Compositions and methods for treatment of liquid cancers

As described below, the present invention features genetically modified immune cells having enhanced anti-neoplasia activity, resistance to immune suppression, and decreased risk of eliciting a graft versus host reaction, or a combination thereof. The present invention also features methods for producing and using these modified immune effector cells.

1. 11. A method for producing a CD7 CAR-expressing immune cell or population of CAR-expressing immune cells having reduced immunogenicity, the method comprising a) contacting a target polynucleotide in an immune cell or population of immune cells with a guide polynucleotide and a base editor comprising a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase domain or a base editor system comprising a guide polynucleotide and a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase domain; b) introducing by nucleobase modification a mutation that reduces or eliminates the expression of a CD7 antigen into the immune cell or the population of immune cells; c) introducing by nucleobase modification a mutation that reduces or eliminates the expression of at least one polypeptide selected from the group consisting of CD3e, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PDC1/PD-1 into the immune cells or the population of immune cells; and d) expressing in the immune cell or population of immune cells a chimeric antigen receptor that targets the CD7 antigen thereby producing a CAR-expressing immune cell or population of CAR-expressing immune cells having reduced immunogenicity, wherein the chimeric antigen receptor that targets the CD7 antigen comprises or consists of SEQ ID NO: 113.
2. 22. The method of claim 1, wherein the immune cell or the population of immune cells produced by the method exhibits fratricide resistance and/or increased anti-neoplasia activity as compared to a corresponding control cell.
3. 33. The method of claim 1 wherein the immune cell or the population of immune produced by the method comprises no detectable translocations.
4. 44. The method of claim 1, wherein the immune cell or the population of immune cells produced by the method comprises less than 1% indels.
5. 55. The method of claim 1, wherein the immune cell or the population of immune cells produced by the method comprises less than 5% of non-target edits.
6. 66. The method of claim 1, wherein the mutation is in an exon, in a splice donor site or a splice acceptor site.

CROSS REFERENCE TO RELATED APPLICATIONS
This application is the U.S. National Stage Application, pursuant to 35 U.S.C. § 371 of International PCT Application No. PCT/US2020/052822, filed Sep. 25, 2020 designating the United States and published in English, which claims priority to and the benefit of U.S. Provisional Application No. 62/907,254, filed Sep. 27, 2019, the entire contents of which are hereby incorporated by reference in its entirety.


SEQUENCE LISTING STATEMENT
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 31, 2020, is named 180802_043301_PCT_SL.txt and is 2,270,064 bytes in size.
BACKGROUND OF THE INVENTION
Autologous and allogeneic immunotherapies are neoplasia treatment approaches in which immune cells expressing chimeric antigen receptors are administered to a subject. To generate an immune cell that expresses a chimeric antigen receptor (CAR), the immune cell is first collected from the subject (autologous) or a donor separate from the subject receiving treatment (allogeneic) and genetically modified to express the chimeric antigen receptor. The resulting cell expresses the chimeric antigen receptor on its cell surface (e.g., CAR T-cell), and upon administration to the subject, the chimeric antigen receptor binds to the marker expressed by the neoplastic cell. This interaction with the neoplasia marker activates the CAR-T cell, which then cell kills the neoplastic cell. But for autologous or allogeneic cell therapy to be effective and efficient, significant conditions and cellular responses, such as T cell signaling inhibition, must be overcome or avoided. For allogeneic cell therapy, graft versus host disease and host rejection of CAR-T cells may provide additional challenges. Editing genes involved in these processes can enhance CAR-T cell function and resistance to immunosuppression or inhibition, but current methodologies for making such edits have the potential to induce large, genomic rearrangements in the CAR-T cell, thereby negatively impacting its efficacy. Thus, there is a significant need for techniques to more precisely modify immune cells, especially CAR-T cells. This application is directed to this and other important needs.
SUMMARY OF THE INVENTION
As described below, the present invention features genetically modified immune cells having enhanced anti-neoplasia activity, resistance to immune suppression, and decreased risk of eliciting a graft versus host reaction, or host versus graft reaction where host CD8+ T cells recognize a graft as non-self (e.g., where a transplant recipient generates an immune response against the transplanted organ), or a combination thereof. In one embodiment, a subject having or having a propensity to develop graft versus host disease (GVHD) is administered a CAR-T cell that lacks or has reduced levels of functional TRAC. In one embodiment, a subject having or having a propensity to develop host versus graft disease (HVGD) is administered a CAR-T cell that lacks or has reduced levels of functional beta2 microglobulin (B2M). The present invention also features methods for producing and using these modified immune cells.
In one aspect, the invention provides a composition comprising two or more immune cells, each immune cell comprising a) a different chimeric antigen receptor that targets an antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123, wherein the immune cells comprise mutations that reduce or eliminate expression of the targeted antigens; and b) one or more mutations that reduce or eliminate the expression of an immunogenic polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In some embodiments, one of the immune cells comprises a chimeric antigen receptor that targets CD5 and another immune cell comprises a chimeric antigen receptor that targets an antigen selected from the group consisting of CD7, CD3, CD33, and CD123. In some embodiments, the composition comprises at least three immune cells each comprising a chimeric antigen receptor that targets a different antigen, wherein the targeted antigens are CD3, CD5, and CD7. In some embodiments, one of the immune cells expresses a chimeric antigen receptor that targets CD7 and another immune cell expresses a chimeric antigen receptor that targets an antigen selected from the group consisting of CD3, CD33, and CD123. In some embodiments, one of the immune cells expresses a chimeric antigen receptor that targets CD3 and another immune cell expresses a chimeric antigen receptor that targets an antigen selected from the group consisting of CD33, and CD123. In some embodiments, one of the immune cells expresses a chimeric antigen receptor that targets CD33 and another immune cell expresses a chimeric antigen receptor that targets CD123. In some embodiments, one immune cell expresses two, three, four or more different chimeric antigen receptors. In some embodiments, one of the chimeric antigen receptors targets CD5 and another chimeric antigen receptor targets an antigen selected from the group consisting of CD7, CD3, CD33, and CD123. In some embodiments, one of the chimeric antigen receptors targets CD7 and another chimeric antigen receptor targets an antigen selected from the group consisting of CD3, CD33, and CD123. In some embodiments, one of the chimeric antigen receptors targets CD3 and another chimeric antigen receptor targets an antigen selected from the group consisting of CD33, and CD123. In some embodiments, one of the chimeric antigen receptors targets CD33 another chimeric antigen receptor targets CD123.
In another aspect, the invention provides a composition comprising at least three immune cells, each comprising a different chimeric antigen receptor, wherein one chimeric antigen receptor targets CD3, one targets CD5, and a third targets CD7, wherein the cells further comprise mutations that reduce or eliminate the expression of the targeted antigens; and further comprise one or more mutations that reduce or eliminate the expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.
In yet another aspect, the invention provides a composition comprising at least two immune cells, each comprising a different chimeric antigen receptor, wherein one chimeric antigen receptor targets CD3 and another targets CD7, wherein the cells further comprise mutations that reduce or eliminate the expression of the targeted antigens; and further comprise one or more mutations that reduce or eliminate the expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.
In one aspect, the invention provides a composition comprising at least two immune cells, each comprising a different chimeric antigen receptor, wherein one chimeric antigen receptor targets CD5 and another targets CD7, wherein the cells further comprise mutations that reduce or eliminate the expression of the targeted antigens; and further comprise one or more mutations that reduce or eliminate the expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.
In another aspect, the invention provides a composition comprising at least two immune cells, each comprising a different chimeric antigen receptor, wherein one chimeric antigen receptor targets CD3 and another targets CD5, wherein the cells further comprise mutations that reduce or eliminate the expression of the targeted antigens; and further comprise one or more mutations that reduce or eliminate the expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.
In yet another aspect, the invention provides a composition comprising at least two immune cells, each comprising a different chimeric antigen receptor, wherein one chimeric antigen receptor targets CD33 and another targets CD123, wherein the cells further comprise mutations that reduce or eliminate the expression of the targeted antigen; and further comprise one or more mutations that reduce or eliminate the expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.
In some embodiments, the mutation is a C to T or A to G mutation that silences the gene or that introduces a stop codon into the gene. In some embodiments, the mutation introduces a premature stop codon or alters a splice donor or acceptor site. In some embodiments, the mutation is generated by a base editor comprising a deaminase domain. In some embodiments, the deaminase is a cytidine or an adenosine deaminase. In some embodiments, the base editor is BE4. In some embodiments, the mutation reduces expression of an encoded polypeptide by about 50% or more relative to a corresponding control cell lacking the mutation. In some embodiments, the composition comprises a population of immune cells. In some embodiments, at least 50% of the population comprises one or more mutations that reduce or eliminate expression of the targeted antigen and/or the immunogenic polypeptide. In some embodiments, the immune cells are fratricide resistant. In some embodiments, the immune cells have increased anti-neoplasia activity. In some embodiments, the immune cells comprise no detectable translocations. In some embodiments, the immune cells comprise less than 1% indels. In some embodiments, the immune cell is a mammalian cell. In some embodiments, the mammalian cell is a human or rodent cell. In some embodiments, the immune cell is a cytotoxic T cell, a regulatory T cell, a T helper cell, a dendritic cell, a B cell, or a NK cell, or a progenitor thereof. In some embodiments, the progenitor is a hematopoietic stem cell.
In one aspect, the invention provides a pharmaceutical composition comprising an effective amount of any of the compositions provided herein and a pharmaceutically acceptable excipient.
In another aspect, the invention provides a base editor system comprising a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase domain, and at least two guide polynucleotides each targeting a different antigen, wherein the antigens are selected from the group consisting of CD5, CD7, CD3, CD33, and CD123. In some embodiments, one guide polynucleotide targets CD5 and another targets an antigen selected from the group consisting of CD7, CD3, CD33, and CD123. In some embodiments, the system comprising three guide polynucleotides each of which targets one of antigens CD3, CD5, and CD7. In some embodiments, one guide polynucleotide targets CD7 and another targets an antigen selected from the group consisting of CD3, CD33, and CD123. In some embodiments, one guide polynucleotide targets CD3 and another targets an antigen selected from the group consisting of CD33, and CD123. In some embodiments, one guide polynucleotide targets CD33 and another targets CD123.
In some embodiments, the guide polynucleotides each comprise a nucleic acid sequence selected from Table 26. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from AGCGACUGCAGAAAGAAGAG (SEQ ID NO: 1) or CAUACCAGCUGAGCCGUCCG (SEQ ID NO: 2). In some embodiments, the fusion protein further comprises one or more uracil glycosylase inhibitors (UGIs) and/or one or more nuclear localization sequences (NLS). In some embodiments, the napDNAbp comprises a Cas9, Cas12a/Cpf1, Cas12b/C2cl, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i, or Cas12j/CasΦ polypeptide or a portion thereof. In some embodiments, the napDNAbp comprises a Cas12 polypeptide or a fragment thereof. In some embodiments, the napDNAbp comprises a Cas9 polypeptide or a fragment thereof. In some embodiments, the Cas9 is a dead Cas9 (dCas9) or a Cas9 nickase (nCas9). In some embodiments, the Cas9 is a modified Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), or a modified Streptococcus pyogenes Cas9 (SpCas9). In some embodiments, the Cas9 comprises an altered protospacer-adjacent motif (PAM) specificity. In some embodiments, the altered PAM has specificity for the nucleic acid sequence 5′-NGC-3′.
In some embodiments, the deaminase domain is capable of deaminating cytidine or adenosine. In some embodiments, the deaminase domain is a cytidine or an adenosine deaminase domain. In some embodiments, the cytidine deaminase is an APOBEC deaminase. In some embodiments, the adenosine deaminase is a TadA variant. In some embodiments, the TadA variant is a TadA*8 variant. In some embodiments, the TadA*8 variant is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24. In some embodiments, the TadA variant is a TadA*9 variant.
In one aspect, the invention provides a pharmaceutical composition comprising any of the base editor systems as provided herein.
In another aspect, the invention provides a polynucleotide encoding any of the base editor systems and guide polynucleotides as provided herein.
In yet another aspect, the invention provides a vector comprising any of the polynucleotides as provided herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector, adenoviral vector, lentiviral vector, herpesvirus vector, or adeno-associated viral vector (AAV).
In one aspect, the invention provides a pharmaceutical composition comprising any of the polynucleotides or any of the vectors as provided herein.
In one aspect, the invention provides a method for producing a CAR-expressing immune cell having reduced immunogenicity. In some embodiments, the method includes expressing in a CAR-expressing immune cell a base editor system comprising a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase domain, and two guide polynucleotides each targeting a polynucleotide encoding a different antigen, wherein the antigens are selected from the group consisting of CD5, CD7, CD3, CD33, and CD123, thereby producing a CAR-expressing immune cell having reduced immunogenicity. In some embodiments, the immune cell expresses or is contacted with a guide polynucleotide that targets a polynucleotide encoding a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.
In some embodiments, the method includes (a) expressing in a CAR-expressing immune cell a base editor system comprising a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase domain; and (b) contacting the CAR-expressing immune cell with at least two guide polynucleotides each targeting a polynucleotide encoding a different antigen, wherein the antigens are selected from the group consisting of CD5, CD7, CD3, CD33, and CD123, thereby producing a CAR-expressing immune cell having reduced immunogenicity.
In some embodiments, the method further includes contacting the CAR-expressing immune cell with a guide polynucleotide that targets a polynucleotide encoding a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In some embodiments, the immune cell expresses a chimeric antigen receptor (CAR) that targets an antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123. In some embodiments, the method further includes introducing a mutation that reduces or eliminates the expression of at least one polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1 into the immune cells, thereby producing a population of CAR-expressing immune cells having reduced immunogenicity.
In one aspect, the invention provides a method for producing a population of CAR-expressing immune cells having reduced immunogenicity. In some embodiments, the method includes a) introducing a mutation that reduces or eliminates the expression of at an antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123 into one immune cell and a different mutation in one of said antigens into a second immune cell; and b) introducing a mutation that reduces or eliminates the expression of at least one polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1 into the immune cells, thereby producing a population of CAR-expressing immune cells having reduced immunogenicity. In some embodiments, the chimeric antigen receptor expressed by the immune cell targets an antigen selected from the group consisting of CD3, CD5, CD7, CD33, and CD123. In some embodiments, an immune cell produced by the method expresses a chimeric antigen receptor that targets a CD3, CD5, and/or CD7 antigen and fails to express or expresses a reduced level of a CD3, CD5, and/or CD7 antigen. In some embodiments, an immune cell produced by the method expresses a chimeric antigen receptor that targets a CD33 and a CD123 antigen and fails to express or expresses a reduced level of a CD33 and a CD123 antigen. In some embodiments, the CAR is a CD5 chimeric antigen receptor (CAR). In some embodiments, the CD5 CAR is encoded by a CD5 CAR construct presented in Table 28.
In another aspect, the invention provides a method for producing an immune cell having reduced immunogenicity. In some embodiments, the method includes a) introducing a mutation in an endogenous CD5 gene sequence or regulatory element that reduces or eliminates expression of CD5; and b) expressing in the cell a CD5 CAR construct presented in Table 28. In some embodiments, the CD5 CAR construct encodes a CD5 CAR polypeptide that comprises or consists of an amino acid sequence selected from the following:








a)



  1 MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW  60


 


 61 VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC 120


 


121 TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC 180


 


181 KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM 240


 


241 GIYYCQQYDESPWTFGGGTKLEMKGSGDPAEPKSPDKTHTCPGQPREPQVYTLPPSRDEL 300


 


301 TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ 360


 


361 QGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWV 420


 


421 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQ 480


 


481 LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE1GMKGE 540


 


541 RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR;                           573


 


b)


  1 MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW  60


 


 61 VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC 120


 


121 TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC 180


 


181 KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM 240


 


241 GIYYCQQYDESPWTFGGGTKLEMKGSGDPATTTPAPRPPTPAPTIASQPLSLRPEACRPA 300


 


301 AGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR 360


 


361 RPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK 420


 


421 RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT 480


 


481 KDTYDALHMQALPPR;                                             495


 


c)


  1 MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW  60


 


 61 VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC 120


 


121 TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC 180


 


181 KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM 240


 


241 GIYYCQQYDESPWTFGGGTKLEMKGSGDPAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPS 300


 


301 PLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTR 360


 


361 KHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP 420


 


421 EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA 480


 


481 LHMQALPPR;                                                   489


 


d)


  1 MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW  60


 


 61 VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC 120


 


121 TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC 180


 


181 KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM 240


 


241 GIYYCQQYDESPWTFGGGTKLEMKGSGDPAEPKSPDKTHTCPGQPREPQVYTLPPSRDEL 300


 


301 TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ 360


 


361 QGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKPTTTPAPRPPTPAPTIASQPLSLRPEA 420


 


421 CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMR 480


 


481 PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL 540


 


541 DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST 600


 


601 ATKDTYDALHMQALPPR;                                           617


 


or


 


e)


  1 MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW  60


 


 61 VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC 120


 


121 TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC 180


 


181 KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM 240


 


241 GIYYCQQYDESPWTFGGGTKLEMKGSGDPATTTPAPRPPTPAPTIASQPLSLRPEACRPA 300


 


301 AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT 360


 


361 TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR 420


 


421 DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD   480


 


481 DALHMQALPPRX.                                                494






In some embodiments, the immune cell produced by the method exhibits fratricide resistance and/or increased anti-neoplasia activity as compared to a corresponding control cell. In some embodiments, the method is carried out in vivo or ex vivo. In some embodiments, the immune cell produced by the method comprises no detectable translocations. In some embodiments, the immune cell produced by the method comprises less than 1% indels. In some embodiments, the immune cell produced by the method comprises less than 5% of non-target edits. In some embodiments, the immune cell produced by the method comprises less than 5% of off-target edits. In some embodiments, the mutation is generated by nucleobase modification. In some embodiments, the mutation is in an exon. In some embodiments, the mutation results in a premature stop codon that reduces or eliminates protein expression. In some embodiments, the mutation is in a splice donor site or a splice acceptor site. In some embodiments, one or more mutations is generated by contacting a target polynucleotide with a base editor system comprising a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp), a deaminase, and one or more guide polynucleotides.
In some embodiments, the deaminase is an adenosine or cytidine deaminase. In some embodiments, the cytidine deaminase is BE4. In some embodiments, the mutation reduces expression of an encoded polypeptide by at least about 50% or more relative to a corresponding control cell lacking the mutation. In some embodiments, the guide polynucleotide comprises a sequence selected from those provided at Table 26. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from AGCGACUGCAGAAAGAAGAG (SEQ ID NO: 1) or CAUACCAGCUGAGCCGUCCG (SEQ ID NO: 2). In some embodiments, each of the one or more guide nucleic acid sequences targets the napDNAbp to a CD5, FAS, LAG-3, CD52, TRAC, B2M, CIITA, TRBC1, TRBC2 and/or a PDC1/PD-1 gene, or regulatory element. In some embodiments, the base editor and one or more guide nucleic acid sequences are introduced into the immune cell via electroporation, nucleofection, cationic lipid-mediated methods, viral transduction, or a combination thereof. In some embodiments, the method further includes expanding the immune cell in culture to generate a population of immune cells. In some embodiments, expression of the antigen or polypeptide is reduced in at least about 50% of the population of immune cells. In some embodiments, the method further includes depleting TCRα/β+ cells from the population of modified immune cells.
In one aspect, the invention provides a CAR-expressing immune cell having reduced immunogenicity produced by any of the methods as provided herein.
In another aspect, the invention provides a pharmaceutical composition comprising any of the immune cells as provided herein. In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable excipient.
In yet another aspect, the invention provides a method for killing a neoplastic cell. In some embodiments, the method includes contacting a neoplastic cell expressing an antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123, with two or more immune cells, each immune cell expressing a different chimeric antigen receptor that targets two of said antigens expressed by the cell, wherein the immune cell comprise one or more mutations that reduce or eliminate expression of the targeted antigen, and one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In some embodiments, the method is carried out in vitro or in vivo. In some embodiments, the neoplastic cell is derived from a neoplasia.
In one aspect, the invention provides a method for treating a neoplasia in a subject. In some embodiments, the method includes administering to the subject two or more immune cells, each immune cell expressing a different chimeric antigen receptor that targets an antigen expressed by a neoplastic cell of the subject, selected from the group consisting of CD5, CD7, CD3, CD33, and CD123, wherein the immune cell comprise one or more mutations that reduce or eliminate expression of the antigen, and each of the immune cells further comprise one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.
In some embodiments, the neoplasia is a hematological cancer. In some embodiments, the neoplasia is a liquid cancer. In some embodiments, the hematological cancer is leukemia, myeloma, and/or lymphoma. In some embodiments, the hematological cancer is a B cell cancer. In some embodiments, the hematological cancer is selected from at least one of the following T-cell acute lymphoblastic leukemia (T-ALL), mycosis fungoides (MF), Sézary syndrome (SS), Peripheral T/NK-cell lymphoma, Anaplastic large cell lymphoma ALK+, Primary cutaneous T-cell lymphoma, T-cell large granular lymphocytic leukemia, Angioimmunoblastic T/NK-cell lymphoma, Hepatosplenic T-cell lymphoma, Primary cutaneous CD30+ lymphoproliferative disorders, Extranodal NK/T-cell lymphoma, Adult T-cell leukemia/lymphoma, T-cell prolymphocytic leukemia, Subcutaneous panniculitis-like T-cell lymphoma, Primary cutaneous gamma-delta T-cell lymphoma, Aggressive NK-cell leukemia, and Enteropathy-associated T-cell lymphoma. In some embodiments, the hematological cancer is a T-cell acute lymphoblastic leukemia (T-ALL) cell. In some embodiments, the hematological cancer is acute myelogenous leukemia (AML).
In another aspect, the invention provides a method for treating a neoplasia in a selected subject. In some embodiments, the method includes administering to the selected subject two or more immune cells, each immune cell expressing a different chimeric antigen receptor that targets an antigen expressed by a neoplastic cell of the subject, wherein the antigen is selected from the group consisting of CD5, CD7, CD3, CD33, and CD123, wherein the immune cell comprise one or more mutations that reduce or eliminate expression of the antigen, and each of the immune cells further comprise one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, wherein the subject is selected as having a neoplasia expressing an antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123. In some embodiments, the hematological cancer is a T-cell acute lymphoblastic leukemia (T-ALL) cell. In some embodiments, the hematological cancer is acute myelogenous leukemia (AML). In some embodiments, the two or more immune cells expressing different chimeric antigen receptors are administered sequentially. In some embodiments, the two or more immune cells expressing different chimeric antigen receptors are administered simultaneously.
In yet another aspect, the invention provides a method for the antigen-dependent killing of a neoplastic cell in a subject. In some embodiments, the method includes administering to a subject having a neoplasia expressing an antigen selected from the group consisting of CD5, CD7, CD3, CD33, and CD123, two or more immune cells, each immune cell expressing a different chimeric antigen receptor that targets one of said antigens, wherein the immune cell comprise one or more mutations that reduce or eliminate expression of the targeted antigen, and one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.
In one aspect, the invention provides a method for the antigen-dependent killing of an acute myelogenous leukemia (AML) cell in a subject. In some embodiments, the method includes administering to a subject having AML expressing CD33 and CD123 antigens, two or more immune cells, each immune cell expressing a different chimeric antigen receptor that targets one of said antigens, wherein the immune cell comprise one or more mutations that reduce or eliminate expression of the targeted antigen, and one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.
In one aspect, the invention provides a method for the antigen-dependent killing of a T-cell acute lymphoblastic leukemia (T-ALL) cell in a subject. In some embodiments, the method includes administering to a subject having T-ALL expressing CD3, CD5, and CD7 antigens, at least three immune cells, each immune cell expressing a different chimeric antigen receptor that targets one of said antigens, wherein the immune cell comprise one or more mutations that reduce or eliminate expression of the targeted antigen, and one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.
In another aspect, the invention provides a method for treating cancer in a selected subject. In some embodiments, the method includes administering to the subject at least two immune cells, each immune cell expressing a chimeric antigen receptor that targets CD33 or CD123 antigens, wherein the immune cells comprise one or more mutations that reduce or eliminate expression of the targeted antigen, and one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, wherein the subject is selected by characterizing the cancer as expressing CD33 and CD123 antigens. In some embodiments, the cancer expressing CD33 and CD123 antigens is AML.
In yet another aspect, the invention provides a method for treating cancer in a selected subject. In some embodiments, the method includes administering to the subject three or more immune cells, each immune cell expressing a different chimeric antigen receptor that targets CD3, CD5, and CD7 antigens, wherein the immune cells comprise one or more mutations that reduce or eliminate expression of the targeted antigen, and one or more mutations that reduce or eliminate expression of a polypeptide selected from the group consisting of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PDT, wherein the subject is selected by characterizing the cancer as expressing CD3, CD5, and CD7 antigens. In some embodiments, the cancer expressing CD3, CD5, and CD7 antigens is T-ALL.
In some embodiments, the immune cells are cytotoxic T cells, regulatory T cells, T helper cells, dendritic cells, B cells, or NK cells. In some embodiments, the subject has been previously treated with lymphodepletion. In some embodiments, the lymphodepletion involves administration of cyclophosphamide, fludarabine, and/or alemtuzumab (Cy/Flu/Campath). In some embodiments, the subject is refractory to chemotherapy or has a high tumor burden. In some embodiments, the subject is subsequently treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT). In some embodiments, the immune cell is derived from a single human donor. In some embodiments, the immune cell is autologous to the subject. In some embodiments, the immune cell is allogenic to the subject. In some embodiments, the subject is a mammalian subject. In some embodiments, the subject is a human or rodent subject. In some embodiments, the subject is a pediatric human subject.
In one aspect, the invention provides a kit including any of the compositions provided herein for use in the treatment of cancer. In another aspect, the invention provides a kit including any of the base editor systems as provided herein for use in generating a CAR-expressing immune cell having reduced immunogenicity. In some embodiments, any of the kits as provided herein include written instructions for the use of the kit.
The description and examples herein illustrate embodiments of the present disclosure in detail. It is to be understood that this disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this disclosure, which are encompassed within its scope.
The practice of some embodiments disclosed herein employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R. I. Freshney, ed. (2010)).
Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and in view of the accompanying drawings as described hereinbelow.
DEFINITIONS
The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).
In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or,” unless stated otherwise, and is understood to be inclusive. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.
The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within one (1) or more than one (1) standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, such as within 5-fold or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” means within an acceptable error range for the particular value should be assumed.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.
By “adenosine deaminase” is meant a polypeptide or fragment thereof capable of catalyzing the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase catalyzing the hydrolytic deamination of adenosine to inosine or deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases (e.g., engineered adenosine deaminases, evolved adenosine deaminases) provided herein may be from any organism, such as a bacterium.
In some embodiments, the deaminase or deaminase domain is a variant of a naturally-occurring deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to a naturally-occurring deaminase. In some embodiments, the adenosine deaminase is from a bacterium, such as, E. coli, S. aureus, B. subtilis, S. typhi, S. putrefaciens, H. influenzae, C. crescentus, or G. sulfurreducens. In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is an E. coli TadA (ecTadA) deaminase or a fragment thereof.
In some embodiments, the ecTadA deaminase is truncated ecTadA. For example, the truncated ecTadA may be missing one or more N-terminal amino acids relative to a full-length ecTadA. In some embodiments, the truncated ecTadA may be missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the truncated ecTadA may be missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the ecTadA deaminase does not comprise an N-terminal methionine. In some embodiments, the TadA deaminase is an N-terminal truncated TadA. In particular embodiments, the TadA is any one of the TadAs described in PCT/US2017/045381, which is incorporated herein by reference in its entirety.
In some embodiments, the TadA deaminase is TadA variant. In some embodiments, the TadA variant is TadA*7.10. In some embodiments, the TadA variant is TadA*8. In some embodiments, the TadA*8 is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24. In some embodiments, the TadA*8 is TadA*8a, TadA*8b, TadA*8c, TadA*8d, or TadA*8e. In some embodiments, the TadA*8 is TadA*8e. In some embodiments, the TadA variant is TadA*9.
By “Adenosine Deaminase Base Editor 8 (ABE8) polypeptide” or “ABE8” is meant a base editor as defined herein comprising an adenosine deaminase variant comprising an alteration at amino acid position 82 and/or 166 of the following reference sequence:









MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGE



 



GWNRAIGLHDPTAHAEIMQGGLVMQNYRLIDATLYVTFEPCVM



 



CAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGRVEITEG



 



ILADECAALLCYFFRMPRQVFNAQKKAQSSTD






In some embodiments, ABE8 comprises further alterations, as described herein, relative to the reference sequence.
By “Adenosine Deaminase Base Editor 8 (ABE8) polynucleotide” is meant a polynucleotide encoding an ABE8.
By “Adenosine Deaminase Base Editor 9 (ABE9) polypeptide” or “ABE9” is meant a base editor as defined herein comprising an adenosine deaminase variant comprising one or more of the following alterations: R21N, R23H, E25F, N38G, L51W, P54C, M70V, Q71M, N72K, Y73S, V82T, M94V, P124W, T133K, D139L, D139M, C146R, and A158K, in the following reference sequence:









        10         20         30



MSEVEFSHEY WMRHALTLAK RARDEREVPV



        40         50         60



GAVLVLNNRV IGEGWNRAIG LHDPTAHAEI



        70         80         90



MALRQGGLVM QNYRLIDATL YVTFEPCVMC



       100        120        130



AGAMIHSRIG RVVFGVRNAK TGAAGSLMDV



       140        150



LHYPGMNHRV EITEGILADE CAALLCYFFR



       160



MPRQVFNAQK KAQSSTD






The relevant bases altered in the reference sequence are shown by underlining and bold font. In some embodiments, ABE9 comprises further alterations, as described herein, relative to the reference sequence. Details of ABE9 base editors are described in International PCT Application No. PCT/2020/049975, which is incorporated herein by reference for its entirety.
By “Adenosine Deaminase Base Editor 9 (ABE9) polynucleotide” is meant a polynucleotide encoding an ABE9.
“Administering” is referred to herein as providing one or more compositions described herein to a patient or a subject. By way of example and without limitation, composition administration, e.g., injection, can be performed by intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. In some embodiments, parenteral administration includes infusing or injecting intravascularly, intravenously, intramuscularly, intraarterially, intrathecally, intratumorally, intradermally, intraperitoneally, transtracheally, subcutaneously, subcuticularly, intraarticularly, subcapsularly, subarachnoidly and intrastemally. Alternatively, or concurrently, administration can be by the oral route.
By “agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
By “alteration” is meant a change (e.g. increase or decrease) in the structure, expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration (e.g., increase or decrease) includes a change in a polynucleotide or polypeptide sequence or a change in expression levels, such as a 10% change, a 25% change, a 40% change, and a 50% change, or greater.
“Allogeneic,” as used herein, refers to cells of the same species that differ genetically to the cell in comparison.
By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
By “analog” is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polynucleotide or polypeptide analog retains the biological activity of a corresponding naturally-occurring polynucleotide or polypeptide, while having certain modifications that enhance the analog's function relative to a naturally occurring polynucleotide or polypeptide. Such modifications could increase the analog's affinity for DNA, efficiency, specificity, protease or nuclease resistance, membrane permeability, and/or half-life, without altering, for example, ligand binding. An analog may include an unnatural nucleotide or amino acid.
By “anti-neoplasia activity” is meant preventing or inhibiting the maturation and/or proliferation of neoplasms.
“Autologous,” as used herein, refers to cells from the same subject.
As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen, and includes polyclonal, monoclonal, genetically engineered, and otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), and antigen binding fragments of antibodies, including, for example, Fab′, F(ab′)2, Fab, Fv, rlgG, and scFv fragments. Unless otherwise indicated, the term “monoclonal antibody” (mAb) is meant to include both intact molecules, as well as antibody fragments (including, for example, Fab and F(ab′)2 fragments) that are capable of specifically binding to a target protein. As used herein, the Fab and F(ab′)2 fragments refer to antibody fragments that lack the Fc fragment of an intact antibody. Examples of these antibody fragments are described herein.
By “B cell maturation antigen, or tumor necrosis factor receptor superfamily member 17 polypeptide, (BCMA)” is meant a protein having at least about 85% amino acid sequence identify to NCBI Accession No. NP_001183 or a fragment thereof that is expressed on mature B lymphocytes. An exemplary BCMA polypeptide sequence is provided below.
>NP_001183.2 tumor necrosis factor receptor superfamily member 17 [Homo sapiens]









>NP_001 183.2 tumor neCrosis faCtor reCeptor



superfamily member 17 [Homo sapiens]



MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQR



 



YCNASVTNSVKGTNAILWTCLGLSLIISLAVFVIMFLLR



 



KINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPR



 



GLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATIL



 



VTTKTNDYCKSLPAALSATEIEKSISAR






This antigen can be targeted in relapsed or refractory multiple myeloma and other hematological neoplasia therapies.
By “B cell maturation antigen, or tumor necrosis factor receptor superfamily member 17, (BCMA) polynucleotide” is meant a nucleic acid molecule encoding a BCMA polypeptide. The BCMA gene encodes a cell surface receptor that recognizes B cell activating factor. An exemplary B2M polynucleotide sequence is provided below.









>NM_001192.2 Homo sapiens TNF reCeptor



superfamily member 17 (TNFRSF17), mRNA



AAGACTCAAACTTAGAAACTTGAATTAGATGTGGTATTCAAAT



 



CCTTAGCTGCCGCGAAGACACAGACAGCCCCCGTAAGAACCCA



 



CGAAGCAGGCGAAGTTCATTGTTCTCAACATTCTAGCTGCTCT



 



TGCTGCATTTGCTCTGGAATTCTTGTAGAGATATTACTTGTCC



 



TTCCAGGCTGTTCTTTCTGTAGCTCCCTTGTTTTCTTTTTGTG



 



ATCATGTTGCAGATGGCTGGGCAGTGCTCCCAAAATGAATATT



 



TTGACAGTTTGTTGCATGCTTGCATACCTTGTCAACTTCGATG



 



TTCTTCTAATACTCCTCCTCTAACATGTCAGCGTTATTGTAAT



 



GCAAGTGTGACCAATTCAGTGAAAGGAACGAATGCGATTCTCT



 



GGACCTGTTTGGGACTGAGCTTAATAATTTCTTTGGCAGTTTT



 



CGTGCTAATGTTTTTGCTAAGGAAGATAAACTCTGAACCATTA



 



AAGGACGAGTTTAAAAACACAGGATCAGGTCTCCTGGGCATGG



 



CTAACATTGACCTGGAAAAGAGCAGGACTGGTGATGAAATTAT



 



TCTTCCGAGAGGCCTCGAGTACACGGTGGAAGAATGCACCTGT



 



GAAGACTGCATCAAGAGCAAACCGAAGGTCGACTCTGACCATT



 



GCTTTCCACTCCCAGCTATGGAGGAAGGCGCAACCATTCTTGT



 



CACCACGAAAACGAATGACTATTGCAAGAGCCTGCCAGCTGCT



 



TTGAGTGCTACGGAGATAGAGAAATCAATTTCTGCTAGGTAAT



 



TAACCATTTCGACTCGAGCAGTGCCACTTTAAAAATCTTTTGT



 



CAGAATAGATGATGTGTCAGATCTCTTTAGGATGACTGTATTT



 



TTCAGTTGCCGATACAGCTTTTTGTCCTCTAACTGTGGAAACT



 



CTTTATGTTAGATATATTTCTCTAGGTTACTGTTGGGAGCTTA



 



ATGGTAGAAACTTCCTTGGTTTCATGATTAAACTCTTTTTTTT



 



CCTGA






By “base editor (BE),” or “nucleobase editor (NBE)” is meant an agent that binds a polynucleotide and has nucleobase modifying activity. In one embodiment, the agent binds the polynucleotide at a specific sequence using a nucleic acid programmable DNA binding protein. In another embodiment, the base editor is an enzyme capable of modifying a cytidine base within a nucleic acid molecule (e.g., DNA). In some embodiments, the base editor is capable of deaminating a base within a nucleic acid molecule. In some embodiments, the base editor is capable of deaminating a base within a DNA molecule. In some embodiments, the base editor is capable of deaminating a cytidine in DNA. In some embodiments, the base editor is a fusion protein comprising a cytidine deaminase or an adenosine deaminase. In some embodiments, the base editor is a Cas9 protein fused to a cytidine deaminase or an adenosine deaminase. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a cytidine deaminase or an adenosine deaminase. In some embodiments, the base editor is fused to an inhibitor of base excision repair, for example, a UGI domain. In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase and an inhibitor of base excision repair, such as a UGI domain.
In some embodiments, the cytidine deaminase or an or an adenosine deaminase nucleobase editor polypeptide comprising the following domains A-B: NH2-[A-B]-COOH, wherein A comprises a cytidine deaminase domain, an adenosine deaminase domain or an active fragment thereof, and wherein B comprises one or more domains having nucleic acid sequence specific binding activity. In one embodiment, the cytidine or adenosine deaminase Nucleobase Editor polypeptide of the previous aspect contains: NH2-[An-Bo]-COOH, wherein A comprises a cytidine deaminase domain, an adenosine deaminase domain, or an active fragment thereof, wherein n is an integer: 1, 2, 3, 4, or 5; and wherein B comprises a domain having nucleic acid sequence specific binding activity; and wherein o is an integer: 1, 2, 3, 4, or 5. In one embodiment, the polypeptide contains one or more nuclear localization sequences. In one embodiment, the polypeptide contains at least one of said nuclear localization sequences is at the N-terminus or C-terminus. In one embodiment, the polypeptide contains the nuclear localization signal is a bipartite nuclear localization signal. In one embodiment, the polypeptide contains one or more domains linked by a linker.
In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the base editor is an adenosine base editor (ABE) and a cytidine base editor (CBE). In some embodiments, the base editor is a nuclease-inactive Cas9 (dCas9) fused to an adenosine deaminase. In some embodiments, the Cas9 is a circular permutant Cas9 (e.g., spCas9 or saCas9). Circular permutant Cas9s are known in the art and described, for example, in Oakes et al., Cell 176, 254-267, 2019. In some embodiments, the base editor is fused to an inhibitor of base excision repair, for example, a UGI domain, or a dISN domain. In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase and an inhibitor of base excision repair, such as a UGI or dISN domain. In other embodiments the base editor is an abasic base editor.
In some embodiments, an adenosine deaminase is evolved from TadA. In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR associated (e.g., Cas or Cpf1) enzyme. In some embodiments, the base editor is a catalytically dead Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a deaminase domain. In some embodiments, the base editor is fused to an inhibitor of base excision repair (BER). In some embodiments, the inhibitor of base excision repair is a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the inhibitor of base excision repair is an inosine base excision repair inhibitor.
In some embodiments, base editors are generated by cloning an adenosine deaminase variant (e.g., TadA*7.10) into a scaffold that includes a circular permutant Cas9 (e.g., spCAS9) and a bipartite nuclear localization sequence. In some embodiments, base editors are generated (e.g. ABE8) by cloning an adenosine deaminase variant (e.g., TadA*8) into a scaffold that includes a circular permutant Cas9 (e.g., spCAS9 or saCAS9) and a bipartite nuclear localization sequence. Circular permutant Cas9s are known in the art and described, for example, in Oakes et al., Cell 176, 254-267, 2019.
In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR associated (e.g., Cas or Cpf1) enzyme. In some embodiments, the base editor is a catalytically dead Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a deaminase domain. In some embodiments, the base editor is fused to an inhibitor of base excision repair (BER). In some embodiments, the inhibitor of base excision repair is a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the inhibitor of base excision repair is an inosine base excision repair inhibitor.
Details of base editors are described in International PCT Application Nos. PCT/2017/045381 (WO 2018/027078) and PCT/US2016/058344 (WO 2017/070632), each of which is incorporated herein by reference for its entirety. Also see Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), and Rees, H. A., et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 December; 19(12):770-788. doi: 10.1038/s41576-018-0059-1, the entire contents of which are hereby incorporated by reference.
By way of example, the adenine base editor (ABE) as used in the base editing compositions, systems and methods described herein has the nucleic acid sequence (8877 base pairs), (Addgene, Watertown, M A.; Gaudelli N M, et al., Nature. 2017 Nov. 23; 551(7681):464-471. doi: 10.1038/nature24644; Koblan L W, et al., Nat Biotechnol. 2018 October; 36(9):843-846. doi: 10.1038/nbt.4172.) as provided below. Polynucleotide sequences having at least 95% or greater identity to the ABE nucleic acid sequence are also encompassed.









ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAA



 



ATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGG



 



GACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCG



 



CTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGG



 



GCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC



 



CACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAA



 



ATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCC



 



ATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTC



 



TATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCT



 



AGAGATCCGCGGCCGCTAATACGACTCACTATAGGGAGAG



 



CCGCCACCATGAAACGGACAGCCGACGGAAGCGAGTTCGA



 



GTCACCAAAGAAGAAGCGGAAAGTCTCTGAAGTCGAGTTT



 



AGCCACGAGTATTGGATGAGGCACGCACTGACCCTGGCAA



 



AGCGAGCATGGGATGAAAGAGAAGTCCCCGTGGGCGCCGT



 



GCTGGTGCACAACAATAGAGTGATCGGAGAGGGATGGAAC



 



AGGCCAATCGGCCGCCACGACCCTACCGCACACGCAGAGA



 



TCATGGCACTGAGGCAGGGAGGCCTGGTCATGCAGAATTA



 



CCGCCTGATCGATGCCACCCTGTATGTGACACTGGAGCCA



 



TGCGTGATGTGCGCAGGAGCAATGATCCACAGCAGGATCG



 



GAAGAGTGGTGTTCGGAGCACGGGACGCCAAGACCGGCGC



 



AGCAGGCTCCCTGATGGATGTGCTGCACCACCCCGGCATG



 



AACCACCGGGTGGAGATCACAGAGGGAATCCTGGCAGACG



 



AGTGCGCCGCCCTGCTGAGCGATTTCTTTAGAATGCGGAG



 



ACAGGAGATCAAGGCCCAGAAGAAGGCACAGAGCTCCACC



 



GACTCTGGAGGATCTAGCGGAGGATCCTCTGGAAGCGAGA



 



CACCAGGCACAAGCGAGTCCGCCACACCAGAGAGCTCCGG



 



CGGCTCCTCCGGAGGATCCTCTGAGGTGGAGTTTTCCCAC



 



GAGTACTGGATGAGACATGCCCTGACCCTGGCCAAGAGGG



 



CACGCGATGAGAGGGAGGTGCCTGTGGGAGCCGTGCTGGT



 



GCTGAACAATAGAGTGATCGGCGAGGGCTGGAACAGAGCC



 



ATCGGCCTGCACGACCCAACAGCCCATGCCGAAATTATGG



 



CCCTGAGACAGGGCGGCCTGGTCATGCAGAACTACAGACT



 



GATTGACGCCACCCTGTACGTGACATTCGAGCCTTGCGTG



 



ATGTGCGCCGGCGCCATGATCCACTCTAGGATCGGCCGCG



 



TGGTGTTTGGCGTGAGGAACGCAAAAACCGGCGCCGCAGG



 



CTCCCTGATGGACGTGCTGCACTACCCCGGCATGAATCAC



 



CGCGTCGAAATTACCGAGGGAATCCTGGCAGATGAATGTG



 



CCGCCCTGCTGTGCTATTTCTTTCGGATGCCTAGACAGGT



 



GTTCAATGCTCAGAAGAAGGCCCAGAGCTCCACCGACTCC



 



GGAGGATCTAGCGGAGGCTCCTCTGGCTCTGAGACACCTG



 



GCACAAGCGAGAGCGCAACACCTGAAAGCAGCGGGGGCAG



 



CAGCGGGGGGTCAGACAAGAAGTACAGCATCGGCCTGGCC



 



ATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACG



 



AGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAA



 



CACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCC



 



CTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGC



 



TGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAA



 



CCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATG



 



GCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGT



 



CCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCC



 



CATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAG



 



AAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGG



 



ACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGC



 



CCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATC



 



GAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGC



 



TGTTCATGCAGCTGGTGCAGACCTACAACCAGCTGTTCGA



 



GGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCC



 



ATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAA



 



ATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCT



 



GTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCC



 



AACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAAC



 



TGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAA



 



CCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTT



 



CTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCG



 



ACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCT



 



GAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAG



 



GACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGC



 



CTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAA



 



CGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAA



 



GAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGG



 



ACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGA



 



CCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATC



 



CCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGC



 



GGCGGCAGGAAGATTTTTAGCCATTCCTGAAGGACAACCG



 



GGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTAC



 



TACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCT



 



GGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAA



 



CTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGC



 



TTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCA



 



ACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTA



 



CTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTG



 



ACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGC



 



AGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCG



 



GAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAG



 



AAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGG



 



AAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCT



 



GCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAG



 



GAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGA



 



CACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAA



 



AACCTATGCGCACCTGTTCGACGACAAAGTGATGAAGCAG



 



CTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCC



 



GGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAA



 



GACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAAC



 



AGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT



 



TTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGG



 



CGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGC



 



CCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGG



 



TGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGA



 



GAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACC



 



CAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGA



 



TCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAA



 



AGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAG



 



CTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACG



 



TGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGA



 



TGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGAC



 



TCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACC



 



GGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAA



 



GAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAG



 



CTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCG



 



AGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCAT



 



CAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCAC



 



GTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACG



 



ACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCAC



 



CCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTC



 



CAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACG



 



CCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCT



 



GATCAAAAAGTACCCTAAGCTGGAAAGGGAGTTCGTGTAC



 



GGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCA



 



AGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTT



 



CTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATT



 



ACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCG



 



AGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGG



 



CCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCC



 



CAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCG



 



GCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGA



 



TAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAG



 



TACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGC



 



TGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACT



 



GAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAA



 



AGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAG



 



CCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAA



 



GCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGG



 



AAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAA



 



ACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTA



 



CCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAG



 



GATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGC



 



ACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTC



 



CAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTG



 



CTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAG



 



AGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAA



 



TCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACC



 



ATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGG



 



ACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGA



 



GACACGGATCGACCTGTCTCAGCTGGGAGGTGACTCTGGC



 



GGCTCAAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCA



 



AGAAGAAGAGGAAAGTCTAACCGGTCATCATCACCATCAC



 



CATTGAGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTT



 



CTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCC



 



TTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCC



 



TAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGT



 



GTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAA



 



GGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGAT



 



GCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCT



 



GGGGCTCGATACCGTCGACCTCTAGCTAGAGCTTGGCGTA



 



ATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCG



 



CTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGT



 



GTAAAGCCTAGGGTGCCTAATGAGTGAGCTAACTCACATT



 



AATTGCGTTGCGCTCACTGGCCGCTTTCCAGTCGGGAAAC



 



CTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGG



 



GGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTC



 



GCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGA



 



GCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCA



 



CAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAA



 



AGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTG



 



CTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATC



 



ACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGAC



 



AGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCC



 



CTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGAT



 



ACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTC



 



TCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTC



 



GTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTC



 



AGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGA



 



GTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCA



 



GCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCG



 



GTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTA



 



CACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAG



 



CCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCG



 



GCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTG



 



CAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAA



 



GATCCTTTGATCTTTTCTACGGGGTCTGACACTCAGTGGA



 



ACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATC



 



AAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA



 



AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGT



 



CTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTC



 



AGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC



 



CCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCAT



 



CTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTC



 



ACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGA



 



AGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCT



 



CCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAG



 



TAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATT



 



GCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGG



 



CTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTAC



 



ATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTC



 



GGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGT



 



TATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTAC



 



TGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAG



 



TACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGAC



 



CGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGC



 



GCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAA



 



CGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGT



 



TGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTG



 



ATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGA



 



GCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAA



 



GGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTT



 



TCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATG



 



AGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAA



 



TAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGA



 



CGTCGACGGATCGGGAGATCGATCTCCCGATCCCCTAGGG



 



TCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAA



 



GCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGA



 



GTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGC



 



TTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGC



 



GTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCG



 



TTGACATTGATTATTGACTAGTTATTAATAGTAATCAATT



 



ACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCG



 



TTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC



 



CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTT



 



CCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT



 



GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACA



 



TCAAGTGTATC






In some embodiments, the base editor is an Adenosine Deaminase Base Editor 8 (ABE8). In some embodiments, the ABE8 is selected from a base editor from Table 13, 14 or 16 infra. In some embodiments, ABE8 contains an adenosine deaminase variant evolved from TadA. In some embodiments, the adenosine deaminase variant of ABE8 is a TadA*8 variant as described in Table 11, 13 or 14 infra. In some embodiments, the adenosine deaminase variant is TadA*7.10 variant (e.g., TadA*8) comprising one or more of an alteration selected from the group of Y147T, Y147R, Q154S, Y123H, V82S, T166R, and/or Q154R. In various embodiments, ABE8 comprises TadA*7.10 variant (e.g., TadA*8) with a combination of alterations selected from the group of. Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. In some embodiments ABE8 is a monomeric construct. In some embodiments, ABE8 is a heterodimeric construct. In some embodiments, the ABE8 base editor comprises the sequence:









MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV



 



IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATL



 



YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV



 



LHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQK



 



KAQSSTD.






By way of example, a cytidine base editor (CBE) as used in the base editing compositions, systems and methods described herein has the following nucleic acid sequence (8877 base pairs), (Addgene, Watertown, M A.; Komor A C, et al., 2017, Sci Adv., 30; 3(8):eaao4774. doi: 10.1126/sciadv.aao4774) as provided below. Polynucleotide sequences having at least 95% or greater identity to the BE4 nucleic acid sequence are also encompassed.








