAKGSSGGG
15,056
MMTVB_P03365


 


PAPGGGGSS
15,057
WDSV_O92815_2mutA


 


GGSPAP
15,058
MMTVB_P03365-Pro


 


GGSGGSGGSGGSGGS
15,059
SFVCP_Q87040-Pro_2mut


 


PAPAPAPAP
15,060
MMTVB_P03365-Pro


 


GGGGG
15,061
HTL1A_P03362


 


GGSGGSGGSGGS
15,062
SFV1_P23074_2mutA


 


GSSGSSGSSGSSGSS
15,063
FOAMV_P14350-Pro_2mut


 


PAPGGSEAAAK
15,064
MMTVB_P03365_2mutB_WS


 


PAPAPAPAP
15,065
SFV1_P23074_2mut


 


PAPGGGGSS
15,066
MMTVB_P03365


 


GGSGSS
15,067
SFV3L_P27401_2mut


 


EAAAKEAAAKEAAAKEAA
15,068
MMTVB_P03365_2mut


AK




 


EAAAKGGSGGG
15,069
HTL3P_Q4U0X6_2mut


 


PAPGGGGSS
15,070
SFVCP_Q87040-Pro_2mutA


 


EAAAKGGGGGS
15,071
MLVAV_P03356


 


GGGGGS
15,072
FOAMV_P14350_2mut


 


GGGEAAAKGGS
15,073
FFV_O93209-Pro_2mutA


 


EAAAKPAPGGG
15,074
MMTVB_P03365_2mutB


 


GGSGGGPAP
15,075
FFV_O93209_2mut


 


GSSEAAAKPAP
15,076
MMTVB_P03365


 


PAPAPAPAPAPAP
15,077
SFV1_P23074_2mut


 


GGSPAPGGG
15,078
MMTVB_P03365-Pro


 


GGSGGGEAAAK
15,079
MMTVB_P03365


 


PAPAP
15,080
SFVCP_Q87040


 


GSSEAAAK
15,081
SFVCP_Q87040


 


GGGGSGGGGSGGGGS
15,082
MMTVB_P03365-Pro


 


GSSGSSGSS
15,083
SFV3L_P27401


 


EAAAKGGSGGG
15,084
MMTVB_P03365-Pro


 


GSSPAP
15,085
SFV1_P23074_2mut


 


GGGEAAAK
15,086
SFV1_P23074-Pro


 


AEAAAKEAAAKEAAAKEA
15,087
MMTVB_P03365-Pro


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


PAPGGS
15,088
HTL1C_P14078_2mut


 


PAPGSSGGS
15,089
SFV1_P23074_2mut


 


PAPEAAAK
15,090
MMTVB_P03365_WS


 


PAPAP
15,091
MMTVB_P03365-Pro


 


EAAAKGGS
15,092
HTL1A_P03362_2mut


 


GGGGSEAAAKGGGGS
15,093
HTL1C_P14078


 


EAAAKGSSGGS
15,094
FOAMV_P14350-Pro


 


PAPGGSGSS
15,095
MMTVB_P03365-Pro


 


PAPGGSEAAAK
15,096
SFV1_P23074_2mut


 


PAPGSSEAAAK
15,097
FFV_O93209-Pro_2mut


 


PAPGSSGGG
15,098
FOAMV_P14350-Pro_2mutA


 


GSSGGGEAAAK
15,099
AVIRE_P03360


 


GGGGGG
15,100
SMRVH_P03364_2mut


 


PAPEAAAKGGG
15,101
MMTVB_P03365-Pro


 


GGGEAAAKGGS
15,102
SFVCP_Q87040_2mutA


 


PAPAPAPAPAP
15,103
SRV2_P51517


 


GSSGSSGSSGSSGSSGSS
15,104
MMTVB_P03365


 


EAAAKGGGPAP
15,105
MLVAV_P03356


 


PAPAPAPAPAP
15,106
FOAMV_P14350-Pro_2mutA


 


PAPGGSEAAAK
15,107
FOAMV_P14350


 


GSSGGGPAP
15,108
HTL32_QOR5R2_2mutB


 


GGGGGSPAP
15,109
HTL3P_Q4U0X6_2mutB


 


GSSGGSGGG
15,110
MMTVB_P03365-Pro


 


PAPAP
15,111
SFVCP_Q87040-Pro


 


GSSGGGPAP
15,112
MMTVB_P03365-Pro


 


GGSGSS
15,113
MMTVB_P03365-Pro_2mut


 


GGSPAPEAAAK
15,114
SFV1_P23074-Pro_2mut


 


EAAAKGGSGGG
15,115
SFV3L_P27401_2mut


 


GGGGSSEAAAK
15,116
MMTVB_P03365_WS


 


GGGGGSGSS
15,117
MMTVB_P03365_2mut


 


GGGGSSGGS
15,118
SFV1_P23074-Pro_2mutA


 


EAAAKGGGGSEAAAK
15,119
MMTVB_P03365_WS


 


PAPGGGEAAAK
15,120
SFV1_P23074-Pro


 


PAPEAAAKGGG
15,121
MMTVB_P03365


 


AEAAAKEAAAKEAAAKEA
15,122
MMTVB_P03365


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GSSGGSEAAAK
15,123
FOAMV_P14350-Pro_2mut


 


GGSPAP
15,124
MLVBM_Q7SVK7_3mut


 


GSSEAAAK
15,125
FOAMV_P14350


 


GSSEAAAK
15,126
MMTVB_P03365-Pro


 


EAAAKGSSGGS
15,127
HTL1A_P03362_2mut


 


GGGEAAAKPAP
15,128
FOAMV_P14350-Pro_2mut


 


EAAAKGGSPAP
15,129
FOAMV_P14350


 


GSSEAAAKPAP
15,130
MMTVB_P03365_WS


 


GSSGSSGSS
15,131
FOAMV_P14350_2mut


 


EAAAKEAAAKEAAAKEAA
15,132
MMTVB_P03365_WS


AK




 


EAAAK
15,133
MMTVB_P03365


 


PAPGSS
15,134
BAEVM_P10272


 


PAPGGS
15,135
FFV_O93209-Pro_2mut


 


GGSGGS
15,136
SFV1_P23074-Pro_2mutA


 


SGGSSGGSSGSETPGTSE
15,137
HTLV2_P03363_2mut


SATPESSGGSSGGSS




 


GGSGGGEAAAK
15,138
MMTVB_P03365_WS


 


PAPGSSGGG
15,139
HTL1A_P03362


 


GGSGGS
15,140
SFV3L_P27401-Pro


 


GSSGSS
15,141
SFV1_P23074-Pro


 


PAPGGSEAAAK
15,142
MMTVB_P03365


 


GSAGSAAGSGEF
15,143
MMTVB_P03365-Pro


 


PAPGGG
15,144
FOAMV_P14350_2mut


 


EAAAKGGSGSS
15,145
MMTVB_P03365_WS


 


GSSGGGEAAAK
15,146
SFV3L_P27401-Pro


 


GGSGGGPAP
15,147
FOAMV_P14350-Pro_2mut


 


PAPAPAPAPAPAP
15,148
WDSV_O92815


 


SGSETPGTSESATPES
15,149
SFVCP_Q87040-Pro_2mutA


 


GGSGGSGGS
15,150
SFV1_P23074


 


GGGGSS
15,151
SFVCP_Q87040_2mut


 


GGGGGSEAAAK
15,152
MMTVB_P03365


 


SGSETPGTSESATPES
15,153
MMTVB_P03365_WS


 


PAPAPAP
15,154
SFV3L_P27401


 


PAPEAAAKGSS
15,155
MMTVB_P03365_2mutB_WS


 


GSSGSSGSSGSSGSS
15,156
SRV2_P51517


 


GGGPAPGSS
15,157
HTL32_Q0R5R2_2mutB


 


GGSGGGGSS
15,158
MMTVB_P03365-Pro


 


SGSETPGTSESATPES
15,159
SRV2_P51517


 


EAAAKGSSGGS
15,160
MMTVB_P03365-Pro


 


GSSPAPEAAAK
15,161
MMTVB_P03365-Pro


 


GSSPAPEAAAK
15,162
SRV2_P51517


 


GGGGSSPAP
15,163
MMTVB_P03365-Pro


 


PAPGGGEAAAK
15,164
SFV1_P23074-Pro_2mutA


 


PAPEAAAKGGS
15,165
MMTVB_P03365


 


GSSGSSGSSGSSGSSGSS
15,166
FOAMV_P14350-Pro


 


GGSPAPGSS
15,167
SFV3L_P27401


 


GGGPAPGGS
15,168
SFV1_P23074-Pro_2mutA


 


GGGPAPGSS
15,169
MMTVB_P03365-Pro


 


EAAAKPAP
15,170
MLVBM_Q7SVK7


 


EAAAKEAAAKEAAAK
15,171
HTL1C_P14078


 


GSSGGSEAAAK
15,172
SRV2_P51517


 


PAPGGGGGS
15,173
SRV2_P51517


 


GGGEAAAK
15,174
FFV_O93209-Pro_2mut


 


EAAAKGGGPAP
15,175
HTL32_Q0R5R2


 


GGSGSSGGG
15,176
MMTVB_P03365


 


PAPEAAAKGSS
15,177
MMTVB_P03365-Pro


 


PAPGGGGGS
15,178
MMTVB_P03365-Pro


 


EAAAKGGGGGS
15,179
MMTVB_P03365_WS


 


GGGGGS
15,180
MMTVB_P03365-Pro


 


GGGGSGGGGSGGGGSGGG
15,181
HTL1C_P14078


GSGGGGS




 


EAAAKGGSPAP
15,182
MMTVB_P03365


 


GGGGSSPAP
15,183
FFV_O93209-Pro_2mut


 


GGGGSSGGS
15,184
MMTVB_P03365-Pro


 


PAPGSSGGS
15,185
MMTVB_P03365-Pro


 


GGGGGS
15,186
SRV2_P51517


 


GGSGSSGGG
15,187
MMTVB_P03365


 


GSSGGSEAAAK
15,188
MMTVB_P03365-Pro


 


EAAAKEAAAKEAAAKEAA
15,189
GALV_P21414


AK




 


GGSEAAAKGGG
15,190
MMTVB_P03365-Pro


 


SGGSSGGSSGSETPGTSE
15,191
MMTVB_P03365-Pro


SATPESSGGSSGGSS




 


GSSEAAAKGGS
15,192
MMTVB_P03365


 


GGGGSGGGGSGGGGSGGG
15,193
HTL3P_Q4U0X6_2mutB


GSGGGGSGGGGS




 


GGGEAAAK
15,194
MMTVB_P03365-Pro


 


PAPAPAPAP
15,195
MMTVB_P03365-Pro


 


PAPGSSGGG
15,196
MMTVB_P03365


 


GSSGSSGSSGSSGSS
15,197
GALV_P21414


 


GGSPAP
15,198
MMTVB_P03365_WS


 


GGGGSGGGGSGGGGSGGG
15,199
MMTVB_P03365-Pro


GSGGGGSGGGGS




 


PAPEAAAK
15,200
MMTVB_P03365-Pro


 


PAPGSSGGG
15,201
SFV1_P23074-Pro_2mutA


 


GGGGGSEAAAK
15,202
MMTVB_P03365_2mutB_WS


 


PAPAPAPAPAP
15,203
MMTVB_P03365-Pro


 


EAAAKGGSGSS
15,204
MMTVB_P03365-Pro


 


EAAAKEAAAKEAAAKEAA
15,205
MLVRD_P11227_3mut


AK




 


PAPAPAPAP
15,206
FOAMV_P14350_2mutA


 


GGGPAPGSS
15,207
SFVCP_Q87040_2mut


 


PAPEAAAKGSS
15,208
SFVCP_Q87040_2mut


 


GGSPAPGGG
15,209
MMTVB_P03365-Pro


 


GGGGSGGGGSGGGGSGGG
15,210
MMTVB_P03365


GS




 


EAAAKGGS
15,211
HTL3P_Q4U0X6_2mut


 


PAPGSSGGS
15,212
MMTVB_P03365_WS


 


GGGGSGGGGS
15,213
MMTVB_P03365


 


GGSGGS
15,214
FOAMV_P14350


 


EAAAKGGGGSEAAAK
15,215
SFVCP_Q87040-Pro_2mut


 


EAAAKEAAAKEAAAKEAA
15,216
MMTVB_P03365-Pro_2mutB


AK




 


PAPGGGEAAAK
15,217
SFVCP_Q87040-Pro


 


GSSGSS
15,218
JSRV_P31623_2mutB


 


EAAAKGGGGGS
15,219
MMTVB_P03365_2mut_WS


 


GSSPAPEAAAK
15,220
MMTVB_P03365-Pro


 


GGGEAAAK
15,221
HTL1C_P14078


 


PAPEAAAKGSS
15,222
HTL32_Q0R5R2_2mutB


 


GGGGSSEAAAK
15,223
MMTVB_P03365-Pro


 


PAPGSSGGS
15,224
MMTVB_P03365-Pro


 


EAAAKGGGGGS
15,225
MMTVB_P03365


 


GGGGSGGGGSGGGGSGGG
15,226
MMTVB_P03365


GS




 


EAAAKGGGGSS
15,227
HTL3P_Q4U0X6_2mut


 


GGGEAAAKGGS
15,228
SFVCP_Q87040-Pro


 


GGGGGSPAP
15,229
MMTVB_P03365-Pro_2mutB


 


GGSGGGEAAAK
15,230
SFV3L_P27401-Pro


 


PAPGGGGGS
15,231
SFV3L_P27401-Pro


 


EAAAKGGGGSEAAAK
15,232
MMTVB_P03365


 


PAPEAAAKGSS
15,233
MMTVB_P03365-Pro


 


GGSEAAAKGGG
15,234
MMTVB_P03365-Pro


 


GGSGGSGGSGGSGGS
15,235
SMRVH_P03364_2mutB


 


GGSGGSGGSGGSGGS
15,236
HTL1L_P0C211_2mut


 


GGGGGG
15,237
WDSV_O92815


 


GGGGGSGSS
15,238
MMTVB_P03365-Pro


 


GGSEAAAKPAP
15,239
SFV3L_P27401-Pro_2mut


 


GGGPAPGSS
15,240
MMTVB_P03365_2mut_WS


 


GGGGGS
15,241
MMTVB_P03365_WS


 


GGSPAPEAAAK
15,242
MMTVB_P03365


 


PAPEAAAKGGS
15,243
HTL1A_P03362


 


EAAAKGGSGSS
15,244
MMTVB_P03365_2mut_WS


 


GGGPAPEAAAK
15,245
SFV3L_P27401-Pro_2mut


 


PAPGGGGSS
15,246
HTL32_QOR5R2_2mut


 


GSSPAPGGG
15,247
HTL3P_Q4U0X6_2mut


 


GGGGSSGGS
15,248
BLVAU_P25059_2mut


 


EAAAKGGGGGS
15,249
HTL1L_P0C211


 


GGSEAAAKGSS
15,250
JSRV_P31623_2mutB


 


GSSGGG
15,251
JSRV_P31623


 


GGSGGSGGSGGS
15,252
MMTVB_P03365-Pro


 


EAAAKPAP
15,253
SFV1_P23074-Pro_2mutA


 


GGGGSSGGS
15,254
MMTVB_P03365_WS


 


GGSGGS
15,255
MMTVB_P03365_WS


 


EAAAKGGGGGS
15,256
MMTVB_P03365-Pro


 


GGGGSGGGGSGGGGSGGG
15,257
MMTVB_P03365


GSGGGGSGGGGS




 


GGSGGSGGS
15,258
MMTVB_P03365


 


GGGGGSEAAAK
15,259
MLVBM_Q7SVK7


 


GGSGSSPAP
15,260
MMTVB_P03365_WS


 


EAAAKEAAAKEAAAK
15,261
JSRV_P31623


 


PAPEAAAKGGS
15,262
MMTVB_P03365-Pro


 


GGSGSSEAAAK
15,263
FOAMV_P14350


 


GGGGGSGSS
15,264
MMTVB_P03365-Pro_2mut


 


GGGPAPGGS
15,265
MMTVB_P03365


 


SGSETPGTSESATPES
15,266
SFVCP_Q87040_2mut


 


GSSPAPGGS
15,267
SFV1_P23074-Pro_2mutA


 


GSSGSSGSSGSSGSS
15,268
MMTVB_P03365


 


EAAAKGGGPAP
15,269
MMTVB_P03365


 


GSSGGG
15,270
MMTVB_P03365_2mut_WS


 


GGGEAAAKPAP
15,271
MMTVB_P03365


 


PAPGGSGGG
15,272
MMTVB_P03365-Pro


 


GSSGGSGGG
15,273
WDSV_O92815_2mut


 


GGSGGG
15,274
HTL32_Q0R5R2_2mut


 


EAAAKGGSPAP
15,275
HTLV2_P03363_2mut


 


GGSPAPEAAAK
15,276
MMTVB_P03365-Pro


 


GSSGGSEAAAK
15,277
MMTVB_P03365_2mut


 


GSAGSAAGSGEF
15,278
MMTVB_P03365_WS


 


PAPGGSGSS
15,279
FFV_O93209


 


GGSEAAAKGGG
15,280
MMTVB_P03365


 


GGSPAPGSS
15,281
MMTVB_P03365-Pro


 


GSSGGSGGG
15,282
SFV3L_P27401


 


PAPEAAAKGGG
15,283
HTL1A_P03362_2mutB


 


GGGEAAAKPAP
15,284
MMTVB_P03365-Pro


 


GGSEAAAK
15,285
HTL32_Q0R5R2_2mutB


 


GGGEAAAKGSS
15,286
MPMV_P07572


 


GGGGGSEAAAK
15,287
MMTVB_P03365-Pro


 


PAPAPAPAPAP
15,288
SFVCP_Q87040-Pro_2mutA


 


PAPAPAPAPAP
15,289
HTL1L_P0C211_2mut


 


GGGGSSGGS
15,290
HTL3P_Q4U0X6


 


PAPGGSEAAAK
15,291
MMTVB_P03365_2mut_WS


 


PAPAPAPAPAP
15,292
HTL1A_P03362


 


EAAAKPAPGGG
15,293
MMTVB_P03365_2mut_WS


 


GGSEAAAK
15,294
MMTVB_P03365_2mut_WS


 


GGGEAAAKGSS
15,295
SFV1_P23074-Pro_2mutA


 


GGSPAPGSS
15,296
MMTVB_P03365-Pro


 


GGSEAAAKPAP
15,297
MLVBM_Q7SVK7


 


PAPEAAAKGGG
15,298
MMTVB_P03365_2mut_WS


 


GSSEAAAKPAP
15,299
MMTVB_P03365-Pro_2mutB


 


GGGGSEAAAKGGGGS
15,300
MMTVB_P03365-Pro_2mut


 


GSSEAAAKGGS
15,301
MMTVB_P03365-Pro_2mutB


 


GSSGSSGSSGSSGSS
15,302
SRV2_P51517_2mutB


 


GGGGGSPAP
15,303
HTL1L_P0C211_2mut


 


GGSEAAAK
15,304
MMTVB_P03365


 


GSSPAPEAAAK
15,305
SMRVH_P03364_2mutB


 


GGGPAPGGS
15,306
HTL1C_P14078_2mut


 


GGSPAPEAAAK
15,307
MMTVB_P03365_WS


 


GGSEAAAKPAP
15,308
HTL1A_P03362_2mut


 


PAPAPAPAP
15,309
HTLV2_P03363_2mut


 


GSSPAPGGG
15,310
MMTVB_P03365


 


GSSGSSGSSGSS
15,311
MMTVB_P03365-Pro


 


GGSEAAAKGSS
15,312
MMTVB_P03365_WS


 


GGSGSSGGG
15,313
MMTVB_P03365_2mutB


 


GSSGSSGSSGSSGSSGSS
15,314
JSRV_P31623_2mutB


 


GGSEAAAKPAP
15,315
MMTVB_P03365-Pro


 


GSSGGSGGG
15,316
HTLV2_P03363_2mut


 


AEAAAKEAAAKEAAAKEA
15,317
WDSV_O92815_2mut


AAKALEAEAAAKEAAAKE




AAAKEAAAKA




 


GGSPAPEAAAK
15,318
MMTVB_P03365


 


GGGGSSEAAAK
15,319
MMTVB_P03365


 


GGSGGGEAAAK
15,320
SFV1_P23074-Pro_2mutA


 


GGGGSEAAAKGGGGS
15,321
WDSV_O92815_2mut


 


GGSGSSEAAAK
15,322
MMTVB_P03365_2mutB_WS


 


GGSEAAAKPAP
15,323
MMTVB_P03365_WS


 


GSSGGGEAAAK
15,324
SFVCP_Q87040-Pro


 


GSSGGS
15,325
SFVCP_Q87040-Pro_2mut


 


GGSEAAAKPAP
15,326
SFVCP_Q87040_2mut


 


GSSGGSEAAAK
15,327
SFVCP_Q87040_2mut


 


GSSPAPEAAAK
15,328
SRV2_P51517_2mutB


 


GGSGGSGGSGGSGGSGGS
15,329
BLVAU_P25059


 


GSSGSSGSSGSSGSS
15,330
HTL1C_P14078_2mut


 


EAAAKGGGGSS
15,331
MMTVB_P03365_2mutB


 


GGGEAAAKGSS
15,332
SFVCP_Q87040-Pro









Example 3: Quantifying Activity of a Gene Editing Polypeptide and Template for Rewriting the Endogenous FAH Locus Achieved in Primary Mouse Hepatocytes
This example demonstrates the use of a gene modifying system containing a gene modifying polypeptide and a template RNA, to convert an A nucleotide to a G nucleotide in the endogenous Fah locus in mouse primary hepatocytes derived from a Fah5981SB mouse. The Fah5981SB mouse model harbors a G to A point mutation in the last nucleotide of exon 8 of the Fah gene, leading to aberrant mRNA splicing and subsequent mRNA degradation, without the production of Fah protein and, and thus serves as a mouse model of hereditary tyrosinemia type I.
In this example, the template RNA contained:

    
    
        (1) a gRNA spacer;
        (2) a gRNA scaffold;
        (3) a heterologous object sequence; and
        (4) a primer binding site (PBS) sequence.
    
    


More specifically, the template RNA (including chemical modification pattern) comprised the following sequences:









FAH1_R14_P12_Heavy



RNACS048-001



(SEQ ID NO: 30421)



mG*mG*mA*rUrGrGrUrCrCrUrCrArUrGrArArCrGr



 



ArCrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr



 



ArCrCrGrCrUrCrCrArGrUrCrGrUrUrCrArUrGrAr



 



G*mG*mA*mC



 



FAH1_R15_P10_Heavy



RNACS049-001



(SEQ ID NO: 30422)



mG*mG*mA*rUrGrGrUrCrCrUrCrArUrGrArArCrGr



 



ArCrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArUr



 



UrArCrCrGrCrUrCrCrArGrUrCrGrUrUrCrArUrG*



 



mA*mG*Mg



 



FAH2_R19_P11_MUT_Heavy



RNACS052-001



(SEQ ID NO: 30423)



mU*mC*mA*rGrArGrGrArArGrCrUrGrGrGrCrCrAr



 



CrCrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr



 



GrArGrCrGrGrUrArArUrGrGrCrUrGrGrUrGrGrCr



 



CrCrArGrC*mU*mU*mC



 



FAH2_R19_P13_MUT_Heavy



RNACS053-001



(SEQ ID NO: 30424)



mU*mC*mA*rGrArGrGrArArGrCrUrGrGrGrCrCrAr



 



CrCrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr



 



GrArGrCrGrGrUrArArUrGrGrCrUrGrGrUrGrGrCr



 



CrCrArGrCrUrU*mC*mC*mU






Additional exemplary template RNAs that could be utilized in this experiment include the following:









FAH1



RNACS050



(SEQ ID NO: 30425)



mG*mG*mA*rUrGrGrUrCrCrUrCrArUrGrArArCrGr



 



ArCrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArGr



 



GrCrArUrUrArCrCrGrCrUrCrCrArGrUrCrGrUrUr



 



CrArUrGrArG*mG*mA*m



C



 



FAH1



RNACS051



(SEQ ID NO: 30426)



mG*mG*mA*rUrGrGrUrCrCrUrCrArUrGrArArCrGr



 



ArCrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArGr



 



GrCrArUrUrArCrCrGrCrUrCrCrArGrUrCrGrUrUr



 



CrArUrG*mA*mG*mG






In the sequences above m=2′-O-methyl ribonucleotide, r=ribose and *=phosphorothioate bond.
The gene modifying polypeptides tested comprised sequence of: RNAV209 (nCas9-RT) and RNAV214 (wtCas9-RT). Specifically, the nCas9-RT and the wtCas9-RT had the following amino acid sequences:









nCas9-RT (RNAV209):



(SEQ ID NO: 30427)



MPAAKRVKLDGGDKKYSIGLDIGTNSVGWAVITDEYKVPS



 



KKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR



 



RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE



 



DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKA



 



DLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLV



 



QTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQL



 



PGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD



 



TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVN



 



TEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKE



 



IFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEEL



 



LVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF



 



YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKS



 



EETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP



 



KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV



 



DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNA



 



SLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDR



 



EMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING



 



IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQ



 



KAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK



 



VMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIK



 



ELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD



 



INRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDN



 



VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLS



 



ELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKL



 



IREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL



 



NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI



 



GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET



 



GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKES



 



ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV



 



EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE



 



VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP



 



SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI



 



IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI



 



IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH



 



QSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESAT



 



PESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSD



 



FPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQ



 



EARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTND



 



YRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTV



 



LDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRL



 



PQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLL



 



AATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKY



 



LGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAG



 



FCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIK



 



QALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW



 



RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM



 



GQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD



 



RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPD



 



LTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIW



 



AKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAF



 



ATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPK



 



RLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTS



 



TLLIENSSPSGGSKRTADGSEFEKRTADGSEFESPKKKAK



 



VE



 



wtCas9-RT (RNAV214-040):



(SEQ ID NO: 30428)



MPAAKRVKLDGGDKKYSIGLDIGTNSVGWAVITDEYKVPS



 



KKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR



 



RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE



 



DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKA



 



DLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLV



 



QTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQL



 



PGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD



 



TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVN



 



TEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKE



 



IFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEEL



 



LVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF



 



YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKS



 



EETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP



 



KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV



 



DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNA



 



SLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDR



 



EMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING



 



IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQ



 



KAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK



 



VMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIK



 



ELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD



 



INRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDN



 



VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLS



 



ELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKL



 



IREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL



 



NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI



 



GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET



 



GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKES



 



ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV



 



EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE



 



VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP



 



SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI



 



IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI



 



IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH



 



QSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESAT



 



PESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSD



 



FPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQ



 



EARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTND



 



YRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTV



 



LDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRL



 



PQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLL



 



AATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKY



 



LGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAG



 



FCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIK



 



QALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW



 



RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM



 



GQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD



 



RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPD



 



LTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIW



 



AKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAF



 



ATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPK



 



RLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTS



 



TLLIENSSPSGGSKRTADGSEFEKRTADGSEFESPKKKAK



 



VE






Underlining indicates the residue that differs between the nickase and wild-type sequences.

The gene modifying system comprising the gene modifying polypeptides listed above and the template RNA described above were transfected into primary mouse hepatocytes. The gene modifying polypeptide and the template RNA were delivered by nucleofection in the RNA format. Specifically, 4 μg of gene modifying polypeptide mRNA were combined with 10 μg of chemically synthesized template RNA in 5 μL of water. The transfection mix was added to 100,000 mouse primary hepatocytes in Buffer P3 [Lonza], and cells were nucleofected using program DG-138. After nucleofection, cells were grown at 37° C., 5% CO2 for 3 days prior to cell lysis and genomic DNA extraction. To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. Conversion of terminal A to G sequence in exon 8 of fah gene indicates successful editing.
As shown in FIG. 2, for FAH2 templates, perfect rewrite levels (conversion of A to G with no unwanted mutations detected) of 4-8% were detected with RNAV209 but not with RNAV214. Indel levels of 4.4 to 6.6% were observed with RNAV209. Furthermore, the amount of WT Fah mRNA was measured using quantitative RT-PCR using primers that bind to exons 7 and 8. As shown in FIG. 3, FAH2 templates result in an increase in the abundance of Fah mRNA relative to WT by up to 12% when FAH2 template is tested with RNAV209 mRNA. These results demonstrate the use of a gene modifying system to reverse a mutation in the Fah gene, resulting in partial restoration of the expression of wild-type Fah mRNA.
Example 4: Quantifying Activity of a Gene Editing Polypeptide and Template In Vivo for Rewriting the Endogenous FAH Locus Achieved in Mouse Liver
This example demonstrates the use of a gene modifying system containing a gene modifying polypeptide and a template RNA, to convert an A nucleotide to a G nucleotide in the Fah5981SB mouse model into the endogenous Fah locus in mouse liver. The Fah5981SB mouse model harbors a G to A point mutation in the last nucleotide of exon 8 of the Fah gene, leading to aberrant mRNA splicing and subsequent mRNA degradation, without the production of Fah protein and serves as a mouse model of hereditary tyrosinemia type I.
In this example, the template RNA contained:

    
    
        (1) a gRNA spacer;
        (2) a gRNA scaffold;
        (3) a heterologous object sequence; and
        (4) a primer binding site (PBS) sequence.
    