   1 ATATGCCAAG TACGCCCCCT ATTGACGTCA ATGACGGTAA ATGGCCCGCC TGGCATTATG



 


  61 CCCAGTACAT GACCTTATGG GACTTTCCTA CTTGGCAGTA CATCTACGTA TTAGTCATCG


 


 121 CTATTACCAT GGTGATGCGG TTTTGGCAGT ACATCAATGG GCGTGGATAG CGGTTTGACT


 


 181 CACGGGGATT TCCAAGTCTC CACCCCATTG ACGTCAATGG GAGTTTGTTT TGGCACCAAA


 


 241 ATCAACGGGA CTTTCCAAAA TGTCGTAACA ACTCCGCCCC ATTGACGCAA ATGGGCGGTA


 


 301 GGCGTGTACG GTGGGAGGTC TATATAAGCA GAGCTGGTTT AGTGAACCGT CAGATCCGCT


 


 361 AGAGATCCGC GGCCGCTAAT ACGACTCACT ATAGGGAGAG CCGCCACCAT GAGCTCAGAG


 


 421 ACTGGCCCAG TGGCTGTGGA CCCCACATTG AGACGGCGGA TCGAGCCCCA TGAGTTTGAG


 


 481 GTATTCTTCG ATCCGAGAGA GCTCCGCAAG GAGACCTGCC TGCTTTACGA AATTAATTGG


 


 541 GGGGGCCGGC ACTCCATTTG GCGACATACA TCACAGAACA CTAACAAGCA CGTCGAAGTC


 


 601 AACTTCATCG AGAAGTTCAC GACAGAAAGA TATTTCTGTC CGAACACAAG GTGCAGCATT


 


 661 ACCTGGTTTC TCAGCTGGAG CCCATGCGGC GAATGTAGTA GGGCCATCAC TGAATTCCTG


 


 721 TCAAGGTATC CCCACGTCAC TCTGTTTATT TACATCGCAA GGCTGTACCA CCACGCTGAC


 


 781 CCCCGCAATC GACAAGGCCT GCGGGATTTG ATCTCTTCAG GTGTGACTAT CCAAATTATG


 


 841 ACTGAGCAGG AGTCAGGATA CTGCTGGAGA AACTTTGTGA ATTATAGCCC GAGTAATGAA


 


 901 GCCCACTGGC CTAGGTATCC CCATCTGTGG GTACGACTGT ACGTTCTTGA ACTGTACTGC


 


 961 ATCATACTGG GCCTGCCTCC TTGTCTCAAC ATTCTGAGAA GGAAGCAGCC ACAGCTGACA


 


1021 TTCTTTACCA TCGCTCTTCA GTCTTGTCAT TACCAGCGAC TGCCCCCACA CATTCTCTGG


 


1081 GCCACCGGGT TGAAATCTGG TGGTTCTTCT GGTGGTTCTA GCGGCAGCGA GACTCCCGGG


 


1141 ACCTCAGAGT CCGCCACACC CGAAAGTTCT GGTGGTTCTT CTGGTGGTTC TGATAAAAAG


 


1201 TATTCTATTG GTTTAGCCAT CGGCACTAAT TCCGTTGGAT GGGCTGTCAT AACCGATGAA


 


1261 TACAAAGTAC CTTCAAAGAA ATTTAAGGTG TTGGGGAACA CAGACCGTCA TTCGATTAAA


 


1321 AAGAATCTTA TCGGTGCCCT CCTATTCGAT AGTGGCGAAA CGGCAGAGGC GACTCGCCTG


 


1381 AAACGAACCG CTCGGAGAAG GTATACACGT CGCAAGAACC GAATATGTTA CTTACAAGAA


 


1441 ATTTTTAGCA ATGAGATGGC CAAAGTTGAC GATTCTTTCT TTCACCGTTT GGAAGAGTCC


 


1501 TTCCTTGTCG AAGAGGACAA GAAACATGAA CGGCACCCCA TCTTTGGAAA CATAGTAGAT


 


1561 GAGGTGGCAT ATCATGAAAA GTACCCAACG ATTTATCACC TCAGAAAAAA GCTAGTTGAC


 


1621 TCAACTGATA AAGCGGACCT GAGGTTAATC TACTTGGCTC TTGCCCATAT GATAAAGTTC


 


1681 CGTGGGCACT TTCTCATTGA GGGTGATCTA AATCCGGACA ACTCGGATGT CGACAAACTG


 


1741 TTCATCCAGT TAGTACAAAC CTATAATCAG TTGTTTGAAG AGAACCCTAT AAATGCAAGT


 


1801 GGCGTGGATG CGAAGGCTAT TCTTAGCGCC CGCCTCTCTA AATCCCGACG GCTAGAAAAC


 


1861 CTGATCGCAC AATTACCCGG AGAGAAGAAA AATGGGTTGT TCGGTAACCT TATAGCGCTC


 


1921 TCACTAGGCC TGACACCAAA TTTTAAGTCG AACTTCGACT TAGCTGAAGA TGCCAAATTG


 


1981 CAGCTTAGTA AGGACACGTA CGATGACGAT CTCGACAATC TACTGGCACA AATTGGAGAT


 


2041 CAGTATGCGG ACTTATTTTT GGCTGCCAAA AACCTTAGCG ATGCAATCCT CCTATCTGAC


 


2101 ATACTGAGAG TTAATACTGA GATTACCAAG GCGCCGTTAT CCGCTTCAAT GATCAAAAGG


 


2161 TACGATGAAC ATCACCAAGA CTTGACACTT CTCAAGGCCC TAGTCCGTCA GCAACTGCCT


 


2221 GAGAAATATA AGGAAATATT CTTTGATCAG TCGAAAAACG GGTACGCAGG TTATATTGAC


 


2281 GGCGGAGCGA GTCAAGAGGA ATTCTACAAG TTTATCAAAC CCATATTAGA GAAGATGGAT


 


2341 GGGACGGAAG AGTTGCTTGT AAAACTCAAT CGCGAAGATC TACTGCGAAA GCAGCGGACT


 


2401 TTCGAGAACG GTAGCATTCC ACATCAAATC CACTTAGGCG AATTGCATGC TATACTTAGA


 


2461 AGGCAGGAGG ATTTTTATCC GTTCCTCAAA GACAATCGTG AAAAGATTGA GAAAATCCTA


 


2521 ACCTTTCGCA TACCTTACTA TGTGGGACCC CTGGCCCGAG GGAACTCTCG GTTCGCATGG


 


2581 ATGACAAGAA AGTCCGAAGA AACGATTACT CCATGGAATT TTGAGGAAGT TGTCGATAAA


 


2641 GGTGCGTCAG CTCAATCGTT CATCGAGAGG ATGACCAACT TTGACAAGAA TTTACCGAAC


 


2701 GAAAAAGTAT TGCCTAAGCA CAGTTTACTT TACGAGTATT TCACAGTGTA CAATGAACTC


 


2751 ACGAAAGTTA AGTATGTCAC TGAGGG+ATG CGTAAACCCG CCTTTCTAAG CGGAGAACAG


 


2821 AAGAAAGCAA TAGTAGATCT GTTATTCAAG ACCAACCGCA AAGTGACAGT TAAGCAATTG


 


2881 AAAGAGGACT ACTTTAAGAA AATTGAATGC TTCGATTCTG TCGAGATCTC CGGGGTAGAA


 


2941 GATCGATTTA ATGCGTCACT TGGTACGTAT CATGACCTCC TAAAGATAAT TAAAGATAAG


 


3001 GACTTCCTGG ATAACGAAGA GAATGAAGAT ATCTTAGAAG ATATAGTGTT GACTCTTACC


 


3061 CTCTTTGAAG ATCGGGAAAT GATTGAGGAA AGACTAAAAA CATACGCTCA CCTGTTCGAC


 


3121 GATAAGGTTA TGAAACAGTT AAAGAGGCGT CGCTATACGG GCTGGGGACG ATTGTCGCGG


 


3181 AAACTTATCA ACGGGATAAG AGACAAGCAA AGTGGTAAAA CTATTCTCGA TTTTCTAAAG


 


3241 AGCGACGGCT TCGCCAATAG GAACTTTATG GAGCTGATCC ATGATGACTC TTTAACCTTC


 


3301 AAAGAGGATA TACAAAAGGC ACAGGTTTCC GGACAAGGGG ACTCATTGCA CGAACATATT


 


3361 GCGAATCTTG CTGGTTCGCC AGCCATCAAA AAGGGCATAC TCCAGACAGT CAAAGTAGTG


 


3421 GATGAGCTAG TTAAGGTCAT GGGACGTCAC AAACCGGAAA ACATTGTAAT CGAGATGGCA


 


3481 CGCGAAAATC AAACGACTCA GAAGGGGCAA AAAAACAGTC GAGAGCGGAT GAAGAGAATA


 


3541 GAAGAGGGTA TTAAAGAACT GGGCAGCCAG ATCTTAAAGG AGCATCCTGT GGAAAATACC


 


3601 CAATTGCAGA ACGAGAAACT TTACCTCTAT TACCTACAAA ATGGAAGGGA CATGTATGTT


 


3661 GATCAGGAAC TGGACATAAA CCGTTTATCT GATTACGACG TCGATCACAT TGTACCCCAA


 


3721 TCCTTTTTGA AGGACGATTC AATCGACAAT AAAGTGCTTA CACGCTCGGA TAAGAACCGA


 


3781 GGGAAAAGTG ACAATGTTCC AAGCGAGGAA GTCGTAAAGA AAATGAAGAA CTATTGGCGG


 


3841 GAGCTCCTAA ATGCGAAACT GATAACGCAA AGAAAGTTCG ATAACTTAAC TAAAGCTGAG


 


3901 AGGGGTGGCT TGTCTGAACT TGACAAGGCC GGATTTATTA AAggTCAGCT CGTGGAAAgC


 


3961 CGCCAAATCA CAAAGCATGT TGCACAGATA CTAGATTCCC GAATGAATAC GAAATACGAC


 


4021 GAGAACGATA AGCTGATTCG GGAAGTCAAA GTAATCACTT TAAAGTCAAA ATTGGTGTCG


 


4081 GACTTCAGAA AGGATTTTCA ATTCTATAAA GTTAGGGAGA TAAATAACTA CCACCATGCG


 


4141 CACGACGCTT ATCTTAATGC CGTCGTAGGG ACCGCACTCA TTAAGAAATA CCCGAAGCTA


 


4201 GAAAGTGAGT TTGTGTATGG TGATTACAAA GTTTATGACG TCCGTAAGAT GATCGCGAAA


 


4261 AGCGAACAGG AGATAGGCAA GGCTACAGCC AAATACTTCT TTTATTCTAA CATTATGAAT


 


4321 TTCTTTAAGA CGGAAATCAC TCTGGCAAAC GGAGAGATAC GCAAACGACC TTTAATTGAA


 


4381 ACCAATGGGG AGACAGGTGA AATCGTATGG GATAAGGGCC GGGACTTCGC GACGGTGAGA


 


4441 AAAGTTTTGT CCATGCCCCA AGTCAACATA GTAAAGAAAA CTGAGGTGCA GACCGGAGGG


 


4501 TTTTCAAAGG AATCGATTCT TCCAAAAAGG AATAGTGATA AGCTCATCGC TCGTAAAAAG


 


4561 GACTGGGACC CGAAAAAGTA CGGTGGCTTC GATAGCCCTA CAGTTGCCTA TTCTGTCCTA


 


4621 GTAGTGGCAA AAGTTGAGAA GGGAAAATCC AAGAAACTGA AGTCAGTCAA AGAATTATTG


 


4681 GGGATAACGA TTATGGAGCG CTCGTCTTTT GAAAAGAACC CCATCGACTT CCTTGAGGCG


 


4741 AAAGGTTACA AGGAAGTAAA AAAGGATCTC ATAATTAAAC TACCAAAGTA TAGTCTGTTT


 


4801 GAGTTAGAAA ATGGCCGAAA ACGGATGTTG GCTAGCGCCG GAGAGCTTCA AAAGGGGAAC


 


4861 GAACTCGCAC TACCGTCTAA ATACGTGAAT TTCCTGTATT TAGCGTCCCA TTACGAGAAG


 


4921 TTGAAAGGTT CACCTGAAGA TAACGAACAG AAGCAACTTT TTGTTGAGCA GCACAAACAT


 


4981 TATCTCGACG AAATCATAGA GCAAATTTCG GAATTCAGTA AGAGAGTCAT CCTAGCTGAT


 


5041 GCCAATCTGG ACAAAGTATT AAGCGCATAC AACAAGCACA GGGATAAACC CATACGTGAG


 


5101 CAGGCGGAAA ATATTATCCA TTTGTTTACT CTTACCAACC TCGGCGCTCC AGCCGCATTC


 


5161 AAGTATTTTG ACACAACGAT AGATCGCAAA CGATACACTT CTACCAAGGA GGTGCTAGAC


 


5221 GCGACACTGA TTCACCAATC CATCACGGGA TTATATGAAA CTCGGATAGA TTTGTCACAG


 


5281 CTTGGGGGTG ACTCTGGTGG TTCTGGAAGA TCTGGTGGTT CTACTAATCT GTCAGATATT


 


5341 ATTGAAAAGG AGACCGGTAA GCAACTGGTT ATCCAGGAAT CCATCCTCAT GCTCCCAGAG


 


5401 GAGGTGGAAG AAGTCATTGG GAACAAGCCG GAAAGCGATA TACTCGTGCA CACCGCCTAC


 


5461 GACGAGAGCA CCGACGAGAA TGTCATGCTT CTGACTAGCG ACGCCCCTGA ATAGAAGCCT


 


5521 TGGGCTCTGG TCATACAGGA TAGCAACGGT GAGAACAAGA TTAAGATGCT CTCTGGTGGT


 


5581 TCTGGAGGAT CTGGTGGTTC TACTAATCTG TCAGATATTA TTGAAAAGGA GACCGGTAAG


 


5641 CAACTGGTTA TCCAGGAATC CATCCTCATG CTCCCAGAGG AGGTGGAAGA AGTCATTGGG


 


5701 AACAAGCCGG AAAGCGATAT ACTCGTGCAC ACCGCCTACG ACGAGAGCAC CGACGAGAAT


 


5761 GTCATGCTTC TGACTAGCGA CGCCCCTGAA TACAAGCCTT GGGCTCTGGT CATACAGGAT


 


5821 AGCAACGGTG AGAACAAGAT TAAGATGCTC TCTGGTGGTT CTCCCAAGAA GAAGAGGAAA


 


5881 GTCTAACCGG TCATCATCAC CATCACCATT GAGTTTAAAC CCGCTGATCA GCCTCGACTG


 


5941 TGCCTTCTAG TTGCCAGCCA TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC TTGACCCTGG


 


6001 AAGGTGCCAC TCCCACTGTC CTTTCCTAAT AAAATGAGGA AATTGCATCG CATTGTCTGA


 


6061 GTAGGTGTCA TTCTATTCTG GGGGGTGGGG TGGGGCAGGA CAGCAAGGGG GAGGATTGGG


 


6121 AAGACAATAG CAGGCATGCT GGGGATGCGG TGGGCTCTAT GGCTTCTGAG GCGGAAAGAA


 


6181 CCAGCTGGGG CTCGATACCG TCGACCTCTA GCTAGAGCTT GGCGTAATCA TGGTCATAGC


 


6241 TGTTTCCTGT GTGAAATTGT TATCCGCTCA CAATTCCACA CAACATACGA GCCGGAAGCA


 


6301 TAAAGTGTAA AGCCTAGGGT GCCTAATGAG TGAGCTAACT CACATTAATT GCGTTGCGCT


 


6361 CACTGCCCGC TTTCCAGTCG GGAAACCTGT CGTGCCAGCT GCATTAATGA ATCGGCCAAC


 


6421 GCGCGGGGAG AGGCGGTTTG CGTATTGGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC


 


6481 TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG GTAATACGGT


 


6541 TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC CAGCAAAAGG


 


6601 CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACG


 


6661 AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT


 


6721 ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA


 


6781 CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT


 


6841 GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC


 


6901 CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA


 


6961 GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG


 


7021 TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGAACAG


 


7081 TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT


 


7141 GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA


 


7201 CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC


 


7261 AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA


 


7321 CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA


 


7381 CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT


 


7441 TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA CGGGAGGGCT


 


7501 TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCG GCTCCAGATT


 


7561 TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT GCAACTTTAT


 


7621 CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA


 


7681 ATAGTTTGCG CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC TCGTCGTTTG


 


7741 GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA TCCCCCATGT


 


7801 TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT AAGTTGGCCG


 


7861 GAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG


 


7921 TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC


 


7981 GGCGACCGAG TTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCA CATAGGAGAA


 


8041 CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTGA AGGATCTTAC


 


8101 CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT


 


8161 TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG


 


8221 GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA TATTATTGAA


 


8281 GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT TAGAAAAATA


 


8341 AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC GACGGATCGG


 


8401 GAGATCGATC TCCCGATCCC CTAGGGTCGA CTCTCAGTAC AATCTGCTCT GATGCCGCAT


 


8461 AGTTAAGCCA GTATCTGCTC CCTGCTTGTG TGTTGGAGGT CGCTGAGTAG TGCGCGAGCA


 


8521 AAATTTAAGC TACAACAAGG CAAGGCTTGA CCGACAATTG CATGAAGAAT CTGCTTAGGG


 


8581 TTAGGCGTTT TGCGCTGCTT CGCGATGTAC GGGCCAGATA TACGCGTTGA CATTGATTAT


 


8641 TGACTAGTTA TTAATAGTAA TCAATTACGG GGTCATTAGT TCATAGCCCA TATATGGAGT


 


8701 TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC


 


8761 CATTGACGTC AATAATGACG TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC


 


8821 GTCAATGGGT GGAGTATTTA CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATC






In some embodiments, the cytidine base editor is BE4 having a nucleic acid sequence selected from one of the following:







Original BE4 nucleic acid sequence:


ATGagctcagagactggcccagtggctgtggaccccacattgagacg


 


gcggatcgagccccatgagtttgaggtattcttcgatccgagagag


 


ctccgcaaggagacctgcctgctttacgaaattaattgggggggc


 


cggcactccatttggcgacatacatcacagaacactaacaagcac


 


gtcgaagtcaacttcatcgagaagttcacgacagaaagatatttc


 


tgtccgaacacaaggtgcagcattacctggtttctcagctggagc


 


cgcgaatgtagtagggccatcactgaattcctgtcaaggtatccc


 


cacatcactctgtttatttacatcgcaaggctgtaccaccacgct


 


gacccccgcaatcgacaaggcctgcgggatttgatctcttcaggt


 


gtgactatccaaattatgactgagcaggagtcaggatactgctgg


 


agaaactttgtgaattatagcccgagtaatgaagcccactggcct


 


aggtatccccatctgtgggtacgactgtacgttcttgaactgtac


 


tgcatcatactgggcctgcctccttgtctcaacattctgagaagg


 


aagcagccacagctgacattctttaccatcgctcttcagtcttgt


 


cattaccagcgactgcccccacacattctctgggccaccgggttg


 


aaatctggtggttcttctggtggttctagcggcagcgagactccc


 


gggacctcagagtccgccacacccgaaagttctggtggttcttct


 


ggtggttctgataaaaagtattctattggtttagccatcggcact


 


aattccgttggatgggctgtcataaccgatgaatacaaagtacct


 


tcaaagaaatttaaggtgttggggaacacagaccgtcattcgatt


 


aaaaagaatcttatcggtgccctcctattcgatagtggcgaaacg


 


gcagaggcgactcgcctgaaacgaaccgctcggagaaggtataca


 


cgtcgcaagaaccgaatatgttacttacaagaaatttttagcaat


 


gagatggccaaagttgacgattctttctttcaccgtttggaagag


 


tccttccttgtcgaagaggacaagaaacatgaacggcaccccatc


 


tttggaaacatagtagatgaggtggcatatcatgaaaagtaccca


 


acgatttatcacctcaaaaaaaagctagttgactcaactgataaa


 


gcggacctgaggttaatctacttggctcttgcccatatgataaag


 


ttccgtgggcactttctcattgagggtgatctaaatccggacaac


 


tcggatgtcgacaaactgttcatccagttagtacaaacctataat


 


cagttgtttgaagagaaccctataaatgcaagtggcgtggatgcg


 


aaggctattcttagcgcccgcctctctaaatcccgacggctagaa


 


aacctgatcgcacaattacccggagagaagaaaaatgggttgttc


 


ggtaaccttatagcgctctcactaggcctgacaccaaattttaag


 


tcgaacttcgacttagctgaagatgccaaattgcagcttagtaag


 


gacacgtacgatgacgatctcgacaatctactggcacaaattgga


 


gatcagtatgcggacttatttttggctgccaaaaaccttagcgat


 


gcaatcctcctatctgacatactgagagttaatactgagattacc


 


aaggcgccgttatccgcttcaatgatcaaaaggtacgatgaacat


 


caccaagacttgacacttctcaaggccctagtccgtcagcaactg


 


cctgagaaatataaggaaatattctttgatcagtcqaaaaacggg


 


tacgcaggttatattgacggcggagcgagtcaagaggaattctac


 


aagtttatcaaacccatattagagaagatggatgggacggaagag


 


ttgcttgtaaaactcaatcgcgaagatctactgcgaaagcagcgg


 


actttcgacaacggtagcattccacatcaaatccacttaggcgaa


 


ttgcatgctatacttagaaggcaggaggatttttatccgttcctc


 


aaagacaatcgtgaaaagattgagaaaatcctaacctttcgcata


 


ccttactatgtgggacccctggcccgagggaactctcggttcgca


 


tggatgacaagaaagtccgaagaaacgattactccatggaatttt


 


gaggaagttgtcgataaaggtgcgtcagctcaatcgttcatcgag


 


aggatgaccaactttgacaagaatttaccgaacgaaaaagtattg


 


cctaagcacagtttactttacgagtatttcacagtgtacaatgaa


 


ctcacgaaagttaagtatgtcactgagggcatgcgtaaacccgcc


 


tttctaagcggagaacagaagaaagcaatagtagatctgttattc


 


aagaccaaccgcaaagtgacagttaagcaattgaaagaggactac


 


tttaagaaaattgaatgcttcgattctgtcgagatctccggagta


 


gaagatcgatttaatgcgtcacttggtacgtatcatgacctccta


 


aagataattaaagataaggacttcctggataacgaagagaatgaa


 


gatatcttagaagatatagtgttgactcttaccctctttgaagat


 


cgggaaatgattgaggaaagactaaaaacatacgctcacctgttc


 


gacgataaggttatgaaacagttaaagaggcgtcgctatacgggc


 


tggggacgattgtcgcggaaacttatcaacgggataagagacaag


 


caaagtggtaaaactattctcgattttctaaagagcgacggcttc


 


gccaataggaactttatgcagctgatccatgatgactctttaacc


 


ttcaaagaggatatacaaaaggcacaggtttccggacaaggggac


 


tcattgcacgaacatattgcgaatcttgctggttcgccagccatc


 


aaaaagggcatactccagacagtcaaagtagtggatgagctagtt


 


aaggtcatgggacgtcacaaaccggaaaacattgtaatcgagatg


 


gcacgcgaaaatcaaacgactcagaaggggcaaaaaaacagtcga


 


gagcggatgaagagaatagaagagggtattaaagaactgggcagc


 


cagatcttaaaggagcatcctgtggaaaatacccaattgcagaac


 


gagaaactttacctctattacctacaaaatggaagggacatgtat


 


gttgatcaggaactggacataaaccgtttatctgattacgacgtc


 


gatcacattgtaccccaatcctttttgaaggacgattcaatcgac


 


aataaagtgcttacacgctcggataagaaccgagggaaaagtgac


 


aatgttccaagcgaggaagtcgtaaagaaaatgaagaactattgg


 


cggcagctcctaaatgcgaaactgataacgcaaagaaagttcgat


 


aacttaactaaagctgagaggggtggcttgtctgaacttgacaag


 


gccggatttattaaacgtcagctcgtggaaacccgccaaatcaca


 


aagcatgttgcacagatactagattcccgaatgaatacgaaatac


 


gacgagaacgataagctgattcgggaagtcaaagtaatcacttta


 


aagtcaaaattggtgtcggacttcagaaaggattttcaattctat


 


aaagttagggagataaataactaccaccatgcgcacgacgcttat


 


cttaatgccgtcgtagggaccgcactcattaagaaatacccgaag


 


ctagaaagtgagtttgtgtatggtgattacaaagtttatgacgtc


 


cgtaagataatcgcgaaaagcaaacaggagataggcaaggctaca


 


gccaaatacttcttttattctaacattatgaatttctttaagacg


 


gaaatcactctggcaaacggagagatacgcaaacgacctttaatt


 


gaaaccaatggggagacaggtgaaatcgtatgggataagggccgg


 


gacttcgcgacggtgagaaaagttttgtccatgccccaagtcaac


 


atagtaaagaaaactgaggtgcagaccggagggttttcaaaggaa


 


tcgattcttccaaaaaggaatagtgataagctcatcgctcgtaaa


 


aaggactgggacccgaaaaagtacggtggcttcgatagccctaca


 


gttgcctattctgtcctagtagtggcaaaagttgagaagggaaaa


 


tccaagaaactgaagtcagtcaaagaattattggggataacgatt


 


atggagcgctcgtcttttgaaaagaaccccatcgacttccttgag


 


gcgaaaggttacaaggaagtaaaaaaggatctcataattaaacta


 


ccaaagtatagtctgtttgagttagaaaatggccgaaaacggatg


 


ttggctagcgccggagagcttcaaaaggggaacgaactcgcacta


 


ccgtctaaatacgtgaatttcctgtatttagcgtcccattacgag


 


aagttgaaaggttcacctgaagataacgaacagaagcaacttttt


 


gttgagcagcacaaacattatctcgacgaaatcatagagcaaatt


 


tcggaattcagtaagagagtcatcctagctgatgccaatctggac


 


aaagtattaagcgcatacaacaagcacagggataaacccatacgt


 


gagcaggcggaaaatattatccatttgtttactcttaccaacctc


 


ggcgctccagccgcattcaagtattttgacacaacgatagatcgc


 


aaacgatacacttctaccaaggaggtgctagacgcgacactgatt


 


caccaatccatcacgggattatatgaaactcggatagatttgtca


 


cagcttgggggtgactctggtggttctggaggatctggtggttct


 


actaatctgtcagatattattgaaaaggagaccggtaagcaactg


 


gttatccaggaatccatcctcatgctcccagaggaggtggaagaa


 


gtcattgggaacaagccggaaagcgatatactcgtgcacaccgcc


 


tacgacgagagcaccgacgagaatgtcatgcttctgactagcgac


 


gcccctgaatacaagccttgggctctggtcatacaggatagcaac


 


ggtgagaacaagattaagatgctctctggtggttctggaggatct


 


gatggttctactaatctgtcagatattattgaaaaggagaccggt


 


aagcaactggttatccaggaatccatcctcatgctcccagaggag


 


gtggaagaagtcattgggaacaagccggaaagcgatatactcgtg


 


cacaccgcctacgacgagagcaccgacgagaatgtcatgcttctg


 


actagcgacgcccctgaatacaagccttgggctctggtcatacag


 


gatagcaacggtgagaacaagattaagatgctctctggtggttct


 