    


More specifically, the template RNA comprised the following sequences:









FAH1_R14_P12_Heavy



RNACS048-001



(SEQ ID NO: 30429)



mG*mG*mA*rUrGrGrUrCrCrUrCrArUrGrArArCrGr



 



ArCrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr



 



ArCrCrGrCrUrCrCrArGrUrCrGrUrUrCrArUrGrAr



 



G*mG*mA*mC



 



FAH1_R15_P10_Heavy



RNACS049-001



(SEQ ID NO: 30430)



mG*mG*mA*rUrGrGrUrCrCrUrCrArUrGrArArCrGr



 



ArCrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArUr



 



UrArCrCrGrCrUrCrCrArGrUrCrGrUrUrCrArUrG*



 



mA*mG*mG



 



FAH2_R19_P11_MUT_Heavy



RNACS052-001



(SEQ ID NO:30431)



mU*mC*mA*rGrArGrGrArArGrCrUrGrGrGrCrCrAr



 



CrCrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr



 



GrArGrCrGrGrUrArArUrGrGrCrUrGrGrUrGrGrCr



 



CrCrArGrC*mU*mU*mC



 



FAH2_R19_P13_MUT_Heavy



RNACS053-001



(SEQ ID NO: 30432)



mU*mC*mA*rGrArGrGrArArGrCrUrGrGrGrCrCrAr



 



CrCrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr



 



GrArGrCrGrGrUrArArUrGrGrCrUrGrGrUrGrGrCr



 



CrCrArGrCrUrU*mC*mC*mU






The gene modifying polypeptides tested comprised a sequence of: RNAV209 and RNAV214, the sequences of which are each provided in Example 3.
The gene modifying system comprising the gene modifying polypeptides and the template RNA described above was formulated in LNP and delivered to mice. Specifically, 2 mg/kg of total RNA equivalent formulated in LNPs, combined at 1:1 (w/w) of template RNA and mRNA, were dosed intravenously in 7 to 9-week-old, mixed gender Fah5981SB mice. Six hours or 6 days post-dosing, animals were sacrificed, and their liver collected for analyses. To determine the expression distribution of the gene modifying polypeptide in the liver, 6-hr liver samples were subjected to immunohistochemistry using an anti-Cas9 antibody. Upon staining, quantification of Cas9-positive hepatocytes was determined by QuPath Markup. As shown in FIG. 4, the expression of the gene modifying polypeptide was observed in 82-91% of hepatocytes.
To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus in the genomic DNA of liver samples collected 6 days post-dosing. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. Conversion of an A nucleotide to a G nucleotide indicates successful editing. As shown in FIG. 5, perfect rewrite levels (conversion of A to G with no unwanted mutations detected) of 0.1%-1.9% were detected across the different groups. Indel levels were in the range of 0.2%-0.4%.
To determine the phenotypic correction caused by the gene editing activity, the restoration of wild-type FAH mRNA was determined by real-time qRT-PCR, and the restoration of Fah protein expression determined by immunohistochemistry using an anti-Fah antibody. As shown in FIG. 6, wild-type mRNA restoration of 0.1%-6%, relative to littermate heterozygous mice, was detected across the different groups. As shown in FIG. 7, Fah protein was detected in 0.1%-7% of liver cross-sectional area across the different groups. These results demonstrate the use of a gene modifying system to reverse a mutation in the Fah gene in an in vivo mouse model for hereditary tyrosinemia type I, resulting in partial restoration of expression of wild-type Fah mRNA and Fah protein.
Example 5. Gene Editing at the TTR Locus in an In Vivo Mouse Model
This Example demonstrates successful delivery of an mRNA and guide using Cas9-mediated gene editing using the protospacer sequence ACACAAAUACCAGUCCAGCG (SEQ ID NO: 37641) that targets the TTR locus using a gene modifying polypeptide and RNA in a C57Blk/6 mouse.
RNAs were prepared as follows. An mRNA encoding a gene modifying polypeptide having the sequence shown in Table 5A1 below was produced by in vitro transcription and the purified mRNA was dissolved in 1 mM sodium citrate, pH 6, to a final concentration of RNA of 1-2 mg/mL. Similarly, a guide RNA having a sequence shown in Table 5A1 below was produced by chemical synthesis and dissolved in water or aqueous buffer, to a final concentration of RNA of 1-2 mg/mL.







TABLE 5A1 







Sequences of Example 5












SEQ





ID




Name
NO
Nucleic acid sequence







Cas9-RT
30433
AUGCCUGCGGCUAAGCGGGUAAAAU



gene

UGGAUGGUGGGGACAAGAAGUACAG



modifying

CAUCGGCCUGGACAUCGGCACCAAC



polypeptide

UCUGUGGGCUGGGCCGUGAUCACCG





ACGAGUACAAGGUGCCCAGCAAGAA





AUUCAAGGUGCUGGGCAACACCGAC





CGGCACAGCAUCAAGAAGAACCUGA





UCGGAGCCCUGCUGUUCGACAGCGG





CGAAACAGCCGAGGCCACCCGGCUG





AAGAGAACCGCCAGAAGAAGAUACA





CCAGACGGAAGAACCGGAUCUGCUA





UCUGCAAGAGAUCUUCAGCAACGAG





AUGGCCAAGGUGGACGACAGCUUCU





UCCACAGACUGGAAGAGUCCUUCCU





GGUGGAAGAGGAUAAGAAGCACGAG





CGGCACCCCAUCUUCGGCAACAUCG





UGGACGAGGUGGCCUACCACGAGAA





GUACCCCACCAUCUACCACCUGAGA





AAGAAACUGGUGGACAGCACCGACA





AGGCCGACCUGCGGCUGAUCUAUCU





GGCCCUGGCCCACAUGAUCAAGUUC





CGGGGCCACUUCCUGAUCGAGGGCG





ACCUGAACCCCGACAACAGCGACGU





GGACAAGCUGUUCAUCCAGCUGGUG





CAGACCUACAACCAGCUGUUCGAGG





AAAACCCCAUCAACGCCAGCGGCGU





GGACGCCAAGGCCAUCCUGUCUGCC





AGACUGAGCAAGAGCAGACGGCUGG





AAAAUCUGAUCGCCCAGCUGCCCGG





CGAGAAGAAGAAUGGCCUGUUCGGA





AACCUGAUUGCCCUGAGCCUGGGCC





UGACCCCCAACUUCAAGAGCAACUU





CGACCUGGCCGAGGAUGCCAAACUG





CAGCUGAGCAAGGACACCUACGACG





ACGACCUGGACAACCUGCUGGCCCA





GAUCGGCGACCAGUACGCCGACCUG





UUUCUGGCCGCCAAGAACCUGUCCG





ACGCCAUCCUGCUGAGCGACAUCCU





GAGAGUGAACACCGAGAUCACCAAG





GCCCCCCUGAGCGCCUCUAUGAUCA





AGAGAUACGACGAGCACCACCAGGA





CCUGACCCUGCUGAAAGCUCUCGUG





CGGCAGCAGCUGCCUGAGAAGUACA





AAGAGAUUUUCUUCGACCAGAGCAA





GAACGGCUACGCCGGCUACAUUGAC





GGCGGAGCCAGCCAGGAAGAGUUCU





ACAAGUUCAUCAAGCCCAUCCUGGA





AAAGAUGGACGGCACCGAGGAACUG





CUCGUGAAGCUGAACAGAGAGGACC





UGCUGCGGAAGCAGCGGACCUUCGA





CAACGGCAGCAUCCCCCACCAGAUC





CACCUGGGAGAGCUGCACGCCAUUC





UGCGGCGGCAGGAAGAUUUUUACCC





AUUCCUGAAGGACAACCGGGAAAAG





AUCGAGAAGAUCCUGACCUUCCGCA





UCCCCUACUACGUGGGCCCUCUGGC





CAGGGGAAACAGCAGAUUCGCCUGG





AUGACCAGAAAGAGCGAGGAAACCA





UCACCCCCUGGAACUUCGAGGAAGU





GGUGGACAAGGGCGCUUCCGCCCAG





AGCUUCAUCGAGCGGAUGACCAACU





UCGAUAAGAACCUGCCCAACGAGAA





GGUGCUGCCCAAGCACAGCCUGCUG





UACGAGUACUUCACCGUGUAUAACG





AGCUGACCAAAGUGAAAUACGUGAC





CGAGGGAAUGAGAAAGCCCGCCUUC





CUGAGCGGCGAGCAGAAAAAGGCCA





UCGUGGACCUGCUGUUCAAGACCAA





CCGGAAAGUGACCGUGAAGCAGCUG





AAAGAGGACUACUUCAAGAAAAUCG





AGUGCUUCGACUCCGUGGAAAUCUC





CGGCGUGGAAGAUCGGUUCAACGCC





UCCCUGGGCACAUACCACGAUCUGC





UGAAAAUUAUCAAGGACAAGGACUU





CCUGGACAAUGAGGAAAACGAGGAC





AUUCUGGAAGAUAUCGUGCUGACCC





UGACACUGUUUGAGGACAGAGAGAU





GAUCGAGGAACGGCUGAAAACCUAU





GCCCACCUGUUCGACGACAAAGUGA





UGAAGCAGCUGAAGCGGCGGAGAUA





CACCGGCUGGGGCAGGCUGAGCCGG





AAGCUGAUCAACGGCAUCCGGGACA





AGCAGUCCGGCAAGACAAUCCUGGA





UUUCCUGAAGUCCGACGGCUUCGCC





AACAGAAACUUCAUGCAGCUGAUCC





ACGACGACAGCCUGACCUUUAAAGA





GGACAUCCAGAAAGCCCAGGUGUCC





GGCCAGGGCGAUAGCCUGCACGAGC





ACAUUGCCAAUCUGGCCGGCAGCCC





CGCCAUUAAGAAGGGCAUCCUGCAG





ACAGUGAAGGUGGUGGACGAGCUCG





UGAAAGUGAUGGGCCGGCACAAGCC





CGAGAACAUCGUGAUCGAAAUGGCC





AGAGAGAACCAGACCACCCAGAAGG





GACAGAAGAACAGCCGCGAGAGAAU





GAAGCGGAUCGAAGAGGGCAUCAAA





GAGCUGGGCAGCCAGAUCCUGAAAG





AACACCCCGUGGAAAACACCCAGCU





GCAGAACGAGAAGCUGUACCUGUAC





UACCUGCAGAAUGGGCGGGAUAUGU





ACGUGGACCAGGAACUGGACAUCAA





CCGGCUGUCCGACUACGAUGUGGAC





CAUAUCGUGCCUCAGAGCUUUCUGA





AGGACGACUCCAUCGACAACAAGGU





GCUGACCAGAAGCGACAAGAAUCGG





GGCAAGAGCGACAACGUGCCCUCCG





AAGAGGUCGUGAAGAAGAUGAAGAA





CUACUGGCGGCAGCUGCUGAACGCC





AAGCUGAUUACCCAGAGAAAGUUCG





ACAAUCUGACCAAGGCCGAGAGAGG





CGGCCUGAGCGAACUGGAUAAGGCC





GGCUUCAUCAAGAGACAGCUGGUGG





AAACCCGGCAGAUCACAAAGCACGU





GGCACAGAUCCUGGACUCCCGGAUG





AACACUAAGUACGACGAGAAUGACA





AGCUGAUCCGGGAAGUGAAAGUGAU





CACCCUGAAGUCCAAGCUGGUGUCC





GAUUUCCGGAAGGAUUUCCAGUUUU





ACAAAGUGCGCGAGAUCAACAACUA





CCACCACGCCCACGACGCCUACCUG





AACGCCGUCGUGGGAACCGCCCUGA





UCAAAAAGUACCCUAAGCUGGAAAG





CGAGUUCGUGUACGGCGACUACAAG





GUGUACGACGUGCGGAAGAUGAUCG





CCAAGAGCGAGCAGGAAAUCGGCAA





GGCUACCGCCAAGUACUUCUUCUAC





AGCAACAUCAUGAACUUUUUCAAGA





CCGAGAUUACCCUGGCCAACGGCGA





GAUCCGGAAGCGGCCUCUGAUCGAG





ACAAACGGCGAAACCGGGGAGAUCG





UGUGGGAUAAGGGCCGGGAUUUUGC





CACCGUGCGGAAAGUGCUGAGCAUG





CCCCAAGUGAAUAUCGUGAAAAAGA





CCGAGGUGCAGACAGGCGGCUUCAG





CAAAGAGUCUAUCCUGCCCAAGAGG





AACAGCGAUAAGCUGAUCGCCAGAA





AGAAGGACUGGGACCCUAAGAAGUA





CGGCGGCUUCGACAGCCCCACCGUG





GCCUAUUCUGUGCUGGUGGUGGCCA





AAGUGGAAAAGGGCAAGUCCAAGAA





ACUGAAGAGUGUGAAAGAGCUGCUG





GGGAUCACCAUCAUGGAAAGAAGCA





GCUUCGAGAAGAAUCCCAUCGACUU





UCUGGAAGCCAAGGGCUACAAAGAA





GUGAAAAAGGACCUGAUCAUCAAGC





UGCCUAAGUACUCCCUGUUCGAGCU





GGAAAACGGCCGGAAGAGAAUGCUG





GCCUCUGCCGGCGAACUGCAGAAGG





GAAACGAACUGGCCCUGCCCUCCAA





AUAUGUGAACUUCCUGUACCUGGCC





AGCCACUAUGAGAAGCUGAAGGGCU





CCCCCGAGGAUAAUGAGCAGAAACA





GCUGUUUGUGGAACAGCACAAGCAC





UACCUGGACGAGAUCAUCGAGCAGA





UCAGCGAGUUCUCCAAGAGAGUGAU





CCUGGCCGACGCUAAUCUGGACAAA





GUGCUGUCCGCCUACAACAAGCACC





GGGAUAAGCCCAUCAGAGAGCAGGC





CGAGAAUAUCAUCCACCUGUUUACC





CUGACCAAUCUGGGAGCCCCUGCCG





CCUUCAAGUACUUUGACACCACCAU





CGACCGGAAGAGGUACACCAGCACC





AAAGAGGUGCUGGACGCCACCCUGA





UCCACCAGAGCAUCACCGGCCUGUA





CGAGACACGGAUCGACCUGUCUCAG





CUGGGAGGUGACUCUGGAGGAUCUA





GCGGAGGAUCCUCUGGCAGCGAGAC





ACCAGGAACAAGCGAGUCAGCAACA





CCAGAGAGCAGUGGCGGCAGCAGCG





GCGGCAGCAGCACCCUAAAUAUAGA





AGAUGAGUAUCGGCUACAUGAGACC





UCAAAAGAGCCAGAUGUUUCUCUAG





GGUCCACAUGGCUGUCUGAUUUUCC





UCAGGCCUGGGCGGAAACCGGGGGC





AUGGGACUGGCAGUUCGCCAAGCUC





CUCUGAUCAUACCUCUGAAAGCAAC





CUCUACCCCCGUGUCCAUAAAACAA





UACCCCAUGUCACAAGAAGCCAGAC





UGGGGAUCAAGCCCCACAUACAGAG





ACUGUUGGACCAGGGAAUACUGGUA





CCCUGCCAGUCCCCCUGGAACACGC





CCCUGCUACCCGUUAAGAAACCAGG





GACUAAUGAUUAUAGGCCUGUCCAG





GAUCUGAGAGAAGUCAACAAGCGGG





UGGAGGACAUCCACCCCACCGUGCC





CAACCCUUACAACCUCUUGAGCGGG





CUCCCACCGUCCCACCAGUGGUACA





CUGUGCUUGAUUUAAAGGAUGCCUU





UUUCUGCCUGAGACUCCACCCCACC





AGUCAGCCUCUCUUCGCCUUUGAGU





GGAGAGAUCCAGAGAUGGGAAUCUC





AGGACAAUUGACCUGGACCAGACUC





CCACAGGGUUUCAAAAACAGUCCCA





CCCUGUUUAAUGAGGCACUGCACAG





AGACCUAGCAGACUUCCGGAUCCAG





CACCCAGACUUGAUCCUGCUACAGU





ACGUGGAUGACUUACUGCUGGCCGC





CACUUCUGAGCUAGACUGCCAACAA





GGUACUCGGGCCCUGUUACAAACCC





UAGGGAACCUCGGGUAUCGGGCCUC





GGCCAAGAAAGCCCAAAUUUGCCAG





AAACAGGUCAAGUAUCUGGGGUAUC





UUCUAAAAGAGGGUCAGAGAUGGCU





GACUGAGGCCAGAAAAGAGACUGUG





AUGGGGCAGCCUACUCCGAAGACCC





CUCGACAACUAAGGGAGUUCCUAGG





GAAGGCAGGCUUCUGUCGCCUCUUC





AUCCCUGGGUUUGCAGAAAUGGCAG





CCCCCCUGUACCCUCUCACCAAACC





GGGGACUCUGUUUAAUUGGGGCCCA





GACCAACAAAAGGCCUAUCAAGAAA





UCAAGCAAGCCCUUCUAACUGCCCC





AGCCCUGGGGUUGCCAGAUUUGACU





AAGCCCUUUGAACUCUUUGUCGACG





AGAAGCAGGGCUACGCCAAAGGUGU





CCUAACGCAAAAACUGGGACCUUGG





CGUCGGCCGGUGGCCUACCUGUCCA





AAAAGCUAGACCCAGUAGCAGCUGG





GUGGCCCCCUUGCCUACGGAUGGUA





GCAGCCAUUGCCGUACUGACAAAGG





AUGCAGGCAAGCUAACCAUGGGACA





GCCACUAGUCAUUCUGGCCCCCCAU





GCAGUAGAGGCACUAGUCAAACAAC





CCCCCGACCGCUGGCUUUCCAACGC





CCGGAUGACUCACUAUCAGGCCUUG





CUUUUGGACACGGACCGGGUCCAGU





UCGGACCGGUGGUAGCCCUGAACCC





GGCUACGCUGCUCCCACUGCCUGAG





GAAGGGCUGCAACACAACUGCCUUG





AUAUCCUGGCCGAAGCCCACGGAAC





CCGACCCGACCUAACGGACCAGCCG





CUCCCAGACGCCGACCACACCUGGU





ACACGGAUGGAAGCAGUCUCUUACA





AGAGGGACAGCGUAAGGCGGGAGCU





GCGGUGACCACCGAGACCGAGGUAA





UCUGGGCUAAAGCCCUGCCAGCCGG





GACAUCCGCUCAGCGGGCUGAACUG





AUAGCACUCACCCAGGCCCUAAAGA





UGGCAGAAGGUAAGAAGCUAAAUGU





UUAUACUGAUAGCCGUUAUGCUUUU





GCUACUGCCCAUAUCCAUGGAGAAA





UAUACAGAAGGCGUGGGUGGCUCAC





AUCAGAAGGCAAAGAGAUCAAAAAU





AAAGACGAGAUCUUGGCCCUACUAA





AAGCCCUCUUUCUGCCCAAAAGACU





UAGCAUAAUCCAUUGUCCAGGACAU





CAAAAGGGACACAGCGCCGAGGCUA





GAGGCAACCGGAUGGCUGACCAAGC





GGCCCGAAAGGCAGCCAUCACAGAG





ACUCCAGACACCUCUACCCUCCUCA





UAGAAAAUUCAUCACCCUCUGGCGG





CUCAAAAAGAACCGCCGACGGCAGC





GAAUUCGAGAAAAGGACGGCGGAUG





GUAGCGAAUUCGAGAGCCCUAAAAA





GAAGGCCAAGGUAGAGUAA



 



guide RNA
30434
mA*mC*mA*CAAAUACCAGUCCAGC





GGUUUUAGAmGmCmUmAmGmAmAmA





mUmAmGmCAAGUUAAAAUAAGGCUA





GUCCGUUAUCAmAmCmUmUmGmAmA





mAmAmAmGmUmGmGmCmAmCmCmGm





AmGmUmCmGmGmUmGmCmU*mU*mU





*mU







m = 2′OMethyl,



*= phosphorothioate linkage






Lipid nanoparticle (LNP) components (ionizable lipid, helper lipid, sterol, PEG) were dissolved in 100% ethanol with the lipid component molar ratios of 47:8:43.5:1.5, respectively. RNA (guide and mRNA) was combined in a 1:1 weight ratio and diluted to a concentration of 0.05-0.2 mg/mL in sodium acetate buffer, pH 5. RNA was formulated into distinct LNPs with a lipid amine to total RNA phosphate (N:P) molar ratio of 4.0. The LNPs were formed by microfluidic or turbulent mixing of the lipid and RNA solutions. A 3:1 ratio of aqueous to organic solvent was maintained during mixing using differential flow rates. After mixing, the LNPs were diluted, collected and buffer exchanged into 50 mM Tris, 9% sucrose buffer using tangential flow filtration. Formulations were concentrated to 1.0 mg/mL or higher then filtered through 0.2 μm sterile filter. The final LNP were stored at −80° C. until further use.
The LNP formulations were delivered intravenously by bolus tail vein injection to C57Blk/6 mice that were approximately 8 weeks old at concentrations ranging from 1-0.1 mg/kg. The expression of the Cas9-RT was measured by 6 hours after injection by euthanizing animals and collecting livers during necropsy. Animals were euthanized at 5 days after injection where liver was collected upon necropsy to which the activity of gene editing of the TTR locus was assessed. Expression of the Cas9-RT gene editing polypeptide in liver was measured by Western blot where Cas9 was detected by a mouse monoclonal antibody (7A9-3A3, Cell Signaling Technology) and GAPDH (Cell Signaling Technology) was used as a loading control. (FIG. 8). Editing of the TTR locus was quantified by Sanger sequencing followed by TIDE analysis of an amplicon of the TTR locus near the binding site of the protospacer. Editing of the TTR locus was observed, as shown in FIG. 9. TTR protein levels in serum were quantified by an ELISA using a standard curve (Aviva Biosciences). TTR protein levels in serum declined in treated animals, as shown in FIG. 10. These experiments demonstrate that the Cas9-RT polypeptide can be expressed in vivo, and can edit the TTR locus, resulting in a decrease in TTR protein levels in serum.
Example 6. Gene Editing at the TTR Locus in an In Vivo Cynomolgus Macaque Model
This Example demonstrates successful delivery of an mRNA and guide using Cas9-mediated gene editing using the protospacer sequence ACACAAAUACCAGUCCAGCG (SEQ ID NO: 37641) that targets the TTR locus using a gene modifying polypeptide and RNA in a cynomolgus model.
RNAs were prepared as follows. An mRNA encoding a gene modifying polypeptide having the sequence shown in Table 6A1 below was produced by in vitro transcription and the purified mRNA was dissolved in 1 mM sodium citrate, pH 6, to a final concentration of RNA of 1-2 mg/mL. Similarly, a guide RNA having a sequence shown in Table 6A1 below was produced by chemical synthesis and dissolved in water or aqueous buffer, to a final concentration of RNA of 1-2 mg/mL.