AAAAGGACGGCGGACGGATCAGAGTTCGAGAGTCCGAAAAAAAAA


 


CGAAAGGTCGAAtaa


 


BE4 Codon Optimization 1 nucleic acid sequence:


ATGTCATCCGAAACCGGGCCAGTGGCCGTAGACCCAACACTCAGG


 


AGGCGGATAGAACCCCATGAGTTTGAAGTGTTCTTCGACCCCAGA


 


GAGCTGCGCAAAGAGACTTGCCTCCTGTATGAAATAAATTGGGGG


 


GGTCGCCATTCAATTTGGAGGCACACTAGCCAGAATACTAACAAA


 


CACGTGGAGGTAAATTTTATCGAGAAGTTTACCACCGAAAGATAC


 


TTTTGCCCCAATACACGGTGTTCAATTACCTGGTTTCTGTCATGG


 


AGTCCATGTGGAGAATGTAGTAGAGCGATAACTGAGTTCCTGTCT


 


CGATATCCTCACGTCACGTTGTTTATATACATCGCTCGGCTTTAT


 


CACCATGCGGACCCGCGGAACAGGCAAGGTCTTCGGGACCTCATA


 


TCCTCTGGGGTGACCATCCAGATAATGACGGAGCAAGAGAGCGGA


 


TACTGCTGGCGAAACTTTGTTAACTACAGCCCAAGCAATGAGGCA


 


CACTGGCCTAGATATCCGCATCTCTGGGTTCGACTGTATGTCCTT


 


GAACTGTACTGCATAATTCTGGGACTTCCGCCATGCTTGAACATT


 


CTGCGGCGGAAACAACCACAGCTGACCTTTTTCACGATTGCTCTC


 


CAAAGTTGTCACTACCAGCGATTGCCACCCCACATCTTGTGGGCT


 


ACTGGACTCAAGTCTGGAGGAAGTTCAGGCGGAAGCAGCGGGTCT


 


GAAACGCCCGGAACCTCAGAGAGCGCAACGCCCGAAAGCTCTGGA


 


GGGTCAAGTGGTGGTAGTGATAAGAAATACTCCATCGGCCTCGCC


 


ATCGGTACGAATTCTGTCGGTTGGGCCGTTATCACCGATGAGTAC


 


AAGGTCCCTTCTAAGAAATTCAAGGTTTTGGGCAATACAGACCGC


 


CATTCTATAAAAAAAAACCTGATCGGCGCCCTTTTGTTTGACAGT


 


GGTGAGACTGCTGAAGCGACTCGCCTGAAGCGAACTGCCAGGAGG


 


CGGTATACGAGGCGAAAAAACCGAATTTGTTACCTCCAGGAGATT


 


TTCTCAAATGAAATGGCCAAGGTAGATGATAGTTTTTTTCACCGC


 


TTGGAAGAAAGTTTTCTCGTTGAGGAGGACAAAAAGCACGAGAGG


 


CACCCAATCTTTGGCAACATAGTCGATGAGGTCGCATACCATGAG


 


AAATATCCTACGATCTATCATCTCCGCAAGAAGCTGGTCGATAGC


 


ACGGATAAAGCTGACCTCCGGCTGATCTACCTTGCTCTTGCTCAC


 


ATGATTAAATTCAGGGGCCATTTCCTGATAGAAGGAGACCTCAAT


 


CCCGACAATTCTGATGTCGACAAACTGTTTATTCAGCTCGTTCAG


 


ACCTATAATCAACTCTTTGAGGAGAACCCCATCAATGCTTCAGGG


 


GTGGACGCAAAGGCCATTTTGTCCGCGCGCTTGAGTAAATCACGA


 


CGCCTCGAGAATTTGATAGCTCAACTGCCGGGTGAGAAGAAAAAC


 


GGGTTGTTTGGGAATCTCATAGCGTTGAGTTTGGGACTTACGCCA


 


AACTTTAAGTCTAACTTTGATTTGGCCGAAGATGCCAAATTGCAG


 


CTGTCCAAAGATACCTATGATGACGACTTGGATAACCTTCTTGCG


 


CAGATTGGTGACCAATACGCGGATCTGTTTCTTGCCGCAAAAAAT


 


CTGTCCGACGCCATACTCTTGTCCGATATACTGCGCGTCAATACT


 


GAGATAACTAAGGCTCCCCTCAGCGCGTCCATGATTAAAAGATAC


 


GATGAGCACCACCAAGATCTCACTCTGTTGAAAGCCCTGGTTCGC


 


CAGCAGCTTCCAGAGAAGTATAAGGAGATATTTTTCGACCAATCT


 


AAAAACGGCTATGCGGGTTACATTGACGGTGGCGCCTCTCAAGAA


 


GAATTCTACAAGTTTATAAAGCCGATACTTGAGAAAATGGACGGT


 


ACAGAGGAATTGTTGGTTAAGCTCAATCGCGAGGACTTGTTGAGA


 


AAGCAGCGCACATTTGACAATGGTAGTATTCCACACCAGATTCAT


 


CTGGGCGAGTTGCATGCCATTCTTAGAAGACAAGAAGATTTTTAT


 


CCGTTTCTGAAAGATAACAGAGAAAAGATTGAAAAGATACTTACC


 


TTTCGCATACCGTATTATGTAGGTCCCCTGGCTAGAGGGAACAGT


 


CGCTTCGCTTGGATGACTCGAAAATCAGAAGAAACAATAACCCCC


 


TGGAATTTTGAAGAAGTGGTAGATAAAGGTGCGAGTGCCCAATCT


 


TTTATTGAGCGGATGACAAATTTTGACAAGAATCTGCCTAACGAA


 


AAGGTGCTTCCCAAGCATTCCCTTTTGTATGAATACTTTACAGTA


 


TATAATGAACTGACTAAAGTGAAGTACGTTACCGAGGGGATGCGA


 


AAGCCAGCTTTTCTCAGTGGCGAGCAGAAAAAAGCAATAGTTGAC


 


CTGCTGTTCAAGACGAATAGGAAGGTTACCGTCAAACAGCTCAAA


 


GAAGATTACTTTAAAAAGATCGAATGTTTTGATTCAGTTGAGATA


 


AGCGGAGTAGAGGATAGATTTAACGCAAGTCTTGGAACTTATCAT


 


GACCTTTTGAAGATCATCAAGGATAAAGATTTTTTGGACAACGAG


 


GAGAATGAAGATATCCTGGAAGATATAGTACTTACCTTGACGCTT


 


TTTGAAGATCGAGAGATGATCGAGGAGCGACTTAAGACGTACGCA


 


CATCTCTTTGACGATAAGGTTATGAAACAATTGAAACGCCGGCGG


 


TATACTGGCTGGGGCAGGCTTTCTCGAAAGCTGATTAATGGTATC


 


CGCGATAAGCAGTCTGGAAAGACAATCCTTGACTTTCTGAAAAGT


 


GATGGATTTGCAAATAGAAACTTTATGCAGCTTATACATGATGAC


 


TCTTTGACGTTCAAGGAAGACATCCAGAAGGCACAGGTATCCGGC


 


CAAGGGGATAGCCTCCATGAACACATAGCCAACCTGGCCGGCTCA


 


CCAGCTATTAAAAAGGGAATATTGCAAACCGTTAAGGTTGTTGAC


 


GAACTCGTTAAGGTTATGGGCCGACACAAACCAGAGAATATCGTG


 


ATTGAGATGGCTAGGGAGAATCAGACCACTCAAAAAGGTCAGAAA


 


AATTCTCGCGAAAGGATGAAGCGAATTGAAGAGGGAATCAAAGAA


 


CTTGGCTCTCAAATTTTGAAAGAGCACCCGGTAGAAAACACTCAG


 


CTGCAGAATGAAAAGCTGTATCTGTATTATCTGCAGAATGGTCGA


 


GATATGTACGTTGATCAGGAGCTGGATATCAATAGGCTCAGTGAC


 


TACGATGTCGACCACATCGTTCCTCAATCTTTCCTGAAAGATGAC


 


TCTATCGACAACAAAGTGTTGACGCGATCAGATAAGAACCGGGGA


 


AAATCCGACAATGTACCCTCAGAAGAAGTTGTCAAGAAGATGAAA


 


AACTATTGGAGACAATTGCTGAACGCCAAGCTCATAACACAACGC


 


AAGTTCGATAACTTGACGAAAGCCGAAAGAGGTGGGTTGTCAGAA


 


TTGGACAAAGCTGGCTTTATTAAGCGCCAATTGGTGGAGACCCGG


 


CAGATTACGAAACACGTAGCACAAATTTTGGATTCACGAATGAAT


 


ACCAAATACGACGAAAACGACAAATTGATACGCGAGGTGAAAGTG


 


ATTACGCTTAAGAGTAAGTTGGTTTCCGATTTCAGGAAGGATTTT


 


CAGTTTTACAAAGTAAGAGAAATAAACAACTACCACCACGCCCAT


 


GATGCTTACCTCAACGCGGTAGTTGGCACAGCTCTTATCAAAAAA


 


TATCCAAAGCTGGAAAGCGAGTTCGTTTACGGTGACTATAAAGTA


 


TACGACGTTCGGAAGATGATAGCCAAATCAGAGCAGGAAATTGGG


 


AAGGCAACCGCAAAATACTTCTTCTATTCAAACATCATGAACTTC


 


TTTAAGACGGAGATTACGCTCGCGAACGGCGAAATACGCAAGAGG


 


CCCCTCATAGAGACTAACGGCGAAACCGGGGAGATCGTATGGGAC


 


AAAGGACGGGACTTTGCGACCGTTAGAAAAGTACTTTCAATGCCA


 


CAAGTGAATATTGTTAAAAAGACAGAAGTACAAACAGGGGGGTTC


 


AGTAAGGAATCCATTTTGCCCAAGCGGAACAGTGATAAATTGATA


 


GCAAGGAAAAAAGATTGGGACCCTAAGAAGTACGGTGGTTTCGAC


 


TCTCCTACCGTTGCATATTCAGTCCTTGTAGTTGCGAAAGTGGAA


 


AAGGGGAAAAGTAAGAAGCTTAAGAGTGTTAAAGAGCTTCTGGGC


 


ATAACCATAATGGAACGGTCTAGCTTCGAGAAAAATCCAATTGAC


 


TTTCTCGAGGCTAAAGGTTACAAGGAGGTAAAAAAGGACCTGATA


 


ATTAAACTCCCAAAGTACAGTCTCTTCGAGTTGGAGAATGGGAGG


 


AAGAGAATGTTGGCATCTGCAGGGGAGCTCCAAAAGGGGAACGAG


 


CTGGCTCTGCCTTCAAAATACGTGAACTTTCTGTACCTGGCCAGC


 


CACTACGAGAAACTCAAGGGTTCTCCTGAGGATAACGAGCAGAAA


 


CAGCTGTTTGTAGAGCAGCACAAGCATTACCTGGACGAGATAATT


 


GAGCAAATTAGTGAGTTCTCAAAAAGAGTAATCCTTGCAGACGCG


 


AATCTGGATAAAGTTCTTTCCGCCTATAATAAGCACCGGGACAAG


 


CCTATACGAGAACAAGCCGAGAACATCATTCACCTCTTTACCCTT


 


ACTAATCTGGGCGCGCCGGCCGCCTTCAAATACTTCGACACCACG


 


ATAGACAGGAAAAGGTATACGAGTACCAAAGAAGTACTTGACGCC


 


ACTCTCATCCACCAGTCTATAACAGGGTTGTACGAAACGAGGATA


 


GATTTGTCCCAGCTCGGCGGCGACTCAGGAGGGTCAGGCGGCTCC


 


GGTGGATCAACGAATCTTTCCGACATAATCGAGAAAGAAACCGGC


 


AAACAGTTGGTGATCCAAGAATCAATCCTGATGCTGCCTGAAGAA


 


GTAGAAGAGGTGATTGGCAACAAACCTGAGTCTGACATTCTTGTC


 


CACACCGCGTATGACGAGAGCACGGACGAGAACGTTATGCTTCTC


 


ACTAGCGACGCCCCTGAGTATAAACCATGGGCGCTGGTCATCCAA


 


GATTCCAATGGGGAAAACAAGATTAAGATGCTTAGTGGTGGGTCT


 


GGAGGGAGCGGTGGGTCCACGAACCTCAGCGACATTATTGAAAAA


 


GAGACTGGTAAACAACTTGTAATACAAGAGTCTATTCTGATGTTG


 


CCTGAAGAGGTGGAGGAGGTGATTGGGAACAAACCGGAGTCTGAT


 


ATACTTGTTCATACCGCCTATGACGAATCTACTGATGAGAATGTG


 


ATGCTTTTAACGTCAGACGCTCCCGAGTACAAACCCTGGGCTCTG


 


GTGATTCAGGACAGCAATGGTGAGAATAAGATTAAAATGTTGAGT


 


GGGGGCTCAAAGCGCACGGCTGACGGTAGCGAATTTGAGAGCCCC


 


AAAAAAAAACGAAAGGTCGAAtaa


 


BE4 Codon Optimization 2 nucleic acid sequence:


ATGAGCAGCGAGACAGGCCCTGTGGCTGTGGATCCTACACTGCGG


 


AGAAGAATCGAGCCCCACGAGTTCGAGGTGTTCTTCGACCCCAGA


 


GAGCTGCGGAAAGAGACATGCCTGCTGTACGAGATCAACTGGGGC


 


GGCAGACACTCTATCTGGCGGCACACAAGCCAGAACACCAACAAG


 


CACGTGGAAGTGAACTTTATCGAGAAGTTTACGACCGAGCGGTAC


 


TTCTGCCCCAACACCAGATGCAGCATCACCTGGTTTCTGAGCTGG


 


TCCCCTTGCGGCGAGTGCAGCAGAGCCATCACCGAGTTTCTGTCC


 


AGATATCCCCACGTGACCCTGTTCATCTATATCGCCCGGCTGTAC


 


CACCACGCCGATCCTAGAAATAGACAGGGACTGCGCGACCTGATC


 


AGCAGCGGAGTGACCATCCAGATCATGACCGAGCAAGAGAGCGGC


 


TACTGCTGGCGGAACTTCGTGAACTACAGCCCCAGCAACGAAGCC


 


CACTGGCCTAGATATCCTCACCTGTGGGTCCGACTGTACGTGCTG


 


GAACTGTACTGCATCATCCTGGGCCTGCCTCCATGCCTGAACATC


 


CTGAGAAGAAAGCAGCCTCAGCTGACCTTCTTCACAATCGCCCTG


 


CAGAGCTGCCACTACCAGAGACTGCCTCCACACATCCTGTGGGCC


 


ACCGGACTTAAGAGCGGAGGATCTAGCGGCGGCTCTAGCGGATCT


 


GAGACACCTGGCACAAGCGAGTCTGCCACACCTGAGAGTAGCGGC


 


GGATCTTCTGGCGGCTCCGACAAGAAGTACTCTATCGGACTGGCC


 


ATCGGCACCAACTCTGTTGGATGGGCCGTGATCACCGACGAGTAC


 


AAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGG


 


CACAGCATCAAGAAGAATCTGATCGGCGCCCTGCTGTTCGACTCT


 


GGCGAAACAGCCGAAGCCACCAGACTGAAGAGAACCGCCAGGCGG


 


AGATACACCCGGCGGAAGAACCGGATCTGCTACCTGCAAGAGATC


 


TTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGA


 


CTGGAAGAGTCCTTCCTGGTGGAAGAGGACAAGAAGCACGAGCGG


 


CACCCCATCTTCGGCAACATCGTGGATGAGGTGGCCTACCACGAG


 


AAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGC


 


ACCGACAAGGCCGACCTGAGACTGATCTACCTGGCTCTGGCCCAC


 


ATGATCAAGTTCCGGGGCCACTTTCTGATCGAGGGCGATCTGAAC


 


CCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAG


 


ACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCTCTGGC


 


GTGGACGCCAAGGCTATCCTGTCTGCCAGACTGAGCAAGAGCAGA


 


AGGCTGGAAAACCTGATCGCCCAGCTGCCTGGCGAGAAGAAGAAT


 


GGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGACTGACCCCT


 


AACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAG


 


CTGAGCAAGGACACCTACGACGACGACCTGGACAATCTGCTGGCC


 


CAGATCGGCGATCAGTACGCCGACTTGTTTCTGGCCGCCAAGAAC


 


CTGTCCGACGCCATCCTGCTGAGCGATATCCTGAGAGTGAACACC


 


GAGATCACAAAGGCCCCTCTGAGCGCCTCTATGATCAAGAGATAC


 


GACGAGCACCACCAGGATCTGACCCTGCTGAAGGCCCTCGTTAGA


 


CAGCAGCTGCCAGAGAAGTACAAAGAGATTTTCTTCGATCAGTCC


 


AAGAACGGCTACGCCGGCTACATTGATGGCGGAGCCAGCCAAGAG


 


GAATTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGC


 


ACCGAGGAACTGCTGGTCAAGCTGAACAGAGAGGACCTGCTGCGG


 


AAGCAGCGGACCTTCGACAATGGCTCTATCCCTCACCAGATCCAC


 


CTGGGAGAGCTGCACGCCATTCTGCGGAGACAAGAGGACTTTTAC


 


CCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACC


 


TTCAGGATCCCCTACTACGTGGGACCACTGGCCAGAGGCAATAGC


 


AGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACACCC


 


TGGAACTTCGAGGAAGTGGTGGACAAGGGCGCCAGCGCTCAGTCC


 


TTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCTAACGAG


 


AAGGTGCTGCCCAAGCACTCCCTGCTGTATGAGTACTTCACCGTG


 


TACAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGA


 


AAGCCCGCCTTTCTGAGCGGCGAGCAGAAAAAGGCCATTGTGGAT


 


CTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAA


 


GAGGACTACTTCAAGAAAATCGAGTGCTTCGACAGCGTGGAAATC


 


AGCGGCGTGGAAGATCGGTTCAATGCCAGCCTGGGCACATACCAC


 


GACCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAACGAA


 


GAGAACGAGGACATTCTCGAGGACATCGTGCTGACCCTGACACTG


 


TTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACATACGCC


 


CACCTGTTCGACGACAAAGTGATGAAGCAACTGAAGCGGAGGCGG


 


TACACAGGCTGGGGCAGACTGTCTCGGAAGCTGATCAACGGCATC


 


CGGGATAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCC


 


GACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGAC


 


AGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGC


 


CAAGGCGATTCTCTGCACGAGCACATTGCCAACCTGGCCGGATCT


 


CCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGAC


 


GAGCTTGTGAAAGTGATGGGCAGACACAAGCCCGAGAACATCGTG


 


ATCGAAATGGCCAGAGAGAACCAGACCACACAGAAGGGCCAGAAG


 


AACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAG


 


CTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAG


 


CTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGACGG


 


GATATGTACGTGGACCAAGAGCTGGACATCAACCGGCTGAGCGAC


 


TACGATGTGGACCATATCGTGCCCCAGAGCTTTCTGAAGGACGAC


 


TCCATCGATAACAAGGTCCTGACCAGAAGCGACAAGAACCGGGGC


 


AAGAGCGATAACGTGCCCTCCGAAGAGGTGGTCAAGAAGATGAAG


 


AACTACTGGCGACAGCTGCTGAACGCCAAGCTGATTACCCAGCGG


 


AAGTTCGATAACCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAA


 


CTTGATAAGGCCGGCTTCATTAAGCGGCAGCTGGTGGAAACCCGG


 


CAGATCACCAAACACGTGGCACAGATTCTGGACTCCCGGATGAAC


 


ACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTC


 


ATCACCCTGAAGTCTAAGCTGGTGTCCGATTTCCGGAAGGATTTC


 


CAGTTCTACAAAGTGCGGGAAATCAACAACTACCATCACGCCCAC


 


GACGCCTACCTGAATGCCGTTGTTGGAACAGCCCTGATCAAGAAG


 


TATCCCAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTG


 


TACGACGTGCGGAAGATGATCGCCAAGAGCGAACAAGAGATCGGC


 


AAGGCTACCGCCAAGTACTTTTTCTACAGCAACATCATGAACTTT


 


TTCAAGACAGAGATCACCCTGGCCAACGGCGAGATCCGGAAAAGA


 


CCCCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGAT


 


AAGGGCAGAGATTTTGCCACAGTGCGGAAAGTGCTGAGCATGCCC


 


CAAGTGAATATCGTGAAGAAAACCGAGGTGCAGACAGGCGGCTTC


 


AGCAAAGAGTCTATCCTGCCTAAGCGGAACAGCGATAAGCTGATC


 


GCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGAT


 


AGCCCTACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAA


 


AAGGGCAAGTCCAAAAAGCTCAAGAGCGTGAAAGAGCTGCTGGGG


 


ATCACCATCATGGAAAGAAGCAGCTTTGAGAAGAACCCGATCGAC


 


TTTCTGGAAGCCAAGGGCTACAAAGAAGTCAAGAAGGACCTCATC


 


ATCAAGCTCCCCAAGTACAGCCTGTTCGAGCTGGAAAATGGCCGG


 


AAGCGGATGCTGGCCTCAGCAGGCGAACTGCAGAAAGGCAATGAA


 


CTGGCCCTGCCTAGCAAATACGTCAACTTCCTGTACCTGGCCAGC


 


CACTATGAGAAGCTGAAGGGCAGCCCCGAGGACAATGAGCAAAAG


 


CAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATC


 


GAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCT


 


AACCTGGATAAGGTGCTGTCTGCCTATAACAAGCACCGGGACAAG


 


CCTATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTG


 


ACCAACCTGGGAGCCCCTGCCGCCTTCAAGTACTTCGACACCACC


 


ATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCC


 


ACACTGATCCACCAGTCTATCACCGGCCTGTACGAAACCCGGATC


 


GACCTGTCTCAGCTCGGCGGCGATTCTGGTGGTTCTGGCGGAAGT


 


GGCGGATCCACCAATCTGAGCGACATCATCGAAAAAGAGACAGGC


 


AAGCAGCTCGTGATCCAAGAATCCATCCTGATGCTGCCTGAAGAG


 


GTTGAGGAAGTGATCGGCAACAAGCCTGAGTCCGACATCCTGGTG


 


CACACCGCCTACGATGAGAGCACCGATGAGAACGTCATGCTGCTG


 


ACAAGCGACGCCCCTGAGTACAAGCCTTGGGCTCTCGTGATTCAG


 


GACAGCAATGGGGAGAACAAGATCAAGATGCTGAGCGGAGGTAGC


 


GGAGGCAGTGGCGGAAGCACAAACCTGTCTGATATCATTGAAAAA


 


GAAACCGGGAAGCAACTGGTCATTCAAGAGTCCATTCTCATGCTC


 


CCGGAAGAAGTCGAGGAAGTCATTGGAAACAAACCCGAGAGCGAT


 


ATTCTGGTCCACACAGCCTATGACGAGTCTACAGACGAAAACGTG


 


ATGCTCCTGACCTCTGACGCTCCCGAGTATAAGCCCTGGGCACTT


 


GTTATCCAGGACTCTAACGGGGAAAACAAAATCAAAATGTTGTCC


 


GGCGGCAGCAAGCGGACAGCCGATGGATCTGAGTTCGAGAGCCCC


 


AAGAAGAAACGGAAGGTgGAGtaa






By “base editing activity” is meant acting to chemically alter a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, e.g., converting target C·G to T·A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, e.g., converting A·T to G·C. In another embodiment, the base editing activity is cytidine deaminase activity, e.g., converting target C·G to T·A and adenosine or adenine deaminase activity, e.g., converting A·T to G·C.
In some embodiments, base editing activity is assessed by efficiency of editing. Base editing efficiency may be measured by any suitable means, for example, by sanger sequencing or next generation sequencing. In some embodiments, base editing efficiency is measured by percentage of total sequencing reads with nucleobase conversion effected by the base editor, for example, percentage of total sequencing reads with target A·T base pair converted to a G·C base pair or target C·G base pair to a T·A base pair. In some embodiments, base editing efficiency is measured by percentage of total cells with nucleobase conversion effected by the base editor, when base editing is performed in a population of cells.
The term “base editor system” refers to a system for editing a nucleobase of a target nucleotide sequence. In various embodiments, the base editor system comprises (1) a polynucleotide programmable nucleotide binding domain (e.g. Cas9); (2) a deaminase domain (e.g. an adenosine deaminase or a cytidine deaminase) for deaminating said nucleobase; and (3) one or more guide polynucleotide (e.g., guide RNA). In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor system is an Adenosine Deaminase Base Editor 8 (ABE8).
In some embodiments, the ABE8 is a monomeric construct. In some embodiments, the ABE8 is ABE8.1-m, ABE8.2-m, ABE8.3-m, ABE8.4-m, ABE8.5-m, ABE8.6-m, ABE8.7-m, ABE8.8-m, ABE8.9-m, ABE8.10-m, ABE8.11-m, ABE8.12-m, ABE8.13-m, ABE8.14-m, ABE8.15-m, ABE8.16-m, ABE8.17-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.21-m, ABE8.22-m, ABE8.23-m, ABE8.24-m. In some embodiments, the ABE8 is a heteromeric construct. In some embodiments, the ABE8 is ABE8.1-d, ABE8.2-d, ABE8.3-d, ABE8.4-d, ABE8.5-d, ABE8.6-d, ABE8.7-d, ABE8.8-d, ABE8.9-d, ABE8.10-d, ABE8.11-d, ABE8.12-d, ABE8.13-d, ABE8.14-d, ABE8.15-d, ABE8.16-d, ABE8.17-d, ABE8.18-d, ABE8.19-d, ABE8.20-d, ABE8.21-d, ABE8.22-d, ABE8.23-d, or ABE8.24-d.
In some embodiments, a base editor system may comprise more than one base editing component. For example, a base editor system may include more than one deaminase. In some embodiments, a base editor system may include one or more cytidine deaminases. In some embodiments, a base editor system may include one or more adenosine deaminases. In some embodiments, a single guide polynucleotide may be utilized to target different deaminases to a target nucleic acid sequence. In some embodiments, a single pair of guide polynucleotides may be utilized to target different deaminases to a target nucleic acid sequence.
The nucleobase components and the polynucleotide programmable nucleotide binding component of a base editor system may be associated with each other covalently or non-covalently, or any combination of associations and interactions thereof. For example, in some embodiments, a deaminase domain can be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain. In some embodiments, a polynucleotide programmable nucleotide binding domain can be fused or linked to a deaminase domain. In some embodiments, a polynucleotide programmable nucleotide binding domain can target a deaminase domain to a target nucleotide sequence by non-covalently interacting with or associating with the deaminase domain. For example, in some embodiments, the nucleobase editing component, e.g., a deaminase domain, can comprise an additional heterologous portion or domain that is capable of interacting with, associating with, or capable of forming a complex with an additional heterologous portion or domain that is part of a polynucleotide programmable nucleotide binding domain. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may be a K Homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a steril alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or a RNA recognition motif.
A base editor system may further comprise a guide polynucleotide component. It should be appreciated that components of the base editor system may be associated with each other via covalent bonds, noncovalent interactions, or any combination of associations and interactions thereof. In some embodiments, a deaminase domain can be targeted to a target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the nucleobase editing component of the base editor system, e.g., a deaminase domain, can comprise an additional heterologous portion or domain (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) that is capable of interacting with, associating with, or capable of forming a complex with a portion or segment (e.g., a polynucleotide motif) of a guide polynucleotide. In some embodiments, the additional heterologous portion or domain (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) can be fused or linked to the deaminase domain. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may be a K Homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or a RNA recognition motif.
In some embodiments, a base editor system can further comprise an inhibitor of base excision repair (BER) component. It should be appreciated that components of the base editor system may be associated with each other via covalent bonds, noncovalent interactions, or any combination of associations and interactions thereof. The inhibitor of BER component may comprise a BER inhibitor. In some embodiments, the inhibitor of BER can be a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the inhibitor of BER can be an inosine BER inhibitor. In some embodiments, the inhibitor of BER can be targeted to the target nucleotide sequence by the polynucleotide programmable nucleotide binding domain. In some embodiments, a polynucleotide programmable nucleotide binding domain can be fused or linked to an inhibitor of BER. In some embodiments, a polynucleotide programmable nucleotide binding domain can be fused or linked to a deaminase domain and an inhibitor of BER. In some embodiments, a polynucleotide programmable nucleotide binding domain can target an inhibitor of BER to a target nucleotide sequence by non-covalently interacting with or associating with the inhibitor of BER. For example, in some embodiments, the inhibitor of BER component can comprise an additional heterologous portion or domain that is capable of interacting with, associating with, or capable of forming a complex with an additional heterologous portion or domain that is part of a polynucleotide programmable nucleotide binding domain.
In some embodiments, the inhibitor of BER can be targeted to the target nucleotide sequence by the guide polynucleotide. For example, in some embodiments, the inhibitor of BER can comprise an additional heterologous portion or domain (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) that is capable of interacting with, associating with, or capable of forming a complex with a portion or segment (e.g., a polynucleotide motif) of a guide polynucleotide. In some embodiments, the additional heterologous portion or domain of the guide polynucleotide (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) can be fused or linked to the inhibitor of BER. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may be a K Homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or a RNA recognition motif.
By “beta-2 microglobulin (B2M) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to UniProt Accession No. P61769 or a fragment thereof and having immunomodulatory activity. An exemplary B2M polypeptide sequence is provided below.









>spP61769B2MG HUMAN Beta-2-miCroglobulin



OS = Homo sapiens OX = 9606 GN = B2M



PE = 1 SV = 1



MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSN



 



FLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWS



 



FYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM






By “beta-2-microglobulin (B2M) polynucleotide” is meant a nucleic acid molecule encoding a B2M polypeptide. The beta-2-microglobulin gene encodes a serum protein associated with the major histocompatibility complex. B2M is involved in non-self recognition by host CD8+ T cells. An exemplary B2M polynucleotide sequence is provided below.









>DQ217933.1 Homo sapiens beta-2-miCroglobin



(B2M) gene, Complete cds



CATGTCATAAATGGTAAGTCCAAGAAAAATACAGGTATTC



 



CCCCCCAAAGAAAACTGTAAAATCGACTTTTTTCTATCTG



 



TACTGTTTTTTATTGGTTTTTAAATTGGTTTTCCAAGTGA



 



GTAAATCAGAATCTATCTGTAATGGATTTTAAATTTAGTG



 



TTTCTCTGTGATGTAGTAAACAAGAAACTAGAGGCAAAAA



 



TAGCCCTGTCCCTTGCTAAACTTCTAAGGCACTTTTCTAG



 



TACAACTCAACACTAACATTTCAGGCCTTTAGTGCCTTAT



 



ATGAGTTTTTAAAAGGGGGAAAAGGGAGGGAGCAAGAGTG



 



TCTTAACTCATACATTTAGGCATAACAATTATTCTCATAT



 



TTTAGTTATTGAGAGGGCTGGTAGAAAAACTAGGTAAATA



 



ATATTAATAATTATAGCGCTTATTAAACACTACAGAACAC



 



TTACTATGTACCAGGCATTGTGGGAGGCTCTCTCTTGTGC



 



ATTATCTCATTTCATTAGGTCCATGGAGAGTATTGCATTT



 



TCTTAGTTTAGGCATGGCCTCCACAATAAAGATTATCAAA



 



AGCCTAAAAATATGTAAAAGAAACCTAGAAGTTATTTGTT



 



GTGCTCCTTGGGGAAGCTAGGCAAATCCTTTCAACTGAAA



 



ACCATGGTGACTTCCAAGATCTCTGCCCCTCCCCATCGCC



 



ATGGTCCACTTCCTCTTCTCACTGTTCCTCTTAGAAAAGA



 



TCTGTGGACTCCACCACCACGAAATGGCGGCACCTTATTT



 



ATGGTCACTTTAGAGGGTAGGTTTTCTTAATGGGTCTGCC



 



TGTCATGTTTAACGTCCTTGGCTGGGTCCAAGGCAGATGC



 



AGTCCAAACTCTCACTAAAATTGCCGAGCCCTTTGTCTTC



 



CAGTGTCTAAAATATTAATGTCAATGGAATCAGGCCAGAG



 



TTTGAATTCTAGTCTCTTAGCCTTTGTTTCCCCTGTCCAT



 



AAAATGAATGGGGGTAATTCTTTCCTCCTACAGTTTATTT



 



ATATATTCACTAATTCATTCATTCATCCATCCATTCGTTC



 



ATTCGGTTTACTGAGTACCTACTATGTGCCAGCCCCTGTT



 



CTAGGGTGGAAACTAAGAGAATGATGTACCTAGAGGGCGC



 



TGGAAGCTCTAAAGCCCTAGCAGTTACTGCTTTTACTATT



 



AGTGGTCGTTTTTTTCTCCCCCCCGCCCCCCGACAAATCA



 



ACAGAACAAAGAAAATTACCTAAACAGCAAGGACATAGGG



 



AGGAACTTCTTGGCACAGAACTTTCCAAACACTTTTTCCT



 



GAAGGGATACAAGAAGCAAGAAAGGTACTCTTTCACTAGG



 



ACCTTCTCTGAGCTGTCCTCAGGATGCTTTTGGGACTATT



 



TTTCTTACCCAGAGAATGGAGAAACCCTGCAGGGAATTCC



 



CAAGCTGTAGTTATAAACAGAAGTTCTCCTTCTGCTAGGT



 



AGCATTCAAAGATCTTAATCTTCTGGGTTTCCGTTTTCTC



 



GAATGAAAAATGCAGGTCCGAGCAGTTAACTGGCTGGGGC



 



ACCATTAGCAAGTCACTTAGCATCTCTGGGGCCAGTCTGC



 



AAAGCGAGGGGGCAGCCTTAATGTGCCTCCAGCCTGAAGT



 



CCTAGAATGAGCGCCCGGTGTCCCAAGCTGGGGCGCGCAC



 



CCCAGATCGGAGGGCGCCGATGTACAGACAGCAAACTCAC



 



CCAGTCTAGTGCATGCCTTCTTAAACATCACGAGACTCTA



 



AGAAAAGGAAACTGAAAACGGGAAAGTCCCTCTCTCTAAC



 



CTGGCACTGCGTCGCTGGCTTGGAGACAGGTGACGGTCCC



 



TGCGGGCCTTGTCCTGATTGGCTGGGCACGCGTTTAATAT



 



AAGTGGAGGCGTCGCGCTGGCGGGCATTCCTGAAGCTGAC



 



AGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTG



 



TGCTCGCGCTACTCTCTCTTTCTGGCCTGGAGGCTATCCA



 



GCGTGAGTCTCTCCTACCCTCCCGCTCTGGTCCTTCCTCT



 



CCCGCTCTGCACCCTCTGTGGCCCTCGCTGTGCTCTCTCG



 



CTCCGTGACTTCCCTTCTCCAAGTTCTCCTTGGTGGCCCG



 



CCGTGGGGCTAGTCCAGGGCTGGATCTCGGGGAAGCGGCG



 



GGGTGGCCTGGGAGTGGGGAAGGGGGTGCGCACCCGGGAC



 



GCGCGCTACTTGCCCCTTTCGGCGGGGAGCAGGGGAGACC



 



TTTGGCCTACGGCGACGGGAGGGTCGGGACAAAGTTTAGG



 



GCGTCGATAAGCGTCAGAGCGCCGAGGTTGGGGGAGGGTT



 



TCTCTTCCGCTCTTTCGCGGGGCCTCTGGCTCCCCCAGCG



 



CAGCTGGAGTGGGGGACGGGTAGGCTCGTCCCAAAGGCGC



 



GGCGCTGAGGTTTGTGAACGCGTGGAGGGGCGCTTGGGGT



 



CTGGGGGAGGCGTCGCCCGGGTAAGCCTGTCTGCTGCGGC



 



TCTGCTTCCCTTAGACTGGAGAGCTGTGGACTTCGTCTAG



 



GCGCCCGCTAAGTTCGCATGTCCTAGCACCTCTGGGTCTA



 



TGTGGGGCCACACCGTGGGGAGGAAACAGCACGCGACGTT



 



TGTAGAATGCTTGGCTGTGATACAAAGCGGTTTCGAATAA



 



TTAACTTATTTGTTCCCATCACATGTCACTTTTAAAAAAT



 



TATAAGAACTACCCGTTATTGACATCTTTCTGTGTGCCAA



 



GGACTTTATGTGCTTTGCGTCATTTAATTTTGAAAACAGT



 



TATCTTCCGCCATAGATAACTACTATGGTTATCTTCTGCC



 



TCTCACAGATGAAGAAACTAAGGCACCGAGATTTTAAGAA



 



ACTTAATTACACAGGGGATAAATGGCAGCAATCGAGATTG



 



AAGTCAAGCCTAACCAGGGCTTTTGCGGGAGCGCATGCCT



 



TTTGGCTGTAATTCGTGCATTTTTTTTTAAGAAAAACGCC



 



TGCCTTCTGCGTGAGATTCTCCAGAGCAAACTGGGCGGCA



 



TGGGCCCTGTGGTCTTTTCGTACAGAGGGCTTCCTCTTTG



 



GCTCTTTGCCTGGTTGTTTCCAAGATGTACTGTGCCTCTT



 



ACTTTCGGTTTTGAAAACATGAGGGGGTTGGGCGTGGTAG



 



CTTACGCCTGTAATCCCAGCACTTAGGGAGGCCGAGGCGG



 



GAGGATGGCTTGAGGTCCGTAGTTGAGACCAGCCTGGCCA



 



ACATGGTGAAGCCTGGTCTCTACAAAAAATAATAACAAAA



 



ATTAGCCGGGTGTGGTGGCTCGTGCCTGTGGTCCCAGCTG



 



CTCCGGTGGCTGAGGCGGGAGGATCTCTTGAGCTTAGGCT



 



TTTGAGCTATCATGGCGCCAGTGCACTCCAGCGTGGGCAA



 



CAGAGCGAGACCCTGTCTCTCAAAAAAGAAAAAAAAAAAA



 



AAAGAAAGAGAAAAGAAAAGAAAGAAAGAAGTGAAGGTTT



 



GTCAGTCAGGGGAGCTGTAAAACCATTAATAAAGATAATC



 



CAAGATGGTTACCAAGACTGTTGAGGACGCCAGAGATCTT



 



GAGCACTTTCTAAGTACCTGGCAATACACTAAGCGCGCTC



 



ACCTTTTCCTCTGGCAAAACATGATCGAAAGCAGAATGTT



 



TTGATCATGAGAAAATTGCATTTAATTTGAATACAATTTA



 



TTTACAACATAAAGGATAATGTATATATCACCACCATTAC



 



TGGTATTTGCTGGTTATGTTAGATGTCATTTTAAAAAATA



 



ACAATCTGATATTTAAAAAAAAATCTTATTTTGAAAATTT



 



CCAAAGTAATACATGCCATGCATAGACCATTTCTGGAAGA



 



TACCACAAGAAACATGTAATGATGATTGCCTCTGAAGGTC



 



TATTTTCCTCCTCTGACCTGTGTGTGGGTTTTGTTTTTGT



 



TTTACTGTGGGCATAAATTAATTTTTCAGTTAAGTTTTGG



 



AAGCTTAAATAACTCTCCAAAAGTCATAAAGCCAGTAACT



 



GGTTGAGCCCAAATTCAAACCCAGCCTGTCTGATACTTGT



 



CCTCTTCTTAGAAAAGATTACAGTGATGCTCTCACAAAAT



 



CTTGCCGCCTTCCCTCAAACAGAGAGTTCCAGGCAGGATG



 



AATCTGTGCTCTGATCCCTGAGGCATTTAATATGTTCTTA



 



TTATTAGAAGCTCAGATGCAAAGAGCTCTCTTAGCTTTTA



 



ATGTTATGAAAAAAATCAGGTCTTCATTAGATTCCCCAAT



 



CCACCTCTTGATGGGGCTAGTAGCCTTTCCTTAATGATAG



 



GGTGTTTCTAGAGAGATATATCTGGTCAAGGTGGCCTGGT



 



ACTCCTCCTTCTCCCCACAGCCTCCCAGACAAGGAGGAGT



 



AGCTGCCTTTTAGTGATCATGTACCCTGAATATAAGTGTA



 



TTTAAAAGAATTTTATACACATATATTTAGTGTCAATCTG



 



TATATTTAGTAGCACTAACACTTCTCTTCATTTTCAATGA



 



AAAATATAGAGTTTATAATATTTTCTTCCCACTTCCCCAT



 



GGATGGTCTAGTCATGCCTCTCATTTTGGAAAGTACTGTT



 



TCTGAAACATTAGGCAATATATTCCCAACCTGGCTAGTTT



 



ACAGCAATCACCTGTGGATGCTAATTAAAACGCAAATCCC



 



ACTGTCACATGCATTACTCCATTTGATCATAATGGAAAGT



 



ATGTTCTGTCCCATTTGCCATAGTCCTCACCTATCCCTGT



 



TGTATTTTATCGGGTCCAACTCAACCATTTAAGGTATTTG



 



CCAGCTCTTGTATGCATTTAGGTTTTGTTTCTTTGTTTTT



 



TAGCTCATGAAATTAGGTACAAAGTCAGAGAGGGGTCTGG



 



CATATAAAACCTCAGCAGAAATAAAGAGGTTTTGTTGTTT



 



GGTAAGAACATACCTTGGGTTGGTTGGGCACGGTGGCTCG



 



TGCCTGTAATCCCAACACTTTGGGAGGCCAAGGCAGGCTG



 



ATCACTTGAAGTTGGGAGTTCAAGACCAGCCTGGCCAACA



 



TGGTGAAATCCCGTCTCTACTGAAAATACAAAAATTAACC



 



AGGCATGGTGGTGTGTGCCTGTAGTCCCAGGAATCACTTG



 



AACCCAGGAGGCGGAGGTTGCAGTGAGCTGAGATCTCACC



 



ACTGCACACTGCACTCCAGCCTGGGCAATGGAATGAGATT



 



CCATCCCAAAAAATAAAAAAATAAAAAAATAAAGAACATA



 



CCTTGGGTTGATCCACTTAGGAACCTCAGATAATAACATC



 



TGCCACGTATAGAGCAATTGCTATGTCCCAGGCACTCTAC



 



TAGACACTTCATACAGTTTAGAAAATCAGATGGGTGTAGA



 



TCAAGGCAGGAGCAGGAACCAAAAAGAAAGGCATAAACAT



 



AAGAAAAAAAATGGAAGGGGTGGAAACAGAGTACAATAAC



 



ATGAGTAATTTGATGGGGGCTATTATGAACTGAGAAATGA



 



ACTTTGAAAAGTATCTTGGGGCCAAATCATGTAGACTCTT



 



GAGTGATGTGTTAAGGAATGCTATGAGTGCTGAGAGGGCA



 



TCAGAAGTCCTTGAGAGCCTCCAGAGAAAGGCTCTTAAAA



 



ATGCAGCGCAATCTCCAGTGACAGAAGATACTGCTAGAAA



 



TCTGCTAGAAAAAAAACAAAAAAGGCATGTATAGAGGAAT



 



TATGAGGGAAAGATACCAAGTCACGGTTTATTCTTCAAAA



 



TGGAGGTGGCTTGTTGGGAAGGTGGAAGCTCATTTGGCCA



 



GAGTGGAAATGGAATTGGGAGAAATCGATGACCAAATGTA



 



AACACTTGGTGCCTGATATAGCTTGACACCAAGTTAGCCC



 



CAAGTGAAATACCCTGGCAATATTAATGTGTCTTTTCCCG



 



ATATTCCTCAGGTACTCCAAAGATTCAGGTTTACTCACGT



 



CATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCT



 



ATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTT



 



ACTGAAGAATGGAGAGAGAATTGAAAAAGTGGAGCATTCA



 



GACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGT



 



ACTACACTGAATTCACCCCCACTGAAAAAGAT



 



GAGTATGCCTGCCGTGTGAACCATGTGACTTTGTCACAGC



 



CCAAGATAGTTAAGTGGGGTAAGTCTTACATTCTTTTGTA



 



AGCTGCTGAAAGTTGTGTATGAGTAGTCATATCATAAAGC



 



TGCTTTGATATAAAAAAGGTCTATGGCCATACTACCCTGA



 



ATGAGTCCCATCCCATCTGATATAAACAATCTGCATATTG



 



GGATTGTCAGGGAATGTTCTTAAAGATCAGATTAGTGGCA



 



CCTGCTGAGATACTGATGCACAGCATGGTTTCTGAACCAG



 



TAGTTTCCCTGCAGTTGAGCAGGGAGCAGCAGCAGCACTT



 



GCACAAATACATATACACTCTTAACACTTCTTACCTACTG



 



GCTTCCTCTAGCTTTTGTGGCAGCTTCAGGTATATTTAGC



 



ACTGAACGAACATCTCAAGAAGGTATAGGCCTTTGTTTGT



 



AAGTCCTGCTGTCCTAGCATCCTATAATCCTGGACTTCTC



 



CAGTACTTTCTGGCTGGATTGGTATCTGAGGCTAGTAGGA



 



AGGGCTTGTTCCTGCTGGGTAGCTCTAAACAATGTATTCA



 



TGGGTAGGAACAGCAGCCTATTCTGCCAGCCTTATTTCTA



 



ACCATTTTAGACATTTGTTAGTACATGGTATTTTAAAAGT



 



AAAACTTAATGTCTTCCTTTTTTTTCTCCACTGTCTTTTT



 



CATAGATCGAGACATGTAAGCAGCATCATGGAGGTAAGTT



 



TTTGACCTTGAGAAAATGTTTTTGTTTCACTGTCCTGAGG



 



ACTATTTATAGACAGCTCTAACATGATAACCCTCACTATG



 



TGGAGAACATTGACAGAGTAACATTTTAGCAGGGAAAGAA



 



GAATCCTACAGGGTCATGTTCCCTTCTCCTGTGGAGTGGC



 



ATGAAGAAGGTGTATGGCCCCAGGTATGGCCATATTACTG



 



ACCCTCTACAGAGAGGGCAAAGGAACTGCCAGTATGGTAT



 



TGCAGGATAAAGGCAGGTGGTTACCCACATTACCTGCAAG



 



GCTTTGATCTTTCTTCTGCCATTTCCACATTGGACATCTC



 



TGCTGAGGAGAGAAAATGAACCACTCTTTTCCTTTGTATA



 



ATGTTGTTTTATTCTTCAGACAGAAGAGAGGAGTTATACA



 



GCTCTGCAGACATCCCATTCCTGTATGGGGACTGTGTTTG



 



CCTCTTAGAGGTTCCCAGGCCACTAGAGGAGATAAAGGGA



 



AACAGATTGTTATAACTTGATATAATGATACTATAATAGA



 



TGTAACTACAAGGAGCTCCAGAAGCAAGAGAGAGGGAGGA



 



ACTTGGACTTCTCTGCATCTTTAGTTGGAGTCCAAAGGCT



 



TTTCAATGAAATTCTACTGCCCAGGGTACATTGATGCTGA



 



AACCCCATTCAAATCTCCTGTTATATTCTAGAACAGGGAA



 



TTGATTTGGGAGAGCATCAGGAAGGTGGATGATCTGCCCA



 



GTCACACTGTTAGTAAATTGTAGAGCCAGGACCTGAACTC



 



TAATATAGTCATGTGTTACTTAATGACGGGGACATGTTCT



 



GAGAAATGCTTACACAAACCTAGGTGTTGTAGCCTACTAC



 



ACGCATAGGCTACATGGTATAGCCTATTGCTCCTAGACTA



 



CAAACCTGTACAGCCTGTTACTGTACTGAATACTGTGGGC



 



AGTTGTAACACAATGGTAAGTATTTGTGTATCTAAACATA



 



GAAGTTGCAGTAAAAATATGCTATTTTAATCTTATGAGAC



 



CACTGTCATATATACAGTCCATCATTGACCAAAACATCAT



 



ATCAGCATTTTTTCTTCTAAGATTTTGGGAGCACCAAAGG



 



GATACACTAACAGGATATACTCTTTATAATGGGTTTGGAG



 



AACTGTCTGCAGCTACTTCTTTTAAAAAGGTGATCTACAC



 



AGTAGAAATTAGACAAGTTTGGTAATGAGATCTGCAATCC



 



AAATAAAATAAATTCATTGCTAACCTTTTTCTTTTCTTTT



 



CAGGTTTGAAGATGCCGCATTTGGATTGGATGAATTCCAA



 



ATTCTGCTTGCTTGCTTTTTAATATTGATATGCTTATACA



 



CTTACACTTTATGCACAAAATGTAGGGTTATAATAATGTT



 



AACATGGACATGATCTTCTTTATAATTCTACTTTGAGTGC



 



TGTCTCCATGTTTGATGTATCTGAGCAGGTTGCTCCACAG



 



GTAGCTCTAGGAGGGCTGGCAACTTAGAGGTGGGGAGCAG



 



AGAATTCTCTTATCCAACATCAACATCTTGGTCAGATTTG



 



AACTCTTCAATCTCTTGCACTCAAAGCTTGTTAAGATAGT



 



TAAGCGTGCATAAGTTAACTTCCAATTTACATACTCTGCT



 



TAGAATTTGGGGGAAAATTTAGAAATATAATTGACAGGAT



 



TATTGGAAATTTGTTATAATGAATGAAACATTTTGTCATA



 



TAAGATTCATATTTACTTCTTATACATTTGATAAAGTAAG



 



GCATGGTTGTGGTTAATCTGGTTTATTTTTGTTCCACAAG



 



TTAAATAAATCATAAAACTTGATGTGTTATCTCTTATATC



 



TCACTCCCACTATTACCCCTTTATTTTCAAACAGGGAAAC



 



AGTCTTCAAGTTCCACTTGGTAAAAAATGTGAACCCCTTG



 



TATATAGAGTTTGGCTCACAGTGTAAAGGGCCTCAGTGAT



 



TCACATTTTCCAGATTAGGAATCTGATGCTCAAAGAAGTT



 



AAATGGCATAGTTGGGGTGACACAGCTGTCTAGTGGGAGG



 



CCAGCCTTCTATATTTTAGCCAGCGTTCTTTCCTGCGGGC



 



CAGGTCATGAGGAGTATGCAGACTCTAAGAGGGAGCAAAA



 



GTATCTGAAGGATTTAATATTTTAGCAAGGAATAGATATA



 



CAATCATCCCTTGGTCTCCCTGGGGGATTGGTTTCAGGAC



 



CCCTTCTTGGACACCAAATCTATGGATATTTAAGTCCCTT



 



CTATAAAATGGTATAGTATTTGCATATAACCTATCCACAT



 



CCTCCTGTATACTTTAAATCATTTCTAGATTACTTGTAAT



 