TABLE 6A1







Sequences of Example 6









Name
SEQ ID NO
Nucleic acid sequence





Cas9-RT gene
30435
AUGCCUGCGGCUAAGCGGGUAAAAUUGGAUGGUGGGGACAAGAAGUA


modifying

CAGCAUCGGCCUGGACAUCGGCACCAACUCUGUGGGCUGGGCCGUGAU


polypeptide

CACCGACGAGUACAAGGUGCCCAGCAAGAAAUUCAAGGUGCUGGGCAA




CACCGACCGGCACAGCAUCAAGAAGAACCUGAUCGGAGCCCUGCUGUU




CGACAGCGGCGAAACAGCCGAGGCCACCCGGCUGAAGAGAACCGCCAG




AAGAAGAUACACCAGACGGAAGAACCGGAUCUGCUAUCUGCAAGAGAU




CUUCAGCAACGAGAUGGCCAAGGUGGACGACAGCUUCUUCCACAGACU




GGAAGAGUCCUUCCUGGUGGAAGAGGAUAAGAAGCACGAGCGGCACCC




CAUCUUCGGCAACAUCGUGGACGAGGUGGCCUACCACGAGAAGUACCC




CACCAUCUACCACCUGAGAAAGAAACUGGUGGACAGCACCGACAAGGC




CGACCUGCGGCUGAUCUAUCUGGCCCUGGCCCACAUGAUCAAGUUCCG




GGGCCACUUCCUGAUCGAGGGCGACCUGAACCCCGACAACAGCGACGU




GGACAAGCUGUUCAUCCAGCUGGUGCAGACCUACAACCAGCUGUUCGA




GGAAAACCCCAUCAACGCCAGCGGCGUGGACGCCAAGGCCAUCCUGUC




UGCCAGACUGAGCAAGAGCAGACGGCUGGAAAAUCUGAUCGCCCAGCU




GCCCGGCGAGAAGAAGAAUGGCCUGUUCGGAAACCUGAUUGCCCUGAG




CCUGGGCCUGACCCCCAACUUCAAGAGCAACUUCGACCUGGCCGAGGA




UGCCAAACUGCAGCUGAGCAAGGACACCUACGACGACGACCUGGACAA




CCUGCUGGCCCAGAUCGGCGACCAGUACGCCGACCUGUUUCUGGCCGC




CAAGAACCUGUCCGACGCCAUCCUGCUGAGCGACAUCCUGAGAGUGAA




CACCGAGAUCACCAAGGCCCCCCUGAGCGCCUCUAUGAUCAAGAGAUA




CGACGAGCACCACCAGGACCUGACCCUGCUGAAAGCUCUCGUGCGGCA




GCAGCUGCCUGAGAAGUACAAAGAGAUUUUCUUCGACCAGAGCAAGAA




CGGCUACGCCGGCUACAUUGACGGCGGAGCCAGCCAGGAAGAGUUCUA




CAAGUUCAUCAAGCCCAUCCUGGAAAAGAUGGACGGCACCGAGGAACU




GCUCGUGAAGCUGAACAGAGAGGACCUGCUGCGGAAGCAGCGGACCUU




CGACAACGGCAGCAUCCCCCACCAGAUCCACCUGGGAGAGCUGCACGC




CAUUCUGCGGCGGCAGGAAGAUUUUUACCCAUUCCUGAAGGACAACCG




GGAAAAGAUCGAGAAGAUCCUGACCUUCCGCAUCCCCUACUACGUGGG




CCCUCUGGCCAGGGGAAACAGCAGAUUCGCCUGGAUGACCAGAAAGAG




CGAGGAAACCAUCACCCCCUGGAACUUCGAGGAAGUGGUGGACAAGGG




CGCUUCCGCCCAGAGCUUCAUCGAGCGGAUGACCAACUUCGAUAAGAA




CCUGCCCAACGAGAAGGUGCUGCCCAAGCACAGCCUGCUGUACGAGUA




CUUCACCGUGUAUAACGAGCUGACCAAAGUGAAAUACGUGACCGAGGG




AAUGAGAAAGCCCGCCUUCCUGAGCGGCGAGCAGAAAAAGGCCAUCGU




GGACCUGCUGUUCAAGACCAACCGGAAAGUGACCGUGAAGCAGCUGAA




AGAGGACUACUUCAAGAAAAUCGAGUGCUUCGACUCCGUGGAAAUCUC




CGGCGUGGAAGAUCGGUUCAACGCCUCCCUGGGCACAUACCACGAUCU




GCUGAAAAUUAUCAAGGACAAGGACUUCCUGGACAAUGAGGAAAACG




AGGACAUUCUGGAAGAUAUCGUGCUGACCCUGACACUGUUUGAGGACA




GAGAGAUGAUCGAGGAACGGCUGAAAACCUAUGCCCACCUGUUCGACG




ACAAAGUGAUGAAGCAGCUGAAGCGGCGGAGAUACACCGGCUGGGGCA




GGCUGAGCCGGAAGCUGAUCAACGGCAUCCGGGACAAGCAGUCCGGCA




AGACAAUCCUGGAUUUCCUGAAGUCCGACGGCUUCGCCAACAGAAACU




UCAUGCAGCUGAUCCACGACGACAGCCUGACCUUUAAAGAGGACAUCC




AGAAAGCCCAGGUGUCCGGCCAGGGCGAUAGCCUGCACGAGCACAUUG




CCAAUCUGGCCGGCAGCCCCGCCAUUAAGAAGGGCAUCCUGCAGACAG




UGAAGGUGGUGGACGAGCUCGUGAAAGUGAUGGGCCGGCACAAGCCCG




AGAACAUCGUGAUCGAAAUGGCCAGAGAGAACCAGACCACCCAGAAGG




GACAGAAGAACAGCCGCGAGAGAAUGAAGCGGAUCGAAGAGGGCAUCA




AAGAGCUGGGCAGCCAGAUCCUGAAAGAACACCCCGUGGAAAACACCC




AGCUGCAGAACGAGAAGCUGUACCUGUACUACCUGCAGAAUGGGGGG




AUAUGUACGUGGACCAGGAACUGGACAUCAACCGGCUGUCCGACUACG




AUGUGGACCAUAUCGUGCCUCAGAGCUUUCUGAAGGACGACUCCAUCG




ACAACAAGGUGCUGACCAGAAGCGACAAGAAUCGGGGCAAGAGCGACA




ACGUGCCCUCCGAAGAGGUCGUGAAGAAGAUGAAGAACUACUGGCGGC




AGCUGCUGAACGCCAAGCUGAUUACCCAGAGAAAGUUCGACAAUCUGA




CCAAGGCCGAGAGAGGCGGCCUGAGCGAACUGGAUAAGGCCGGCUUCA




UCAAGAGACAGCUGGUGGAAACCCGGCAGAUCACAAAGCACGUGGCAC




AGAUCCUGGACUCCCGGAUGAACACUAAGUACGACGAGAAUGACAAGC




UGAUCCGGGAAGUGAAAGUGAUCACCCUGAAGUCCAAGCUGGUGUCCG




AUUUCCGGAAGGAUUUCCAGUUUUACAAAGUGCGCGAGAUCAACAACU




ACCACCACGCCCACGACGCCUACCUGAACGCCGUCGUGGGAACCGCCCU




GAUCAAAAAGUACCCUAAGCUGGAAAGCGAGUUCGUGUACGGCGACUA




CAAGGUGUACGACGUGCGGAAGAUGAUCGCCAAGAGCGAGCAGGAAAU




CGGCAAGGCUACCGCCAAGUACUUCUUCUACAGCAACAUCAUGAACUU




UUUCAAGACCGAGAUUACCCUGGCCAACGGCGAGAUCCGGAAGCGGCC




UCUGAUCGAGACAAACGGCGAAACCGGGGAGAUCGUGUGGGAUAAGG




GCCGGGAUUUUGCCACCGUGCGGAAAGUGCUGAGCAUGCCCCAAGUGA




AUAUCGUGAAAAAGACCGAGGUGCAGACAGGCGGCUUCAGCAAAGAGU




CUAUCCUGCCCAAGAGGAACAGCGAUAAGCUGAUCGCCAGAAAGAAGG




ACUGGGACCCUAAGAAGUACGGCGGCUUCGACAGCCCCACCGUGGCCU




AUUCUGUGCUGGUGGUGGCCAAAGUGGAAAAGGGCAAGUCCAAGAAA




CUGAAGAGUGUGAAAGAGCUGCUGGGGAUCACCAUCAUGGAAAGAAG




CAGCUUCGAGAAGAAUCCCAUCGACUUUCUGGAAGCCAAGGGCUACAA




AGAAGUGAAAAAGGACCUGAUCAUCAAGCUGCCUAAGUACUCCCUGUU




CGAGCUGGAAAACGGCCGGAAGAGAAUGCUGGCCUCUGCCGGCGAACU




GCAGAAGGGAAACGAACUGGCCCUGCCCUCCAAAUAUGUGAACUUCCU




GUACCUGGCCAGCCACUAUGAGAAGCUGAAGGGCUCCCCCGAGGAUAA




UGAGCAGAAACAGCUGUUUGUGGAACAGCACAAGCACUACCUGGACGA




GAUCAUCGAGCAGAUCAGCGAGUUCUCCAAGAGAGUGAUCCUGGCCGA




CGCUAAUCUGGACAAAGUGCUGUCCGCCUACAACAAGCACCGGGAUAA




GCCCAUCAGAGAGCAGGCCGAGAAUAUCAUCCACCUGUUUACCCUGAC




CAAUCUGGGAGCCCCUGCCGCCUUCAAGUACUUUGACACCACCAUCGA




CCGGAAGAGGUACACCAGCACCAAAGAGGUGCUGGACGCCACCCUGAU




CCACCAGAGCAUCACCGGCCUGUACGAGACACGGAUCGACCUGUCUCA




GCUGGGAGGUGACUCUGGAGGAUCUAGCGGAGGAUCCUCUGGCAGCGA




GACACCAGGAACAAGCGAGUCAGCAACACCAGAGAGCAGUGGCGGCAG




CAGCGGCGGCAGCAGCACCCUAAAUAUAGAAGAUGAGUAUCGGCUACA




UGAGACCUCAAAAGAGCCAGAUGUUUCUCUAGGGUCCACAUGGCUGUC




UGAUUUUCCUCAGGCCUGGGCGGAAACCGGGGGCAUGGGACUGGCAGU




UCGCCAAGCUCCUCUGAUCAUACCUCUGAAAGCAACCUCUACCCCCGU




GUCCAUAAAACAAUACCCCAUGUCACAAGAAGCCAGACUGGGGAUCAA




GCCCCACAUACAGAGACUGUUGGACCAGGGAAUACUGGUACCCUGCCA




GUCCCCCUGGAACACGCCCCUGCUACCCGUUAAGAAACCAGGGACUAA




UGAUUAUAGGCCUGUCCAGGAUCUGAGAGAAGUCAACAAGCGGGUGG




AGGACAUCCACCCCACCGUGCCCAACCCUUACAACCUCUUGAGCGGGC




UCCCACCGUCCCACCAGUGGUACACUGUGCUUGAUUUAAAGGAUGCCU




UUUUCUGCCUGAGACUCCACCCCACCAGUCAGCCUCUCUUCGCCUUUG




AGUGGAGAGAUCCAGAGAUGGGAAUCUCAGGACAAUUGACCUGGACCA




GACUCCCACAGGGUUUCAAAAACAGUCCCACCCUGUUUAAUGAGGCAC




UGCACAGAGACCUAGCAGACUUCCGGAUCCAGCACCCAGACUUGAUCC




UGCUACAGUACGUGGAUGACUUACUGCUGGCCGCCACUUCUGAGCUAG




ACUGCCAACAAGGUACUCGGGCCCUGUUACAAACCCUAGGGAACCUCG




GGUAUCGGGCCUCGGCCAAGAAAGCCCAAAUUUGCCAGAAACAGGUCA




AGUAUCUGGGGUAUCUUCUAAAAGAGGGUCAGAGAUGGCUGACUGAG




GCCAGAAAAGAGACUGUGAUGGGGCAGCCUACUCCGAAGACCCCUCGA




CAACUAAGGGAGUUCCUAGGGAAGGCAGGCUUCUGUCGCCUCUUCAUC




CCUGGGUUUGCAGAAAUGGCAGCCCCCCUGUACCCUCUCACCAAACCG




GGGACUCUGUUUAAUUGGGGCCCAGACCAACAAAAGGCCUAUCAAGAA




AUCAAGCAAGCCCUUCUAACUGCCCCAGCCCUGGGGUUGCCAGAUUUG




ACUAAGCCCUUUGAACUCUUUGUCGACGAGAAGCAGGGCUACGCCAAA




GGUGUCCUAACGCAAAAACUGGGACCUUGGCGUCGGCCGGUGGCCUAC




CUGUCCAAAAAGCUAGACCCAGUAGCAGCUGGGUGGCCCCCUUGCCUA




CGGAUGGUAGCAGCCAUUGCCGUACUGACAAAGGAUGCAGGCAAGCUA




ACCAUGGGACAGCCACUAGUCAUUCUGGCCCCCCAUGCAGUAGAGGCA




CUAGUCAAACAACCCCCCGACCGCUGGCUUUCCAACGCCCGGAUGACU




CACUAUCAGGCCUUGCUUUUGGACACGGACCGGGUCCAGUUCGGACCG




GUGGUAGCCCUGAACCCGGCUACGCUGCUCCCACUGCCUGAGGAAGGG




CUGCAACACAACUGCCUUGAUAUCCUGGCCGAAGCCCACGGAACCCGA




CCCGACCUAACGGACCAGCCGCUCCCAGACGCCGACCACACCUGGUACA




CGGAUGGAAGCAGUCUCUUACAAGAGGGACAGCGUAAGGCGGGAGCUG




CGGUGACCACCGAGACCGAGGUAAUCUGGGCUAAAGCCCUGCCAGCCG




GGACAUCCGCUCAGCGGGCUGAACUGAUAGCACUCACCCAGGCCCUAA




AGAUGGCAGAAGGUAAGAAGCUAAAUGUUUAUACUGAUAGCCGUUAU




GCUUUUGCUACUGCCCAUAUCCAUGGAGAAAUAUACAGAAGGCGUGGG




UGGCUCACAUCAGAAGGCAAAGAGAUCAAAAAUAAAGACGAGAUCUU




GGCCCUACUAAAAGCCCUCUUUCUGCCCAAAAGACUUAGCAUAAUCCA




UUGUCCAGGACAUCAAAAGGGACACAGCGCCGAGGCUAGAGGCAACCG




GAUGGCUGACCAAGCGGCCCGAAAGGCAGCCAUCACAGAGACUCCAGA




CACCUCUACCCUCCUCAUAGAAAAUUCAUCACCCUCUGGCGGCUCAAA




AAGAACCGCCGACGGCAGCGAAUUCGAGAAAAGGACGGCGGAUGGUAG




CGAAUUCGAGAGCCCUAAAAAGAAGGCCAAGGUAGAGUAA


 


guide RNA
30436
mA*mC*mA*CAAAUACCAGUCCAGCGGUUUUAGAmGmCmUmAmGmAmA




mAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUm




GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmG




mCmU*mU*mU*mU





m = 2′OMethyl, * = phosphorothioate linkage






Lipid nanoparticle (LNP) components (ionizable lipid, helper lipid, sterol, PEG) were dissolved in 100% ethanol with the lipid component molar ratios of 47:8:43.5:1.5, respectively. RNA (guide and mRNA) was combined in a 1:1 weight ratio and diluted to a concentration of 0.05-0.2 mg/mL in sodium acetate buffer, pH 5. RNA was formulated into distinct LNPs with a lipid amine to total RNA phosphate (N:P) molar ratio of 4.0. The LNPs were formed by microfluidic or turbulent mixing of the lipid and RNA solutions. A 3:1 ratio of aqueous to organic solvent was maintained during mixing using differential flow rates. After mixing, the LNPs were diluted, collected and buffer exchanged into 50 mM Tris, 9% sucrose buffer using tangential flow filtration. Formulations were concentrated to 1.0 mg/mL or higher then filtered through 0.2 μm sterile filter. The final LNP were stored at −80° C. until further use. The LNP formulations were delivered intravenously by infusion over the course of 1 hour at 2 mg/kg where the volume of the infusion was 5 ml/kg. Cynomolgus macaques from mainland Asia were given dexamethasone 2 mg/kg bolus via intramuscular injection 1.5-2 h prior to intravenous infusion using a syringe pump. Animals were monitored after infusion and the expression of the Cas9-RT was measured by laparoscopic biopsies taken from the liver 8-12 h, 24 h, and 48 h after infusion. Animals were euthanized 14 days after infusion and liver was harvested by dividing the organ up into 8 different segments to which the activity of gene editing of the TTR locus was assessed. Expression of the Cas9-RT gene editing polypeptide in liver was quantified by capillary electrophoresis western blot using the ProteinSimple Jess system (bio-techne) where Cas9 was detected by a mouse monoclonal antibody (7A9-3A3, Cell Signaling Technology). Relative expression of the Cas9-RT gene editing polypeptide was measured by an area under curve analysis, as shown in FIG. 11. Editing of the TTR locus was quantified by amplicon-sequencing of the TTR locus near the binding site of the protospacer. Editing of the TTR locus was observed, as shown in FIG. 12. These experiments demonstrate that the Cas9-RT polypeptide can be expressed in vivo in a non-human primate model and can edit the TTR locus.
Example 7: Evaluating Rewrite Efficiency of Exemplary Template RNAs and Second Strand-Nicking gRNAs with an Exemplary Gene Modifying Polypeptide in Correcting an F263S Mutation in a Murine Pah Gene in Murine Hepatocyte Cells
This example describes the use of exemplary gene modifying systems containing either of two gene modifying polypeptides and template RNAs comprising varied lengths of heterologous object sequences and PBS sequences to quantify the activity of template RNAs for correction of an F263S mutation (corresponding to a T835C base change) in the murine Pah gene. In this example, a template RNA contained:

    
    
        (1) a gRNA spacer;
        (2) a gRNA scaffold;
        (3) a heterologous object sequence; and
        (4) a primer binding site (PBS) sequence.
    
    


Exemplary template RNAs generated are given in Table E1. Two different versions of each template RNA were produced and tested (EM and HM), one with a first level and distributions of nucleotide modifications (e.g., phosphorothioate linkages denoted by an asterisk and/or 2′-O-methyl groups denoted by an ‘m’ preceding a nucleotide) and one with a second level and distribution.
In Examples 7-8, unless otherwise noted the HM version of a template RNA was used in the described experiments.







TABLE E1







Exemplary Template RNAs and Sequences













SEQ


RNACS
Name
Sequence
ID NO





RNACS1
EM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrArGrCrUrArGrArAr
30437


205
_R19_P9
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrGrCrCrUrUrCrCrGrArGrUrCrUr





UrCrCrArCrUrGrCrArCrArC*mA*mG*mU



 


RNACS1
EM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrArGrCrUrArGrArAr
30438


256
_R29_P8
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrGrGrGrUrGrGrCrCrUrGrGrCrCr





UrUrCrCrGrArGrUrCrUrUrCrCrArCrUrGrCrArCrA*mC*mA*mG



 


RNACS1
EM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrArGrCrUrArGrArAr
30439


184
_R15_P10
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUrCrCrGrArGrUrCrUrUrCrCrAr





CrUrGrCrArCrArCrA*mG*mU*mA



 


RNACS1
EM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrArGrCrUrArGrArAr
30440


216
_R21_P8
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUrGrGrCrCrUrUrCrCrGrArGrUr





CrUrUrCrCrArCrUrGrCrArCrA*mC*mA*mG



 


RNACS1
EM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrArGrCrUrArGrArAr
30441


215
_R21_P9
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUrGrGrCrCrUrUrCrCrGrArGrUr





CrUrUrCrCrArCrUrGrCrArCrArC*mA*mG*mU



 


RNACS1
EM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrArGrCrUrArGrArAr
30442


204
_R19_P10
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrGrCrCrUrUrCrCrGrArGrUrCrUr





UrCrCrArCrUrGrCrArCrArCrA*mG*mU*mA



 


RNACS1
EM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrArGrCrUrArGrArAr
30443


195
_R17_P9
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrCrUrUrCrCrGrArGrUrCrUrUrCr





CrArCrUrGrCrArCrArC*mA*mG*mU



 


RNACS1
EM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrArGrCrUrArGrArAr
30444


206
_R19_P8
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrGrCrCrUrUrCrCrGrArGrUrCrUr





UrCrCrArCrUrGrCrArCrA*mC*mA*mG



 


RNACS1
EM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrArGrCrUrArGrArAr
30445


166
_R11_P8
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrArGrUrCrUrUrCrCrArCrUrGrCr





ArCrA*mC*mA*mG



 


RNACS1
EM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrArGrCrUrArGrArAr
30446


196
_R17_P8
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrCrUrUrCrCrGrArGrUrCrUrUrCr





CrArCrUrGrCrArCrA*mC*mA*mG



 


RNACS1
EM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrArGrCrUrArGrArAr
30447


214
_R21_P10
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUrGrGrCrCrUrUrCrCrGrArGrUr





CrUrUrCrCrArCrUrGrCrArCrArCrA*mG*mU*mA



 


RNACS1
EM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrArGrCrUrArGrArAr
30448


186
_R15_P8
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUrCrCrGrArGrUrCrUrUrCrCrAr





CrUrGrCrArCrA*mC*mA*mG



 


RNACS1
EM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrArGrCrUrArGrArAr
30449


185
_R15_P9
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUrCrCrGrArGrUrCrUrUrCrCrAr





CrUrGrCrArCrArC*mA*mG*mU



 


RNACS1
EM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrArGrCrUrArGrArAr
30450


174
_R13_P10
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrCrGrArGrUrCrUrUrCrCrArCrUr





GrCrArCrArCrA*mG*mU*mA



 


RNACS1
EM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrArGrCrUrArGrArAr
30451


175
_R13_P9
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrCrGrArGrUrCrUrUrCrCrArCrUr





GrCrArCrArC*mA*mG*mU



 


RNACS4
EM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrArGrCrUrArGrArAr
30452


25
2.1
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUrUrCrCrGrArGrUrCrUrUrCrCr





ArCrUrGrCrArCrArCrArGrUrA*mC*mA*mU



 


RNACS1
EM_mPKU6
mG*mC*mC*rUrArArUrGrUrArCrUrGrUrGrUrGrCrArGrGrUrUrUrUrArGrArGrCrUrArGrArAr
30453


375
_R18_P9
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrCrUrUrCrCrGrArGrUrCrUrUrCr





CrArCrUrGrCrArCrArCrA*mG*mU*mA



 


RNACS1
EM_mPKU6
mG*mC*mC*rUrArArUrGrUrArCrUrGrUrGrUrGrCrArGrGrUrUrUrUrArGrArGrCrUrArGrArAr
30454


385
_R20_P9
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrGrCrCrUrUrCrCrGrArGrUrCrUr





UrCrCrArCrUrGrCrArCrArCrA*mG*mU*mA



 


RNACS1
EM_mPKU6
mG*mC*mC*rUrArArUrGrUrArCrUrGrUrGrUrGrCrArGrGrUrUrUrUrArGrArGrCrUrArGrArAr
30455


355
_R14_P9
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrCrGrArGrUrCrUrUrCrCrArCrUr





GrCrArCrArCrA*mG*mU*mA



 


RNACS3
EM_mPKU6
mG*mC*mC*rUrArArUrGrUrArCrUrGrUrGrUrGrCrArGrGrUrUrUrUrArGrArGrCrUrArGrArAr
30456


664
1.2
ArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUr





GrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrCrCrUrUrCrCrGrArGrUrCrUrUr





CrCrArCrUrGrCrArCrArCrArGrUrArC*mA*mU*mU



 


RNACS3
HM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30457


665
_R19_P9
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCrCrUrUrCrCrGrArGrUrCrUr





UrCrCrArCrUrGrCrArCrArC*mA*mG*mU



 


RNACS3
HM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30458


666
_R29_P8
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrGrGrUrGrGrCrCrUrGrGrCrCr





UrUrCrCrGrArGrUrCrUrUrCrCrArCrUrGrCrArCrA*mC*mA*mG



 


RNACS3
HM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30459


460
_R15_P10
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrCrCrGrArGrUrCrUrUrCrCrAr





CrUrGrCrArCrArCrA*mG*mU*mA



 


RNACS3
HM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30460


667
_R21_P8
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGrGrCrCrUrUrCrCrGrArGrUr





CrUrUrCrCrArCrUrGrCrArCrA*mC*mA*mG



 


RNACS3
HM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30461


668
_R21_P9
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGrGrCrCrUrUrCrCrGrArGrUr





CrUrUrCrCrArCrUrGrCrArCrArC*mA*mG*mU



 


RNACS3
HM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30462


669
_R19_P10
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCrCrUrUrCrCrGrArGrUrCrUr





UrCrCrArCrUrGrCrArCrArCrA*mG*mU*mA



 


RNACS2
HM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30463


302
_R17_P9
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUrUrCrCrGrArGrUrCrUrUrCr





CrArCrUrGrCrArCrArC*mA*mG*mU



 


RNACS3
HM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30464


670
_R19_P8
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCrCrUrUrCrCrGrArGrUrCrUr





UrCrCrArCrUrGrCrArCrA*mC*mA*mG



 


RNACS1
HM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30465


855
_R11_P8
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArGrUrCrUrUrCrCrArCrUrGrCr





ArCrA*mC*mA*mG



 


RNACS3
HM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30466


671
_R17_P8
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUrUrCrCrGrArGrUrCrUrUrCr





CrArCrUrGrCrArCrA*mC*mA*mG



 


RNACS3
HM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30467


672
_R21_P10
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGrGrCrCrUrUrCrCrGrArGrUr





CrUrUrCrCrArCrUrGrCrArCrArCrA*mG*mU*mA



 


RNACS1
HM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30468


862
_R15_P8
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrCrCrGrArGrUrCrUrUrCrCrAr





CrUrGrCrArCrA*mC*mA*mG



 


RNACS2
HM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30469


103
_R15_P9
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrCrCrGrArGrUrCrUrUrCrCrAr





CrUrGrCrArCrArC*mA*mG*mU



 


RNACS2
HM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30470


104
_R13_P10
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGrArGrUrCrUrUrCrCrArCrUr





GrCrArCrArCrA*mG*mU*mA



 


RNACS2
HM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30471


099
_R13_P9
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGrArGrUrCrUrUrCrCrArCrUr





GrCrArCrArC*mA*mG*mU



 


RNACS3
HM_mPKU5
mC*mC*mU*rArArUrGrUrArCrUrGrUrGrUrGrCrArGrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30472


673
2.1
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUrCrCrGrArGrUrCrUrUrCrCr





ArCrUrGrCrArCrArCrArGrUrA*mC*mA*mU



 


RNACS1
HM_mPKU6
mG*mC*mC*rUrArArUrGrUrArCrUrGrUrGrUrGrCrArGrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30473


869
_R18_P9
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUrUrCrCrGrArGrUrCrUrUrCr





CrArCrUrGrCrArCrArCrA*mG*mU*mA



 


RNACS3
HM_mPKU6
mG*mC*mC*rUrArArUrGrUrArCrUrGrUrGrUrGrCrArGrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30474


674
_R20_P9
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCrCrUrUrCrCrGrArGrUrCrUr





UrCrCrArCrUrGrCrArCrArCrA*mG*mU*mA



 


RNACS2
HM_mPKU6
mG*mC*mC*rUrArArUrGrUrArCrUrGrUrGrUrGrCrArGrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30475


303
_R14_P9
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGrArGrUrCrUrUrCrCrArCrUr





GrCrArCrArCrA*mG*mU*mA



 


RNACS3
HM_mPKU6
mG*mC*mC*rUrArArUrGrUrArCrUrGrUrGrUrGrCrArGrGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30476


675
1.2
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCrUrUrCrCrGrArGrUrCrUrUr





CrCrArCrUrGrCrArCrArCrArGrUrArC*mA*mU*mU









Table E1A shows the sequences of E1 without chemical modifications. In some embodiments, the sequences of Table E1A may be used without chemical modifications, or with one or more chemical modifications.








TABLE E1A







Table E1 Sequences without Chemical Modifications













SEQ ID


RNACS
Name
Sequence
NO





RNACS
EM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37166


1205
5_R19_P9
GAAAAAGUGGCACCGAGUCGGUGCGCCUUCCGAGUCUUCCACUGCACACAGU



 


RNACS
EM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37167


1256
5_R29_P8
GAAAAAGUGGCACCGAGUCGGUGCGGGUGGCCUGGCCUUCCGAGUCUUCCACUGCACACAG



 


RNACS
EM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37168


1184
5_R15_P10
GAAAAAGUGGCACCGAGUCGGUGCUCCGAGUCUUCCACUGCACACAGUA



 


RNACS
EM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37169


1216
5_R21_P8
GAAAAAGUGGCACCGAGUCGGUGCUGGCCUUCCGAGUCUUCCACUGCACACAG



 


RNACS
EM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37170


1215
5_R21_P9
GAAAAAGUGGCACCGAGUCGGUGCUGGCCUUCCGAGUCUUCCACUGCACACAGU



 


RNACS
EM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37171


1204
5_R19_P10
GAAAAAGUGGCACCGAGUCGGUGCGCCUUCCGAGUCUUCCACUGCACACAGUA



 


RNACS
EM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37172


1195
5_R17_P9
GAAAAAGUGGCACCGAGUCGGUGCCUUCCGAGUCUUCCACUGCACACAGU



 


RNACS
EM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37173


1206
5_R19_P8
GAAAAAGUGGCACCGAGUCGGUGCGCCUUCCGAGUCUUCCACUGCACACAG



 


RNACS
EM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37174


1166
5_R11_P8
GAAAAAGUGGCACCGAGUCGGUGCAGUCUUCCACUGCACACAG



 


RNACS
EM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37175


1196
5_R17_P8
GAAAAAGUGGCACCGAGUCGGUGCCUUCCGAGUCUUCCACUGCACACAG



 


RNACS
EM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37176


1214
5_R21_P10
GAAAAAGUGGCACCGAGUCGGUGCUGGCCUUCCGAGUCUUCCACUGCACACAGUA



 


RNACS
EM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37177


1186
5_R15_P8
GAAAAAGUGGCACCGAGUCGGUGCUCCGAGUCUUCCACUGCACACAG



 


RNACS
EM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37178


1185
5_R15_P9
GAAAAAGUGGCACCGAGUCGGUGCUCCGAGUCUUCCACUGCACACAGU



 


RNACS
EM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37179


1174
5_R13_P10
GAAAAAGUGGCACCGAGUCGGUGCCGAGUCUUCCACUGCACACAGUA



 


RNACS
EM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37180


1175
5_R13_P9
GAAAAAGUGGCACCGAGUCGGUGCCGAGUCUUCCACUGCACACAGU



 


RNACS
EM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37181


425
5_2.1
GAAAAAGUGGCACCGAGUCGGUGCUUCCGAGUCUUCCACUGCACACAGUACAU



 


RNACS
EM_mPKU
GCCUAAUGUACUGUGUGCAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37182


1375
6_R18_P9
GAAAAAGUGGCACCGAGUCGGUGCCUUCCGAGUCUUCCACUGCACACAGUA



 


RNACS
EM_mPKU
GCCUAAUGUACUGUGUGCAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37183


1385
6_R20_P9
GAAAAAGUGGCACCGAGUCGGUGCGCCUUCCGAGUCUUCCACUGCACACAGUA



 


RNACS
EM_mPKU
GCCUAAUGUACUGUGUGCAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37184


1355
6_R14_P9
GAAAAAGUGGCACCGAGUCGGUGCCGAGUCUUCCACUGCACACAGUA



 


RNACS
EM_mPKU
GCCUAAUGUACUGUGUGCAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37185


3664
6_1.2
GAAAAAGUGGCACCGAGUCGGUGCCCUUCCGAGUCUUCCACUGCACACAGUACAUU



 


RNACS
HM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37186


3665
5_R19_P9
GAAAAAGUGGCACCGAGUCGGUGCGCCUUCCGAGUCUUCCACUGCACACAGU



 


RNACS
HM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37187


3666
5_R29_P8
GAAAAAGUGGCACCGAGUCGGUGCGGGUGGCCUGGCCUUCCGAGUCUUCCACUGCACACAG



 


RNACS
HM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37188


3460
5_R15_P10
GAAAAAGUGGCACCGAGUCGGUGCUCCGAGUCUUCCACUGCACACAGUA



 


RNACS
HM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37189


3667
5_R21_P8
GAAAAAGUGGCACCGAGUCGGUGCUGGCCUUCCGAGUCUUCCACUGCACACAG



 


RNACS
HM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37190


3668
5_R21_P9
GAAAAAGUGGCACCGAGUCGGUGCUGGCCUUCCGAGUCUUCCACUGCACACAGU



 


RNACS
HM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37191


3669
5_R19_P10
GAAAAAGUGGCACCGAGUCGGUGCGCCUUCCGAGUCUUCCACUGCACACAGUA



 


RNACS
HM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37192


2302
5_R17_P9
GAAAAAGUGGCACCGAGUCGGUGCCUUCCGAGUCUUCCACUGCACACAGU



 


RNACS
HM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37193


3670
5_R19_P8
GAAAAAGUGGCACCGAGUCGGUGCGCCUUCCGAGUCUUCCACUGCACACAG



 


RNACS
HM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37194


1855
5_R11_P8
GAAAAAGUGGCACCGAGUCGGUGCAGUCUUCCACUGCACACAG



 


RNACS
HM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37195


3671
5_R17_P8
GAAAAAGUGGCACCGAGUCGGUGCCUUCCGAGUCUUCCACUGCACACAG



 


RNACS
HM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37196


3672
5_R21_P10
GAAAAAGUGGCACCGAGUCGGUGCUGGCCUUCCGAGUCUUCCACUGCACACAGUA



 


RNACS
HM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37197


1862
5_R15_P8
GAAAAAGUGGCACCGAGUCGGUGCUCCGAGUCUUCCACUGCACACAG



 


RNACS
HM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37198


2103
5_R15_P9
GAAAAAGUGGCACCGAGUCGGUGCUCCGAGUCUUCCACUGCACACAGU



 


RNACS
HM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37199


2104
5_R13_P10
GAAAAAGUGGCACCGAGUCGGUGCCGAGUCUUCCACUGCACACAGUA



 


RNACS
HM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37200


2099
5_R13_P9
GAAAAAGUGGCACCGAGUCGGUGCCGAGUCUUCCACUGCACACAGU



 


RNACS
HM_mPKU
CCUAAUGUACUGUGUGCAGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37201


3673
5_2.1
GAAAAAGUGGCACCGAGUCGGUGCUUCCGAGUCUUCCACUGCACACAGUACAU



 


RNACS
HM_mPKU
GCCUAAUGUACUGUGUGCAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37202


1869
6_R18_P9
GAAAAAGUGGCACCGAGUCGGUGCCUUCCGAGUCUUCCACUGCACACAGUA



 


RNACS
HM_mPKU
GCCUAAUGUACUGUGUGCAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37203


3674
6_R20_P9
GAAAAAGUGGCACCGAGUCGGUGCGCCUUCCGAGUCUUCCACUGCACACAGUA



 


RNACS
HM_mPKU
GCCUAAUGUACUGUGUGCAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37204


2303
6_R14_P9
GAAAAAGUGGCACCGAGUCGGUGCCGAGUCUUCCACUGCACACAGUA



 


RNACS
HM_mPKU
GCCUAAUGUACUGUGUGCAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
37205


3675
6_1.2
GAAAAAGUGGCACCGAGUCGGUGCCCUUCCGAGUCUUCCACUGCACACAGUACAUU









In this example, exemplary gene modifying polypeptide RNAV209 was examined. Gene modifying polypeptide 1 (RNAV209) comprised two NLS sequences (e.g., a c-Myc NLS and an SV40 NLS), an exemplary endonuclease domain comprising nCas9 which comprised an N863A mutation relative to wildtype Cas9, an exemplary RT domain comprising a MoMLV RT sequence (comprising D200N, T306K, W313F, T330P, and L603W mutations relative to a wildtype MoMLV RT sequence), and an exemplary flexible linker connecting the RT and endonuclease domains and comprising the amino acid sequence of SEQ ID NO: 6. The amino acid sequence of RNAV209 is given by SEQ ID NO: 30477.