ACCTAATACAATGTAAATGCTATGCAAATAGTTGTTATTG



 



TTTAAGGAATAATGACAAGAAAAAAAAGTCTGTACATGCT



 



CAGTAAAGACACAACCATCCCTTTTTTTCCCCAGTGTTTT



 



TGATCCATGGTTTGCTGAATCCACAGATGTGGAGCCCCTG



 



GATACGGAAGGCCCGCTGTACTTTGAATGACAAATAACAG



 



ATTTAAA






The term “Cas9” or “Cas9 domain” refers to an RNA-guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). A Cas9 nuclease is also referred to sometimes as a Casn1 nuclease or a CRISPR(“clustered regularly interspaced short palindromic repeat”)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., et al., Charpentier E. Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., et al., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., et al. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.
A nuclease-inactivated Cas9 protein may interchangeably be referred to as a “dCas9” protein (for nuclease-“dead” Cas9) or catalytically inactive Cas9. Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell. 28; 152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28; 152(5):1173-83 (2013)). In some embodiments, dCas9 corresponds to, or comprises in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. In some embodiments, a dCas9 domain comprises D10A and an H840A mutation or corresponding mutations in another Cas9. In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase, referred to as an “nCas9” protein (for “nickase” Cas9). It should be appreciated that additional Cas9 proteins (e.g., a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9), including variants and homologs thereof, are within the scope of this disclosure. Exemplary Cas9 proteins include, without limitation, those provided herein. In some embodiments, the Cas9 protein is a nuclease dead Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease active Cas9.
In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as “Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild type Cas9. In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9.
In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.
In some embodiments, Cas9 refers to Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1) or Neisseria. meningitidis (NCBI Ref: YP_002342100.1) or to a Cas9 from any other organism.
In some embodiments, the Cas9 is from Neisseria meningitidis (Nme). In some embodiments, the Cas9 is Nme1, Nme2 or Nme3. In some embodiments, the PAM-interacting domains for Nme1, Nme2 or Nme3 are N4GAT, N4CC, and N4CAAA, respectively (see e.g., Edraki, A., et al., A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing, Molecular Cell (2018)).
In some embodiments, Cas9 fusion proteins as provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas9 sequence, but only one or more fragments thereof. For example, in some embodiments, a Cas9 fusion protein provided herein comprises a Cas9 fragment, wherein the fragment binds crRNA and tracrRNA or sgRNA, but does not comprise a functional nuclease domain, e.g., in that it comprises only a truncated version of a nuclease domain or no nuclease domain at all.
Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments will be apparent to those of skill in the art.
In some embodiments, Cas9 refers to a Cas9 from archaea (e.g. nanoarchaea), which constitute a domain and kingdom of single-celled prokaryotic microbes. In some embodiments, Cas9 refers to CasX or CasY, which have been described in, for example, Burstein et al., “New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017 Feb. 21. doi: 10.1038/cr.2017.21, the entire contents of which is hereby incorporated by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems were discovered, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. In some embodiments, Cas9 refers to CasX, or a variant of CasX. In some embodiments, Cas9 refers to a CasY, or a variant of CasY. It should be appreciated that other RNA-guided DNA binding proteins may be used as a nucleic acid programmable DNA binding protein (napDNAbp), and are within the scope of this disclosure.
In particular embodiments, napDNAbps useful in the methods of the invention include circular permutants, which are known in the art and described, for example, by Oakes et al., Cell 176, 254-267, 2019.
Non-limiting examples of a polynucleotide programmable nucleotide binding domain which can be incorporated into a base editor include a CRISPR protein-derived domain, a restriction nuclease, a meganuclease, TAL nuclease (TALEN), and a zinc finger nuclease (ZFN).
In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) or any of the fusion proteins provided herein may be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to a naturally-occurring CasX or CasY protein. In some embodiments, the napDNAbp is a naturally-occurring CasX or CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to any CasX or CasY protein described herein. It should be appreciated that CasX and CasY from other bacterial species may also be used in accordance with the present disclosure.
The term “Cas12b” or “Cas12b domain” refers to an RNA-guided nuclease comprising a Cas12b/C2c1 protein, or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas12b, and/or the gRNA binding domain of Cas12b). contents of each of which are incorporated herein by reference). Cas12b orthologs have been described in various species, including, but not limited to, Alicyclobacillus acidoterrestris, Alicyclobacillus acidophilus (Teng et al., Cell Discov. 2018 Nov. 27; 4:63), Bacillus hisashi, and Bacillus sp. V3-13. Additional suitable Cas12b nucleases and sequences will be apparent to those of skill in the art based on this disclosure.
In some embodiments, proteins comprising Cas12b or fragments thereof are referred to as “Cas12b variants.” A Cas12b variant shares homology to Cas12b, or a fragment thereof. For example, a Cas12b variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild type Cas12b. In some embodiments, the Cas12b variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild type Cas12b. In some embodiments, the Cas12b variant comprises a fragment of Cas12b (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas12b. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas12b. Exemplary Cas12b polypeptides are listed herein.
By “Cbl proto-oncogene B (CBLB) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to GenBank Accession No. ABC86700.1 or a fragment thereof that is involved in the regulation of immune responses. An exemplary CBLB polypeptide sequence is provided below.







>ABC86700.1 CBL-B [Homo sapiens]


MANSMNGRNPGGRGGNPRKGRILGIIDAIQDAVGPPKQAAADRRTVEKTW


 


KLMDKVVRLCQNPKLQLKNSPPYILDILPDTYQHLRLILSKYDDNQKLAQ


 


LSENEYFKIYIDSLMKKSKRAIRLFKEGKERMYEEQSQDRRNLTKLSLIF


 


SHMLAEIKAIFPNGQFQGDNFRITKADAAEFWRKFFGDKTIVPWKVFRQC


 


LHEVHQISSGLEAMALKSTIDLTCNDYISVFEFDIFTRLFQPWGSILRNW


 


NFLAVTHPGYMAFLTYDEVKARLQKYSTKPGSYIFRLSCTRLGQWAIGYV


 


TGDGNILQTIPHNKPLFQALIDGSREGFYLYPDGRSYNPDLTGLCEPTPH


 


DHIKVTQEQYELYCEMGSTFQLCKICAENDKDVKIEPCGHLMCTSCLTAW


 


QESDGQGCPFCRCEIKGTEPIIVDPFDPRDEGSRCCSIIDPFGMPMLDLD


 


DDDDREESLMMNRLANVRKCTDRQNSPVTSPGSSPLAQRRKPQPDPLQIP


 


HLSLPPVPPRLDLIQKGIVRSPCGSPTGSPKSSPCMVRKQDKPLPAPPPP


 


LRDPPPPPPERPPPIPPDNRLSRHIHHVESVPSRDPPMPLEAWCPRDVFG


 


TNQLVGCRLLGEGSPKPGITASSNVNGRHSRVGSDPVLMRKHRRHDLPLE


 


GAKVFSNGHLGSEEYDVPPRLSPPPPVTTLLPSIKCTGPLANSLSEKTRD


 


PVEEDDDEYKIPSSHPVSLNSQPSHCHNVKPPVRSCDNGHCMLNGTHGPS


 


SEKKSNIPDLSIYLKGDVFDSASDPVPLPPARPPTRDNPKHGSSLNRTPS


 


DYDLLIPPLGEDAFDALPPSLPPPPPPARHSLIEHSEPPGSSSRPSSGQD


 


LFLLPSDPFVDLASGQVPLPPARRLPGENVKTNRTSQDYDQLPSCSDGSQ


 


APARPPKPRPRRTAPEIHHRKPHGPEAALENVDAKIAKLMGEGYAFEEVK


 


RALEIAQNNVEVARSILREFAFPPPVSPRLNL






By “Cbl proto-oncogene B (CBLB) polynucleotide” is meant a nucleic acid molecule encoding a CBLB polypeptide. The CBLB gene encodes an E3 ubiquitin ligase. An exemplary CBLB nucleic acid sequence is provided below.







>DQ349203.1 Homo sapiens CBL-B mRNA, complete cds


ATGGCAAACTCAATGAATGGCAGAAACCCTGGTGGTCGAGGAGGAAATCC


 


CCGAAAAGGTCGAATTTTGGGTATTATTGATGCTATTCAGGATGCAGTTG


 


GACCCCCTAAGCAAGCTGCCGCAGATCGCAGGACCGTGGAGAAGACTTGG


 


AAGCTCATGGACAAAGTGGTAAGACTGTGCCAAAATCCCAAACTTCAGTT


 


GAAAAATAGCCCACCATATATACTTGATATTTTGCCTGATACATATCAGC


 


ATTTACGACTTATATTGAGTAAATATGATGACAACCAGAAACTTGCCCAA


 


CTCAGTGAGAATGAGTACTTTAAAATCTACATTGATAGCCTTATGAAAAA


 


GTCAAAACGGGCAATAAGACTCTTTAAAGAAGGCAAGGAGAGAATGTATG


 


AAGAACAGTCACAGGACAGACGAAATCTCACAAAACTGTCCCTTATCTTC


 


AGTCACATGCTGGCAGAAATCAAAGCAATCTTTCCCAATGGTCAATTCCA


 


GGGAGATAACTTTCGTATCACAAAAGCAGATGCTGCTGAATTCTGGAGAA


 


AGTTTTTTGGAGACAAAACTATCGTACCATGGAAAGTATTCAGACAGTGC


 


CTTCATGAGGTCCACCAGATTAGCTCTGGCCTGGAAGCAATGGCTCTAAA


 


ATCAACAATTGATTTAACTTGCAATGATTACATTTCAGTTTTTGAATTTG


 


ATATTTTTACCAGGCTGTTTCAGCCTTGGGGCTCTATTTTGCGGAATTGG


 


AATTTCTTAGCTGTGACACATCCAGGTTACATGGCATTTCTCACATATGA


 


TGAAGTTAAAGCACGACTACAGAAATATAGCACCAAACCCGGAAGCTATA


 


TTTTCCGGTTAAGTTGCACTCGATTGGGACAGTGGGCCATTGGCTATGTG


 


ACTGGGGATGGGAATATCTTACAGACCATACCTCATAACAAGCCCTTATT


 


TCAAGCCCTGATTGATGGCAGCAGGGAAGGATTTTATCTTTATCCTGATG


 


GGAGGAGTTATAATCCTGATTTAACTGGATTATGTGAACCTACACCTCAT


 


GACCATATAAAAGTTACACAGGAACAATATGAATTATATTGTGAAATGGG


 


CTCCACTTTTCAGCTCTGTAAGATTTGTGCAGAGAATGACAAAGATGTCA


 


AGATTGAGCCTTGTGGGCATTTGATGTGCACCTCTTGCCTTACGGCATGG


 


CAGGAGTCGGATGGTCAGGGCTGCCCTTTCTGTCGTTGTGAAATAAAAGG


 


AACTGAGCCCATAATCGTGGACCCCTTTGATCCAAGAGATGAAGGCTCCA


 


GGTGTTGCAGCATCATTGACCCCTTTGGCATGCCGATGCTAGACTTGGAC


 


GACGATGATGATCGTGAGGAGTCCTTGATGATGAATCGGTTGGCAAACGT


 


CCGAAAGTGCACTGACAGGCAGAACTCACCAGTCACATCACCAGGATCCT


 


CTCCCCTTGCCCAGAGAAGAAAGCCACAGCCTGACCCACTCCAGATCCCA


 


CATCTAAGCCTGCCACCCGTGCCTCCTCGCCTGGATCTAATTCAGAAAGG


 


CATAGTTAGATCTCCCTGTGGCAGCCCAACGGGTTCACCAAAGTCTTCTC


 


CTTGCATGGTGAGAAAACAAGATAAACCACTCCCAGCACCACCTCCTCCC


 


TTAAGAGATCCTCCTCCACCGCCACCTGAAAGACCTCCACCAATCCCACC


 


AGACAATAGACTGAGTAGACACATCCATCATGTGGAAAGCGTGCCTTCCA


 


GAGACCCGCCAATGCCTCTTGAAGCATGGTGCCCTCGGGATGTGTTTGGG


 


ACTAATCAGCTTGTGGGATGTCGACTCCTAGGGGAGGGCTCTCCAAAACC


 


TGGAATCACAGCGAGTTCAAATGTCAATGGAAGGCACAGTAGAGTGGGCT


 


CTGACCCAGTGCTTATGCGGAAACACAGACGCCATGATTTGCCTTTAGAA


 


GGAGCTAAGGTCTTTTCCAATGGTCACCTTGGAAGTGAAGAATATGATGT


 


TCCTCCCCGGCTTTCTCCTCCTCCTCCAGTTACCACCCTCCTCCCTAGCA


 


TAAAGTGTACTGGTCCGTTAGCAAATTCTCTTTCAGAGAAAACAAGAGAC


 


CCAGTAGAGGAAGATGATGATGAATACAAGATTCCTTCATCCCACCCTGT


 


TTCCCTGAATTCACAACCATCTCATTGTCATAATGTAAAACCTCCTGTTC


 


GGTCTTGTGATAATGGTCACTGTATGCTGAATGGAACACATGGTCCATCT


 


TCAGAGAAGAAATCAAACATCCCTGACTTAAGCATATATTTAAAGGGAGA


 


TGTTTTTGATTCAGCCTCTGATCCCGTGCCATTACCACCTGCCAGGCCTC


 


CAACTCGGGACAATCCAAAGCATGGTTCTTCACTCAACAGGACGCCCTCT


 


GATTATGATCTTCTCATCCCTCCATTAGGTGAAGATGCTTTTGATGCCCT


 


CCCTCCATCTCTCCCACCTCCCCCACCTCCTGCAAGGCATAGTCTCATTG


 


AACATTCAAAACCTCCTGGCTCCAGTAGCCGGCCATCCTCAGGACAGGAT


 


CTTTTTCTTCTTCCTTCAGATCCCTTTGTTGATCTAGCAAGTGGCCAAGT


 


TCCTTTGCCTCCTGCTAGAAGGTTACCAGGTGAAAATGTCAAAACTAACA


 


GAACATCACAGGACTATGATCAGCTTCCTTCATGTTCAGATGGTTCACAG


 


GCACCAGCCAGACCCCCTAAACCACGACCGCGCAGGACTGCACCAGAAAT


 


TCACCACAGAAAACCCCATGGGCCTGAGGCGGCATTGGAAAATGTCGATG


 


CAAAAATTGCAAAACTCATGGGAGAGGGTTATGCCTTTGAAGAGGTGAAG


 


AGAGCCTTAGAGATAGCCCAGAATAATGTCGAAGTTGCCCGGAGCATCCT


 


CCGAGAATTTGCCTTCCCTCCTCCAGTATCCCCACGTCTAAATCTATAG






By “chimeric antigen receptor” or “CAR” is meant a synthetic or engineered receptor comprising an extracellular antigen binding domain joined to one or more intracellular signaling domains (e.g., T cell signaling domain) that confers specificity for an antigen onto an immune effector cell. In some embodiments, the CAR includes a transmembrane domain.
By “chimeric antigen receptor T cell” or “CAR-T cell” is meant a T cell expressing a CAR that has antigen specificity determined by the antibody-derived targeting domain of the CAR. As used herein, “CAR-T cells” includes T cells or NK cells. As used herein, “CAR-T cells” includes cells engineered to express a CAR or a T cell receptor (TCR). In some embodiments, CAR-T cells can be T helper CD4+ and/or T effector CD8+ cells, optionally in defined proportions. Methods of making CARs (e.g., for treatment of cancer) are publicly available (see, e.g., Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol. 6:47, 2013; Haso et al., (2013) Blood, 121, 1165-1174; PCT Pubs. WO2012/079000, WO2013/059593; and U.S. Pub. 2012/0213783, each of which is incorporated by reference herein in its entirety).
By “class II, major histocornpatibility complex, transactivator (CIITA)” is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession No. NP_001273331.1 or a fragment thereof and having immunomodulatory activity. An exemplary amino acid sequence is provided below.







>NP_001273331.1 MHC class II transactivator


isoform 1 [Homo sapiens]


MRCLAPRPAGSYLSEPQGSSQCATMELGPLEGGYLELLNSDADPLCLYHF


 


YDQMDLAGEEEIELYSEPDTDTINCDQFSRLLCDMEGDEETREAYANIAE


 


LDQYVFQDSQLEGLSKDIFIEHIGPDEVIGESMEMPAEVGQKSQKRPFPE


 


ELPADLKHWKPAEPPTVVTGSLLVGPVSDCSTLPCLPLPALFNQEPASGQ


 


MRLEKTDQIPMPFSSSSLSCLNLPEGPIQFVPTISTLPHGLWQISEAGTG


 


VSSIFIYHGEVPQASQVPPPSGFTVHGLPTSPDRPGSTSPFAPSATDLPS


 


MPEPALTSRANMTEHKTSPTQCPAAGEVSNKLPKWPEPVEQFYRSLQDTY


 


GAEPAGPDGILVEVDLVQARLERSSSKSLERELATPDWAERQLAQGGLAE


 


VLLAAKEHRRPRETRVIAVLGKAGOGKSYWAGAVSRAWACGRLPQYDFVF


 


SVPCHCLNRPGDAYGLQDLLFSLGPQPLVAADEVFSHILKRPDRVLLILD


 


GFEELEAQDGFLHSTCGPAPAEPCSLRGLLAGLFQKKLLRGCTLLLTARP


 


RGRLVQSLSKADALFELSGFSMEQAQAYVMRYFESSGMTEHQDRALTLLR


 


DRPLLLSHSHSPTLCRAVCQLSEALLELGEDAKLPSTLTGLYVGLLGRAA


 


LDSPPGALAELAKLAWELGRRHQSTLQEDQFPSADVRTWAMAKGLVQHPP


 


RAAESELAFPSFLLQCFLGALWLALSGEIKDKELPQYLALTPRKKRPYDN


 


WLEGVPRFLAGLIFQPPARCLGALLGPSAAASVDRKQKVLARYLKRLQPG


 


TLRARQLLELLHCAHEAEEAGIWQHVVQELPGRLSFLGTRLTPPDAHVLG


 


KALEAAGQDFSLDLRSTGICPSGLGSLVGLSCVTRFRAALSDTVALWESL


 


QQHGETKLLQAAEEKFTIEPFKAKSLKDVEDLGKLVQTQRTRSSSEDTAG


 


ELPAVRDLKKLEFALGPVSGPQAFPKLVRILTAFSSLQHLDLDALSENKI


 


GDEGVSQLSATFPQLKSLETLNLSQNNITDLGAYKLAEALPSLAASLLRL


 


SLYNNCICDVGAESLARVLPDMVSLRVMDVQYNKFTAAGAQQLAASLRRC


 


PHVETLAMWTPTIPFSVQEHLQQQDSRISLR






By “class II, major histocornpatibility complex, transactivator (CIITA)” is meant a nucleic acid encoding a CIITA polypeptide. An exemplary CIITA nucleic acid sequence is provided below.








>NM_001286402.1 Homo sapiens class II major histocompatibility complex transactivator



(CIITA), transcript variant 1, mRNA


GGTTAGTGATGAGGCTAGTGATGAGGCTGTGTGCTTCTGAGCTGGGCATCCGAAGGCATCCTTG


 


GGGAAGCTGAGGGCACGAGGAGGGGCTGCCAGACTCCGGGAGCTGCTGCCTGGCTGGGATTCCT


 


ACACAATGCGTTGCCTGGCTCCACGCCCTGCTGGGTCCTACCTGTCAGAGCCCCAAGGCAGCTC


 


ACAGTGTGCCACCATGGAGTTGGGGCCCCTAGAAGGTGGCTACCTGGAGCTTCTTAACAGCGAT


 


GCTGACCCCCTGTGCCTCTACCACTTCTATGACCAGATGGACCTGGCTGGAGAAGAAGAGATTG


 


AGCTCTACTCAGAACCCGACACAGACACCATCAACTGCGACCAGTTCAGCAGGCTGTTGTGTGA


 


CATGGAAGGTGATGAAGAGACCAGGGAGGCTTATGCCAATATCGCGGAACTGGACCAGTATGTC


 


TTCCAGGACTCCCAGCTGGAGGGCCTGAGCAAGGACATTTTCATAGAGCACATAGGACCAGATG


 


AAGTGATCGGTGAGAGTATGGAGATGCCAGCAGAAGTTGGGCAGAAAAGTCAGAAAAGACCCTT


 


CCCAGAGGAGCTTCCGGCAGACCTGAAGCACTGGAAGCCAGCTGAGCCCCCCACTGTGGTGACT


 


GGCAGTCTCCTAGTGGGACCAGTGAGCGACTGCTCCACCCTGCCCTGCCTGCCACTGCCTGCGC


 


TGTTCAACCAGGAGCCAGCCTCCGGCCAGATGCGCCTGGAGAAAACCGACCAGATTCCCATGCC


 


TTTCTCCAGTTCCTCGTTGAGCTGCCTGAATCTCCCTGAGGGACCCATCCAGTTTGTCCCCACC


 


ATCTCCACTCTGCCCCATGGGCTCTGGCAAATCTCTGAGGCTGGAACAGGGGTCTCCAGTATAT


 


TCATCTACCATGGTGAGGTGCCCCAGGCCAGCCAAGTACCCCCTCCCAGTGGATTCACTGTCCA


 


CGGCCTCCCAACATCTCCAGACCGGCCAGGCTCCACCAGCCCCTTCGCTCCATCAGCCACTGAC


 


CTGCCCAGCATGCCTGAACCTGCCCTGACCTCCCGAGCAAACATGACAGAGCACAAGACGTCCC


 


CCACCCAATGCCCGGCAGCTGGAGAGGTCTCCAACAAGCTTCCAAAATGGCCTGAGCCGGTGGA


 


GCAGTTCTACCGCTCACTGCAGGACACGTATGGTGCCGAGCCCGCAGGCCCGGATGGCATCCTA


 


GTGGAGGTGGATCTGGTGCAGGCCAGGCTGGAGAGGAGCAGCAGCAAGAGCCTGGAGCGGGAAC


 


TGGCCACCCCGGACTGGGCAGAACGGCAGCTGGCCCAAGGAGGCCTGGCTGAGGTGCTGTTGGC


 


TGCCAAGGAGCACCGGCGGCCGCGTGAGACACGAGTGATTGCTGTGCTGGGCAAAGCTGGTCAG


 


GGCAAGAGCTATTGGGCTGGGGCAGTGAGCCGGGCCTGGGCTTGTGGCCGGCTTCCCCAGTACG


 


ACTTTGTCTTCTCTGTCCCCTGCCATTGCTTGAACCGTCCGGGGGATGCCTATGGCCTGCAGGA


 


TCTGCTCTTCTCCCTGGGCCCACAGCCACTCGTGGCGGCCGATGAGGTTTTCAGCCACATCTTG


 


AAGAGACCTGACCGCGTTCTGCTCATCCTAGACGGCTTCGAGGAGCTGGAAGCGCAAGATGGCT


 


TCCTGCACAGCACGTGCGGACCGGCACCGGCGGAGCCCTGCTCCCTCCGGGGGCTGCTGGCCGG


 


CCTTTTCCAGAAGAAGCTGCTCCGAGGTTGCACCCTCCTCCTCACAGCCCGGCCCCGGGGCCGC


 


CTGGTCCAGAGCCTGAGCAAGGCCGACGCCCTATTTGAGCTGTCCGGCTTCTCCATGGAGCAGG


 


CCCAGGCATACGTGATGCGCTACTTTGAGAGCTCAGGGATGACAGAGCACCAAGACAGAGCCCT


 


GACGCTCCTCCGGGACCGGCCACTTCTTCTCAGTCACAGCCACAGCCCTACTTTGTGCCGGGCA


 


GTGTGCCAGCTCTCAGAGGCCCTGCTGGAGCTTGGGGAGGACGCCAAGCTGCCCTCCACGCTCA


 


CGGGACTCTATGTCGGCCTGCTGGGCCGTGCAGCCCTCGACAGCCCCCCCGGGGCCCTGGCAGA


 


GCTGGCCAAGCTGGCCTGGGAGCTGGGCCGCAGACATCAAAGTACCCTACAGGAGGACCAGTTC


 


CCATCCGCAGACGTGAGGACCTGGGCGATGGCCAAAGGCTTAGTCCAACACCCACCGCGGGCCG


 


CAGAGTCCGAGCTGGCCTTCCCCAGCTTCCTCCTGCAATGCTTCCTGGGGGCCCTGTGGCTGGC


 


TCTGAGTGGCGAAATCAAGGACAAGGAGCTCCCGCAGTACCTAGCATTGACCCCAAGGAAGAAG


 


AGGCCCTATGACAACTGGCTGGAGGGCGTGCCACGCTTTCTGGCTGGGCTGATCTTCCAGCCTC


 


CCGCCCGCTGCCTGGGAGCCCTACTCGGGCCATCGGCGGCTGCCTCGGTGGACAGGAAGCAGAA


 


GGTGCTTGCGAGGTACCTGAAGCGGCTGCAGCCGGGGACACTGCGGGCGCGGCAGCTGCTGGAG


 


CTGCTGCACTGCGCCCACGAGGCCGAGGAGGCTGGAATTTGGCAGCACGTGGTACAGGAGCTCC


 


CCGGCCGCCTCTCTTTTCTGGGCACCCGCCTCACGCCTCCTGATGCACATGTACTGGGCAAGGC


 


CTTGGAGGCGGCGGGCCAAGACTTCTCCCTGGACCTCCGCAGCACTGGCATTTGCCCCTCTGGA


 


TTGGGGAGCCTCGTGGGACTCAGCTGTGTCACCCGTTTCAGGGCTGCCTTGAGCGACACGGTGG


 