(SEQ ID NO: 30477)



MPAAKRVKLDGGDKKYSIGLDIGTNSVGWAVITDEYKVPS



 



KKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR



 



RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE



 



DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKA



 



DLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLV



 



QTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQL



 



PGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD



 



TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVN



 



TEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKE



 



IFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEEL



 



LVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF



 



YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKS



 



EETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP



 



KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV



 



DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNA



 



SLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDR



 



EMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING



 



IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQ



 



KAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK



 



VMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIK



 



ELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD



 



INRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDN



 



VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLS



 



ELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKL



 



IREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL



 



NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI



 



GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET



 



GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKES



 



ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV



 



EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE



 



VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP



 



SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI



 



IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI



 



IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH



 



QSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGTSESAT



 



PESSGGSSGGSSTLNIEDEYRLHETSKEPDVSLGSTWLSD



 



FPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQ



 



EARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTND



 



YRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTV



 



LDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRL



 



PQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLL



 



AATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKY



 



LGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAG



 



FCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIK



 



QALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPW



 



RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTM



 



GQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD



 



RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPD



 



LTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIW



 



AKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAF



 



ATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPK



 



RLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTS



 



TLLIENSSPSGGSKRTADGSEFEKRTADGSEFESPKKKAK



 



VE






Mouse primary hepatocytes were prepared for administration of a gene modifying system as follows. The liver from animals under anesthesia were cannulated at the vena cava and perfused with 1 mg/mL Liberase digestion buffer. Upon digestion, the entire liver was dissected, cells were released into suspension in media and remaining connective tissue was eliminated by filtration with 70 μm cell strainer. Viable hepatocytes were further purified from dead cells and non-parenchymal cells by centrifugation in a 40% Percoll solution.
A gene modifying system comprising a (i) RNAV209 gene modifying polypeptide described herein, and (ii) a template RNA of any of Table E1 was nucleofected into prepared mouse primary hepatocytes in RNA format. Specifically, 4 μg of gene modifying polypeptide mRNA was combined with 10 μg of template RNAs in a total volume of 5 μL. The mRNA and template RNAs are added to 20 μL P3 buffer containing 100,000 primary hepatocytes cells and cells were nucleofected. After nucleofection, cells were grown at 37° C., 5% CO2 for 3 days prior to cell lysis and genomic DNA extraction. To analyze gene editing activity, primers flanking the Pah mutation site were used to amplify across the locus. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. FIG. 13 shows a graph of the rewriting rate (the rate at which the target mutation was corrected) in primary mouse hepatocytes nucleofected with the indicated template RNA and RNAV209 gene modifying polypeptide as measured by Amp-SEQ. Rewriting was seen with a number of exemplary template RNAs.
Higher rewriting rates can increase the impact of administered gene modifying systems and could decrease the amount of a gene modifying system required to achieve a therapeutic effect. Generation of a second nick to the second strand proximal to the template RNA specified nick may increase rewriting rate, e.g., by biasing cellular DNA repair toward incorporation of the mutation correction. To test the ability of a second nick to increase rewriting, exemplary second strand-targeting gRNAs were screened by administration with the top performing template RNA from FIG. 13 (mPKU5_R11_P8) and RNAV209 to mouse primary hepatocytes prepared as described above (FIG. 14).







TABLE E2







Exemplary second strand-targeting


gRNAs (ngRNAs)











Descrip-

SEQ ID


RNACS
tion
Sequence
NO





RNACS
mPKU_
UAUAAAAAGCCUUGAGUUUUGUUUU
30478


2100
ngRNA
AGAGCUAGAAAUAGCAAGUUAAAAU




1
AAGGCUAGUCCGUUAUCAACUUGAA





AAAGUGGCACCGAGUCGGUGCUUUU



 


RNACS
mPKU_
CUGUCGUCUCGAGAUUUCUUGUUUU
30479


2101
ngRNA
AGAGCUAGAAAUAGCAAGUUAAAAU




5
AAGGCUAGUCCGUUAUCAACUUGAA





AAAGUGGCACCGAGUCGGUGCUUUU









The presence of second strand-targeting gRNAs increased the rewriting activity of the exemplary RNAV209/mPKU5_R11_P8 gene modifying system. Two second strand-targeting gRNAs were selected for further evaluation: ngRNA1 (specifying a second strand nick more than 100 bp from the mutation to be corrected) and ngRNA5 (specifying a second strand nick less than 40 bp from the mutation to be corrected). Template RNAs from Table E1 were combined with RNAV209 and either ngRNA1 or ngRNA5 and rewrite efficiency was evaluated as above (FIG. 15). ngRNA5 enhanced rewrite efficiency relative to the absence of second strand-targeting gRNA when combined with a number of template RNAs.
These results demonstrate that an exemplary gene modifying system comprising RNAV209 and any of a variety of template RNAs can correct a mutation in a murine PAH gene in murine hepatocytes, and that the efficiency of rewriting can be enhanced by the addition of a second nick specified by a second strand-nicking gRNA.
Example 8: Comparing Exemplary Template RNAs and Gene Modifying Polypeptides that Correct an F263S Mutation in a Murine Pah Gene in Marine Model Animals
This example describes the use of exemplary gene modifying systems containing either of two gene modifying polypeptides and template RNAs comprising varied lengths and compositions of heterologous object sequences and PBS sequences to quantify the activity of template RNAs for correction of an F263S mutation (corresponding to a T835C base change) in the murine Pah gene. The enhancing effect of inclusion of a second strand-targeting gRNA was also examined. In this example, a template RNA contained:

    
    
        (1) a gRNA spacer;
        (2) a gRNA scaffold;
        (3) a heterologous object sequence; and
        (4) a primer binding site (PBS) sequence.

Exemplary template RNAs tested were template RNAs RNACS1855, RNACS1862, RNACS2103, RNACS2104, and RNACS2099, shown in Table E1. The exemplary second strand-targeting gRNA was RNACS2101, shown in Table E2.

    
    


Exemplary gene modifying polypeptide RNAV209 was compared to exemplary gene modifying polypeptide 2 (RNAIVT338). RNAIVT338 comprised two NLS sequences (e.g., a c-Myc NLS and an SV40 NLS), an exemplary endonuclease domain comprising Cas9, an exemplary RT domain comprising an AVIRE RT sequence, and an exemplary linker between the RT and endonuclease domains and comprising the amino acid sequence of SEQ ID NO: 217. The amino acid sequence of RNAIVT338 is given by SEQ ID NO: 30480.









(SEQ ID NO: 30480)



DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRH



 



SIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY



 



LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGN



 



IVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHM



 



IKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI



 



NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL



 



IALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ



 



IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM



 



IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG



 



YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK



 



QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE



 



KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEV



 



VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVY



 



NELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV



 



KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII



 



KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAH



 



LFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILD



 



FLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH



 



EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVI



 



EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV



 



ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHI



 



VPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKN



 



YWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL



 



VETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK



 



LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY



 



PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSN



 



IMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFA



 



TVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA



 



RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVK



 



ELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY



 



SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH



 



YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI



 



LADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP



 



AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID



 



LSQLGGDGGAEAAAKEAAAKEAAAKEAAAKALEAEAAAKE



 



AAAKEAAAKEAAAKAGGTAPLEEEYRLFLEAPIQNVTLLE



 



QWKREIPKVWAEINPPGLASTQAPIHVQLLSTALPVRVRQ



 



YPITLEAKRSLRETIRKFRAAGILRPVHSPWNTPLLPVRK



 



SGTSEYRMVQDLREVNKRVETIHPTVPNPYTLLSLLPPDR



 



IWYSVLDLKDAFFCIPLAPESQLIFAFEWADAEEGESGQL



 



TWTRLPQGFKNSPTLFNEALNRDLQGFRLDHPSVSLLQYV



 



DDLLIAADTQAACLSATRDLLMTLAELGYRVSGKKAQLCQ



 



EEVTYLGFKIHKGSRSLSNSRTQAILQIPVPKTKRQVREF



 



LGKIGYCRLFIPGFAELAQPLYAATRPGNDPLVWGEKEEE



 



AFQSLKLALTQPPALALPSLDKPFQLFVEETSGAAKGVLT



 



QALGPWKRPVAYLSKRLDPVAAGWPRCLRAIAAAALLTRE



 



ASKLTFGQDIEITSSHNLESLLRSPPDKWLTNARITQYQV



 



LLLDPPRVRFKQTAALNPATLLPETDDTLPIHHCLDTLDS



 



LTSTRPDLTDQPLAQAEATLFTDGSSYIRDGKRYAGAAVV



 



TLDSVIWAEPLPIGTSAQKAELIALTKALEWSKDKSVNIY



 



TDSRYAFATLHVHGMIYRERGWLTAGGKAIKNAPEILALL



 



TAVWLPKRVAVMHCKGHQKDDAPTSTGNRRADEVAREVAI



 



RPLSTQATISAGKRTADGSEFEKRTADGSEFESPKKKAKV



 



E 






The gene modifying system comprising either RNAV209 or RNAIVT338 gene modifying polypeptide and a template RNA described above was formulated in LNP and delivered to mice. Specifically, approximately 1.6 or 2.4 mg/kg of total RNA equivalent formulated in LNPs, combined at 1:1 (w/w) of template RNA and mRNA, were dosed intravenously in 8 to 10-week-old, mixed gender ENU2 mice (0.8 mg/kg each of template RNA and mRNA, optionally with an additional 0.8 mg/kg of ngRNA for second strand-targeting experiments) in a 10 ml/kg bolus. Mice were administered a first dose at time 0 (t=0), and a second dose at t=24 hours. Six hours or 7 days post-dosing (as used herein post-dosing refers to time since the first dose), animals were sacrificed, and their liver and plasma collected for analyses. To determine the expression distribution of the gene modifying polypeptide in the liver, 6-hr liver samples were subjected to immunohistochemistry using an anti-Cas9 antibody. Upon staining, quantification of Cas9-positive hepatocytes was determined by QuPath Markup. As shown in FIG. 16, the expression of the gene modifying polypeptide was observed in 70-80% of hepatocytes. 6-hour liver samples were further analyzed by Western blot using an anti-Cas9 antibody. As shown in FIG. 17, expression of the gene modifying polypeptide was observed in liver from all treated animals. These results show that both RNAV209 and RNAIVT338 gene modifying polypeptides are expressed in murine liver at 6 hours post-dosing.
7-day plasma samples were analyzed by LC/MS to determine the level of phenylalanine present. ENU2 mice harbor a mutation in the murine PAH gene that inactivates the PAH enzyme, resulting in sharply higher Phe levels in plasma than healthy wildtype mice.
Phenylalanine was extracted from mouse plasma using protein precipitation and then analyzed by a LC-MS/MS system equipped with a Shimadzu Nexera UPLC (LC-40) coupled to a Sciex API 7500 mass spectrometer. Surrogate analyte (13C2,15N-Phenylalanine) was used for phenylalanine quantitation. Equivalence of ionization for naturally occurring and surrogate compounds was established prior to and after analytical run. Data were collected in the positive ion mode with three MRM transitions, 169.1 to 123.1 (13C2,15N-Phenylalanine), 166.1 to 120.1 (Phenylalanine) and 172.1 to 126.0 (13C6-Phenylalanine, internal standard). Extracted samples were injected onto an Acquity UPLC BEH C18 column (1.7 μm, 2.1×50 mm) and eluted using a gradient of 0% to 15% of mobile phase B at a flow rate of 0.8 mL/min with a total run time of 3 min per injection. Mobile phase A is 100:2:0.1 H2O:Formic acid (FA): Trifluoroacetic acid (TFA) and mobile phase B is 95:5:2:0.1 ACN:H2O:FA:TFA. Data were processed using Sciex OS software.
The results showed that Phe levels decreased in plasma from all treated mice, consistent with correction of the PAH deactivating point mutation using either gene modifying polypeptide and either of the template RNAs examined (FIG. 18). Treatment with LNP containing RNAIVT338 decreased Phe levels more than treatment with LNP containing RNAV209, with RNAIVT338 decreasing Phe levels by approximately 65% relative to saline compared to RNAV209 decreasing Phe levels by approximately 40%. Similarly, treatment using template RNACS1855 decreased Phe levels more than treatment with template RNACS1862, independent of which gene modifying polypeptide was administered. RNACS1855 contains a shorter reverse transcriptase binding sequence (11 nucleotides) than template RNACS1862 (15 nucleotides).
7-day liver samples were further analyzed using Amp-Seq to determine % rewriting and % INDEL in target liver cells (FIG. 19A and FIG. 19B). To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus in the genomic DNA of liver samples. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. Conversion of a C nucleotide to a T nucleotide at position 835 in the PAH gene indicates successful editing. Rewriting was observed from all treated mice, demonstrating that the gene modifying systems tested successfully corrected the PAH mutation (FIG. 19A). Treatment with LNP containing RNAIVT338 resulted in a higher rewrite % than treatment with LNP containing RNAV209, with approximately twice the rewrite % observed when using RNAIVT338 relative to RNAV209. Similarly, treatment using template RNACS1855 resulted in a higher rewrite % than treatment with template RNACS1862, independent of which gene modifying polypeptide was used. Treatment with LNP containing RNAIVT338 resulted in a lower INDEL % than treatment with LNP containing RNAV209, with almost half the INDEL % observed when using RNAIVT338 relative to RNAV209 (FIG. 19B). Treatment using template RNACS1855 resulted in a lower INDEL % than treatment with template RNACS1862, independent of which gene modifying polypeptide was used. Based on RNAIVT338's improved performance over RNAV209, additional template RNAs were tested in combination with RNAIVT338. ENU2 mice were treated as described above using a gene modifying system comprising RNAIVT338 and one of template RNAs RNACS1855, RNACS1862, RNACS2103, RNACS2104, and RNACS2099, and liver and plasma samples were taken from sacrificed mice at 7 days post-dosing (FIGS. 20A, 20B, and 20C). Rewriting was observed in all treated samples, with strong correlation between % rewriting in liver samples and Phe level reduction in plasma samples. INDEL % was much lower (approximately 25 to 35 times lower) than rewriting %, and correlated with rewriting % level (FIG. 20C). The results demonstrate that rewriting a disease-associated mutation using a gene modifying system containing RNAIVT338 can correct the disease-associated mutation and achieve the desired therapeutic effect (e.g., reduction in Phe level), and that RNAIVT338 achieves rewriting with a number of template RNAs. The results further demonstrate that the rewriting occurs with low levels of INDEL generation.
To determine whether second strand-nicking can enhance rewriting efficiency and improve Phe-reducing therapeutic effect, ENU2 mice were treated as described above with exemplary gene modifying system containing RNAIVT338, template RNA, as well as ngRNA5 which directs a second strand nick less than 40 bp from the target PAH mutation, and liver and plasma samples were analyzed as described above (FIGS. 21A and 21B). Administering a gene modifying system including a second strand-targeting gRNA increased % rewriting in liver and decreased plasma Phe levels relative to gene modifying systems not containing a second strand-targeting gRNA. Over 40% of the liver cells sampled contained the rewrite modification after mice were treated with a gene modifying system containing gene modifying polypeptide RNAIVT338 and an exemplary template RNA and second strand nicking gRNA (FIG. 21A), and this was accompanied by serum Phe levels of approximately 100 μM, physiologically normal for mice (FIG. 21B). Indel percent, while measurably higher when the second strand-targeting gRNA was included, remained low relative to rewriting percent. These results show adding a second strand nick proximal to the mutation to be corrected (e.g., biasing cellular DNA repair to favor correction of the mutation) can increase rewriting and substantially increase the therapeutic effect. These results further show that unintended modifications (e.g., indels) are not significantly increased by second strand nicking.
Across in vivo murine samples, percent rewriting observed in liver samples should correlate inversely with Phe plasma levels if a gene modifying system is effectively correcting a PAH-inactivating, hyperphenylalaninemia (HPA)-inducing mutation. To confirm this, Phe level in plasma samples was plotted over % rewriting for all plasma and liver samples obtained from treated ENU2 mice (FIG. 22). The results show a clear inverse correlation; as % rewriting increases, plasma Phe levels decrease. Plotted as dotted lines are literature-recognized physiologically normal Phe levels and mild-HPA Phe levels in mice (see, e.g., Ahmed et al. Mol Ther Methods Clin Dev. 2020 Mar. 13; 17:568-580 or Bruinenberg et al. Front Behav Neurosci. 2016; 10: 233). The results show that treatment with nearly all combinations of gene modifying polypeptides, template RNAs, and second strand-targeting gRNAs reduce Phe levels to below mild HPA levels and to normal Phe levels in many cases.
Example 9: Evaluating Safety and Efficacy of Exemplary Template RNAs and Gene Modifying Polypeptides that Insert a Silent Mutation in a Primate PAH Gene in Non-Human Primate Model Animals
This example describes the use of exemplary gene modifying systems containing an exemplary gene modifying polypeptide (e.g., RNAIVT338, described above), exemplary template RNA (e.g., described in Tables 4A-4D), and optionally an exemplary second strand-nicking gRNA (e.g., described in Tables 4A-4D) to demonstrate the safety of the gene modifying systems, translation activity of the gene modifying polypeptide, and rewriting activity in a group of non-human primates (NHPs). In some embodiments, the NHPs are Cynomolgus macaques. In some embodiments, the NHPs are Rhesus macaques.
One, two, or more studies are performed. The studies treat NHPs using exemplary gene modifying systems and determine the safety of administration of the systems and/or evaluate the efficacy of the gene modifying system (e.g., percent rewriting, writing accuracy (e.g., indel and imperfect rewriting), and/or phenotypic correction (e.g., PAH protein/mRNA expression and/or plasma Phe level)).
NHPs are distributed into two treated groups, with at least 3 individuals per treated group. Pre-dosing liver biopsy samples are taken at approximately 7 days pre-dosing. Doses of gene modifying system containing mRNA encoding RNAIVT338, exemplary template RNA, and optionally exemplary second strand-nicking gRNA are formulated in LNPs, combined at 1:1 (w/w) of template RNA and mRNA (and optionally 1:1:1 w/w/w of template RNA, mRNA, and gRNA). On Day 1, a single dose is administered intravenously at 3 mg/kg. A single-dose liver biopsy sample is collected from treated individuals one week after the single dose (Day 8). One week after the single-dose liver biopsy sample is taken, the multi-dose phase of the study begins, with two or more (e.g., three) additional doses administered on an alternating day schedule (e.g., on days 15, 17, and 19). A multi-dose liver biopsy sample is collected from treated individuals one week after the last dose of the multi-dose phase (e.g., day 26).
Liver samples are analyzed by Amp-Seq to determine % rewriting, LC/MS to determine Phe levels, and/or immunohistochemistry (e.g., in situ hybridization and/or Western blot) to determine expression of the exemplary gene modifying polypeptide. Plasma samples are obtained from blood draws at each biopsy time point, as well as at shorter time points (e.g., less than 7 days post-dosing). Plasma samples are analyzed, e.g., by LC/MS, for Phe levels. Rewriting will be observed in liver cells after a single dose, with increasing rewriting % observed after multiple doses are administered. Plasma Phe levels will be observed to decrease after a single dose, with more significant decreases observed after multiple doses are administered and correlating with rewriting %.
Example 10: Demonstrating Use of Different Exemplary Template RNA Spacers to Target Macaca fascicularis PAH Gene
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide and template RNAs comprising one of four different gRNA spacer sequences combined with a variety of heterologous object sequences and PBS sequences to induce a series of phenylketonuria relevant genetic alterations in HEK293T cells modified to contain a Macaca fascicularis PAH gene. This example demonstrates gene modification to:

    
    
        (1) Introduce a silent C to A mutation in the codon homologous to that encoding human R408 (CtoA)
        (2) Introduce the C to A mutation of (1) and additionally a PAM/seed killing mutation that inhibits retargeting by the gene modifying polypeptide after editing (CtoA+PSkill).
        (3) Introduce the C to A mutation of (1) and additional silent substitutions promoting favorable DNA repair incorporating the C to A mutation (CtoA+subs).
        (4) Introduce a T to C mutation, similar to that needed to correct PKU-associated R408W in human PAH (TtoC).
    
    


In this example, a template RNA contained:

    
    
        (1) a gRNA spacer;
        (2) a gRNA scaffold;
        (3) a heterologous object sequence; and
        (4) a primer binding site (PBS) sequence.
    
    


Exemplary template RNAs contained one of the following four spacer sequences:









cPKU4:



(SEQ ID NO: 30481)



GGGUCAUAGCGAACUGAGAA



 



cPKU5.1:



(SEQ ID NO: 30482)



UAGCGAACUGAGAAGGGCCG



 



cPKU5.2:



(SEQ ID NO: 30483)



AGCGAACUGAGAAGGGCCGA



 



cPKU6:



(SEQ ID NO: 30484)



ACUUUGCUGCCACAAUCCCU






The exemplary gene modifying polypeptide was RNAIVT338 (described in Example 8).
mRNA encoding RNAIVT338 and template RNA were transfected into HEK293T cells containing a genomic modification which inserted the M fascicularis PAH gene. After transfection, HEK293T cells were cultured for at least 4 days and then assayed for rewriting by isolating genomic DNA, conducting PCR using primers flanking the M fascicularis PAH gene, and then sequencing the amplicons using Amp-Seq.
FIG. 23 shows a graph of percent rewriting for the 4 different mutation types using the template RNAs containing the four spacer sequences under evaluation (where each dot represents that column's spacer combined with a particular of RT and PBS sequence). CtoA, CtoA+PSkill, CtoA+subs, and TtoC mutations were observed when utilizing each of the spacer sequences tested, but with different rewriting % levels. For example: a large number of template RNAs containing spacer cPKU5.2 yielded high rewriting % for CtoA+PSkill mutation and TtoC mutation; a large number of template RNAs containing spacer cPKU6 yielded high rewriting % for CtoA, CtoA+PSkill, and CtoA+subs mutations; a large number of template RNAs containing spacer cPKU5.2 yielded high rewriting % for CtoA+PSkill and CtoA+subs mutations. The results show that gene modifying systems comprising an exemplary gene modifying polypeptide and template RNAs containing four different exemplary spacer sequences enabled 4 different PKU-relevant genetic modifications to a primate PAH gene.
Example 11: Evaluating Template RNA/Second Strand-Targeting gRNA Dose Response in Correction of an F263S Mutation in a Murine Pah Gene in Murine Model Animals
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide, either of two template RNAs (each comprising different spacers, lengths and compositions of heterologous object sequences, and PBS sequences), and a second strand-targeting gRNA to evaluate the dose response of template RNA and second strand-targeting gRNA level to rewriting activity (the correction of an F263S mutation (corresponding to a T835C base change) in the murine Pah gene). Two different concentrations of template RNA and second strand-targeting gRNA (1.2 milligrams per kilogram body weight (mpk) and 2.4 mpk) were evaluated. In this example, a template RNA contained:

    
    
        (1) a gRNA spacer;
        (2) a gRNA scaffold;
        (3) a heterologous object sequence; and
        (4) a primer binding site (PBS) sequence.
    
    


Exemplary template RNAs tested were template RNAs RNACS1855 and RNACS2303, shown in Table E1. The exemplary second strand-targeting gRNA was ngRNA5 (also referred to herein as RNACS2101), shown in Table E2. The exemplary gene modifying polypeptide used was RNAIVT338 (as described above and corresponding to the amino acid sequence of SEQ ID NO: 30480.
The gene modifying system was formulated in LNP and delivered to mice. Specifically, 1.2 or 2.4 mpk of total RNA equivalent formulated in LNPs, combined at 1:1 (w/w) of template RNA and mRNA or 1:1:1 (w/w/w) when including ngRNA, were dosed intravenously in 8 to 10-week-old, mixed gender ENU2 mice (0.8 mg/kg each of template RNA and mRNA, optionally with an additional 0.8 mg/kg of ngRNA for second strand-targeting experiments; or 0.4 mg/kg each of template RNA and mRNA, optionally with an additional 0.4 mg/kg of ngRNA for second strand-targeting experiments) in a 10 ml/kg bolus. Mice were administered a first dose at time 0 (t=0) and a second dose 24 hours later (t=24). 7 days post-dosing, animals were sacrificed, and their liver and plasma collected for analyses.
7-day liver samples were analyzed using Amp-Seq to determine % rewriting in target liver cells (FIG. 24A). To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus in the genomic DNA of liver samples. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. Conversion of a C nucleotide to a T nucleotide at position 835 in the PAH gene indicates successful editing. Rewriting was observed from all treated mice except for RNACS2303 without second strand-targeting gRNA. This is consistent with the interpretation that RNACS2303 is less effective at facilitating rewriting at this locus than RNACS1855 in the absence of a second strand-targeting gRNA, and that the presence of a second strand-targeting gRNA increases rewriting activity. As the dose of template RNA and second strand-targeting gRNA increased, so did the % rewriting. This suggests that the amount of template RNA and/or second strand-targeting gRNA can be adjusted to achieve a desired (e.g., sufficient) level of rewriting activity corresponding to a therapeutic outcome. Amplicon sequencing was also used to evaluate the degree to which INDELs were introduced into the liver sample DNA (FIG. 24B). % INDEL increased with template RNA and second strand-targeting gRNA dosage, correlating with % rewriting. The results suggest that the amount of template RNA and/or second strand-targeting gRNA can be adjusted to decrease (e.g., minimize) the % INDEL while achieving a desired (e.g., sufficient) level of rewriting activity corresponding to a therapeutic outcome.
7-day plasma samples were also analyzed by LC/MS to determine the level of phenylalanine present. ENU2 mice harbor a mutation (F263S) in the murine PAH gene that inactivates the PAH enzyme, resulting in sharply higher Phe levels in plasma than healthy wildtype mice. The ENU2 mutation is described by The Jackson Laboratory (jax.org/strain/002232) as a T835C missense mutation in the murine PAH gene, however the mutation is also described in the literature as being a T788C mutation (see, e.g., Harding, C. O. Mol Front J. 2019 December; 3(2):110-121; or Pecimonova, M. Genes (Basel). 2019 Jun. 15; 10(6):459). The Examples explicitly contemplate both positions whenever reference is made to either nucleotide mutation herein. Phenylalanine was extracted from mouse plasma using protein precipitation and then analyzed by a LC-MS/MS system equipped with a Shimadzu Nexera UPLC (LC-40) coupled to a Sciex API 7500 mass spectrometer. Surrogate analyte (13C2,15N-Phenylalanine) was used for phenylalanine quantitation. Equivalence of ionization for naturally occurring and surrogate compounds was established prior to and after analytical run. Data were collected in the positive ion mode with three MRM transitions, 169.1 to 123.1 (13C2,15N-Phenylalanine), 166.1 to 120.1 (Phenylalanine) and 172.1 to 126.0 (13C6-Phenylalanine, internal standard). Extracted samples were injected onto an Acquity UPLC BEH C18 column (1.7 μm, 2.1×50 mm) and eluted using a gradient of 0% to 15% of mobile phase B at a flow rate of 0.8 mL/min with a total run time of 3 min per injection. Mobile phase A is 100:2:0.1 H2O:Formic acid (FA): Trifluoroacetic acid (TFA) and mobile phase B is 95:5:2:0.1 ACN:H2O:FA:TFA. Data were processed using Sciex OS software.
The results showed that Phe levels decreased in plasma from all treated mice except for RNACS2303 without second strand-targeting gRNA. This is consistent with the interpretation that RNACS2303 is less effective at facilitating rewriting at this locus than RNACS1855 in the absence of a second strand-targeting gRNA, and that the presence of a second strand-targeting gRNA increases rewriting activity. The results are consistent with correction of the PAH deactivating point mutation using the exemplary gene modifying polypeptide and either of the template RNAs examined (FIG. 24C). Treatment with LNP containing RNACS1855 decreased Phe levels more than treatment with LNP containing RNACS2303. Addition of a second strand-targeting gRNA and increasing the dosage of template RNA and second strand-targeting gRNA caused Phe levels to decrease more precipitously in RNACS2303 treated samples. The addition of a second strand-targeting gRNA and increasing the dosage of template RNA and second strand-targeting gRNA caused only small additional Phe levels decreases in RNACS1855 treated samples. These results suggest that a higher dose and/or second strand-targeting gRNA may be necessary to reduce plasma Phe levels to wild-type levels for some template RNAs, but that for highly effective template RNAs a lower dose or omission of second strand-targeting gRNA may be sufficient to reduce plasma Phe to wild-type levels. When considered together (FIGS. 24A-24C), the results show that increasing the template RNA and second strand-targeting gRNA dose increases the % rewriting, as well as % INDEL, and that for highly effective template RNAs a lower dose or omission of second strand-targeting gRNA (despite lower % rewriting) provides a nearly wild-type plasma Phe phenotype.
Example 12: Evaluating Rewriting Activity of Exemplary Human Template RNAs in hPAH Mice
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide and template RNAs comprising varied spacers, lengths and compositions of heterologous object sequences, and PBS sequences to quantify the activity of template RNAs for correction of an R408W mutation (corresponding to a C>T base change) in a human PAH (hPAH) gene in vivo in mice modified to carry R408W hPAH.
In this example, a template RNA contained:

    
    
        (1) a gRNA spacer;
        (2) a gRNA scaffold;
        (3) a heterologous object sequence; and
        (4) a primer binding site (PBS) sequence.
    
    


Exemplary template RNAs generated are given in Table E3. Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2′-O-methyl groups denoted by an ‘m’ preceding a nucleotide. The exemplary gene modifying polypeptide is RNAIVT338, comprising the amino acid sequence of SEQ ID NO: 30480.







TABLE E3







Exemplary Template RNAs and Sequences













SEQ


RNACS
Name
Sequence
ID NO





RNACS
hPKU3_R
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmAmAm
30485


4047
17_P9
AmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUm





GmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGrCrCrCr





UrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU3_R
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmAmAmA
30486


1747
19_P9
mUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrUrCrGrGrCrCrC





rUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU4_R
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmAmAmA
30487


3878
16_P9
mUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGrCrCrCrUrU





rCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU4_R
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmAmAmA
30488


3877
16_P10
mUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGrCrCrCrUrU





rCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU5_R
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUrUrUrArGrAmGmCmUmAmGmAmAmA
30489


4135
10_P9
mUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGrCrCrCrUrU





rC*mU*mC*mA



 


RNACS
hPKU5_R
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUrUrUrArGrAmGmCmUmAmGmAmAmA
30490


4134
10_P10
mUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGrCrCrCrUrU





rCrU*mC*mA*mG



 


RNACS
hPKU5_R
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUrUrUrArGrAmGmCmUmAmGmAmAmA
30491


2300
10_P11
mUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGrCrCrCrUrU





rCrUrC*mA*mG*mU



 


RNACS
hPKU5_R
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUrUrUrArGrAmGmCmUmAmGmAmAmA
30492


2299
12_P10
mUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrUrCrGrGrCrCrC





rUrUrCrU*mC*mA*mG



 


RNACS
hPKU5_R
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUrUrUrArGrAmGmCmUmAmGmAmAmA
30493


4142
12_P11
mUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrUrCrGrGrCrCrC





rUrUrCrUrC*mA*mG*mU



 


RNACS
hPKU5_R
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUrUrUrArGrAmGmCmUmAmGmAmAmA
30494


4173
18_P8
mUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCrUrGrCrCrArCrArArUrArCrCrU





rCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU3_R
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmAmAmA
30495


4045
17_P11
mUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGrCrCrCrUrU





rCrUrCrArGrUrUrCrGrC*mU*mA*mC



 


RNACS
hPKU3_R
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmAmAmA
30496


4048
17_P8
mUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGrCrCrCrUrU





rCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU4_R
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmAmAmA
30497


1763
18_P9
mUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrUrCrGrGrCrCrC





rUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU4_R
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmAmAmA
30498


3907
22_P9
mUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGrCrCrArCrArArUrArCrCrUrCrG





rGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU6_R
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmA
30499


3643
11_P11
mUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArGrGrGrCrCrGrArGrGrUrArUrU





rGrUrGrG*mC*mA*mG



 


RNACS
hPKU6_R
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmA
30500


1792
9_P11
mUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrGrGrCrCrGrArGrGrUrArUrUrGrU





rGrG*mC*mA*mG









Table E3A shows the sequences of E3 without chemical modifications. In some embodiments, the sequences of Table E3A may be used without chemical modifications, or with one or more chemical modifications.








TABLE E3A







Table E3 Sequences without Chemical Modifications













SEQ ID


RNACS
Name
Sequence
NO





RNACS4047
hPKU3_R1
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
37206



7_P9
UGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCGCU



 


RNACS1747
hPKU3_R1
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
37207



9_P9
UGAAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGUUCGCU



 


RNACS3878
hPKU4_R1
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
37208



6_P9
UGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS3877
hPKU4_R1
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
37209



6_P10
UGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCGCU



 


RNACS4135
hPKU5_R1
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
37210



0_P9
UGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCA



 


RNACS4134
0_P10
UGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAG
37211



hPKU5_R1
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU



 


RNACS2300
hPKU5_R1
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
37212



0_P11
UGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGU



 


RNACS2299
hPKU5_R1
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
37213



2_P10
UGAAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAG



 


RNACS4142
hPKU5_R1
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
37214



2_P11
UGAAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGU



 


RNACS4173
hPKU5_R1
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
37215



8_P8
UGAAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGGCCCUUCUC



 


RNACS4045
hPKU3_R1
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
37216



7_P11
UGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCGCUAC



 


RNACS4048
hPKU3_R1
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
37217



7_P8
UGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS1763
hPKU4_R1
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
37218



8_P9
UGAAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS3907
hPKU4_R2
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
37219



2_P9
UGAAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS3643
hPKU6_R1
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
37220



1_P11
UGAAAAAGUGGCACCGAGUCGGUGCAAGGGCCGAGGUAUUGUGGCAG



 


RNACS1792
hPKU6_R9
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
37221



P11
UGAAAAAGUGGCACCGAGUCGGUGCGGGCCGAGGUAUUGUGGCAG









The gene modifying system comprising RNAIVT338 gene modifying polypeptide and a template RNA described above were formulated in LNP and delivered to mice. Specifically, approximately 1.6 mg/kg of total RNA equivalent formulated in LNPs (4:1 N:P ratio for mRNA, and 3:1 N:P ratio for tgRNA), combined at 1:1 (w/w) of template RNA and mRNA, were dosed intravenously in 8 to 10-week-old, mixed gender hPAH mice (0.8 mg/kg each of template RNA and mRNA) in a 10 ml/kg bolus. Mice were administered a dose at time 0 (t=0). 7 days post-dosing (as used herein post-dosing refers to time since the first dose), animals were sacrificed, and their liver and plasma are collected for analyses.
7-day plasma samples were analyzed by LC/MS to determine the level of phenylalanine present. hPAH trangenic mice harbor a mutation in the human PAH gene that inactivates the PAH enzyme, resulting in sharply higher Phe levels in plasma than healthy wildtype mice. Successful rewriting of the hPAH transgene would be expected to result in a decrease in Phe levels in plasma.
Phenylalanine was extracted from mouse plasma using protein precipitation and was analyzed by a LC-MS/MS system equipped with a Shimadzu Nexera UPLC (LC-40) coupled to a Sciex API 7500 mass spectrometer. Surrogate analyte (13C2,15N-Phenylalanine) was used for phenylalanine quantitation. Equivalence of ionization for naturally occurring and surrogate compounds was established prior to and after analytical run. Data were collected in the positive ion mode with three MRM transitions, 169.1 to 123.1 (13C2,15N-Phenylalanine), 166.1 to 120.1 (Phenylalanine) and 172.1 to 126.0 (13C6-Phenylalanine, internal standard). Extracted samples were injected onto an Acquity UPLC BEH C18 column (1.7 μm, 2.1×50 mm) and eluted using a gradient of 0% to 15% of mobile phase B at a flow rate of 0.8 mL/min with a total run time of 3 min per injection. Mobile phase A was 100:2:0.1 H2O:Formic acid (FA): Trifluoroacetic acid (TFA) and mobile phase B was a 95:5:2:0.1 ACN:H2O:FA:TFA. Data were processed using Sciex OS software.
FIG. 35 shows a graph of Phe levels in plasma from treated mice. The results show that Phe levels decreased in hPAH mice treated with exemplary gene modifying systems. The results further showed that Phe levels decreased most in mice treated with hPKU5 template RNAs, with RNACS4134 showing the steepest decrease in Phe levels. These results show that exemplary gene modifying systems targeting mutant hPAH in vivo can achieve editing that results in clinically-relevant phenotype changes.
7-day liver samples were analyzed using Amp-Seq to determine % rewriting and % INDELs in target liver cells. To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus in the genomic DNA of liver samples. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. Conversion of a C nucleotide to a T nucleotide at position 1222 in exon 12 in the human PAH gene indicated successful editing.
FIG. 36A shows a graph of % rewriting level for each of the tested template RNAs. The results showed that hPKU5-spacer-containing template RNAs showed the highest rewriting activity, with RNACS4134 showing the highest rewriting activity of about 4.6%. FIG. 36B shows a graph of % INDEL activity for each of the tested template RNAs. The results showed that all tested template RNAs had very low levels of INDEL generation in hPAH in mouse liver cells, including hPKU5 template RNAs. The results show that exemplary gene modifying systems can be used to specifically correct a clinically relevant mutation in hPAH in vivo in mice.
Example 13: Evaluating Rewriting Activity of Exemplary Human Template RNAs and Second Strand-Targeting gRNAs in Several Cellular Systems
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide, template RNAs comprising varied spacers, lengths and compositions of heterologous object sequences, and PBS sequences, and either of two second strand-targeting gRNAs to evaluate the activity of template RNAs and second strand-targeting gRNAs to: 1) produce a R408W mutation into wild-type hPAH in primary human hepatocytes, 2) produce an W408R mutation to correct the R408W mutation in hPAH in CRISPR gene-edited iPSC hepatoblast cells, and 3) produce an W408R mutation to correct the R408W mutation in hPAH transgenic mice (described in Example 12). Combinations of template RNAs and second strand-targeting gRNAs (e.g., sequences, e.g., spacer sequences) that provide high rewriting activity in one or more of (1)-(3) may also provide rewriting activity in therapeutic template RNAs and second strand-targeting gRNAs designed for correcting pathogenic hPAH mutations in a human subject.
In this example, a template RNA contained:

    
    
        (1) a gRNA spacer;
        (2) a gRNA scaffold;
        (3) a heterologous object sequence; and
        (4) a primer binding site (PBS) sequence.
    
    


Exemplary template RNAs generated and used in iPSC hepatoblast cells and to be used in hPAH transgenic mice are given in Table E5. Exemplary template RNAs generated for use in primary human hepatocytes are given in Table E4. Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2′-O-methyl groups denoted by an ‘m’ preceding a nucleotide. The exemplary gene modifying polypeptide is RNAIVT338, comprising the amino acid sequence of SEQ ID NO: 30480. Exemplary second strand-targeting gRNAs for use with hPKU3, hPKU4, and hPKU5 template RNAs are RNACS1809 and RNACS1810, whereas exemplary second strand-targeting gRNAs for use with hPKU6 template RNAs are RNACS1812, RNACS1834, and RNACS1831, the nucleic acid sequence and chemical modifications of which are given here.









RNACS1809:



(SEQ ID NO: 30501)



mG*mU*mG*rCrCrCrUrUrCrArCrUrCrArArGrCrCr



 



UrGrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU



 



*mU*mU



 



RNACS1810:



(SEQ ID NO: 30502)



mU*mU*mC*rArCrUrCrArArGrCrCrUrGrUrGrGrUr



 



UrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU



 



*mU*mU



 



RNACS1812:



(SEQ ID NO: 30503)



mG*mU*mC*rCrArArGrArCrCrUrCrArArUrCrCrUr



 



UrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU



 



*mU*mU



 



RNACS1834:



(SEQ ID NO: 30504)



mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCr



 



CrArGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU



 



*mU*mU



 



RNACS1831:



(SEQ ID NO: 30505)



mU*mG*mA*rGrArArGrGrGrCrCrGrArGrGrUrArUr



 



UrGrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU



 



*mU*mU






Unmodified versions of these sequences are shown in Table BB below. In some embodiments, the sequences used in this table can be used without chemical modifications.








TABLE BB







RNACS1809, RNACS1810, RNACS1812,


RNACS1834, and RNACS1831


without nucleotide modifications.













SEQ



Name
Sequence
ID NO







RNAC
GUGCCCUUCACUCAAGCCUG
37222



S1809
GUUUUAGAGCUAGAAAUAGC





AAGUUAAAAUAAGGCUAGUC





CGUUAUCAACUUGAAAAAGU





GGCACCGAGUCGGUGCUUUU




 



RNAC
UUCACUCAAGCCUGUGGUUU
37223



S1810
GUUUUAGAGCUAGAAAUAGC





AAGUUAAAAUAAGGCUAGUC





CGUUAUCAACUUGAAAAAGU





GGCACCGAGUCGGUGCUUUU




 



RNAC
GUCCAAGACCUCAAUCCUUU
37224



S1812
GUUUUAGAGCUAGAAAUAGC





AAGUUAAAAUAAGGCUAGUC





CGUUAUCAACUUGAAAAAGU





GGCACCGAGUCGGUGCUUUU




 



RNAC
UAGCGAACUGAGAAGGGCCA
37225



S1834
GUUUUAGAGCUAGAAAUAGC





AAGUUAAAAUAAGGCUAGUC





CGUUAUCAACUUGAAAAAGU





GGCACCGAGUCGGUGCUUUU




 



RNAC
UGAGAAGGGCCGAGGUAUUG
37226



S1831
GUUUUAGAGCUAGAAAUAGC





AAGUUAAAAUAAGGCUAGUC





CGUUAUCAACUUGAAAAAGU





GGCACCGAGUCGGUGCUUUU

















TABLE E4







Exemplary Template RNAs and Sequences for Mutation Installation










RN


SEQ


ACS


ID


#
Name
Sequence
NO






hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmA
30506



4_R18
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P9
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrU





rUrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 



hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmA
30507



4_R16
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P9
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrUrG





rGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 



hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmA
30508



4_R16
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P10
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrUrG





rGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 



hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmA
30509



4_R18
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P10
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrU





rUrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 



hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmA
30510



4_R18
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P11
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrU





rUrGrGrCrCrCrUrUrCrUrCrArGrUrUrCrG*mC*mU*mA



 



hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmA
30511



4_R18
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P8
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrU





rUrGrGrCrCrCrUrUrCrUrCrArGrU*mU*mC*mG



 



hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmA
30512



4_R20
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P9
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCrArCrArArUrArC





rCrUrUrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 



hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmA
30514



4_R24
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P9
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCrUrGrCrCrArCrA





rArUrArCrCrUrUrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 



hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmA
30515



4_R22
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P9
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGrCrCrArCrArArU





rArCrCrUrUrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 



hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmA
30516



3_R17
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P8
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrUrG





rGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 



hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmA
30517



3_R19
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P8
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrU





rUrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 



hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmA
30518



3_R17
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P9
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrUrG





rGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 



hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmA
30519



3_R17
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P10
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrUrG





rGrCrCrCrUrUrCrUrCrArGrUrUrCrG*mC*mU*mA



 



hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmA
30520



3_R19
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P9
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrU





rUrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 



hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmA
30521



3_R23
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P8
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGrCrCrArCrArArU





rArCrCrUrUrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 



hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmA
30522



3_R19
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P10
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrU





rUrGrGrCrCrCrUrUrCrUrCrArGrUrUrCrG*mC*mU*mA



 



hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmA
30523



3_R17
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P12
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrUrG





rGrCrCrCrUrUrCrUrCrArGrUrUrCrGrCrU*mA*mC*mG



 



hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmA
30524



3_R17
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P11
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrUrG





rGrCrCrCrUrUrCrUrCrArGrUrUrCrGrC*mU*mA*mC



 



hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmA
30525



3_R19
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm




P11
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrU





rUrGrGrCrCrCrUrUrCrUrCrArGrUrUrCrGrC*mU*mA*mC



 


RN
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmA
30526


ACS
6_R9P
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm



3649
12
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrGrGrCrCrArArGrG





rUrArUrUrGrUrGrGrC*mA*mG*mC



 


RN
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmA
30527


ACS
6_R17
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm



3650
P11
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrUrGrArGrArArG





rGrGrCrCrArArGrGrUrArUrUrGrUrGrG*mC*mA*mG



 


RN
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmA
30528


ACS
6_R13
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm



3651
P9
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArGrArArGrGrGrCrC





rArArGrGrUrArUrUrGrU*mG*mG*mC



 


RN
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmA
30529


ACS
6_R11
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm



3652
P11
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArGrGrGrCrCrArA





rGrGrUrArUrUrGrUrGrG*mC*mA*mG



 


RN
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmA
30530


ACS
6_R15
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm



3653
P11
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGrArGrArArGrGrG





rCrCrArArGrGrUrArUrUrGrUrGrG*mC*mA*mG



 


RN
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmA
30531


ACS
6_R13
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm



3654
P10
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArGrArArGrGrGrCrC





rArArGrGrUrArUrUrGrUrG*mG*mC*mA



 


RN
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmA
30532


ACS
6_R11
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm



3655
P13
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArGrGrGrCrCrArA





rGrGrUrArUrUrGrUrGrGrCrA*mG*mC*mA



 


RN
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmA
30533


ACS
6_R9P
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm



1799
11
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrGrGrCrCrArArGrG





rUrArUrUrGrUrGrG*mC*mA*mG



 


RN
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmA
30534


ACS
6_R13
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm



3656
P13
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArGrArArGrGrGrCrC





rArArGrGrUrArUrUrGrUrGrGrCrA*mG*mC*mA



 


RN
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmA
30535


ACS
6_R11
mAmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAm



3657
P9
CmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArGrGrGrCrCrArA





rGrGrUrArUrUrGrU*mG*mG*mC
















TABLE E5







Further Exemplary Template RNAs and Sequences for Mutation Correction













SEQ





ID


RNACS#
Name
Sequence
NO





RNACS
hPKU
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30536


4947
5_R12
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P10
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrUrCr





GrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30537


2300
5_R10
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P11
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGr





CrCrCrUrUrCrUrC*mA*mG*mU



 


RNACS
hPKU
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30538


2301
5_R12
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P12
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrUrCr





GrGrCrCrCrUrUrCrUrCrA*mG*mU*mU



 


RNACS
hPKU
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30539


4134
5_R10
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P10
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGr





CrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30540


4172
5_R18
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P10
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCrUrGrCrCrArCrArAr





UrArCrCrUrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30541


4142
5_R12
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P11
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrUrCr





GrGrCrCrCrUrUrCrUrC*mA*mG*mU



 


RNACS
hPKU
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30542


4163
5_R16
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P9
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGrCrCrArCrArArUrAr





CrCrUrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30543


4173
5_R18
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P9
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCrUrGrCrCrArCrArAr





UrArCrCrUrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30544


4181
5_R20
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P10
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUrGrCrUrGrCrCrArCr





ArArUrArCrCrUrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30545


4135
5_R10
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P9
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGr





CrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30546


1763
4_R18
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P9
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrUrCr





GrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30547


3878
4_R16
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P9
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGr





CrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30548


3877
4_R16
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P10
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGr





CrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30549


3887
4_R18
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P10
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrUrCr





GrGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30550


3886
4_R18
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P11
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrUrCr





GrGrCrCrCrUrUrCrUrCrArGrUrUrCrG*mC*mU*mA



 


RNACS
hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30551


3888
4_R18
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P8
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrUrCr





GrGrCrCrCrUrUrCrUrCrArGrU*mU*mC*mG



 


RNACS
hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30552


3897
4_R20
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P9
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCrArCrArArUrArCrCr





UrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30553


3868
4_R14
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P9
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrArCrCrUrCrGrGrCrCr





CrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30554


3917
4_R24
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P9
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCrUrGrCrCrArCrArAr





UrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30555


3907
4_R22
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P9
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGrCrCrArCrArArUrAr





CrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30556


4048
3_R17
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P8
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGr





CrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30557


4057
3_R19
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P8
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrUrCr





GrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30558


4047
3_R17
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P9
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGr





CrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30559


4046
3_R17
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P10
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGr





CrCrCrUrUrCrUrCrArGrUrUrCrG*mC*mU*mA



 


RNACS
hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30560


1747
3_R19
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P9
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrUrCr





GrGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30561


4077
3_R23
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P8
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGrCrCrArCrArArUrAr





CrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrUmC*mG*mC



 


RNACS
hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30562


4056
3_R19
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P10
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrUrCr





GrGrCrCrCrUrUrCrUrCrArGrUrUrCrG*mC*mU*mA



 


RNACS
hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30563


4044
3_R17
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P12
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGr





CrCrCrUrUrCrUrCrArGrUrUrCrGrCrU*mA*mC*mG



 


RNACS
hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30564


4045
3_R17
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P11
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArUrArCrCrUrCrGrGr





CrCrCrUrUrCrUrCrArGrUrUrCrGrC*mU*mA*mC



 


RNACS
hPKU
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUrUrUrArGrAmGmCmUmAmGmAm
30565


4055
3_R19
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P11
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrUrCr





GrGrCrCrCrUrUrCrUrCrArGrUrUrCrGrC*mU*mA*mC



 


RNACS
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmAm
30566


3640
6_R9P
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




12
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrGrGrCrCrGrArGrGrUr





ArUrUrGrUrGrGrC*mA*mG*mC



 


RNACS
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmAm
30567


3641
6_R17
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P11
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCrUrGrArGrArArGrGr





GrCrCrGrArGrGrUrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmAm
30568


3642
6_R13
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P9
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArGrArArGrGrGrCrCrGr





ArGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmAm
30569


3643
6_R11
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P11
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArGrGrGrCrCrGrArGr





GrUrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmAm
30570


3644
6_R15
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P11
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGrArGrArArGrGrGrCr





CrGrArGrGrUrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmAm
30571


3645
6_R13
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P10
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArGrArArGrGrGrCrCrGr





ArGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmAm
30572


3646
6_R11
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P13
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArGrGrGrCrCrGrArGr





GrUrArUrUrGrUrGrGrCrA*mG*mC*mA



 


RNACS
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmAm
30573


1792
6_R9P
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




11
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrGrGrCrCrGrArGrGrUr





ArUrUrGrUrGrG*mC*mA*mG



 


RNACS
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmAm
30574


3647
6_R13
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P13
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArGrArArGrGrGrCrCrGr





ArGrGrUrArUrUrGrUrGrGrCrA*mG*mC*mA



 


RNACS
hPKU
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUrUrUrArGrAmGmCmUmAmGmAm
30575


3648
6_R11
AmAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCm




P9
UmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArArGrGrGrCrCrGrArGr





GrUrArUrUrGrU*mG*mG*mC









Table E5A shows the sequences of E5 without chemical modifications. In some embodiments, the sequences of Table E5 may be used without chemical modifications, or with one or more chemical modifications.








TABLE E5A







Table E5 Sequences without Chemical Modifications













SEQ


RNACS#
Name
Sequence
ID NO





RNACS
hPKU5_
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37227


4947
R12P10
UUGAAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAG



 


RNACS
hPKU5_
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37228


2300
R10P11
UUGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGU



 


RNACS
hPKU5_
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37229


2301
R12P12
UUGAAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGUU



 


RNACS
hPKU5_
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37230


4134
R10P10
UUGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAG



 


RNACS
hPKU5_
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37231


4172
R18P10
UUGAAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGGCCCUUCUCAG



 


RNACS
hPKU5_
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37232


4142
R12P11
UUGAAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGU



 


RNACS
hPKU5_
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37233


4163
R16P9
UUGAAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGGCCCUUCUCA



 


RNACS
hPKU5_
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37234


4173
R18P8
UUGAAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGGCCCUUCUC



 


RNACS
hPKU5_
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37235


4181
R20P10
UUGAAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACAAUACCUCGGCCCUUCUCAG



 


RNACS
hPKU5_
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37236


4135
R10P9
UUGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCA



 


RNACS
hPKU4_
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37237


1763
R18P9
UUGAAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS
hPKU4_
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37238


3878
R16P9
UUGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS
hPKU4_
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37239


3877
R16P10
UUGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCGCU



 


RNACS
hPKU4_
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37240


3887
R18P10
UUGAAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGUUCGCU



 


RNACS
hPKU4_
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37241


3886
R18P11
UUGAAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGUUCGCUA



 


RNACS
hPKU4_
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37242


3888
R18P8
UUGAAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGUUCG



 


RNACS
hPKU4_
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37243


3897
R20P9
UUGAAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS
hPKU4_
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37244


3868
R14P9
UUGAAAAAGUGGCACCGAGUCGGUGCUACCUCGGCCCUUCUCAGUUCGC



 


RNACS
hPKU4_
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37245


3917
R24P9
UUGAAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS
hPKU4_
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37246


3907
R22P9
UUGAAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS
hPKU3_
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37247


4048
R17P8
UUGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS
hPKU3_
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37248


4057
R19P8
UUGAAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS
hPKU3_
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37249


4047
R17P9
UUGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCGCU



 


RNACS
hPKU3_
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37250


4046
R17P10
UUGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCGCUA



 


RNACS
hPKU3_
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37251


1747
R19P9
UUGAAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGUUCGCU



 


RNACS
hPKU3_
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37252


4077
R23P8
UUGAAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS
hPKU3_
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37253


4056
R19P10
UUGAAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGUUCGCUA



 


RNACS
hPKU3_
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37254


4044
R17P12
UUGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCGCUACG



 


RNACS
hPKU3_
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37255


4045
R17P11
UUGAAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCGCUAC



 


RNACS
hPKU3_
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37256


4055
R19P11
UUGAAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGUUCGCUAC



 


RNACS
hPKU6_
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37257


3640
R9P12
UUGAAAAAGUGGCACCGAGUCGGUGCGGGCCGAGGUAUUGUGGCAGC



 


RNACS
hPKU6_
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37258


3641
R17P11
UUGAAAAAGUGGCACCGAGUCGGUGCACUGAGAAGGGCCGAGGUAUUGUGGCAG



 


RNACS
hPKU6_
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37259


3642
R13P9
UUGAAAAAGUGGCACCGAGUCGGUGCAGAAGGGCCGAGGUAUUGUGGC



 


RNACS
hPKU6_
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37260


3643
R11P11
UUGAAAAAGUGGCACCGAGUCGGUGCAAGGGCCGAGGUAUUGUGGCAG



 


RNACS
hPKU6_
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37261


3644
R15P11
UUGAAAAAGUGGCACCGAGUCGGUGCUGAGAAGGGCCGAGGUAUUGUGGCAG



 


RNACS
hPKU6_
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37262


3645
R13P10
UUGAAAAAGUGGCACCGAGUCGGUGCAGAAGGGCCGAGGUAUUGUGGCA



 


RNACS
hPKU6_
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37263


3646
R11P13
UUGAAAAAGUGGCACCGAGUCGGUGCAAGGGCCGAGGUAUUGUGGCAGCA



 


RNACS
hPKU6_
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37264


1792
R9P11
UUGAAAAAGUGGCACCGAGUCGGUGCGGGCCGAGGUAUUGUGGCAG



 


RNACS
hPKU6_
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37265


3647
R13P13
UUGAAAAAGUGGCACCGAGUCGGUGCAGAAGGGCCGAGGUAUUGUGGCAGCA



 


RNACS
hPKU6_
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
37266


3648
R11P9
UUGAAAAAGUGGCACCGAGUCGGUGCAAGGGCCGAGGUAUUGUGGC









The gene modifying system comprising mRNA encoding the gene modifying polypeptide listed above, a template RNA listed above, and a second strand-targeting gRNA described above were transfected into primary human hepatocytes. The gene modifying polypeptide, template RNA, and second strand-targeting gRNA were delivered by nucleofection in the RNA format. Specifically, 4 μg of gene modifying polypeptide mRNA were combined with 10 μg of chemically synthesized template RNA in 5 μL of water. The transfection mix was added to 100,000 primary hepatocytes in Buffer P3 [Lonza], and cells were nucleofected using program DG-138. After nucleofection, cells were grown at 37° C., 5% CO2 for 3 days prior to cell lysis and genomic DNA extraction. To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. Conversion of C nucleotide to T nucleotide indicates successful editing.
FIG. 25A shows a heatmap of % rewriting for each combination of template RNA and second strand-targeting RNA. The results show that many combinations of exemplary template RNAs and exemplary second strand-targeting gRNAs facilitated installation of the R408W mutation into hPAH gene of primary human hepatocytes.
The gene modifying system comprising mRNA encoding the gene modifying polypeptide listed above, a template RNA listed above, and a second strand-targeting gRNA described above were transfected into hepatoblasts differentiated from CRISPR-edited iPSCs containing the R408W mutation in the human PAH gene. Briefly PAH iPSCs are dissociated into single cells and then replated onto Geltex-coated plates. The iPSCs were then differentiated into definitive endoderm cells by treatment with Activin A, FGF2, and ChIR for 7 days. Definitive endoderm cells are then further patterned into foregut endoderm cells by activation of the BMP4 and FGF2 signaling pathway for an additional 6 days. Lastly, foregut endoderm cells were patterned into hepatoblast cells by treatment with oncostamin M, dexamethasone, hepatocyte growth factors, and ChIR for 12 days. Hepatoblasts were then sub-cultured onto collagen1-coated plates and expanded in media containing FGF19, Dexamethazone, ChIR, and SB431542 prior to transfection. The gene modifying polypeptide, template RNA, and second strand-targeting gRNA were delivered by nucleofection in the RNA format. Specifically, 4 μg of gene modifying polypeptide mRNA were combined with 10 μg of chemically synthesized template RNA, with or without 10 μg of second strand-targeting gRNA, in 5 μL of water. The transfection mix was added to 100,000 iPSCs in Buffer P3 [Lonza], and cells were nucleofected using program DG-138. After nucleofection, cells were grown at 37° C., 5% CO2 for 3 days prior to cell lysis and genomic DNA extraction. To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. Conversion of T>C indicates successful editing.
FIG. 25B shows a heatmap of % rewriting for each combination of template RNA and second strand-targeting RNA. The results show that many combinations of exemplary template RNAs and exemplary second strand-targeting gRNAs facilitated a corrective W408R mutation in the mutant hPAH gene of iPSC hepatoblasts. In particular, template RNAs RNACS1747, RNACS3877, RNACS4135, RNACS4134, RNACS2300, RNACS2299, RNACS4142, RNACS4173, RNACS4045, RNACS4048, and RNACS1763 showed the highest rewriting activity in this experiment.
The gene modifying system comprising mRNA encoding the gene modifying polypeptide listed above, a template RNA listed above, and a second strand-targeting gRNA described above were nucleofected in primary mouse hepatocytes from transgenic animals harboring the humanized region of exon 12 of the human PAH gene and contain the R408W PAH mutation. The gene modifying polypeptide, template RNA, and second strand-targeting gRNA were delivered by nucleofection in the RNA format. Specifically, 4 μg of gene modifying polypeptide mRNA were combined with 10 μg of chemically synthesized template RNA in 5 μL of water. The transfection mix was added to 100,000 primary hepatocytes in Buffer P3 [Lonza], and cells were nucleofected using program DG-138. After nucleofection, cells were grown at 37° C., 5% CO2 for 3 days prior to cell lysis and genomic DNA extraction. To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. Conversion of C nucleotide to T nucleotide indicates successful editing.
FIG. 25C shows a heatmap of % rewriting for each combination of template RNA and second strand-targeting RNA. The results show that many combinations of exemplary template RNAs and exemplary second strand-targeting gRNAs facilitated a corrective W408R mutation in the mutant hPAH gene of primary mouse hepatocytes. In particular, template RNAs RNACS4134 and RNACS1792 showed the highest rewriting activity in this experiment. In particular, template RNAs RNACS4134 and RNACS1792 showed high rewriting activity in combination with second strand-targeting gRNA RNACS1812 (about 17% and about 14% editing, respectively).
The gene modifying system comprising the gene modifying polypeptide listed above, a template RNA listed above, and a second strand-targeting gRNA listed above is formulated in LNP and delivered to mice. Specifically, approximately 2.4 mg/kg of total RNA equivalent formulated in LNPs (4:1 N:P ratio for mRNA, and 3:1 N:P ratio for tgRNA/gRNA), combined at 1:1:1 (w/w) of template RNA:mRNA:gRNA, are dosed intravenously in 8 to 10-week-old, mixed gender hPAH mice (0.8 mg/kg each of template RNA, mRNA, and gRNA) in a 10 ml/kg bolus. Mice are administered a dose at time 0 (t=0). 7 days post-dosing (as used herein post-dosing refers to time since the first dose), animals are sacrificed, and their liver and plasma are collected for analyses.
7-day plasma samples are analyzed by LC/MS to determine the level of phenylalanine present. Using CRISPR, hPAH transgenic mice were generated with a humanized region of exon 12 of the human PAH gene and contain the R408W PAH mutation that inactivates the PAH enzyme, resulting in sharply higher Phe levels in plasma than healthy wildtype mice. Successful rewriting of the hPAH transgene is expected to result in a decrease in Phe levels in plasma.]]
Phenylalanine is extracted from mouse plasma using protein precipitation and is analyzed by a LC-MS/MS system equipped with a Shimadzu Nexera UPLC (LC-40) coupled to a Sciex API 7500 mass spectrometer. Surrogate analyte (13C2,15N-Phenylalanine) is used for phenylalanine quantitation. Equivalence of ionization for naturally occurring and surrogate compounds is established prior to and after analytical run. Data are collected in the positive ion mode with three MRM transitions, 169.1 to 123.1 (13C2,15N-Phenylalanine), 166.1 to 120.1 (Phenylalanine) and 172.1 to 126.0 (13C6-Phenylalanine, internal standard). Extracted samples are injected onto an Acquity UPLC BEH C18 column (1.7 μm, 2.1×50 mm) and eluted using a gradient of 0% to 15% of mobile phase B at a flow rate of 0.8 mL/min with a total run time of 3 min per injection. Mobile phase A is 100:2:0.1 H2O:Formic acid (FA): Trifluoroacetic acid (TFA) and mobile phase B is 95:5:2:0.1 ACN:H2O:FA:TFA. Data are processed using Sciex OS software.
The results will show that Phe levels decreased in plasma from mice with exemplary gene modifying systems targeting hPAH.
7-day liver samples are analyzed using Amp-Seq to determine % rewriting in target liver cells. To analyze gene editing activity, primers flanking the target insertion site locus are used to amplify across the locus in the genomic DNA of liver samples. Amplicons are analyzed via short read sequencing using an Illumina MiSeq. Conversion of a T nucleotide to a C nucleotide at position 1222 in the PAH gene indicates successful editing. The results will show that rewriting is observed in mice treated with exemplary gene modifying systems targeting hPAH.
Example 14: Evaluating the Rewriting Activity Over Time and Short-Term Safety of Exemplary Murine Template RNAs and Second Strand-Targeting gRNAs in an ENU2 Mouse Model
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide, an exemplary template RNA, and exemplary second strand-targeting gRNA to evaluate the stability of rewriting over time and the short term safety of gene modifying systems used to correct the F263S mutation in ENU2 mice.
In this example, a template RNA contained:

    
    
        (1) a gRNA spacer;
        (2) a gRNA scaffold;
        (3) a heterologous object sequence; and
        (4) a primer binding site (PBS) sequence.
    