CGCTGTGGGAGTCCCTGCAGCAGCATGGGGAGACCAAGCTACTTCAGGCAGCAGAGGAGAAGTT


 


CACCATCGAGCCTTTCAAAGCCAAGTCCCTGAAGGATGTGGAAGACCTGGGAAAGCTTGTGCAG


 


ACTCAGAGGACGAGAAGTTCCTCGGAAGACACAGCTGGGGAGCTCCCTGCTGTTCGGGACCTAA


 


AGAAACTGGAGTTTGCGCTGGGCCCTGTCTCAGGCCCCCAGGCTTTCCCCAAACTGGTGCGGAT


 


CCTCACGGCCTTTTCCTCCCTGCAGCATCTGGACCTGGATGCGCTGAGTGAGAACAAGATCGGG


 


GACGAGGGTGTCTCGCAGCTCTCAGCCACCTTCCCCCAGCTGAAGTCCTTGGAAACCCTCAATC


 


TGTCCCAGAACAACATCACTGACCTGGGTGCCTACAAACTCGCCGAGGCCCTGCCTTCGCTCGC


 


TGCATCCCTGCTCAGGCTAAGCTTGTACAATAACTGCATCTGCGACGTGGGAGCCGAGAGCTTG


 


GCTCGTGTGCTTCCGGACATGGTGTCCCTCCGGGTGATGGACGTCCAGTACAACAAGTTCACGG


 


CTGCCGGGGCCCAGCAGCTCGCTGCCAGCCTTCGGAGGTGTCCTCATGTGGAGACGCTGGCGAT


 


GTGGACGCCCACCATCCCATTCAGTGTCCAGGAACACCTGCAACAACAGGATTCACGGATCAGC


 


CTGAGATGATCCCAGCTGTGCTCTGGACAGGCATGTTCTCTGAGGACACTAACCACGCTGGACC


 


TTGAACTGGGTACTTGTGGACACAGCTCTTCTCCAGGCTGTATCCCATGAGCCTCAGCATCCTG


 


GCACCCGGCCCCTGCTGGTTCAGGGTTGGCCCCTGCCCGGCTGCGGAATGAACCACATCTTGCT


 


CTGCTGACAGACACAGGCCCGGCTCCAGGCTCCTTTAGCGCCCAGTTGGGTGGATGCCTGGTGG


 


CAGCTGCGGTCCACCCAGGAGCCCCGAGGCCTTCTCTGAAGGACATTGCGGACAGCCACGGCCA


 


GGCCAGAGGGAGTGACAGAGGCAGCCCCATTCTGCCTGCCCAGGCCCCTGCCACCCTGGGGAGA


 


AAGTACTTCTTTTTTTTTATTTTTAGACAGAGTCTCACTGTTGCCCAGGCTGGCGTGCAGTGGT


 


GCGATCTGGGTTCACTGCAACCTCCGCCTCTTGGGTTCAAGCGATTCTTCTGCTTCAGCCTCCC


 


GAGTAGCTGGGACTACAGGCACCCACCATCATGTCTGGCTAATTTTTCATTTTTAGTAGAGACA


 


GGGTTTTGCCATGTTGGCCAGGCTGGTCTCAAACTCTTGACCTCAGGTGATCCACCCACCTCAG


 


CCTCCCAAAGTGCTGGGATTACAAGCGTGAGCCACTGCACCGGGCCACAGAGAAAGTACTTCTC


 


CACCCTGCTCTCCGACCAGACACCTTGACAGGGCACACCGGGCACTCAGAAGACACTGATGGGC


 


AACCCCCAGCCTGCTAATTCCCCAGATTGCAACAGGCTGGGCTTCAGTGGCAGCTGCTTTTGTC


 


TATGGGACTCAATGCACTGACATTGTTGGCCAAAGCCAAAGCTAGGCCTGGCCAGATGCACCAG


 


CCCTTAGCAGGGAAACAGCTAATGGGACACTAATGGGGCGGTGAGAGGGGAACAGACTGGAAGC


 


ACAGCTTCATTTCCTGTGTCTTTTTTCACTACATTATAAATGTCTCTTTAATGTCACAGGCAGG


 


TCCAGGGTTTGAGTTCATACCCTGTTACCATTTTGGGGTACCCACTGCTCTGGTTATCTAATAT


 


GTAACAAGCCACCCCAAATCATAGTGGCTTAAAACAACACTCACATTTA






In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
By “cytotoxic T-lymphocyte associated protein 4 (CTLA-4) polypeptide” is meant a protein having at least about 85% sequence identity to NCBI Accession No. EAW70354.1 or a fragment thereof. An exemplary amino acid sequence is provided below:







>EAW70354.1 cytotoxic T-lymphocyte-associated


protein 4 [Homo sapiens]


MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASS


 


RGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDD


 


SICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIY


 


VIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGV


 


YVKMPPTEPECEKQFQPYFIPIN






By “cytotoxic T-lymphocyte associated protein 4 (CTLA-4) polynucleotide” is meant a nucleic acid molecule encoding a CTLA-4 polypeptide. The CTLA-4 gene encodes an immunoglobulin superfamily and encodes a protein which transmits an inhibitory signal to T cells. An exemplary CTLA-4 nucleic acid sequence is provided below.







>BC074842.2 Homo sapiens cytotoxic T-lymphocyte-


associated protein 4, mRNA (cDNA clone MGC:


104099 IMAGE: 30915552), complete cds


GACCTGAACACCGCTCCCATAAAGCCATGGCTTGCCTTGGATTTCAGCGG


 


CACAAGGCTCAGCTGAACCTGGCTACCAGGACCTGGCCCTGCACTCTCCT


 


GTTTTTTCTTCTCTTCATCCCTGTCTTCTGCAAAGCAATGCACGTGGCCC


 


AGCCTGCTGTGGTACTGGCCAGCAGCCGAGGCATCGCCAGCTTTGTGTGT


 


GAGTATGCATCTCCAGGCAAAGCCACTGAGGTCCGGGTGACAGTGCTTCG


 


GCAGGCTGACAGCCAGGTGACTGAAGTCTGTGCGGCAACCTACATGATGG


 


GGAATGAGTTGACCTTCCTAGATGATTCCATCTGCACGGGCACCTCCAGT


 


GGAAATCAAGTGAACCTCACTATCCAAGGACTGAGGGCCATGGACACGGG


 


ACTCTACATCTGCAAGGTGGAGCTCATGTACCCACCGCCATACTACCTGG


 


GCATAGGCAACGGAACCCAGATTTATGTAATTGATCCAGAACCGTGCCCA


 


GATTCTGACTTCCTCCTCTGGATCCTTGCAGCAGTTAGTTCGGGGTTGTT


 


TTTTTATAGCTTTCTCCTCACAGCTGTTTCTTTGAGCAAAATGCTAAAGA


 


AAAGAAGCCCTCTTACAACAGGGGTCTATGTGAAAATGCCCCCAACAGAG


 


CCAGAATGTGAAAAGCAATTTCAGCCTTATTTTATTCCCATCAATTGAGA


 


AACCATTATGAAGAAGAGAGTCCATATTTCAATTTCCAAGAGCTGAGG






By “cluster of differentiation 2 (CD2) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession No. NP_001758.2 or fragment thereof and having immunomodulatory activity. An exemplary amino acid sequence is provided below.







>NP_001758.2 T-cell surface antigen CD2 isoform 2


precursor [Homo sapiens]








1
MSFPCKFVAS FLLIFNVSSK GAVSKEITNA LETWGALGQD


 



INLDIPSFQM SDDIDDIKWE


 


61
KTSDKKKIAQ FRKEKETFKE KDTYKLFKNG TLKIKHLKTD


 



DQDIYKVSIY DTKGKNVLEK


 


121
IFDLKIQERV SKPKISWTCI NTTLTCEVMN GTDPELNLYQ


 



DGKHLKLSQR VITHKWTTSL


 


181
SAKFKCTAGN KVSKESSVEP VSCPEKGLDI YLIIGICGGG


 



SLLMVFVALL VFYITKRKKQ


 


241
RSRRNDEELE TRAHRVATEE RGRKPHQIPA STPQNPATSQ


 



HPPPPPGHRS QAPSHRPPPP


 


301
GHRVQHQPQK RPPAPSGTQV HQQKGPPLPR PRVQPKPPHG


 



AAENSLSPSS N






The CD2 cytoplasmic domain (amino acid residues 235-351) is shown in bold font. The architecture of an exemplary CD2 polypeptide from Homo Sapiens is shown in FIG. 4.

By “Cluster of Differentiation 2 (CD2) polynucleotide” is meant a nucleic acid encoding a CD2 polypeptide. An exemplary CD2 nucleic acid sequence is provided below. >







NM_001767.5 Homo sapiens CD2 molecule (CD2),


transcript variant 2, mRNA








1
agtctcactt cagttccttt tgcatgaaga gctcagaatc


 



aaaagaggaa accaacccct


 


61
aagatgagct ttccatgtaa atttgtagcc agcttccttc


 



tgattttcaa tgtttcttcc


 


121
aaaggtgcag tctccaaaga gattacgaat gccttggaaa


 



cctggggtgc cttgggtcag


 


181
gacatcaact tggacattcc tagttttcaa atgagtgatg


 



atattgacga tataaaatgg


 


241
gaaaaaactt cagacaagaa aaagattgca caattcagaa


 



aagagaaaga gactttcaag


 


301
gaaaaagata catataagct atttaaaaat ggaactctga


 



aaattaagca tctgaagacc


 


361
gatgatcagg atatctacaa ggtatcaata tatgatacaa


 



aaggaaaaaa tgtgttggaa


 


421
aaaatatttg atttgaagat tcaagagagg gtctcaaaac


 



caaagatctc ctggacttgt


 


481
atcaacacaa ccctgacctg tgaggtaatg aatggaactg


 



accccgaatt aaacctgtat


 


541
caagatggga aacatctaaa actttctcag agggtcatca


 



cacacaagtg gaccaccagc


 


601
ctgagtgcaa aattcaagtg cacagcaggg aacaaagtca


 



gcaaggaatc cagtgtcgag


 


661
cctgtcagct gtccagagaa aggtctggac atctatctca


 



tcattggcat atgtggagga


 


721
ggcagcctct tgatggtctt tgtggcactg ctcgttttct


 



atatcaccaa aaggaaaaaa


 


781
cagaggagtc ggagaaatga tgaggagctg gagacaagag


 



cccacagagt agctactgaa


 


841
gaaaggggcc ggaagcccca ccaaattcca gcttcaaccc


 



ctcagaatcc agcaacttcc


 


901
caacatcctc ctccaccacc tggtcatcgt tcccaggcac


 



ctagtcatcg tcccccgcct


 


961
cctggacacc gtgttcagca ccagcctcag aagaggcctc


 



ctgctccgtc gggcacacaa


 


1021
gttcaccagc agaaaggccc gcccctcccc agacctcgag


 



ttcagccaaa acctccccat


 


1081
ggggcagcag aaaactcatt gtccccttcc tctaattaaa


 



aaagatagaa actgtctttt


 


1141
tcaataaaaa gcactgtgga tttctgccct cctgatgtgc


 



atatccgtac ttccatgagg


 


1201
tgttttctgt gtgcagaaca ttgtcacctc ctgaggctgt


 



gggccacagc cacctctgca


 


1261
tcttcgaact cagccatgtg gtcaacatct ggagtttttg


 



gtctcctcag agagctccat


 


1321
cacaccagta aggagaagca atataagtgt gattgcaaga


 



atggtagagg accgagcaca


 


1381
gaaatcttag agatttcttg tcccctctca ggtcatgtgt


 



agatgcgata aatcaagtga


 


1441
ttggtgtgcc tgggtctcac tacaagcagc ctatctgctt


 



aagagactct ggagtttctt


 


1501
atgtgccctg gtggacactt gcccaccatc ctgtgagtaa


 



aagtgaaata aaagctttga


 


1561
ctaga






By “cluster of differentiation 5 (CD5) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession No. NP_001333385.1 or fragment thereof and having immunomodulatory activity. An exemplary amino acid sequence is provided below.







>NP_001333385.1 T-cell surface glycoprotein CD5


isoform 2 [Homo sapiens]


MVCSQSWGRSSKQWEDPSQASKVCQRLNCGVPLSLGPFLVTYTPQSSIIC


 


YGQLGSFSNCSHSRNDMCHSLGLTCLEPQKTTPPTTRPPPTTTPEPTAPP


 


RLQLVAQSGGQHCAGVVEFYSGSLGGTISYEAQDKTQDLENFLCNNLQCG


 


SFLKHLPETEAGRAQDPGEPREHQPLPIQWKIQNSSCTSLEHCFRKIKPQ


 


KSGRVLALLCSGFQPKVQSRLVGGSSICEGTVEVRQGAQWAALCDSSSAR


 


SSLRWEEVCREQQCGSVNSYRVLDAGDPTSRGLFCPHQKLSQCHELWERN


 


SYCKKVFVTCQDPNPAGLAAGTVASIILALVLLVVLLVVCGPLAYKKLVK


 


KFRQKKQRQWIGPTGMNQNMSFHRNHTATVRSHAENPTASHVDNEYSQPP


 


RNSHLSAYPALEGALHRSSMQPDNSSDSDYDLHGAQRL






By “cluster of differentiation 5 (CD5) polynucleotide” is meant a nucleic acid encoding a CD5 polypeptide. An exemplary CD5 nucleic acid sequence is provided below.







>NM_001346456.1 Homo sapiens CDS molecule (CD5),


transcript variant 2, mRNA








1
gagtcttgct gatgctcccg gctgaataaa ccccttcctt


 



ctttaacttg gtgtctgagg


 


61
ggttttgtct gtggcttgtc ctgctacatt tcttggttcc


 



ctgaccagga agcaaagtga


 


121
ttaacggaca gttgaggcag ccccttaggc agcttaggcc


 



tgccttgtgg agcatccccg


 


181
cggggaactc tggccagctt gagcgacacg gatcctcaga


 



gagctcccag gtaggcaatt


 


241
gcaccagtgg aatgcctcgt cagagcagtg catggcaggc


 



ccctgtggag gatcaacgca


 


301
gtggctgaac acagggaagg aactggcact tggagtccgg


 



acaactgaaa cttgtcgctt


 


361
cctgcctcgg acggctcagc tggtatgacc cagatttcca


 



ggcaaggctc acccgttcca


 


421
actcgaagtg ccagggccag ctggaggtct acctcaagga


 



cggatggcac atggtttgca


 


481
gccagagctg gggccggagc tccaagcagt gggaggaccc


 



cagtcaagcg tcaaaagtct


 


541
gccagcggct gaactgtggg gtgcccttaa gccttggccc


 



cttccttgtc acctacacac


 


601
ctcagagctc aatcatctgc tacggacaac tgggctcctt


 



ctccaactgc agccacagca


 


661
gaaatgacat gtgtcactct ctgggcctga cctgcttaga


 



accccagaag acaacacctc


 


721
caacgacaag gcccccgccc accacaactc cagagcccac


 



agctcctccc aggctgcagc


 


781
tggtggcaca gtctggcggc cagcactgtg ccggcgtggt


 



ggagttctac agcggcagcc


 


841
tggggggtac catcagctat gaggcccagg acaagaccca


 



ggacctggag aacttcctct


 


901
gcaacaacct ccagtgtggc tccttcttga agcatctgcc


 



agagactgag gcaggcagag


 


961
cccaagaccc aggggagcca cgggaacacc agcccttgcc


 



aatccaatgg aagatccaga


 


1021
actcaagctg tacctccctg gagcattgct tcaggaaaat


 



caagccccag aaaagtggcc


 


1081
gagttcttgc cctcctttgc tcaggtttcc agcccaaggt


 



gcagagccgt ctggtggggg


 


1141
gcagcagcat ctgtgaaggc accgtggagg tgcgccaggg


 



ggctcagtgg gcagccctgt


 


1201
gtgacagctc ttcagccagg agctcgctgc ggtgggagga


 



ggtgtgccgg gagcagcagt


 


1261
gtggcagcgt caactcctat cgagtgctgg acgctggtga


 



cccaacatcc cgggggctct


 


1321
tctgtcccca tcagaagctg tcccagtgcc acgaactttg


 



ggagagaaat tcctactgca


 


1381
agaaggtgtt tgtcacatgc caggatccaa accccgcagg


 



cctggccgca ggcacggtgg


 


1441
caagcatcat cctggccctg gtgctcctgg tggtgctgct


 



ggtcgtgtgc ggcccccttg


 


1501
cctacaagaa gctagtgaag aaattccgcc agaagaagca


 



gcgccagtgg attggcccaa


 


1561
cgggaatgaa ccaaaacatg tctttccatc gcaaccacac


 



ggcaaccgtc cgatcccatg


 


1621
ctgagaaccc cacagcctcc cacgtggata acgaatacag


 



ccaacctccc aggaactccc


 


1681
acctgtcagc ttatccagct ctggaagggg ctctgcatcg


 



ctcctccatg cagcctgaca


 


1741
actcctccga cagtgactat gatctgcatg gggctcagag


 



gctgtaaaga actgggatcc


 


1801
atgagcaaaa agccgagagc cagacctgtt tgtcctgaga


 



aaactgtccg ctcttcactt


 


1861
gaaatcatgt ccctatttct accccggcca gaacatggac


 



agaggccaga agccttccgg


 


1921
acaggcgctg ctgccccgag tggcaggcca gctcacactc


 



tgctgcacaa cagctcggcc


 


1981
gcccctccac ttgtggaagc tgtggtgggc agagccccaa


 



aacaagcagc cttccaacta


 


2041
gagactcggg ggtgtctgaa gggggccccc tttccctgcc


 



cgctggggag cggcgtctca


 


2101
gtgaaatcgg ctttctcctc agactctgtc cctggtaagg


 



agtgacaagg aagctcacag


 


2161
ctgggcgagt gcattttgaa tagttttttg taagtagtgc


 



ttttcctcct tcctgacaaa


 


2221
tcgagcgctt tggcctcttc tgtgcagcat ccacccctgc


 



ggatccctct ggggaggaca


 


2281
ggaaggggac tcccggagac ctctgcagcc gtggtggtca


 



gaggctgctc acctgagcac


 


2341
aaagacagct ctgcacattc accgcagctg ccagccaggg


 



gtctgggtgg gcaccaccct


 


2401
gacccacagc gtcaccccac tccctctgtc ttatgactcc


 



cctccccaac cccctcatct


 


2461
aaagacacct tcctttccac tggctgtcaa gcccacaggg


 



caccagtgcc acccagggcc


 


2521
cggcacaaag gggcgcctag taaaccttaa ccaacttggt


 



tttttgcttc acccagcaat


 


2581
taaaagtccc aagctgaggt agtttcagtc catcacagtt


 



catcttctaa cccaagagtc


 


2641
agagatgggg ctggtcatgt tcctttggtt tgaataactc


 



ccttgacgaa aacagactcc


 


2701
tctagtactt ggagatcttg gacgtacacc taatcccatg


 



gggcctcagc ttccttaact


 


2761
gcaagtgaga agaggaggtc tacccaggag cctcgggtct


 



gatcaaggga gaggccaggc


 


2821
gcagctcact gcggcggctc cctaagaagg tgaagcaaca


 



tgggaacaca tcctaagaca


 


2881
ggtcctttct ccacgccatt tgatgctgta tctcctggga


 



gcacaggcat caatggtcca


 


2941
agccgcataa taagtctgga agagcaaaag ggagttacta


 



ggatatgggg tgggctgctc


 


3001
ccagaatctg ctcagctttc tgcccccacc aacaccctcc


 



aaccaggcct tgccttctga


 


3061
gagcccccgt ggccaagccc aggtcacaga tcttcccccg


 



accatgctgg gaatccagaa


 


3121
acagggaccc catttgtctt cccatatctg gtggaggtga


 



gggggctcct caaaagggaa


 


3181
ctgagaggct gctcttaggg agggcaaagg ttcgggggca


 



gccagtgtct cccatcagtg


 


3241
ccttttttaa taaaagctct ttcatctata gtttggccac


 



catacagtgg cctcaaagca


 


3301
accatggcct acttaaaaac caaaccaaaa ataaagagtt


 



tagttgagga gaaaaaaaaa


 


3361
aaaaaaaaaa aaaaaa






By “Cluster of Differentiation 7 (CD7) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence: NP_006128.1 or a fragment thereof and having immunomodulatory activity. An exemplary amino acid sequence is provided below.







>NP_006128.1 T-cell antigen CD7 precursor [Homo


sapiens]








1
MAGPPRLLLL PLLLALARGL PGALAAQEVQ QSPHCTTVPV


 



GASVNITCST SGGLRGIYLR


 


61
QLGPOPQDII YYEDGVVPTT DRRFRGRLDF SGSQDNLTIT


 



MHRLQLSDTG TYTCQAITEV


 


121
NVYGSGTLVL VTEEQSQGWH RCSDAPPRAS ALPAPPTGSA


 



LPDPQTASAL PDPPAASALP


 


181
AALAVISFLL GLGLGVACVL ARTQIKKLCS WRDKNSAACV


 



VYEDMSHSRC NTLSSPNQYQ






By “Cluster of Differentiation 7 (CD7) polynucleotide” is meant a nucleic acid molecule encoding a CD7 polypeptide. An exemplary CD7 nucleic acid sequence is provided below.







>NM_006137.7 Homo sapiens CD7 molecule (CD7), mRNA








1
ctctctgagc tctgagcgcc tgcggtctcc tgtgtgctgc


 



tctctgtggg gtcctgtaga


 


61
cccagagagg ctcagctgca ctcgcccggc tgggagagct


 



gggtgtgggg aacatggccg


 


121
ggcctccgag gctcctgctg ctgcccctgc ttctggcgct


 



ggctcgcggc ctgcctgggg


 


181
ccctggctgc ccaagaggtg cagcagtctc cccactgcac


 



gactgtcccc gtgggagcct


 


241
ccgtcaacat cacctgctcc accagcgggg gcctgcgtgg


 



gatctacctg aggcagctcg


 


301
ggccacagcc ccaagacatc atttactacg aggacggggt


 



ggtgcccact acggacagac


 


361
ggttccgggg ccgcatcgac ttctcagggt cccaggacaa


 



cctgactatc accatgcacc


 


421
gcctgcagct gtcggacact ggcacctaca cctgccaggc


 



catcacggag gtcaatgtct


 


481
acggctccgg caccctggtc ctggtgacag aggaacagtc


 



ccaaggatgg cacagatgct


 


541
cggacgcccc accaagggcc tctgccctcc ctgccccacc


 



gacaggctcc gccatccctg


 


601
acccgcagac agcctctgcc ctccctgacc cgccagcagc


 



ctctgccctc cctgcggccc


 


661
tggcggtgat ctccttcctc ctcgggctgg gcctgggggt


 



ggcgtgtgtg ctggcgagga


 


721
cacagataaa gaaactgtgc tcgtggcggg ataagaattc


 



ggcagcatgt gtggtgtacg


 


781
aggacatgtc gcacagccgc tgcaacacgc tgtcctcccc


 



caaccagtac cagtgaccca


 


841
gtgggcccct gcacgtcccg cctgtggtcc ccccagcacc


 



ttccctgccc caccatgccc


 


901
cccaccctgc cacacccctc accctgctgt cctcccacgg


 



ctgcagcaga gtttgaaggg


 


961
cccagccgtg cccagctcca agcagacaca caggcagtgg


 



ccaggcccca cggtgcttct


 


1021
cagtggacaa tgatgcctcc tccgggaagc cttccctgcc


 



cagcccacgc cgccaccggg


 


1081
aggaagcctg actgtccttt ggctgcatct cccgaccatg


 



gccaaggagg gcttttctgt


 


1141
gggatgggcc tgggcacgcg gccctctcct gtcagtgccg


 



gcccacccac cagcaggccc


 


1201
ccaaccccca ggcagcccgg cagaggacgg gaggagacca


 



gtcccccacc cagccgtacc


 


1261
agaaataaag gcttctgtgc ttcc






By “Cluster of Differentiation 33 (CD33) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence: NP_001763.3 or a fragment thereof. CD33 is also known as Siglec-3. An exemplary amino acid sequence is provided below.







> NP_001763.3 myeloid cell surface antigen CD33


isoform 1 precursor [Homo sapiens]








1
MPLLLLLPLL WAGALAMDPN FWLQVQESVT VQEGLCVLVP


 



CTFFHPIPYY DKNSPVHGYW


 


61
FREGAIISRD SPVATNKLDQ EVQEETQGRF RLLGDPSRNN


 



CSLSIVDARR RDNGSYFFRM


 


121
ERGSTKYSYK SPQLSVHVTD LTHRPKILIP GTLEPGHSKN


 



LTCSVSWACE QGTPPIFSWL


 


181
SAAPTSLGPR TTHSSVLIIT PRPQDHGTNL TCQVKFAGAG


 



VTTERTIQLN VTVVPQNPTT


 


241
GIFPGDGSGK QETRAGVVHG AIGGAGVTAL LALCLCLIFF


 



IVKTHRRKAA RTAVGRNDTH


 


301
PTTGSASPKH QKKSKLHGPT ETSSCSGAAP TVEMDEELHY


 



ASLNFHGMNP SKDTSTEYSE


 


361
VRTQ






By “Cluster of Differentiation 33 (CD33) polynucleotide” is meant a nucleic acid molecule encoding a CD33 polypeptide. An exemplary CD33 nucleic acid sequence is provided below.