    


The exemplary template RNA tested was template RNA RNACS1855 shown in Table E1. The exemplary second strand-targeting gRNA was ngRNA5 (also referred to herein as RNACS2101), shown in Table E2. The exemplary gene modifying polypeptide used was RNAIVT338 (as described above and corresponding to the amino acid sequence of SEQ ID NO: 30480).
The gene modifying system was formulated in LNP and delivered to mice. [[Specifically, 2.4 mpk of total RNA equivalent formulated in LNPs, combined at 1:1 (w/w) of template RNA and mRNA, were dosed intravenously in 8 to 10-week-old, mixed gender ENU2 mice (0.8 mg/kg each of template RNA and mRNA with an additional 0.8 mg/kg of ngRNA in a 10 ml/kg bolus. Mice were administered a dose at time 0 (t=0). 7 days and 28 days post-dosing, animals were sacrificed, and their liver, brain, plasma collected for analyses.
7-day and 28-day liver samples were analyzed using Amp-Seq to determine % rewriting in target liver cells (FIG. 26A). To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus in the genomic DNA of liver samples. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. Conversion of a C nucleotide to a T nucleotide at position 835 in the PAH gene indicated successful editing. Rewriting was observed from all treated mice at the 7 d and 28 d timepoints, and the % rewriting at the 7 d and 28 d timepoints were very similar. Amplicon sequencing was also used to evaluate the degree to which INDELs were introduced into the liver sample DNA (FIG. 26B). As seen in other data described herein, the % INDEL was significantly lower than the % rewriting, and the % INDEL at the 7 d and 28 d timepoints were very similar.
7-day and 28-day plasma and brain samples were also analyzed by LC/MS to determine the level of phenylalanine present. ENU2 mice harbor a mutation in the murine PAH gene that inactivates the PAH enzyme, resulting in sharply higher Phe levels in plasma and brain than healthy wildtype mice.
Phenylalanine was extracted from mouse plasma and brain using protein precipitation and then analyzed by a LC-MS/MS system equipped with a Shimadzu Nexera UPLC (LC-40) coupled to a Sciex API 7500 mass spectrometer. Surrogate analyte (13C2,15N-Phenylalanine) was used for phenylalanine quantitation. Equivalence of ionization for naturally occurring and surrogate compounds was established prior to and after analytical run. Data were collected in the positive ion mode with three MRM transitions, 169.1 to 123.1 (13C2,15N-Phenylalanine), 166.1 to 120.1 (Phenylalanine) and 172.1 to 126.0 (13C6-Phenylalanine, internal standard). Extracted samples were injected onto an Acquity UPLC BEH C18 column (1.7 μm, 2.1×50 mm) and eluted using a gradient of 0% to 15% of mobile phase B at a flow rate of 0.8 mL/min with a total run time of 3 min per injection. Mobile phase A is 100:2:0.1 H2O:Formic acid (FA): Trifluoroacetic acid (TFA) and mobile phase B is 95:5:2:0.1 ACN:H2O:FA:TFA. Data were processed using Sciex OS software.
The results showed that Phe levels decreased in plasma from all treated mice, consistent with correction of the PAH deactivating point mutation using the gene modifying polypeptide and template RNAs examined (FIG. 27). The plasma Phe levels were consistently low at the 7 d and 28d timepoints, showing that treated mice achieved stable Phe levels comparable to strain-matched wildtype mice at least out to 28d. When considered together (FIGS. 26A-27), the results show that the exemplary gene modifying systems can be used to specifically rewrite the mPAH gene of ENU2 mice, resulting in stable genetic modification and stable therapeutic phenotypic change.
The results showed that Phe levels decreased in brain from all treated mice, consistent with correction of the PAH deactivating point mutation using the gene modifying polypeptide and template RNAs examined (FIG. 28). The brain Phe levels were consistently low at the 7 d and 28d timepoints, showing that treated mice achieved stable Phe levels comparable to strain-matched wildtype mice at least out to 28d. When considered together (FIGS. 26A-26B and 28), the results show that the exemplary gene modifying systems can be used to specifically rewrite the mPAH gene of ENU2 mice, resulting in stable genetic modification and stable therapeutic phenotypic change.
FIG. 29 shows a graph plotting correlation of plasma and brain Phe levels from samples used to generate FIGS. 27 and 28. The results show that plasma Phe level and brain Phe levels strongly correlate, with samples derived from saline treated ENU2 mice having higher Phe levels in plasma and in brain and samples derived from ENU2 mice treated with exemplary gene modifying systems as described herein having much lower Phe levels in plasma and in brain, similar to samples from saline treated wildtype mice.
At 90 days post dosing, animals are sacrificed, and their liver, brain, and plasma collected for analyses. The above analyses of % rewriting, % INDEL, and plasma Phe level are repeated with 90 d liver, brain, and plasma samples. The results will show comparable rewriting and INDEL levels to 7 d and 28 d samples from treated mice, and comparable Phe plasma and brain levels to 7 d and 28 d samples from treated mice.
Example 15: Evaluating Rewriting Activity of Exemplary Template RNAs and Second Strand-Targeting gRNAs in Cynomolgus Macaque Hepatocytes
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide, template RNAs comprising varied spacers, lengths and compositions of heterologous object sequences, and PBS sequences, and either of two second strand-targeting gRNAs to evaluate the activity of template RNAs and second strand-targeting gRNAs to produce a R408 silent mutation (C to A) or P407 silent mutation (T to C) mutation into wild-type PAH in primary cyno hepatocytes. Sequences (e.g., spacer sequences) of template RNAs or combinations of sequences of template RNAs and second strand-targeting gRNAs that provide high rewriting activity in a comparable model system may also provide rewriting activity in therapeutic template RNAs and second strand-targeting gRNAs designed for correcting pathogenic hPAH mutations in a human subject.
In this example, a template RNA contained:

    
    
        (1) a gRNA spacer;
        (2) a gRNA scaffold;
        (3) a heterologous object sequence; and
        (4) a primer binding site (PBS) sequence.
    
    


Exemplary template RNAs generated and used are given in Table EX. Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2′-O-methyl groups denoted by an ‘m’ preceding a nucleotide. The exemplary gene modifying polypeptide is RNAIVT338, comprising the amino acid sequence of SEQ ID NO: 30480. Exemplary second strand-targeting gRNAs for use with cPKU4, cPKU5.1, and cPKU5.2 template RNAs are RNACS1809 and RNACS1810, whereas exemplary second strand-targeting gRNAs for use with cPKU6 template RNAs are RNACS1906, RNACS1812, and RNACS1813, the nucleic acid sequence and chemical modifications of which are given here.
RNACS1809:









RNACS1809:



(SEQ ID NO: 30576)



mG*mU*mG*rCrCrCrUrUrCrArCrUrCrArArGrCrCr



 



UrGrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU



 



*mU*mU



 



RNACS1810:



(SEQ ID NO: 30577)



mU*mU*mC*rArCrUrCrArArGrCrCrUrGrUrGrGrUr



 



UrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU



 



*mU*mU



 



RNACS1812:



(SEQ ID NO: 30578)



mG*mU*mC*rCrArArGrArCrCrUrCrArArUrCrCrUr



 



UrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU



 



*mU*mU



 



RNACS1813:



(SEQ ID NO: 30579)



mU*mG*mU*rCrCrArArGrArCrCrUrCrArArUrCrCr



 



UrUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU



 



*mU*mU



 



RNACS1906:



(SEQ ID NO: 30580)



mC*mC*mU*rCrArArUrCrCrUrUrUrGrGrGrUrGrUr



 



ArUrGrUrUrUrUrArGrAmGmCmUmAmGmAmAmAmUmAm



 



GmCrArArGrUrUrArArArArUrArArGrGrCrUrArGr



 



UrCrCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAm



 



GmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU



 



*mU*mU













TABLE EX







Further Exemplary Template RNAs and Sequences for use in C. macaques













SEQ





ID


RNACS#
Name
Sequence
NO





RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30581


2611
_P9R16
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




_CtoA-
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrCrCrCrUrArGrGrCrCrCrUrUrUrUrCrArGrUrU*mC*mG*mC



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30582


2620
_P9R18
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




_CtoA-
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrCrCrCrUrArGrGrCrCrCrUrUrUrUrCrArGrUrU*mC*mG*





mC



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30583


2610
_P10R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




6_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrCrCrCrUrArGrGrCrCrCrUrUrUrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30584


2602
_P9R14
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




_CtoA-
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrCr





CrCrUrArGrGrCrCrCrUrUrUrUrCrArGrUrU*mC*mG*mC



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30585


2618
_P11R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




8_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrCrCrCrUrArGrGrCrCrCrUrUrUrUrCrArGrUrUrCrG*mC





*mU*mA



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30586


2609
_P11R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




6_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrCrCrCrUrArGrGrCrCrCrUrUrUrUrCrArGrUrUrCrG*mC*mU*





mA



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30587


2619
_P10R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




8_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrCrCrCrUrArGrGrCrCrCrUrUrUrUrCrArGrUrUrC*mG*m





C*mU



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30588


2606
_P14R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




6_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrCrCrCrUrArGrGrCrCrCrUrUrUrUrCrArGrUrUrCrGrCrUrA*





mU*mG*mA



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30589


2638
_P9R22
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




_CtoA-
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrCrCrCrUrArGrGrCrCrCrUrUrUrUrCrArGrUr





U*mC*mG*mC



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30590


2607
_P13R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




6_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrCrCrCrUrArGrGrCrCrCrUrUrUrUrCrArGrUrUrCrGrCrU*mA





*mU*mG



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30591


2648
_P8R24
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




_CtoA-
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrCrCrCrUrArGrGrCrCrCrUrUrUrUrCrAr





GrU*mU*mC*mG



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30592


2689
1_P12R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




12_Cto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




A-
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr




PSkill
ArArUrCrCrCrUrArGrArCrCrCrUrUrCrUrCrA*mG*mU*mU



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30593


2718
1_P10R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




18_Cto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




A-
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr




PSkill
UrGrCrCrArCrArArUrCrCrCrUrArGrArCrCrCrUrUrCrU*mC*m





A*mG



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30594


2691
1_P10R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




12_Cto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




A-
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr




PSkill
ArArUrCrCrCrUrArGrArCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30595


2690
1_P11R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




12_Cto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




A-
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr




PSkill
ArArUrCrCrCrUrArGrArCrCrCrUrUrCrUrC*mA*mG*mU



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30596


2717
1_P11R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




18_Cto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




A-
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr




PSkill
UrGrCrCrArCrArArUrCrCrCrUrArGrArCrCrCrUrUrCrUrC*mA





*mG*mU



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30597


2681
1_P11R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




10_
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




CtoA-
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr




PSkill
UrCrCrCrUrArGrArCrCrCrUrUrCrUrC*mA*mG*mU



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30598


2679
1_P13R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




10_
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




CtoA-
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr




PSkill
UrCrCrCrUrArGrArCrCrCrUrUrCrUrCrArG*mU*mU*mC



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30599


2680
1_P12R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




10_
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




CtoA-
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr




PSkill
UrCrCrCrUrArGrArCrCrCrUrUrCrUrCrA*mG*mU*mU



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30600


2709
1_P10R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




16_Cto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




A-
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr




PSkill
CrCrArCrArArUrCrCrCrUrArGrArCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30601


2692
1_P9R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




-PSkill
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




2_CtoA
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrCrCrCrUrArGrArCrCrCrUrUrC*mU*mC*mA



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30602


2736
1_P10R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




22_Cto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




A-
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr




PSkill
UrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrArGrArCrCrCrUrUr





CrU*mC*mA*mG



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30603


2738
1_P8R2
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




2_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





UrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrArGrArCrCrCrUrU*





mC*mU*mC



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30604


2782
2_P9R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




9_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrArArUrArCrCrUrArGrGrCrCrCrUrU*mC*m





U*mC



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30605


2773
2_P9R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




7_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrArGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30606


2764
2_P9R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




5_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrArGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30607


2781
2_P10R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




19_Cto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




A-
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr




PSkill
GrCrUrGrCrCrArCrArArUrArCrCrUrArGrGrCrCrCrUrUrC*mU





*mC*mA



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30608


2765
2_P8R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




5_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrArGrGrCrCrCrU*mU*mC*mU



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30609


2763
2_P10R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




15_Cto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




A-
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr




PSkill
CrCrArCrArArUrArCrCrUrArGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30610


2762
2_P11R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




15_Cto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




A-
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr




PSkill
CrCrArCrArArUrArCrCrUrArGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30611


2802
2_P7R2
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




3_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrArGrGrCrCrC*





mU*mU*mC



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30612


2775
2_P7R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




7_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrArGrGrCrCrC*mU*mU*mC



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30613


2800
2_P9R2
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




3_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrArGrGrCrCrCr





UrU*mC*mU*mC



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30614


2740
2_P15R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




11_Cto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




A-
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr




PSkill
ArArUrArCrCrUrArGrGrCrCrCrUrUrCrUrCrArGrU*mU*mC*mG



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30615


2758
2_P15R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




15_Cto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




A-
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr




PSkill
CrCrArCrArArUrArCrCrUrArGrGrCrCrCrUrUrCrUrCrArGrU*





mU*mC*mG



AC
_P10R1
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30616


2817
1_CtoA
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




cPKU6
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





GrGrGrUrCrUrArGrGrGrArUrUrGrUrG*mG*mC*mA



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30617


2818
_P9R11_
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




CtoA-
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





GrGrGrUrCrUrArGrGrGrArUrUrGrU*mG*mG*mC



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30618


2816
_P11R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




1_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





GrGrGrUrCrUrArGrGrGrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30619


2835
_P10R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




5_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





ArGrArArGrGrGrUrCrUrArGrGrGrArUrUrGrUrG*mG*mC*mA



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30620


2826
_P10R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




3_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArGr





ArArGrGrGrUrCrUrArGrGrGrArUrUrGrUrG*mG*mC*mA



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30621


2845
_P9R17_
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




CtoA-
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





UrGrArGrArArGrGrGrUrCrUrArGrGrGrArUrUrGrU*mG*mG*mC



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30622


2836
_P9R15_
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




CtoA-
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





ArGrArArGrGrGrUrCrUrArGrGrGrArUrUrGrU*mG*mG*mC



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30623


2827
_P9R13_
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




CtoA-
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArGr





ArArGrGrGrUrCrUrArGrGrGrArUrUrGrU*mG*mG*mC



 


RNACS
_P11R1
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30624


2825
3_CtoA
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




cPKU6
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArGr





ArArGrGrGrUrCrUrArGrGrGrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30625


2819
_P8R11_
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




CtoA-
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





GrGrGrUrCrUrArGrGrGrArUrUrG*mU*mG*mG



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30626


2813
_P14R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




1_CtoA
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




-PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





GrGrGrUrCrUrArGrGrGrArUrUrGrUrGrGrCrArG*mC*mA*mA



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30627


2820
_P7R11_
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




CtoA-
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




PSkill
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





GrGrGrUrCrUrArGrGrGrArUrU*mG*mU*mG



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30628


3178
_P9R15_
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArUr





CrCrCrCrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30629


3175
_P12R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




5_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArUr





CrCrCrCrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrCrGrC*mU*mA*





mU



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30630


3177
_P10R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




5_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArUr





CrCrCrCrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30631


3174
_P13R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




5_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArUr





CrCrCrCrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrCrGrCrU*mA*m





U*mG



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30632


3176
_P11R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




5_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArUr





CrCrCrCrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrCrG*mC*mU*mA



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30633


3173
_P14R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




5_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArUr





CrCrCrCrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrCrGrCrUrA*mU





*mG*mA



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30634


3179
_P8R15_
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArUr





CrCrCrCrCrGrGrCrCrCrUrUrCrUrCrArGrU*mU*mC*mG



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30635


3172
_P15R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




5_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArUr





CrCrCrCrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrCrGrCrUrArU*





mG*mA*mC



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30636


3187
_P9R17_
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrAr





ArUrCrCrCrCrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30637


3186
_P10R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




7_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrAr





ArUrCrCrCrCrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*





mU



 


RNACS
cPKU4
mG*mG*mG*rUrCrArUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30638


3204
_P10R2
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




1_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





CrArCrArArUrCrCrCrCrCrGrGrCrCrCrUrUrCrUrCrArGrUrUr





C*mG*mC*mU



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30639


3276
1_P10R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




15_Tto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




C
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





CrArArUrCrCrCrCrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30640


3277
1_P9R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




15_Tto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




C
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





CrArArUrCrCrCrCrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30641


3278
1_P8R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




5_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





CrArCrArArUrCrCrCrCrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30642


3249
1_P10R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




9_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArUr





CrCrCrCrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30643


3275
1_P11R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




15_Tto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




C
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





CrArCrArArUrCrCrCrCrCrGrGrCrCrCrUrUrCrUrC*mA*mG*mU



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30644


3303
1_P10R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




21_Tto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




C
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





UrGrCrUrGrCrCrArCrArArUrCrCrCrCrCrGrGrCrCrCrUrUrCr





U*mC*mA*mG



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30645


3250
1_P9R9_
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArUr





CrCrCrCrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30646


3304
1_P9R2
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




1_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





UrGrCrUrGrCrCrArCrArArUrCrCrCrCrCrGrGrCrCrCrUrUrC*





mU*mC*mA



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30647


3248
1_P11R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




9_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArUr





CrCrCrCrCrGrGrCrCrCrUrUrCrUrC*mA*mG*mU



 


RNACS
1_P12R
mAmUmAmGmCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrC
30648


3274
15_TtoC
rCrGrUrUrArUrCrAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmC




cPKU5.
mCmGmAmGmUmCmGmGmUmGmCrGrCrCrArCrArArUrCrCrCrCrCrG





rGrCrCrCrUrUrCrUrCrA*mG*mU*mUmU*mA*mG*rCrGrArArCrU





rGrArGrArArGrGrGrCrCrGrGrUrUrUrUrArGrAmGmCmUmAmGmA





mA



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30649


3295
1_P9R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




9_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrUrGrCrCrArCrArArUrCrCrCrCrCrGrGrCrCrCrUrUrC*mU*m





C*mA



 


RNACS
cPKU5.
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrGrGrUrUr
30650


3294
1_P10R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




19_Tto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




C
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrUrGrCrCrArCrArArUrCrCrCrCrCrGrGrCrCrCrUrUrCrU*mC





*mA*mG



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30651


3349
2_P9R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




4_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





CrArCrArArUrCrCrCrCrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30652


3347
2_P11R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




14_Tto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




C
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





CrArCrArArUrCrCrCrCrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30653


3385
2_P9R2
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




2_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





UrUrUrGrCrUrGrCrCrArCrArArUrCrCrCrCrCrGrGrCrCrCrUr





U*mC*mU*mC



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30654


3384
2_P10R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




22_Tto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




C
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





UrUrUrGrCrUrGrCrCrArCrArArUrCrCrCrCrCrGrGrCrCrCrUr





UrC*mU*mC*mA



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30655


3377
2_P8R2
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




0_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





UrGrCrUrGrCrCrArCrArArUrCrCrCrCrCrGrGrCrCrCrU*mU*m





C*mU



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30656


3350
2_P8R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




4_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





CrArCrArArUrCrCrCrCrCrGrGrCrCrCrU*mU*mC*mU



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30657


3378
2_P7R2
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




0_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





UrGrCrUrGrCrCrArCrArArUrCrCrCrCrCrGrGrCrCrC*mU*mU*





mC



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30658


3320
2_P11R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




8_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArUr





CrCrCrCrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30659


3348
2_P10R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




14_Tto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




C
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





CrArCrArArUrCrCrCrCrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30660


3360
2_P7R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




6_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrCrCrArCrArArUrCrCrCrCrCrGrGrCrCrC*mU*mU*mC



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30661


3331
2_P9R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




0_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrAr





ArUrCrCrCrCrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
cPKU5.
mA*mG*mC*rGrArArCrUrGrArGrArArGrGrGrCrCrGrArGrUrUr
30662


3328
2_P12R
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




10_Tto
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




C
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrAr





ArUrCrCrCrCrCrGrGrCrCrCrUrUrCrUrC*mA*mG*mU



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30663


3392
_P11R8_
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrGr





CrCrGrGrGrGrGrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30664


3391
_P12R8_
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrGr





CrCrGrGrGrGrGrArUrUrGrUrGrUrGrGrC*mA*mG*mC



 


RNACS
cPKU6_
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30665


3390
P13R8_
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrGr





CrCrGrGrGrGrGrArUrUrGrUrGrGrCrA*mG*mC*mA



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30666


3401
_P11R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




0_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArGr





GrGrCrCrGrGrGrGrGrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30667


3393
_P10R8_
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrGr





CrCrGrGrGrGrGrArUrUrGrUrG*mG*mC*mA



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30668


3402
_P10R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




0_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArGr





GrGrCrCrGrGrGrGrGrArUrUrGrUrG*mG*mC*mA



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30669


3400
_P12R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




0_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArGr





GrGrCrCrGrGrGrGrGrArUrUrGrUrGrGrC*mA*mG*mC



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30670


3389
_P14R8_
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrGr





CrCrGrGrGrGrGrArUrUrGrUrGrGrCrArG*mC*mA*mA



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30671


3403
_P9R10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArGr





GrGrCrCrGrGrGrGrGrArUrUrGrU*mG*mG*mC



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30672


3394
_P9R8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrGr





CrCrGrGrGrGrGrArUrUrGrU*mG*mG*mC



 


RNACS
cPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrCrCrCrUrGrUrUr
30673


3410
_P11R1
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




2_TtoC
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArGrGrGrCrCrGrGrGrGrGrArUrUrGrUrGrG*mC*mA*mG









The gene modifying system comprising mRNA encoding the gene modifying polypeptide listed above, a template RNA listed above, and a second strand-targeting gRNA described above were transfected into primary cyno hepatocytes. The gene modifying polypeptide, template RNA, and second strand-targeting gRNA were delivered by nucleofection in the RNA format. Specifically, 4 μg of gene modifying polypeptide mRNA were combined with 4 μg of chemically synthesized template RNA and 4 ug of nicking gRNA in 5 μL of water. The transfection mix was added to 100,000 primary hepatocytes in Buffer P3 [Lonza], and cells were nucleofected using program DG-138. After nucleofection, cells were grown at 37° C., 5% CO2 for 3 days prior to cell lysis and genomic DNA extraction. To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. Conversion of T to C or C to A nucleotide indicated successful editing.
FIGS. 30A-30H show heatmaps of % rewriting for each combination of template RNA and second strand-targeting RNA, with FIGS. 30A-30D showing C to A rewriting and FIGS. 30E-30H showing T to C rewriting and grouped by the spacer of the template RNA. The results show that many combinations of exemplary template RNAs and exemplary second strand-targeting gRNAs facilitated installation of the silent mutations at R408 or P407 positions into cPAH gene of primary cyno hepatocytes. The results showed that for C to A editing, cPKU6 template RNAs showed the highest % rewriting compared to other spacers. For this edit, the second strand-targeting gRNA RNACS1906 showed the highest % rewriting combined with cPKU6 template RNAs (e.g., RNACS2827 template RNA combined with RNACS1906 showed 43% rewriting). The results showed that for T to C editing, cPKU5.2 template RNAs showed the highest % rewriting compared to other spacers, followed by cPKU6 template RNAs. For this edit, the second strand-targeting gRNA RNACS1810 showed the highest % rewriting combined with cPKU5.2 template RNAs (e.g., RNACS3349 template RNA combined with RNACS1810 showed 57% rewriting), whereas the second strand-targeting gRNA RNACS1906 showed the highest % rewriting combined with cPKU6 template RNAs.
Example 16: Evaluating Impact of Different Silent Substitutions on Rewriting Activity in Human iPSC-Derived Hepatoblasts
This example describes the use of exemplary gene modifying systems containing a gene modifying polypeptide and template RNAs comprising four different spacers (hPKU3, hPKU4, hPKU5, and hPKU6), five lengths of heterologous object sequences, and three lengths of PBS sequences, wherein the template RNAs comprised one of five different silent substitutions. The example describes evaluation of the activity of template RNAs containing said silent substitutions to produce an W408R mutation to correct the R408W mutation in hPAH in CRISPR gene-edited iPSC-derived hepatoblast cells.
In this example, a template RNA contained:

    
    
        (1) a gRNA spacer;
        (2) a gRNA scaffold;
        (3) a heterologous object sequence; and
        (4) a primer binding site (PBS) sequence.

Exemplary template RNAs generated and used are given in Table E6. Nucleotide modifications are noted as follows: phosphorothioate linkages denoted by an asterisk, 2′-O-methyl groups denoted by an ‘m’ preceding a nucleotide. The exemplary gene modifying polypeptide is RNAIVT338, comprising the amino acid sequence of SEQ ID NO: 30480.

    
    









TABLE E6







Exemplary Template RNAs with Various Silent Substitutions













SEQ ID


RNACS#
Name
IDT Notation
NO





RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30674


4741
R25 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrAr





GrUrUrCrG*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30675


4742
R25 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrAr





GrUrUrCrG*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30676


4743
R25 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrCrCrCrUrCrGrGrCrCrCrUrUrCrUrCrAr





GrUrUrCrG*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30677


4744
R25 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrAr





GrUrUrCrG*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30678


4745
R25 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrAr





GrUrUrCrG*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30679


4746
R25 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrAr





GrUrUrCrG*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30680


4747
R23 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUr





UrCrG*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30681


4748
R23 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUr





UrCrG*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30682


4749
R23 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrCrCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUr





UrCrG*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30683


4750
R23 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCr




sub4
ArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrAr





UrCrAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUm





CmGmGmUmGmCrUrGrCrCrArCrArArUrArCrCrUrCrGrCrCrCrCr





UrUrCrUrCrArGrUrUrCrG*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30684


4751
R23 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUr





UrCrG*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30685


4752
R23 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUr





UrCrG*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30686


4753
R21 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrCr





G*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30687


4754
R21 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrCr





G*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30688


4755
R21 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrCrCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrCr





G*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30689


4756
R21 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUrUrCr





G*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30690


4757
R21 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUrUrCr





G*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30691


4758
R21 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUrUrCr





G*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30692


4759
R19 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrCrG*mC





*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30693


4760
R19 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrCrG*mC





*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30694


4761
R19 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrCrCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrCrG*mC





*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30695


4762
R19 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmC





mAmCmCmGmAmGmUmCmGmGmUmGmCrArCrArArUrArCrCrUrCrGrC





rCrCrCrUrUrCrUrCrArGrUrUrCrG*mC*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30696


4763
R19 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUrUrCrG*mC





*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30697


4764
R19 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUrUrCrG*mC





*mU*mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30698


4765
R17 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrCrG*mC*mU*





mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30699


4766
R17 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrCrG*mC*mU*





mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30700


4767
R17 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrCrCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrCrG*mC*mU*





mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30701


4768
R17 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUrUrCrG*mC*mU*





mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30702


4769
R17 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUrUrCrG*mC*mU*





mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30703


4770
R17 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUrUrCrG*mC*mU*





mA



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30704


4771
R25 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrAr





GrUrUrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30705


4772
R25 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrAr





GrUrUrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30706


4773
R25 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrCrCrCrUrCrGrGrCrCrCrUrUrCrUrCrAr





GrUrUrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30707


4774
R25 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrAr





GrUrUrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30708


4775
R25 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrAr





GrUrUrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30709


4776
R25 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrAr





GrUrUrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30710


4777
R23 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUr





UrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30711


4778
R23 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUr





UrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30712


4779
R23 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmC





mAmCmCmGmAmGmUmCmGmGmUmGmCrUrGrCrCrArCrArArUrCrCrC





rUrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30713


4780
R23 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUr





UrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30714


4781
R23 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUr





UrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30715


4782
R23 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUr





UrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30716


4783
R21 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*





mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30717


4784
R21 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*





mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30718


4785
R21 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrCrCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*





mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30719


4786
R21 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUrUrC*





mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30720


4787
R21 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUrUrC*





mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30721


4788
R21 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUrUrC*





mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30722


4789
R19 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*m





C*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30723


4790
R19 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*m





C*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30724


4791
R19 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrCrCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*m





C*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30725


4792
R19 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUrUrC*mG*m





C*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30726


4793
R19 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUrUrC*mG*m





C*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30727


4794
R19 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUrUrC*mG*m





C*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30728


4795
R17 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30729


4796
R17 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30730


4797
R17 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrCrCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30731


4798
R17 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30732


4799
R17 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30733


4800
R17 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30734


4801
R25 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrAr





GrUrU*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30735


4802
R25 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrAr





GrUrU*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30736


4803
R25 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrCrCrCrUrCrGrGrCrCrCrUrUrCrUrCrAr





GrUrU*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30737


4804
R25 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrAr





GrUrU*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30738


4805
R25 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrAr





GrUrU*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30739


4806
R25 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrAr





GrUrU*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30740


4807
R23 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUr





U*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30741


4808
R23 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUr





U*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30742


4809
R23 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrCrCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUr





U*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30743


4810
R23 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUr





U*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30744


4811
R23 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUr





U*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30745


4812
R23 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUr





U*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30746


4813
R21 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC





*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30747


4814
R21 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC





*mG*mc



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30748


4815
R21 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrCrCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC





*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30749


4816
R21 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUrU*mC





*mG*mc



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30750


4817
R21 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUrU*mC





*mG*mc



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30751


4818
R21 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUrU*mC





*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30752


4819
R19 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*





mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30753


4820
R19 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUrUmC*mG*m





C



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30754


4821
R19 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrCrCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*





mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30755


4822
R19 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*





mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30756


4823
R19 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*





mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30757


4824

UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCr




R19 P8
ArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrAr




sub8
UrCrAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUm





CmGmGmUmGmCrArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUr





CrArGrUrU*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30758


4825
R17 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30759


4826
R17 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30760


4827
R17 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrCrCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30761


4828
R17 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30762


4829
R17 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU3
mU*mG*mG*rGrUrCrGrUrArGrCrGrArArCrUrGrArGrArGrUrUr
30763


4830
R17 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30764


4831
R24 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrAr





GrUrUrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30765


4832
R24 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrAr





GrUrUrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30766


4833
R24 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrAr





GrUrUrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30767


4834
R24 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrCrCrCrArUrUrCrUrCrAr





GrUrUrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30768


4835
R24 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrAr





GrUrUrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30769


4836
R24 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrAr





GrUrUrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30770


4837
R22 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUr





UrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30771


4838
R22 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUr





UrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30772


4839
R22 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUr





UrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30773


4840
R22 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrCrCrCrArUrUrCrUrCrArGrUr





UrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30774


4841
R22 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUr





UrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30775


4842
R22 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUr





UrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30776


4843
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*





mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30777


4844
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*





mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30778


4845
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUrUrC*





mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30779


4846
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrCrCrCrArUrUrCrUrCrArGrUrUrC*





mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30780


4847
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUrUrC*





mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30781


4848
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUrUrC*





mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30782


4849
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*m





C*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30783


4850
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*m





C*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30784


4851
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUrUrC*mG*m





C*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30785


4852
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrCrCrCrArUrUrCrUrCrArGrUrUrC*mG*m





C*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30786


4853
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUrUrC*mG*m





C*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30787


4854
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUrUrC*mG*m





C*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30788


4855
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30789


4856
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30790


4857
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30791


4858
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCr




sub5
ArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrAr





UrCrAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUm





CmGmGmUmGmCrArArUrArCrCrUrCrGrCrCrCrArUrUrCrUrCrAr





GrUrUrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30792


4859
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30793


4860
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUrUrC*mG*mC*mU



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30794


4861
R24 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrAr





GrUrU*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30795


4862
R24 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrAr





GrUrU*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30796


4863
R24 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrAr





GrUrU*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30797


4864
R24 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrCrCrCrArUrUrCrUrCrAr





GrUrU*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30798


4865
R24 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrAr





GrUrU*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30799


4866
R24 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrAr





GrUrU*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30800


4867
R22 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUr





U*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30801


4868
R22 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUr





U*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30802


4869
R22 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUr





U*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30803


4870
R22 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrCrCrCrArUrUrCrUrCrArGrUr





U*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30804


4871
R22 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUr





U*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30805


4872
R22 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUr





U*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30806


4873
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC





*mG*mc



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30807


4874
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCr




sub1
ArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrAr





UrCrAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUm





CmGmGmUmGmCrCrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUr





CrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30808


4875
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUrU*mC





*mG*mc



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30809


4876
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrCrCrCrArUrUrCrUrCrArGrUrU*mC





*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30810


4877
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUrU*mC





*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30811


4878
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUrU*mC





*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30812


4879
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*





mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30813


4880
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*





mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30814


4881
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUrUmC*mG*m





C



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30815


4882
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrCrCrCrArUrUrCrUrCrArGrUrU*mC*mG*





mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30816


4883
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*





mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30817


4884
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUrU*mC*mG*





mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30818


4885
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30819


4886
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30820


4887
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30821


4888
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrUrCrGrCrCrCrArUrUrCrUrCrArGrUrUmC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30822


4889
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30823


4890
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrUrU*mC*mG*mC



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30824


4891
R24 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrAr





GrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30825


4892
R24 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrAr





GrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30826


4893
R24 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrAr





GrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30827


4894
R24 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrCrCrCrArUrUrCrUrCrAr





GrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30828


4895
R24 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrAr





GrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30829


4896
R24 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrAr





GrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30830


4897
R22 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrU*





mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30831


4898
R22 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrU*





mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30832


4899
R22 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrU*





mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30833


4900
R22 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrCrCrCrArUrUrCrUrCrArGrU*





mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30834


4901
R22 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrU*





mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30835


4902
R22 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrU*





mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30836


4903
R20 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrU*mU*m





C*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30837


4904
R20 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrU*mU*m





C*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30838


4905
R20 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCr




sub4
ArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrAr





UrCrAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUm





CmGmGmUmGmCrCrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUr





CrUrCrArGrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30839


4906
R20 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrCrCrCrArUrUrCrUrCrArGrU*mU*m





C*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30840


4907
R20 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrU*mU*m





C*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30841


4908
R20 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrU*mU*m





C*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30842


4909
R18 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30843


4910
R18 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30844


4911
R18 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30845


4912
R18 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrCrCrCrArUrUrCrUrCrArGrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30846


4913
R18 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30847


4914
R18 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30848


4915
R16 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrUrCrGrGrCrCrCrUrUrCrUrCrArGrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30849


4916
R16 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrGrCrCrCrUrUrCrUrCrArGrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30850


4917
R16 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrUrCrGrCrCrCrCrUrUrCrUrCrArGrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30851


4918
R16 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrUrCrGrCrCrCrArUrUrCrUrCrArGrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30852


4919
R16 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrCrCrCrCrUrUrCrUrCrArGrU*mU*mC*mG



 


RNACS
hPKU4
mG*mG*mG*rUrCrGrUrArGrCrGrArArCrUrGrArGrArArGrUrUr
30853


4920
R16 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





UrArCrCrGrCrGrCrCrCrArUrUrCrUrCrArGrU*mU*mC*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30854


4923
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrU*





mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30855


4924
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrU*





mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30856


4925
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrArArUrCrCrCrUrCrGrGrCrCrCrUrUrCrU*





mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30857


4926
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrA




sub3
rArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArU





rCrAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmC





mGmGmUmGmCrUrUrGrCrUrGrCrCrArCrArArUrCrCrCrGrCrGrG





rCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30858


4927
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrU*





mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30859


4928
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrGrArUrArCrCrUrCrGrGrCrCrCrUrUrCrU*





mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30860


4929
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrU*mC*m





A*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30861


4930
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrU*mC*m





A*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30862


4931
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrCrCrCrUrCrGrGrCrCrCrUrUrCrU*mC*m





A*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30863


4932
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub3
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrCrCrCrGrCrGrGrCrCrCrUrUrCrU*mC*m





A*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30864


4933
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrU*mC*m





A*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30865


4934
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrGrArUrArCrCrUrCrGrGrCrCrCrUrUrCrU*mC*m





A*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30866


4935
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30867


4936
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30868


4937
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrCrCrCrUrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30869


4938
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub3
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrCrCrCrGrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30870


4939
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30871


4940
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrGrArUrArCrCrUrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30872


4941
R14 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30873


4942
R14 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30874


4943
R14 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrCrCrCrUrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30875


4944
R14 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub3
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrCrCrCrGrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30876


4945
R14 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30877


4946
R14 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrGrArUrArCrCrUrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30878


4947
R12 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30879


4948
R12 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30880


4949
R12 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrCrCrCrUrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30881


4950
R12 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub3
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrCrCrCrGrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30882


4951
R12 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrCrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30883


4952
R12 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





GrArUrArCrCrUrCrGrGrCrCrCrUrUrCrU*mC*mA*mG



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30884


4953
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrC*mU





*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30885


4954
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrC*mU





*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30886


4955
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrArArUrCrCrCrUrCrGrGrCrCrCrUrUrC*mU





*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30887


4956
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub3
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrArArUrCrCrCrGrCrGrGrCrCrCrUrUrC*mU





*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30888


4957
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrC*mU





*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30889


4958
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrGrArUrArCrCrUrCrGrGrCrCrCrUrUrC*mU





*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30890


4959
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrC*mU*mC*





mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30891


4960
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrC*mU*mC*





mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30892


4961
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrCrCrCrUrCrGrGrCrCrCrUrUrC*mU*mC*





mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30893


4962
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub3
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrCrCrCrGrCrGrGrCrCrCrUrUrC*mU*mC*





mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30894


4963
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrC*mU*mC*





mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30895


4964
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrGrArUrArCrCrUrCrGrGrCrCrCrUrUrC*mU*mC*





mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30896


4965
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30897


4966
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30898


4967
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrCrCrCrUrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30899


4968
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub3
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrCrCrCrGrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30900


4969
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30901


4970
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrGrArUrArCrCrUrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30902


4971
R14 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30903


4972
R14 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30904


4973
R14 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrCrCrCrUrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30905


4974
R14 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub3
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrCrCrCrGrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30906


4975
R14 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrCrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30907


4976
R14 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrGrArUrArCrCrUrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30908


4977
R12 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30909


4978
R12 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30910


4979
R12 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrCrCrCrUrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30911


4980
R12 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub3
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrCrCrCrGrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30912


4981
R12 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrCrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30913


4982
R12 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





GrArUrArCrCrUrCrGrGrCrCrCrUrUrC*mU*mC*mA



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30914


4983
R20 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrU*mC*m





U*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30915


4984
R20 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrU*mC*m





U*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30916


4985
R20 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrArArUrCrCrCrUrCrGrGrCrCrCrUrU*mC*m





U*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30917


4986
R20 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub3
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrArArUrCrCrCrGrCrGrGrCrCrCrUrU*mC*m





U*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30918


4987
R20 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrU*mC*m





U*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30919


4988
R20 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrUr





GrCrUrGrCrCrArCrGrArUrArCrCrUrCrGrGrCrCrCrUrU*mC*m





U*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30920


4989
R18 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30921


4990
R18 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30922


4991
R18 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrCrCrCrUrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30923


4992
R18 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub3
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrCrCrCrGrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30924


4993
R18 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30925


4994
R18 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrCr





UrGrCrCrArCrGrArUrArCrCrUrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30926


4995
R16 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30927


4996
R16 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrGrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30928


4997
R16 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrCrCrCrUrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30929


4998
R16 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub3
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrCrCrCrGrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30930


4999
R16 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrArArUrArCrCrUrCrGrCrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30931


5000
R16 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrUrGr





CrCrArCrGrArUrArCrCrUrCrGrGrCrCrCrUrUmC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30932


5001
R14 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30933


5002
R14 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrGrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30934


5003
R14 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrCrCrCrUrCrGrGrCrCrCrUrUmC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30935


5004
R14 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub3
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrCrCrCrGrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30936


5005
R14 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrArArUrArCrCrUrCrGrCrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30937


5006
R14 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrCr





ArCrGrArUrArCrCrUrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30938


5007
R12 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30939


5008
R12 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrGrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30940


5009
R12 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub2
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrCrCrCrUrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30941


5010
R12 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub3
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrCrCrCrGrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS

mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30942


5011
hPKU5
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




R12 P8
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm




sub4
AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





ArArUrArCrCrUrCrGrCrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU5
mU*mA*mG*rCrGrArArCrUrGrArGrArArGrGrGrCrCrArGrUrUr
30943


5012
R12 P8
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArCr





GrArUrArCrCrUrCrGrGrCrCrCrUrU*mC*mU*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30944


5013
R20 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArGrGrGrCrCrGrArGrGrUrArUrUrGrUrGr





G*mC*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30945


5014
R20 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArGrGrGrCrCrGrGrGrGrUrArUrUrGrUrGr





G*mC*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30946


5015
R20 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArCrGrGrCrCrGrGrGrGrUrArUrUrGrUrGr





G*mC*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30947


5016
R20 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArCrGrGrArCrGrGrGrGrUrArUrUrGrUrGr





G*mC*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30948


5017
R20 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub6
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArGrGrGrCrCrGrCrGrGrUrArUrUrGrUrGr





G*mC*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30949


5018
R20 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArGrGrGrArCrGrCrGrGrUrArUrUrGrUrGr





G*mC*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30950


5019
R18 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArGrGrGrCrCrGrArGrGrUrArUrUrGrUrGrG*mC





*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30951


5020
R18 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArGrGrGrCrCrGrGrGrGrUrArUrUrGrUrGrG*mC





*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30952


5021
R18 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArCrGrGrCrCrGrGrGrGrUrArUrUrGrUrGrG*mC





*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30953


5022
R18 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArCrGrGrArCrGrGrGrGrUrArUrUrGrUrGrG*mC





*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30954


5023
R18 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub6
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArGrGrGrCrCrGrCrGrGrUrArUrUrGrUrGrG*mC





*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30955


5024
R18 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArGrGrGrArCrGrCrGrGrUrArUrUrGrUrGrG*mC





*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30956


5025
R16 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArGrGrGrCrCrGrArGrGrUrArUrUrGrUrGrG*mC*mA*





mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30957


5026
R16 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArGrGrGrCrCrGrGrGrGrUrArUrUrGrUrGrG*mC*mA*





mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30958


5027
R16 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArCrGrGrCrCrGrGrGrGrUrArUrUrGrUrGrG*mC*mA*





mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30959


5028
R16 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArCrGrGrArCrGrGrGrGrUrArUrUrGrUrGrG*mC*mA*





mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30960


5029
R16 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub6
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArGrGrGrCrCrGrCrGrGrUrArUrUrGrUrGrG*mC*mA*





mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30961


5030
R16 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArGrGrGrArCrGrCrGrGrUrArUrUrGrUrGrG*mC*mA*





mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30962


5031
R14 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrA




sub0
rArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArU





rCrAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmC





mAmCmCmGmAmGmUmCmGmGmUmGmCrGrArGrArArGrGrGrCrCrGrA





rGrGrUrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30963


5032
R14 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





GrArArGrGrGrCrCrGrGrGrGrUrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30964


5033
R14 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





GrArArCrGrGrCrCrGrGrGrGrUrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30965


5034
R14 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





GrArArCrGrGrArCrGrGrGrGrUrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30966


5035
R14 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub6
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





GrArArGrGrGrCrCrGrCrGrGrUrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30967


5036
R14 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





GrArArGrGrGrArCrGrCrGrGrUrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30968


5037
R12 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArGrGrGrCrCrGrArGrGrUrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30969


5038
R12 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArGrGrGrCrCrGrGrGrGrUrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30970


5039
R12 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArCrGrGrCrCrGrGrGrGrUrArUrUrGrUrGrG*mC*mA*mG



RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30971


5040
R12 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArCrGrGrArCrGrGrGrGrUrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30972


5041
R12 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub6
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArGrGrGrCrCrGrCrGrGrUrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30973


5042
R12 P11
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArGrGrGrArCrGrCrGrGrUrArUrUrGrUrGrG*mC*mA*mG



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30974


5043
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArGrGrGrCrCrGrArGrGrUrArUrUrGrUrG*





mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30975


5044
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArGrGrGrCrCrGrGrGrGrUrArUrUrGrUrG*





mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30976


5045
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArCrGrGrCrCrGrGrGrGrUrArUrUrGrUrG*





mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30977


5046
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArCrGrGrArCrGrGrGrGrUrArUrUrGrUrG*





mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30978


5047
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub6
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArGrGrGrCrCrGrCrGrGrUrArUrUrGrUrG*





mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30979


5048
R20 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArGrGrGrArCrGrCrGrGrUrArUrUrGrUrG*





mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30980


5049
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArGrGrGrCrCrGrArGrGrUrArUrUrGrUrG*mG*m





C*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30981


5050
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArGrGrGrCrCrGrGrGrGrUrArUrUrGrUrG*mG*m





C*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30982


5051
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArCrGrGrCrCrGrGrGrGrUrArUrUrGrUrG*mG*m





C*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30983


5052
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArCrGrGrArCrGrGrGrGrUrArUrUrGrUrG*mG*m





C*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30984


5053
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub6
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArGrGrGrCrCrGrCrGrGrUrArUrUrGrUrG*mG*m





C*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30985


5054
R18 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArGrGrGrArCrGrCrGrGrUrArUrUrGrUrG*mG*m





C*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30986


5055
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArGrGrGrCrCrGrArGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30987


5056
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArGrGrGrCrCrGrGrGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30988


5057
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArCrGrGrCrCrGrGrGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30989


5058
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArCrGrGrArCrGrGrGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30990


5059
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub6
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArGrGrGrCrCrGrCrGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30991


5060
R16 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArGrGrGrArCrGrCrGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30992


5061
R14 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





GrArArGrGrGrCrCrGrArGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30993


5062
R14 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





GrArArGrGrGrCrCrGrGrGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30994


5063
R14 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





GrArArCrGrGrCrCrGrGrGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30995


5064
R14 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





GrArArCrGrGrArCrGrGrGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30996


5065
R14 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub6
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





GrArArGrGrGrCrCrGrCrGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30997


5066
R14 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





GrArArGrGrGrArCrGrCrGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30998


5067
R12 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArGrGrGrCrCrGrArGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
30999


5068
R12 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArGrGrGrCrCrGrGrGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31000


5069
R12 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArCrGrGrCrCrGrGrGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31001


5070
R12 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArCrGrGrArCrGrGrGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31002


5071
R12 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub6
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArGrGrGrCrCrGrCrGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31003


5072
R12 P10
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArGrGrGrArCrGrCrGrGrUrArUrUrGrUrG*mG*mC*mA



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31004


5073
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArGrGrGrCrCrGrArGrGrUrArUrUrGrU*mG





*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31005


5074
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArGrGrGrCrCrGrGrGrGrUrArUrUrGrU*mG





*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31006


5075
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArCrGrGrCrCrGrGrGrGrUrArUrUrGrU*mG





*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31007


5076
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArCrGrGrArCrGrGrGrGrUrArUrUrGrU*mG





*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31008


5077
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub6
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArGrGrGrCrCrGrCrGrGrUrArUrUrGrU*mG





*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31009


5078
R20 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrGr





ArArCrUrGrArGrArArGrGrGrArCrGrCrGrGrUrArUrUrGrU*mG





*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31010


5079
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArGrGrGrCrCrGrArGrGrUrArUrUrGrU*mG*mG*





mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31011


5080
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArGrGrGrCrCrGrGrGrGrUrArUrUrGrU*mG*mG*





mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31012


5081
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArCrGrGrCrCrGrGrGrGrUrArUrUrGrU*mG*mG*





mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31013


5082
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArCrGrGrArCrGrGrGrGrUrArUrUrGrU*mG*mG*





mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31014


5083
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub6
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArGrGrGrCrCrGrCrGrGrUrArUrUrGrU*mG*mG*





mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31015


5084
R18 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrArAr





CrUrGrArGrArArGrGrGrArCrGrCrGrGrUrArUrUrGrU*mG*mG*





mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31016


5085
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArGrGrGrCrCrGrArGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31017


5086
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArGrGrGrCrCrGrGrGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31018


5087
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArCrGrGrCrCrGrGrGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31019


5088
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArCrGrGrArCrGrGrGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31020


5089
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub6
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArGrGrGrCrCrGrCrGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31021


5090
R16 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrCrUr





GrArGrArArGrGrGrArCrGrCrGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31022


5091
R14 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





GrArArGrGrGrCrCrGrArGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31023


5092
R14 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





GrArArGrGrGrCrCrGrGrGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31024


5093
R14 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





GrArArCrGrGrCrCrGrGrGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31025


5094
R14 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





GrArArCrGrGrArCrGrGrGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31026


5095
R14 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub6
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





GrArArGrGrGrCrCrGrCrGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31027


5096
R14 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





GrArArGrGrGrArCrGrCrGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31028


5097
R12 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub0
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArGrGrGrCrCrGrArGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31029


5098
R12 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub1
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArGrGrGrCrCrGrGrGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31030


5099
R12 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub4
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArCrGrGrCrCrGrGrGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31031


5100
R12 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub5
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArCrGrGrArCrGrGrGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31032


5101
R12 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub6
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArGrGrGrCrCrGrCrGrGrUrArUrUrGrU*mG*mG*mC



 


RNACS
hPKU6
mA*mC*mU*rUrUrGrCrUrGrCrCrArCrArArUrArCrCrUrGrUrUr
31033


5102
R12 P9
UrUrArGrAmGmCmUmAmGmAmAmAmUmAmGmCrArArGrUrUrArArAr




sub7
ArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAmAmCmUmUmGm





AmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCrGrAr





ArGrGrGrArCrGrCrGrGrUrArUrUrGrU*mG*mG*mC









Table E6A shows the sequences of E6 without chemical modifications. In some embodiments, the sequences of Table E6A may be used without chemical modifications, or with one or more chemical modifications.








TABLE E6A







Table E6 Sequences without Chemical Modifications













SEQ ID


RNACS#
Name
IDT Notation
NO





RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37267


741
P10 sub0
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGGCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37268


742
P10 sub1
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGGCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37269


743
P10 sub2
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUCCCUCGGCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37270


744
P10 sub4
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGCCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37271


745
P10 sub7
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGCCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37272


746
P10 sub8
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGCCCAUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37273


747
P10 sub0
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGGCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37274


748
P10 sub1
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGGCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37275


749
P10 sub2
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUCCCUCGGCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37276


750
P10 sub4
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGCCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37277


751
P10 sub7
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGCCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37278


752
P10 sub8
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGCCCAUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37279


753
P10 sub0
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGGCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37280


754
P10 sub1
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGGCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37281


755
P10 sub2
AAAAAGUGGCACCGAGUCGGUGCCCACAAUCCCUCGGCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37282


756
P10 sub4
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGCCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37283


757
P10 sub7
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGCCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37284


758
P10 sub8
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGCCCAUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37285


759
P10 sub0
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37286


760
P10 sub1
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGGCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37287


761
P10 sub2
AAAAAGUGGCACCGAGUCGGUGCACAAUCCCUCGGCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37288


762
P10 sub4
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGCCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37289


763
P10 sub7
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGCCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37290


764
P10 sub8
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGCCCAUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37291


765
P10 sub0
AAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37292


766
P10 sub1
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGGCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37293


767
P10 sub2
AAAAAGUGGCACCGAGUCGGUGCAAUCCCUCGGCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37294


768
P10 sub4
AAAAAGUGGCACCGAGUCGGUGCAAUACCUCGCCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37295


769
P10 sub7
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGCCCCUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37296


770
P10 sub8
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGCCCAUUCUCAGUUCGCUA



 


RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37297


771
P9 sub0
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37298


772
P9 sub1
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37299


773
P9 sub2
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUCCCUCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37300


774
P9 sub4
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37301


775
P9 sub7
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37302


776
P9 sub8
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGCCCAUUCUCAGUUCGCU



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37303


777
P9 sub0
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37304


778
P9 sub1
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37305


779
P9 sub2
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUCCCUCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37306


780
P9 sub4
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37307


781
P9 sub7
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37308


782
P9 sub8
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGCCCAUUCUCAGUUCGCU



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37309


783
P9 sub0
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37310


784
P9 sub1
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37311


785
P9 sub2
AAAAAGUGGCACCGAGUCGGUGCCCACAAUCCCUCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37312


786
P9 sub4
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37313


787
P9 sub7
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37314


788
P9 sub8
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGCCCAUUCUCAGUUCGCU



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37315


789
P9 sub0
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37316


790
P9_sub1
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37317


791
P9 sub2
AAAAAGUGGCACCGAGUCGGUGCACAAUCCCUCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37318


792
P9 sub4
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37319


793
P9 sub7
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37320


794
P9 sub8
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGCCCAUUCUCAGUUCGCU



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37321


795
P9 sub0
AAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37322


796
P9 sub1
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37323


797
P9 sub2
AAAAAGUGGCACCGAGUCGGUGCAAUCCCUCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37324


798
P9 sub4
AAAAAGUGGCACCGAGUCGGUGCAAUACCUCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37325


799
P9 sub7
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37326


800
P9 sub8
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGCCCAUUCUCAGUUCGCU



 


RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37327


801
P8 sub0
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37328


802
P8 sub1
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37329


803
P8 sub2
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUCCCUCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37330


804
P8 sub4
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37331


805
P8 sub7
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R25
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37332


806
P8 sub8
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGCCCAUUCUCAGUUCGC



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37333


807
P8 sub0
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37334


808
P8 sub1
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37335


809
P8 sub2
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUCCCUCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37336


810
P8 sub4
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37337


811
P8 sub7
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R23
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37338


812
P8 sub8
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGCCCAUUCUCAGUUCGC



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37339


813
P8 sub0
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37340


814
P8 sub1
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37341


815
P8 sub2
AAAAAGUGGCACCGAGUCGGUGCCCACAAUCCCUCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37342


816
P8 sub4
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37343


817
P8 sub7
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R21
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37344


818
P8 sub8
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGCCCAUUCUCAGUUCGC



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37345


819
P8 sub0
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37346


820
P8 sub1
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37347


821
P8 sub2
AAAAAGUGGCACCGAGUCGGUGCACAAUCCCUCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37348


822
P8 sub4
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37349


823
P8 sub7
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R19
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37350


824
P8 sub8
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGCCCAUUCUCAGUUCGC



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37351


825
P8 sub0
AAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37352


826
P8 sub1
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37353


827
P8 sub2
AAAAAGUGGCACCGAGUCGGUGCAAUCCCUCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37354


828
P8 sub4
AAAAAGUGGCACCGAGUCGGUGCAAUACCUCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37355


829
P8 sub7
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU3 R17
UGGGUCGUAGCGAACUGAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37356


830
P8 sub8
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGCCCAUUCUCAGUUCGC



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37357


831
P10 sub0
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37358


832
P10 sub1
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37359


833
P10 sub4
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37360


834
P10 sub5
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGCCCAUUCUCAGUUCGCU



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37361


835
P10 sub7
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37362


836
P10 sub8
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGCCCAUUCUCAGUUCGCU



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37363


837
P10 sub0
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37364


838
P10 sub1
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37365


839
P10 sub4
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37366


840
P10 sub5
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGCCCAUUCUCAGUUCGCU



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37367


841
P10 sub7
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37368


842
P10 sub8
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGCCCAUUCUCAGUUCGCU



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37369


843
P10 sub0
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37370


844
P10 sub1
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37371


845
P10 sub4
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37372


846
P10 sub5
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGCCCAUUCUCAGUUCGCU



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37373


847
P10 sub7
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37374


848
P10 sub8
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGCCCAUUCUCAGUUCGCU



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37375


849
P10 sub0
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37376


850
P10 sub1
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37377


851
P10 sub4
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37378


852
P10 sub5
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGCCCAUUCUCAGUUCGCU



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37379


853
P10 sub7
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37380


854
P10 sub8
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGCCCAUUCUCAGUUCGCU



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37381


855
P10 sub0
AAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37382


856
P10 sub1
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGGCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37383


857
P10 sub4
AAAAAGUGGCACCGAGUCGGUGCAAUACCUCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37384


858
P10 sub5
AAAAAGUGGCACCGAGUCGGUGCAAUACCUCGCCCAUUCUCAGUUCGCU



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37385


859
P10 sub7
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGCCCCUUCUCAGUUCGCU



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37386


860
P10 sub8
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGCCCAUUCUCAGUUCGCU



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37387


861
P9 sub0
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37388


862
P9 sub1
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37389


863
P9 sub4
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37390


864
P9 sub5
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGCCCAUUCUCAGUUCGC



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37391


865
P9 sub7
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37392


866
P9 sub8
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGCCCAUUCUCAGUUCGC



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37393


867
P9 sub0
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37394


868
P9 sub1
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37395


869
P9 sub4
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37396


870
P9 sub5
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGCCCAUUCUCAGUUCGC



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37397


871
P9 sub7
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37398


872
P9 sub8
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGCCCAUUCUCAGUUCGC



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37399


873
P9 sub0
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37400


874
P9 sub1
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37401


875
P9 sub4
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37402


876
P9 sub5
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGCCCAUUCUCAGUUCGC



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37403


877
P9 sub7
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37404


878
P9 sub8
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGCCCAUUCUCAGUUCGC



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37405


879
P9 sub0
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37406


880
P9 sub1
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37407


881
P9 sub4
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37408


882
P9 sub5
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGCCCAUUCUCAGUUCGC



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37409


883
P9 sub7
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37410


884
P9 sub8
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGCCCAUUCUCAGUUCGC



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37411


885
P9 sub0
AAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37412


886
P9 sub1
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGGCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37413


887
P9 sub4
AAAAAGUGGCACCGAGUCGGUGCAAUACCUCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37414


888
P9 sub5
AAAAAGUGGCACCGAGUCGGUGCAAUACCUCGCCCAUUCUCAGUUCGC



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37415


889
P9 sub7
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGCCCCUUCUCAGUUCGC



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37416


890
P9 sub8
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGCCCAUUCUCAGUUCGC



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37417


891
P8 sub0
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGGCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37418


892
P8 sub1
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGGCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37419


893
P8 sub4
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGCCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37420


894
P8 sub5
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGCCCAUUCUCAGUUCG



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37421


895
P8 sub7
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGCCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R24
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37422


896
P8 sub8
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGCCCAUUCUCAGUUCG



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37423


897
P8 sub0
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGGCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37424


898
P8 sub1
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGGCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37425


899
P8 sub4
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGCCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37426


900
P8 sub5
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGCCCAUUCUCAGUUCG



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37427


901
P8 sub7
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGCCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R22
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37428


902
P8 sub8
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGCCCAUUCUCAGUUCG



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37429


903
P8 sub0
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGGCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37430


904
P8 sub1
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGGCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37431


905
P8 sub4
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGCCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37432


906
P8 sub5
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGCCCAUUCUCAGUUCG



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37433


907
P8 sub7
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGCCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R20
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37434


908
P8 sub8
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGCCCAUUCUCAGUUCG



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37435


909
P8 sub0
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37436


910
P8 sub1
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGGCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37437


911
P8 sub4
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGCCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37438


912
P8 sub5
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGCCCAUUCUCAGUUCG



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37439


913
P8 sub7
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGCCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R18
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37440


914
P8 sub8
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGCCCAUUCUCAGUUCG



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37441


915
P8 sub0
AAAAAGUGGCACCGAGUCGGUGCAAUACCUCGGCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37442


916
P8 sub1
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGGCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37443


917
P8 sub4
AAAAAGUGGCACCGAGUCGGUGCAAUACCUCGCCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37444


918
P8 sub5
AAAAAGUGGCACCGAGUCGGUGCAAUACCUCGCCCAUUCUCAGUUCG



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37445


919
P8 sub7
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGCCCCUUCUCAGUUCG



 


RNACS4
hPKU4 R16
GGGUCGUAGCGAACUGAGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37446


920
P8 sub8
AAAAAGUGGCACCGAGUCGGUGCAAUACCGCGCCCAUUCUCAGUUCG



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37447


923
P10 sub0
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACAAUACCUCGGCCCUUCUCAG



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37448


924
P10 sub1
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACAAUACCGCGGCCCUUCUCAG



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37449


925
P10 sub2
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACAAUCCCUCGGCCCUUCUCAG



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37450


926
P10 sub3
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACAAUCCCGCGGCCCUUCUCAG



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37451


927
P10 sub4
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACAAUACCUCGCCCCUUCUCAG



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37452


928
P10 sub7
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACGAUACCUCGGCCCUUCUCAG



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37453


929
P10 sub0
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGGCCCUUCUCAG



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37454


930
P10 sub1
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGGCCCUUCUCAG



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37455


931
P10 sub2
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUCCCUCGGCCCUUCUCAG



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37456


932
P10 sub3
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUCCCGCGGCCCUUCUCAG



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37457


933
P10 sub4
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGCCCCUUCUCAG



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37458


934
P10 sub7
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACGAUACCUCGGCCCUUCUCAG



 


RNACS4
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37459


935
P10 sub0
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGGCCCUUCUCAG



 


RNACS4
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37460


936
P10 sub1
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGGCCCUUCUCAG



 


RNACS4
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37461


937
P10 sub2
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUCCCUCGGCCCUUCUCAG



 


RNACS4
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37462


938
P10 sub3
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUCCCGCGGCCCUUCUCAG



 


RNACS4
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37463


939
P10 sub4
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGCCCCUUCUCAG



 


RNACS4
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37464


940
P10 sub7
AAAAAGUGGCACCGAGUCGGUGCUGCCACGAUACCUCGGCCCUUCUCAG



 


RNACS4
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37465


941
P10 sub0
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGGCCCUUCUCAG



 


RNACS4
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37466


942
P10 sub1
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGGCCCUUCUCAG



 


RNACS4
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37467


943
P10 sub2
AAAAAGUGGCACCGAGUCGGUGCCCACAAUCCCUCGGCCCUUCUCAG



 


RNACS4
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37468


944
P10 sub3
AAAAAGUGGCACCGAGUCGGUGCCCACAAUCCCGCGGCCCUUCUCAG



 


RNACS4
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37469


945
P10 sub4
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGCCCCUUCUCAG



 


RNACS4
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37470


946
P10 sub7
AAAAAGUGGCACCGAGUCGGUGCCCACGAUACCUCGGCCCUUCUCAG



 


RNACS4
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37471


947
P10 sub0
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCAG



 


RNACS4
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37472


948
P10 sub1
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGGCCCUUCUCAG



 


RNACS4
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37473


949
P10 sub2
AAAAAGUGGCACCGAGUCGGUGCACAAUCCCUCGGCCCUUCUCAG



 


RNACS4
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37474


950
P10 sub3
AAAAAGUGGCACCGAGUCGGUGCACAAUCCCGCGGCCCUUCUCAG



 


RNACS4
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37475


951
P10 sub4
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGCCCCUUCUCAG



 


RNACS4
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37476


952
P10 sub7
AAAAAGUGGCACCGAGUCGGUGCACGAUACCUCGGCCCUUCUCAG



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37477


953
P9 sub0
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACAAUACCUCGGCCCUUCUCA



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37478


954
P9 sub1
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACAAUACCGCGGCCCUUCUCA



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37479


955
P9 sub2
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACAAUCCCUCGGCCCUUCUCA



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37480


956
P9 sub3
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACAAUCCCGCGGCCCUUCUCA



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37481


957
P9 sub4
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACAAUACCUCGCCCCUUCUCA



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37482


958
P9 sub7
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACGAUACCUCGGCCCUUCUCA



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37483


959
P9 sub0
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGGCCCUUCUCA



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37484


960
P9 sub1
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGGCCCUUCUCA



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37485


961
P9 sub2
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUCCCUCGGCCCUUCUCA



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37486


962
P9 sub3
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUCCCGCGGCCCUUCUCA



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37487


963
P9 sub4
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGCCCCUUCUCA



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37488


964
P9 sub7
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACGAUACCUCGGCCCUUCUCA



 


RNACS4
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37489


965
P9 sub0
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGGCCCUUCUCA



 


RNACS4
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37490


966
P9 sub1
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGGCCCUUCUCA



 


RNACS4
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37491


967
P9 sub2
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUCCCUCGGCCCUUCUCA



 


RNACS4
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37492


968
P9 sub3
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUCCCGCGGCCCUUCUCA



 


RNACS4
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37493


969
P9 sub4
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGCCCCUUCUCA



 


RNACS4
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37494


970
P9 sub7
AAAAAGUGGCACCGAGUCGGUGCUGCCACGAUACCUCGGCCCUUCUCA



 


RNACS4
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37495


971
P9 sub0
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGGCCCUUCUCA



 


RNACS4
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37496


972
P9 sub1
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGGCCCUUCUCA



 


RNACS4
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37497


973
P9 sub2
AAAAAGUGGCACCGAGUCGGUGCCCACAAUCCCUCGGCCCUUCUCA



 


RNACS4
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37498


974
P9 sub3
AAAAAGUGGCACCGAGUCGGUGCCCACAAUCCCGCGGCCCUUCUCA



 


RNACS4
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37499


975
P9 sub4
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGCCCCUUCUCA



 


RNACS4
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37500


976
P9 sub7
AAAAAGUGGCACCGAGUCGGUGCCCACGAUACCUCGGCCCUUCUCA



 


RNACS4
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37501


977
P9 sub0
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUCA



 


RNACS4
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37502


978
P9 sub1
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGGCCCUUCUCA



 


RNACS4
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37503


979
P9 sub2
AAAAAGUGGCACCGAGUCGGUGCACAAUCCCUCGGCCCUUCUCA



 


RNACS4
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37504


980
P9 sub3
AAAAAGUGGCACCGAGUCGGUGCACAAUCCCGCGGCCCUUCUCA



 


RNACS4
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37505


981
P9 sub4
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGCCCCUUCUCA



 


RNACS4
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37506


982
P9 sub7
AAAAAGUGGCACCGAGUCGGUGCACGAUACCUCGGCCCUUCUCA



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37507


983
P8 sub0
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACAAUACCUCGGCCCUUCUC



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37508


984
P8 sub1
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACAAUACCGCGGCCCUUCUC



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37509


985
P8 sub2
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACAAUCCCUCGGCCCUUCUC



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37510


986
P8 sub3
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACAAUCCCGCGGCCCUUCUC



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37511


987
P8 sub4
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACAAUACCUCGCCCCUUCUC



 


RNACS4
hPKU5 R20
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37512


988
P8 sub7
AAAAAGUGGCACCGAGUCGGUGCUUGCUGCCACGAUACCUCGGCCCUUCUC



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37513


989
P8 sub0
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGGCCCUUCUC



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37514


990
P8 sub1
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCGCGGCCCUUCUC



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37515


991
P8 sub2
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUCCCUCGGCCCUUCUC



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37516


992
P8 sub3
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUCCCGCGGCCCUUCUC



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37517


993
P8 sub4
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACAAUACCUCGCCCCUUCUC



 


RNACS4
hPKU5 R18
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37518


994
P8 sub7
AAAAAGUGGCACCGAGUCGGUGCGCUGCCACGAUACCUCGGCCCUUCUC



 


RNACS4
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37519


995
P8 sub0
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGGCCCUUCUC



 


RNACS4
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37520


996
P8 sub1
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCGCGGCCCUUCUC



 


RNACS4
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37521


997
P8 sub2
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUCCCUCGGCCCUUCUC



 


RNACS4
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37522


998
P8 sub3
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUCCCGCGGCCCUUCUC



 


RNACS4
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37523


999
P8 sub4
AAAAAGUGGCACCGAGUCGGUGCUGCCACAAUACCUCGCCCCUUCUC



 


RNACS5
hPKU5 R16
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37524


000
P8 sub7
AAAAAGUGGCACCGAGUCGGUGCUGCCACGAUACCUCGGCCCUUCUC



 


RNACS5
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37525


001
P8 sub0
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGGCCCUUCUC



 


RNACS5
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37526


002
P8 sub1
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCGCGGCCCUUCUC



 


RNACS5
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37527


003
P8 sub2
AAAAAGUGGCACCGAGUCGGUGCCCACAAUCCCUCGGCCCUUCUC



 


RNACS5
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37528


004
P8 sub3
AAAAAGUGGCACCGAGUCGGUGCCCACAAUCCCGCGGCCCUUCUC



 


RNACS5
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37529


005
P8 sub4
AAAAAGUGGCACCGAGUCGGUGCCCACAAUACCUCGCCCCUUCUC



 


RNACS5
hPKU5 R14
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37530


006
P8 sub7
AAAAAGUGGCACCGAGUCGGUGCCCACGAUACCUCGGCCCUUCUC



 


RNACS5
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37531


007
P8 sub0
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGGCCCUUCUC



 


RNACS5
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37532


008
P8 sub1
AAAAAGUGGCACCGAGUCGGUGCACAAUACCGCGGCCCUUCUC



 


RNACS5
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37533


009
P8 sub2
AAAAAGUGGCACCGAGUCGGUGCACAAUCCCUCGGCCCUUCUC



 


RNACS5
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37534


010
P8 sub3
AAAAAGUGGCACCGAGUCGGUGCACAAUCCCGCGGCCCUUCUC



 


RNACS5
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37535


011
P8 sub4
AAAAAGUGGCACCGAGUCGGUGCACAAUACCUCGCCCCUUCUC



 


RNACS5
hPKU5 R12
UAGCGAACUGAGAAGGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG
37536


012
P8 sub7
AAAAAGUGGCACCGAGUCGGUGCACGAUACCUCGGCCCUUCUC



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37537


013
P11 sub0
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAAGGGCCGAGGUAUUGUGGCAG



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37538


014
P11 sub1
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAAGGGCCGGGGUAUUGUGGCAG



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37539


015
P11 sub4
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAACGGCCGGGGUAUUGUGGCAG



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37540


016
P11 sub5
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAACGGACGGGGUAUUGUGGCAG



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37541


017
P11 sub6
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAAGGGCCGCGGUAUUGUGGCAG



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37542


018
P11 sub7
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAAGGGACGCGGUAUUGUGGCAG



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37543


019
P11 sub0
AAAAGUGGCACCGAGUCGGUGCAACUGAGAAGGGCCGAGGUAUUGUGGCAG



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37544


020
P11 sub1
AAAAGUGGCACCGAGUCGGUGCAACUGAGAAGGGCCGGGGUAUUGUGGCAG



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37545


021
P11 sub4
AAAAGUGGCACCGAGUCGGUGCAACUGAGAACGGCCGGGGUAUUGUGGCAG



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37546


022
P11 sub5
AAAAGUGGCACCGAGUCGGUGCAACUGAGAACGGACGGGGUAUUGUGGCAG



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37547


023
P11 sub6
AAAAGUGGCACCGAGUCGGUGCAACUGAGAAGGGCCGCGGUAUUGUGGCAG



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37548


024
P11 sub7
AAAAGUGGCACCGAGUCGGUGCAACUGAGAAGGGACGCGGUAUUGUGGCAG



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37549


025
P11 sub0
AAAAGUGGCACCGAGUCGGUGCCUGAGAAGGGCCGAGGUAUUGUGGCAG



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37550


026
P11 sub1
AAAAGUGGCACCGAGUCGGUGCCUGAGAAGGGCCGGGGUAUUGUGGCAG



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37551


027
P11 sub4
AAAAGUGGCACCGAGUCGGUGCCUGAGAACGGCCGGGGUAUUGUGGCAG



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37552


028
P11 sub5
AAAAGUGGCACCGAGUCGGUGCCUGAGAACGGACGGGGUAUUGUGGCAG



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37553


029
P11 sub6
AAAAGUGGCACCGAGUCGGUGCCUGAGAAGGGCCGCGGUAUUGUGGCAG



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37554


030
P11 sub7
AAAAGUGGCACCGAGUCGGUGCCUGAGAAGGGACGCGGUAUUGUGGCAG



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37555


031
P11 sub0
AAAAGUGGCACCGAGUCGGUGCGAGAAGGGCCGAGGUAUUGUGGCAG



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37556


032
P11 sub1
AAAAGUGGCACCGAGUCGGUGCGAGAAGGGCCGGGGUAUUGUGGCAG



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37557


033
P11 sub4
AAAAGUGGCACCGAGUCGGUGCGAGAACGGCCGGGGUAUUGUGGCAG



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37558


034
P11 sub5
AAAAGUGGCACCGAGUCGGUGCGAGAACGGACGGGGUAUUGUGGCAG



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37559


035
P11 sub6
AAAAGUGGCACCGAGUCGGUGCGAGAAGGGCCGCGGUAUUGUGGCAG



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37560


036
P11 sub7
AAAAGUGGCACCGAGUCGGUGCGAGAAGGGACGCGGUAUUGUGGCAG



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37561


037
P11 sub0
AAAAGUGGCACCGAGUCGGUGCGAAGGGCCGAGGUAUUGUGGCAG



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37562


038
P11 sub1
AAAAGUGGCACCGAGUCGGUGCGAAGGGCCGGGGUAUUGUGGCAG



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37563


039
P11 sub4
AAAAGUGGCACCGAGUCGGUGCGAACGGCCGGGGUAUUGUGGCAG



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37564


040
P11 sub5
AAAAGUGGCACCGAGUCGGUGCGAACGGACGGGGUAUUGUGGCAG



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37565


041
P11 sub6
AAAAGUGGCACCGAGUCGGUGCGAAGGGCCGCGGUAUUGUGGCAG



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37566


042
P11 sub7
AAAAGUGGCACCGAGUCGGUGCGAAGGGACGCGGUAUUGUGGCAG



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37567


043
P10 sub0
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAAGGGCCGAGGUAUUGUGGCA



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37568


044
P10 sub1
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAAGGGCCGGGGUAUUGUGGCA



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37569


045
P10 sub4
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAACGGCCGGGGUAUUGUGGCA



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37570


046
P10 sub5
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAACGGACGGGGUAUUGUGGCA



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37571


047
P10 sub6
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAAGGGCCGCGGUAUUGUGGCA



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37572


048
P10 sub7
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAAGGGACGCGGUAUUGUGGCA



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37573


049
P10 sub0
AAAAGUGGCACCGAGUCGGUGCAACUGAGAAGGGCCGAGGUAUUGUGGCA



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37574


050
P10 sub1
AAAAGUGGCACCGAGUCGGUGCAACUGAGAAGGGCCGGGGUAUUGUGGCA



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37575


051
P10 sub4
AAAAGUGGCACCGAGUCGGUGCAACUGAGAACGGCCGGGGUAUUGUGGCA



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37576


052
P10 sub5
AAAAGUGGCACCGAGUCGGUGCAACUGAGAACGGACGGGGUAUUGUGGCA



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37577


053
P10 sub6
AAAAGUGGCACCGAGUCGGUGCAACUGAGAAGGGCCGCGGUAUUGUGGCA



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37578


054
P10 sub7
AAAAGUGGCACCGAGUCGGUGCAACUGAGAAGGGACGCGGUAUUGUGGCA



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37579


055
P10 sub0
AAAAGUGGCACCGAGUCGGUGCCUGAGAAGGGCCGAGGUAUUGUGGCA



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37580


056
P10 sub1
AAAAGUGGCACCGAGUCGGUGCCUGAGAAGGGCCGGGGUAUUGUGGCA



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37581


057
P10 sub4
AAAAGUGGCACCGAGUCGGUGCCUGAGAACGGCCGGGGUAUUGUGGCA



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37582


058
P10 sub5
AAAAGUGGCACCGAGUCGGUGCCUGAGAACGGACGGGGUAUUGUGGCA



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37583


059
P10 sub6
AAAAGUGGCACCGAGUCGGUGCCUGAGAAGGGCCGCGGUAUUGUGGCA



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37584


060
P10 sub7
AAAAGUGGCACCGAGUCGGUGCCUGAGAAGGGACGCGGUAUUGUGGCA



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37585


061
P10 sub0
AAAAGUGGCACCGAGUCGGUGCGAGAAGGGCCGAGGUAUUGUGGCA



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37586


062
P10 sub1
AAAAGUGGCACCGAGUCGGUGCGAGAAGGGCCGGGGUAUUGUGGCA



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37587


063
P10 sub4
AAAAGUGGCACCGAGUCGGUGCGAGAACGGCCGGGGUAUUGUGGCA



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37588


064
P10 sub5
AAAAGUGGCACCGAGUCGGUGCGAGAACGGACGGGGUAUUGUGGCA



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37589


065
P10 sub6
AAAAGUGGCACCGAGUCGGUGCGAGAAGGGCCGCGGUAUUGUGGCA



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37590


066
P10 sub7
AAAAGUGGCACCGAGUCGGUGCGAGAAGGGACGCGGUAUUGUGGCA



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37591


067
P10 sub0
AAAAGUGGCACCGAGUCGGUGCGAAGGGCCGAGGUAUUGUGGCA



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37592


068
P10 sub1
AAAAGUGGCACCGAGUCGGUGCGAAGGGCCGGGGUAUUGUGGCA



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37593


069
P10 sub4
AAAAGUGGCACCGAGUCGGUGCGAACGGCCGGGGUAUUGUGGCA



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37594


070
P10 sub5
AAAAGUGGCACCGAGUCGGUGCGAACGGACGGGGUAUUGUGGCA



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37595


071
P10 sub6
AAAAGUGGCACCGAGUCGGUGCGAAGGGCCGCGGUAUUGUGGCA



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37596


072
P10 sub7
AAAAGUGGCACCGAGUCGGUGCGAAGGGACGCGGUAUUGUGGCA



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37597


073
P9 sub0
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAAGGGCCGAGGUAUUGUGGC



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37598


074
P9 sub1
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAAGGGCCGGGGUAUUGUGGC



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37599


075
P9 sub4
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAACGGCCGGGGUAUUGUGGC



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37600


076
P9 sub5
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAACGGACGGGGUAUUGUGGC



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37601


077
P9 sub6
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAAGGGCCGCGGUAUUGUGGC



 


RNACS5
hPKU6 R20
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37602


078
P9 sub7
AAAAGUGGCACCGAGUCGGUGCCGAACUGAGAAGGGACGCGGUAUUGUGGC



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37603


079
P9 sub0
AAAAGUGGCACCGAGUCGGUGCAACUGAGAAGGGCCGAGGUAUUGUGGC



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37604


080
P9 sub1
AAAAGUGGCACCGAGUCGGUGCAACUGAGAAGGGCCGGGGUAUUGUGGC



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37605


081
P9 sub4
AAAAGUGGCACCGAGUCGGUGCAACUGAGAACGGCCGGGGUAUUGUGGC



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37606


082
P9 sub5
AAAAGUGGCACCGAGUCGGUGCAACUGAGAACGGACGGGGUAUUGUGGC



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37607


083
P9 sub6
AAAAGUGGCACCGAGUCGGUGCAACUGAGAAGGGCCGCGGUAUUGUGGC



 


RNACS5
hPKU6 R18
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37608


084
P9 sub7
AAAAGUGGCACCGAGUCGGUGCAACUGAGAAGGGACGCGGUAUUGUGGC



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37609


085
P9 sub0
AAAAGUGGCACCGAGUCGGUGCCUGAGAAGGGCCGAGGUAUUGUGGC



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37610


086
P9 sub1
AAAAGUGGCACCGAGUCGGUGCCUGAGAAGGGCCGGGGUAUUGUGGC



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37611


087
P9 sub4
AAAAGUGGCACCGAGUCGGUGCCUGAGAACGGCCGGGGUAUUGUGGC



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37612


088
P9 sub5
AAAAGUGGCACCGAGUCGGUGCCUGAGAACGGACGGGGUAUUGUGGC



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37613


089
P9 sub6
AAAAGUGGCACCGAGUCGGUGCCUGAGAAGGGCCGCGGUAUUGUGGC



 


RNACS5
hPKU6 R16
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37614


090
P9 sub7
AAAAGUGGCACCGAGUCGGUGCCUGAGAAGGGACGCGGUAUUGUGGC



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37615


091
P9 sub0
AAAAGUGGCACCGAGUCGGUGCGAGAAGGGCCGAGGUAUUGUGGC



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37616


092
P9 sub1
AAAAGUGGCACCGAGUCGGUGCGAGAAGGGCCGGGGUAUUGUGGC



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37617


093
P9 sub4
AAAAGUGGCACCGAGUCGGUGCGAGAACGGCCGGGGUAUUGUGGC



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37618


094
P9 sub5
AAAAGUGGCACCGAGUCGGUGCGAGAACGGACGGGGUAUUGUGGC



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37619


095
P9 sub6
AAAAGUGGCACCGAGUCGGUGCGAGAAGGGCCGCGGUAUUGUGGC



 


RNACS5
hPKU6 R14
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37620


096
P9 sub7
AAAAGUGGCACCGAGUCGGUGCGAGAAGGGACGCGGUAUUGUGGC



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37621


097
P9 sub0
AAAAGUGGCACCGAGUCGGUGCGAAGGGCCGAGGUAUUGUGGC



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37622


098
P9 sub1
AAAAGUGGCACCGAGUCGGUGCGAAGGGCCGGGGUAUUGUGGC



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37623


099
P9 sub4
AAAAGUGGCACCGAGUCGGUGCGAACGGCCGGGGUAUUGUGGC



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37624


100
P9 sub5
AAAAGUGGCACCGAGUCGGUGCGAACGGACGGGGUAUUGUGGC



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37625


101
P9 sub6
AAAAGUGGCACCGAGUCGGUGCGAAGGGCCGCGGUAUUGUGGC



 


RNACS5
hPKU6 R12
ACUUUGCUGCCACAAUACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA
37626


102
P9 sub7
AAAAGUGGCACCGAGUCGGUGCGAAGGGACGCGGUAUUGUGGC









The gene modifying system comprising mRNA encoding the gene modifying polypeptide listed above and a template RNA listed above were transfected into human hepatoblasts differentiated from CRISPR-edited iPSCs containing the R408W mutation in the human PAH gene. Briefly PAH iPSCs are dissociated into single cells and then replated onto Geltex-coated plates. The iPSCs were then differentiated into definitive endoderm cells by treatment with Activin A, FGF2, and ChIR for 7 days. Definitive endoderm cells are then further patterned into foregut endoderm cells by activation of the BMP4 and FGF2 signaling pathway for an additional 6 days. Lastly, foregut endoderm cells were patterned into hepatoblast cells by treatment with oncostamin M, dexamethasone, hepatocyte growth factors, and ChIR for 12 days. Hepatoblasts were then sub-cultured onto collagen1-coated plates and expanded in media containing FGF19, Dexamethazone, ChIR, and SB431542 prior to transfection. The gene modifying polypeptide and template RNA were delivered by nucleofection in the RNA format. Specifically, 4 μg of gene modifying polypeptide mRNA were combined with 10 μg of chemically synthesized template RNA, in 5 μL of water. The transfection mix was added to 100,000 iPSCs in Buffer P3 [Lonza], and cells were nucleofected using program DG-138. After nucleofection, cells were grown at 37° C., 5% CO2 for 3 days prior to cell lysis and genomic DNA extraction. To analyze gene editing activity, primers flanking the target insertion site locus were used to amplify across the locus. Amplicons were analyzed via short read sequencing using an Illumina MiSeq. Conversion of T>C indicates successful editing.
As shown by FIG. 31A, treating hepatoblasts with gene modifying systems comprising exemplary hPKU3 template RNAs comprising a variety of silent substitutions (FIG. 31B) resulted in editing at the target locus. The results show that silent substitution 4 (sub4) resulted in comparable rewriting % as the template RNA without silent substitutions, whereas other silent substitutions resulted in lower rewriting %.
As shown by FIG. 32A, treating hepatoblasts with gene modifying systems comprising exemplary hPKU4 template RNAs comprising a variety of silent substitutions (FIG. 32B) resulted in editing at the target locus. The results show that silent substitution 4 (sub4) resulted in comparable or higher rewriting % as the template RNA without silent substitutions, whereas other silent substitutions resulted in lower rewriting %.
As shown by FIG. 33A, treating hepatoblasts with gene modifying systems comprising exemplary hPKU5 template RNAs comprising a variety of silent substitutions (FIG. 33B) resulted in editing at the target locus. The results show that silent substitution 4 (sub4) resulted in comparable or higher rewriting % as the template RNA without silent substitutions, whereas other silent substitutions resulted in lower rewriting %.
As shown by FIG. 34A, treating hepatoblasts with gene modifying systems comprising exemplary hPKU6 template RNAs comprising a variety of silent substitutions (FIG. 34B) resulted in editing at the target locus. The results show that silent substitutions 5, 6, and 7 (sub5, sub6, and sub7, respectively) resulted in much higher rewriting % as the template RNA without silent substitutions, which had a very low rewriting %. These results demonstrate that silent substitutions can rescue the low rewriting activity of exemplary hPKU6 template RNAs.
Taken together, these results demonstrate that silent substitutions can increase the rewriting activity of exemplary template RNAs that target a therapeutically relevant human locus, and in some cases rescue otherwise low rewriting activity.
It should be understood that for all numerical bounds describing some parameter in this application, such as “about,” “at least,” “less than,” and “more than,” the description also necessarily encompasses any range bounded by the recited values. Accordingly, for example, the description “at least 1, 2, 3, 4, or 5” also describes, inter alia, the ranges 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, et cetera.
For all patents, applications, or other reference cited herein, such as non-patent literature and reference sequence information, it should be understood that they are incorporated by reference in their entirety for all purposes as well as for the proposition that is recited. Where any conflict exists between a document incorporated by reference and the present application, this application will control. All information associated with reference gene sequences disclosed in this application, such as GeneIDs or accession numbers (typically referencing NCBI accession numbers), including, for example, genomic loci, genomic sequences, functional annotations, allelic variants, and reference mRNA (including, e.g., exon boundaries or response elements) and protein sequences (such as conserved domain structures), as well as chemical references (e.g., PubChem compound, PubChem substance, or PubChem Bioassay entries, including the annotations therein, such as structures and assays, et cetera), are hereby incorporated by reference in their entirety.
Headings used in this application are for convenience only and do not affect the interpretation of this application.







LENGTHY TABLES




The patent contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (https://seqdata.uspto.gov/docdetail?docId=US12544458B2). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).