> NM_001772.4 Homo sapiens CD33 molecule (CD33),


transcript variant 1, mRNA








1
ctgctcacac aggaagccct ggaagctgct tcctcagaca


 



tgccgctgct gctactgctg


 


61
cccctgctgt gggcaggggc cctggctatg gatccaaatt


 



tctggctgca agtgcaggag


 


121
tcagtgacgg tacaggaggg tttgtgcgtc ctagtgccct


 



gcactttctt ccatcccata


 


181
ccctactacg acaagaactc cccagttcat ggttactggt


 



tccgggaagg agccattata


 


241
tccagggact ctccagtggc cacaaacaag ctagatcaag


 



aagtacagga ggagactcag


 


301
ggcagattcc gcctccttgg ggatcccagt aggaacaact


 



gctccctgag catcgtagac


 


361
gccaggagga gggataatgg ttcatacttc tttcggatgg


 



agagaggaag taccaaatac


 


421
agttacaaat ctccccagct ctctgtgcat gtgacagact


 



tgacccacag gcccaaaatc


 


481
ctcatccctg gcactctaga acccggccac tccaaaaacc


 



tgacctgctc tgtgtcctgg


 


541
gcctgtgagc agggaacacc cccgatcttc tcctggttgt


 



cagctgcccc cacctccctg


 


601
ggccccagga ctactcactc ctcggtgctc ataatcaccc


 



cacggcccca ggaccacggc


 


661
accaacctga cctctcaggt gaagttcgct ggagctggtg


 



tgactacgga gagaaccacc


 


721
cagctcaacg tcacctatgt tccacagaac ccaacaactg


 



gtatctttcc aggagatggc


 


181
tcagggaaac aagagaccag agcaggagtg gttcatgggg


 



ccattggagg agctggtgtt


 


841
acagccctgc tcgctctttg tctctgcctc atcttcttca


 



tagtgaagac ccacaggagg


 


901
aaagcagcca ggacagcagt gggcaggaat gacacccacc


 



ctaccacagg gtcagcctcc


 


961
ccgaaacacc agaagaagtc caagttacat ggccccactg


 



aaacctcaag ctgttcaggt


 


1021
gccgccccta ctgtggagat ggatgaggag ctgcattatg


 



cttccctcaa ctttcatggg


 


1081
atgaatcctt ccaaggacac ctccaccgaa tactcagagg


 



tcaggaccca gtgaggaacc


 


1141
cacaagagca tcaggctcag ctagaagatc cacatcctct


 



acaggtcggg gaccaaaggc


 


1201
tgattcttgg agatttaaca ccccacaggc aatgggttta


 



tagacattat gtgagtttcc


 


1261
tgctatatta acatcatctt agactttgca agcagagagt


 



cgtggaatca aatctgtgct


 


1321
ctttcatttg ctaagtgtat gatgtcacac aagctcctta


 



accttccatg tctccatttt


 


1381
cttctctgtg aagtaggtat aagaagtcct atctcatagg


 



gatgctgtga gcattaaata


 


1441
aaggtacaca tggaaaacac ca






By “Cluster of Differentiation 52 (CD52) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence: NP_001794.2 or a fragment thereof. CD52 is also known as CAMPATH-1. An exemplary amino acid sequence is provided below.







> NP_001794.2 CAMPATH-1 antigen precursor [Homo


sapiens}








1
MKRFLFLLLT ISLLVMVQIQ TGLSGQNDTS QTSSPSASSN


 



ISGGIFLFFV ANAIIHLFCG


 


61
S






By “Cluster of Differentiation 52 (CD52) polynucleotide” is meant a nucleic acid molecule encoding a CD52 polypeptide. An exemplary CD52 nucleic acid sequence is provided below.







>NM_001803.3 Homo sapiens CD52 molecule (CD52),


mRNA








1
agacagccct gagatcacct aaaaagctgc taccaagaca


 



gccacgaaga tcctaccaaa


 


61
atgaagcgct tcctcttcct cctactcacc atcagcctcc


 



tggttatggt acagatacaa


 


121
actggactct caggacaaaa cgacaccagc caaaccagca


 



gcccctcagc atccagcaac


 


181
ataagcggag gcattttcct tttcttcgtg gccaatgcca


 



taatccacct cttctgcttc


 


241
agttgaggtg acacgtctca gccttagccc tgtgccccct


 



gaaacagctg ccaccatcac


 


301
tcgcaagaga atcccctcca tctttgggag gggttgatgc


 



cagacatcac caggttgtag


 


361
aagttgacag gcagtgccat gggggcaaca gccaaaatag


 



gggggtaatg atgtaggggc


 


421
caagcagtgc ccagctgggg gtcaataaag ttacccttgt


 



acttgca






By “Cluster of Differentiation 123 (CD123) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence: NP_002174.1 or a fragment thereof. CD123 is also known as the interleukin-3 receptor. An exemplary amino acid sequence is provided below.







>NP_002174.1 interleukin-3 receptor subunit alpha


isoform 1 precursor [Homo sapiens]








1
MVLLWLTLLL IALPCLLQTK EDPNPPITNL RMKAKAQQLT


 



WDLNRNVTDI ECVKDADYSM


 


61
PAVNNSYCQF GAISLCEVTN YTVRVANPPF STWILFPENS


 



GKPWAGAENL TCWIHDVDFL


 


121
SCSWAVGPGA PADCQYDLYL NVANRRQQYE CLHYKTDAQG


 



TRIGCRFDDI SRLSSGSQSS


 


181
HILVRGRSAA FGIPCTDKFV VFSQIEILTP PNMTAKCNKT


 



HSFMHWKMRS HFNRKFRYEL


 


241
QIQKRMQPVI TEQVRDRTSF QLLNPGTYTV QIRARERVYE


 



FLSAWSTPQR FECDQEEGAN


 


301
TRAWRTSLLI ALGTLLALVC VFVICRRYLV MQRLFPRIPH


 



MKDPIGDSFQ NDKLVVWEAG


 


361
KAGLEECLVT EVQVVQKT






By “Cluster of Differentiation 123 (CD123) polynucleotide” is meant a nucleic acid molecule encoding a CD123 polypeptide. An exemplary CD123 nucleic acid sequence is provided below.







>NM_002183.4 Homo sapiens interleukin 3 receptor


subunit alpha (IL3RA), transcript variant 1, mRNA








1
cttcggtttc tcttcgggga aagctgcttt cagcgcacac


 



gggaagatat cagaaacatc


 


61
ctaggatcag gacaccccag atcttctcaa ctggaaccac


 



gaaggctgtt tcttccacac


 


121
agtactttga tctccattta agcaggcacc tctgtcctgc


 



gttccggagc tgcgttcccg


 


181
atggtcctcc tttggctcac gctgctcctg atcgccctgc


 



cctgtctcct gcaaacgaag


 


241
gaagatccaa acccaccaat cacgaaccta aggatgaaag


 



caaaggctca gcagttgacc


 


301
tgggacctta acagaaatgt gaccgatatc gagtgtgtta


 



aagacgccga ctattctatg


 


361
ccggcagtga acaatagcta ttgccagttt ggagcaattt


 



ccttatgtga agtgaccaac


 


421
tacaccgtcc gagtggccaa cccaccattc tccacgtgga


 



tcctcttccc tgagaacagt


 


481
gggaagcctt gggcaggtgc ggagaatctg acctgctgga


 



ttcatgacgt ggatttcttg


 


541
agctgcagct gggcggtagg cccgggggcc cccgcggacg


 



tccagtacga cctgtacttg


 


601
aacgttgcca acaggcgtca acagtacgag tgtcttcact


 



acaaaacgga tgctcaggga


 


661
acacgtatcg ggtgtcgttt cgatgacatc tctcgactct


 



ccagcggttc tcaaagttcc


 


721
cacatcctgg tgcggggcag gagcgcagcc ttcggtatcc


 



cctgcacaga taagtttgtc


 


781
gtcttttcac agattgagat attaactcca cccaacatga


 



ctgcaaagtg taataagaca


 


841
cattccttta tgcactggaa aatgagaagt catttcaatc


 



gcaaatttcg ctatgagctt


 


901
cagatacaaa agagaatgca gcctgtaatc acagaacagg


 



tcagagacag aacctccttc


 


961
cagctactca atcctggaac gtacacagta caaataagag


 



cccgggaaag agtgtatgaa


 


1021
ttcttgagcg cctggagcac cccccagcgc ttcgagtgcg


 



accaggagga gggcgcaaac


 


1081
acacgtgcct ggcggacgtc gctgctgatc gcgctgggga


 



cgccgctggc cctggtctgt


 


1141
gtcttcgtga tctgcagaag gtatctggtg atgcagagac


 



tctttccccg catccctcac


 


1201
atgaaagacc ccatcggtga cagcttccaa aacgacaagc


 



tggtggtctg ggaggcgggc


 


1261
aaagccggcc tggaggagtg tctggtgact gaagtacagg


 



tcgtgcagaa aacttgagac


 


1321
tggggttcag ggcttgtggg ggtctgcctc aatctccctg


 



gacgggccag gcgcctgcac


 


1381
agactggctg ctggacctgc gcacgcagcc caggaatgga


 



cattcctaac gggtggtggg


 


1441
catgggagat gcctgtgtaa tttcgtccga agctgccagg


 



aagaagaaca gaactttgtg


 


1501
tgtttatttc atgataaagt gatttttttt tttttaaccc


 



a






By “Cluster of Differentiation 137 (CD137) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence: NP_001552.2 or a fragment thereof. CD137 is also known as 4-1BB. An exemplary amino acid sequence is provided below.







>NP_001552.2 Tumor necrosis factor receptor super-


family member 9 precursor [Homo sapiens]








1
MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN


 



RNQICSPCPP NSFSSAGGQR


 


61
TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS


 



MCEQDCKQGQ ELTKKGCKDC


 


121
CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP


 



SPADLSPGAS SVTPPAPARE


 


181
PGHSPQIISF FLALTSTALL FLLFFLTLRE SVVKRGRKKL


 



LYIFKQPFMR PVQTTQEEDG


 


241
CSCRFPEEEE GGCEL






By “Cluster of Differentiation 137 (CD137) polynucleotide” is meant a nucleic acid molecule encoding a CD137 polypeptide. An exemplary CD137 nucleic acid sequence is provided below.








>NM_001561.6 Homo sapiens TNF receptor superfamily member 9 (TNFRSF9), mRNA










1
gcagaagcct gaagaccaag gagtggaaag ttctccggca gccctgagat ctcaagagtg



 


61
acatttgtga gaccagctaa tttgattaaa attctcttgg aatcagcttt gctagtatca


 


121
tacctgtgcc agatttcatc atgggaaaca gctgttacaa catagtagcc actctgttgc


 


181
tggtcctcaa ctttgagagg acaagatcat tgcaggatcc ttgtagtaac tgcccagctg


 


241
gtacattctg tgataataac aggaatcaga tttgcagtcc ctgtcctcca aatagtttct


 


301
ccagcgcagg tggacaaagg acctgtgaca tatgcaggca gtgtaaaggt gttttcagga


 


361
ccaggaagga gtgttcctcc accagcaatg cagagtgtga ctgcactcca gggtttcact


 


441
gcctgggggc aggatgcagc atgtgtgaac aggattgtaa acaaggtcaa gaactgacaa


 


481
aaaaaggttg taaagactgt tgctttggga catttaacga tcagaaacgt ggcatctgtc


 


541
gaccctggac aaactgttct ttggatggaa agtctgtgct tgtgaatggg acgaaggaga


 


601
gggacgtggt ctgtggacca tctccagccg acctctctcc gggagcatcc tctgtgaccc


 


661
cgcctgcccc tgcgagagag ccaggacact ctccgcagat catctccttc tttcttgcgc


 


721
tgacgtcgac tgcgttgctc ttcctgctgt tcttcctcac gctccgtttc tctgttgtta


 


781
aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga ccagtacaaa


 


841
ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa ggaggatgtg


 


901
aactgtgaaa tggaagtcaa tagggctgtt gggactttct tgaaaagaag caaggaaata


 


961
tgagtcatcc gctatcacag ctttcaaaag caagaacacc atcctacata atacccagga


 


1021
ttcccccaac acacgttctt ttctaaatgc caatgagttg gcctttaaaa atgcaccact


 


1081
tttttttttt ttttgacagg gtctcactct gtcacccagg ctggagtgca gtggcaccac


 


1141
catggctctc tgcagccttg acctctggga gctcaagtga tcctcctgcc tcagtctcct


 


1201
gagtagctgg aactacaagg aagggccacc acacctgact aacttttttg ttttttgttt


 


1261
ggtaaagatg gcatttcacc atgttgtaca ggctggtctc aaactcctag gttcactttg


 


1321
gcctcccaaa gtgctgggat tacagacatg aactgccagg cccggccaaa ataatgcacc


 


1381
acttttaaca gaacagacag atgaggacag agctggtgat aaaaaaaaaa aaaaaaaagc


 


1441
attttctaga taccacttaa caggtttgag ctagtttttt tgaaatccaa agaaaattat


 


1501
agtttaaatt caattacata gtccagtggt ccaactataa ttataatcaa aatcaatgca


 


1561
ggtttgtttt ttggtgctaa tatgacatat gacaataagc cacgaggtgc agtaagtacc


 


1621
cgactaaagt ttccgtgggt tctgtcatgt aacacgacat gctccaccgt caggggggag


 


1681
tatgagcaga gtgcctgagt ttagggtcaa ggaeaaaaaa cctcaggcct ggaggaagtt


 


1741
ttggaaagag ttcaagtgtc tgtatatcct atggtcttct ccatcctcac accttctgcc


 


1801
tttgtcctgc tcccttttaa gccaggttac attctaaaaa ttcttaactt ttaacataat


 


1861
attttatacc aaagccaata aatgaactgc atatgatagg tatgaagtac agtgagaaaa


 


1921
ttaacacctg tgagctcatt gtcctaccac agcactagag tgggggccgc caaactccca


 


1981
tggccaaacc tggtgcacca tttgcctttg tttgtctgtt ggtttgcttg agacagtctt


 


2041
gctctgttgc ccaggctgga atggagtggc tattcacagg cacaatcata gcacacttta


 


2101
gccttaaact cctgggctca agtgatccac ccgcctcagt ctcccaagta gctgggatta


 


2161
caggtgcaaa cctggcatgc ctgccattgt ttggcttatg atctaaggat agctttttaa


 


2221
attttattca ttttattttt ttttgagaca gtgtctcact atgtctccca ggctggagta


 


2281
cagtggtaca atcttggatc accgcctccc agtttcaagt gatctccctg cctcagcctc


 


2341
ctaagtagct gggactacag gtatgtgcca ccacgcctgg ctaattttta tatttttagt


 


2401
agagacgggg tttcaccatg ttgtccaggc tggtctcaaa ctcctgacct caggtgatct


 


2461
gcccacctct gcctcccaaa gtgctgggat tacaggcatg agccaccatg cctggccatt


 


2521
tcttacactt ttgtatgaca tgcctattgc aagcttgcgt gcctctgtcc catgttattt


 


2581
tactctggga tttaggtgga gggagcagct tctatttgga acattggcca tcgcatggca


 


2641
aatgggtatc tgtcacttct gctcctattt agttggttct actataacct ttagagcaaa


 


2701
tcctgcagcc aagccaggca tcaatagggc agaaaagtat attctgtaaa taggggtgag


 


2761
gagaagatat ttctgaacaa tagtetactg cagtaccaaa ttgcttttca aagtggctgt


 


2821
tctaatgtac tcccgtcagt catataagtg tcatgtaagt atcccattga tccacatcct


 


2881
tgctaccctc tggtactatc aggtgccctt aattttgcca agccagtggg tatagaatga


 


2941
gatctcactg tggtcttagt ttgcatttgc ttggttactg atgagcacct tgtcaaatat


 


3001
ttatatacca tttgtgttta tttttttaaa taaaatgctt gctcatgctt ttttgcccat


 


3061
ttgcaaaaaa acttggggcc gggtgcagtg gctcatgcct gtagtcccag ctctttggga


 


3121
ggccaaggtg ggcagatcgc ttgagcccag gagttcgaga ccagccttgg caacatggcg


 


3181
aaaccctgtc tttacaaaaa atacaaaaat tagccgggtg tggtggtgtg cacctgaagt


 


3241
cccagctact cagtaggttc gctttgagcc tgggaggcag aggttgcagt gagctgggac


 


3301
cgcatcacta cacttcagcc tgggcaacag agaaaaacct tttctcagaa acaaacaaac


 


3361
ccaaatgtgg ttgtttgtcc tgattcctaa aaggtcttta tgtattctag ataataatct


 


3421
ttggtcagtt atatgtgtta aaaaatatct tctttgtggc caggcacggt agctcacacc


 


3481
tgtaatccca gcactttgcg gggctgaggt gggtggatca tctgaggtca agagttcaag


 


3541
atcagcctgg ccaacacagt gaaaccccat ctctactaaa catgtacaaa acttagctgg


 


3601
gtatggtggc gggtgcctgt aaccccagct gctccagagg ctgtggcaga agaatcgctt


 


3661
gaacccagga ggcagaggtt gcagcgagcc aagattgtgc cattgcactc cagactgggt


 


3721
gacaagagtg aaattctgcc tatctatcta tctatctatc tatatctata tatatatata


 


3781
tatatatcct ttgtaattta tttttccctt tttaaaattt tttataaaat tcttttttat


 


3841
ttttattttt agcagaggtg aggtttctga gatttcatta tgttgcccag gctggtcttg


 


3901
aactcctgag ctcaagtgat cctcccacct cagccttcca aagtgctgga attgcagaca


 


3961
tgagccaccg cgcccctcct gtttttctct aattaatggt gtctttcttt gtctttctgg


 


4021
taataagcaa aaagttcttc atttgatttg gttaaattta taactgtttt ctcatatggt


 


4081
taacattttt tcttgcctgg ctaaagaaat ccttttctgc ccaatactat aaagaggttt


 


4141
gcccacattt tattccaaaa gttttaagtt ttgtctttca tcttgaagtc taatgtatca


 


4201
ggaactggct tttgtgcctg ttgggaggta gtgatccaat tccatgtctt gcatgtaggt


 


4261
aaccactggt ccctgcgcca tgtattcaat acgtcgtctt tctcctgcgg gtctgcaatc


 


4321
tcacctacca tccatcaagt ttccataggg ccatgggtct gcttctgggc tccctgttct


 


4381
gttccattgt caatttgtct atcctgtgcc actatcacac tgtgtttatt acaatagctt


 


4441
tgtaacagct ctcgatatcc ggtaggacat ctccctccac cttctttttc tacttcagaa


 


4501
gtgtcttagc taggtcaggc acggtggctc acgcctgtaa tcccagcact ttgggaggcc


 


4561
gacgcggatg gatcacctga ggtcaggagt tttgagacag cctggccaac atggtgaaac


 


4621
cccatctcta ctaaaaaata caaaaattag tcaggcatgg tggcatgtgc ctgtaatccc


 


4681
agctatttgg gaggctgagg ccggagaatt gcttgaaccc ggggggcgga ggttgcagtg


 


4741
agccgagatc gtaccattgc actccagcct gggtgacaga gcgaaactct gtctcaggaa


 


4801
aaaaaagaaa agagatgtct tggttattct tggttcttta ttattcaata taaattttag


 


4861
aagctgaatt tgaaaagatt tggattggaa tttcattaaa tctacaggtc aatttaggga


 


4921
gagttgataa ttttacagaa ttgagtcatc tggtgttcca ataagaataa gagaacaatt


 


4981
attggctgta caattcttgc caaatagtag gcaaagcaaa gcttaggaag tatactggtg


 


5041
ccatttcagg aacaaagcta ggtgcgaata tttttgtctt tctgaatcat gatgctgtaa


 


5101
gttctaaagt gatttctcct cttggctttg gacacatggt gtttaattac ctactgctga


 


5161
ctatccacaa acagaaagag actggtcatg ccccacaggg ttggggtatc caagataatg


 


5221
gagcgaggct ctcatgtgtc ctaggttaca caccgaaaat ccacagttta ttctgtgaag


 


5281
aaaggaggct atgtttatga tacagactgt gatattttta tcatagccta ttctggtatc


 


5341
atgtgcaaaa gctataaatg aaaaacacag gaacttggca tgtgagtcat tgctccccct


 


5401
aaatgacaat taataaggaa ggaacattga gacagaataa aatgatcccc ttctgggttt


 


5461
aatttagaaa gttccataat taggtttaat agaaataaat gtaaatttct atgattaaaa


 


5521
ataaattagc acatttaggg atacacaaat tataaatcat tttctaaatg ctaaaaacaa


 


5581
gctcaggttt ttttcagaag aaagttttaa ttttttttct ttagtggaag atatcactct


 


5641
gacggaaagt tttgatgtga ggggcggatg actataaagt gggcatcttc ccccacagga


 


5701
agatgtttcc atctgtgggt gagaggtgcc caccgcagct agggcaggtt acatgtgccc


 


5761
tgtgtgtggt aggacttgga gagtgatctt tatcaacgtt tttatttaaa agactatcta


 


5821
ataaaacaca aaactatgat gttcacagga aaaaaagaat aagaaaaaaa ga






By “Cluster of Differentiation 247 (CD247) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Reference Sequence: NP_932170.1 or a fragment thereof. CD137 is also known as CD3(. An exemplary amino acid sequence is provided below.







>NP_932170.1 T-cell surface glycoprotein CD3 zeta


chain isoform 1 precursor [Homo sapiens]








1
MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF


 



IYGVILTALF LRVKFSRSAD


 


61
APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP


 



QRRKNPQEGL YNELQKDKMA


 


121
EAYSEIGMKG ERRRGKGHDG LYQGLSTATK DTYDALHMQA


 



LPPR






By “Cluster of Differentiation 247 (CD247) polynucleotide” is meant a nucleic acid molecule encoding a CD247 polypeptide. An exemplary CD247 nucleic acid sequence is provided below.







>NM_NM_198053.3 Homo sapiens CD247 molecule


(CD247), transcript variant 1, mRNA








1
aaccgtcccg gccaccgctg cctcagcctc tgcctcccag


 



cctctttctg agggaaagga


 


61
caagatgaag tggaaggcgc ttttcaccgc ggccatcctg


 



caggcacagt tgccgattac


 


121
agaggcacag agctttggcc tgctggatcc caaactctgc


 



tacctgctgg atggaatcct


 


181
cttcatctat ggtgtcattc tcactgcctt gttcctgaga


 



gtgaagttca gcaggagcgc


 


241
agacgccccc gagtaccagc agggccagaa ccagctctat


 



aacgagctca atctaggacg


 


301
aagagaggag tacgatgttt tggacaagag acgtggccgg


 



gaccctgaga tggggggaaa


 


361
gccgcagaga aggaagaacc ctcaggaagg cctgtacaat


 



gaactgcaga aagataagat


 


421
ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc


 



aggaggqgca aggggcacga


 


481
tggcctttac cagggtctca gtacagccac caaggacacc


 



tacgacgccc ttcacatgca


 


541
ggccctgccc cctcgctaac agccagggga tttcaccact


 



caaaggccag acctgcagac


 


601
gcccagatta tgagacacag gatgaagcat ttacaacccg


 



gttcactctt ctcagccact


 


661
gaagtattcc cctttatgta caggatgctt tggttatatt


 



tagctccaaa ccttcacaca


 


721
cagactgttg tccctgcact ctttaaggga gtgtactccc


 



agggcttacg gccctggcct


 


781
tgggccctct ggtttgcogg tggtgcaggt agacctgtct


 



cctggcggtt cctcgttctc


 


841
cctgggaggc gggcgcactg cctctcacag ctgagttgtt


 



gagtctgttt tgtaaagtcc


 


901
ccagagaaag cgcagatgct agcacatgcc ctaatgtctg


 



tatcactctg tgtctgagtg


 


961
gcttcactcc tgctgtaaat ttggcttctg ttgtcacctt


 



cacctccttt caaggtaact


 


1021
gtactgggcc atgttgtgcc tccctggtga gagggccggg


 



cagaggggca gatggaaagg


 


1081
agcctaggcc aggtgcaacc agggagctgc aggggcatgg


 



gaaggtgggc gggcagggga


 


1141
gggtcagcca gggcctgcga gggcagcggg agcctccctg


 



cctcaggcct ctgtgccgca


 


1201
ccattgaact gtaccatgtg ctacaggggc cagaagatga


 



acagactgac cttgatgagc


 


1261
tgtgcacaaa gtggcataaa aaacatgtgg ttacacagtg


 



tgaataaagt gctgcggagc


 


1321
aagaggaggc cgttgattca cttcacgctt tcagcgaatg


 



acaaaatcat ctttgtgaag


 


1381
gcctcgcagg aagacccaac acatgggacc tataactgcc


 



cagcggacag tggcaggaca


 


1441
ggaaaaaccc gtcaatgtac taggatactg ctgcgtcatt


 



acagggcaca ggccatggat


 


1501
ggaaaacgct ctctactctg ctttttttct actgttttaa


 



tttatactgg catgctaaag


 


1561
ccttcctatt ttgcataata aatgcttcag tgaaaatgca






“Co-administration” or “co-administered” refers to administering two or more therapeutic agents or pharmaceutical compositions during a course of treatment. Such co-administration can be simultaneous administration or sequential administration. Sequential administration of a later-administered therapeutic agent or pharmaceutical composition can occur at any time during the course of treatment after administration of the first pharmaceutical composition or therapeutic agent.
The term “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and Schirmer, R. H., Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids can be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and Schirmer, R. H., supra). Non-limiting examples of conservative mutations include amino acid substitutions of amino acids, for example, lysine for arginine and vice versa such that a positive charge can be maintained; glutamic acid for aspartic acid and vice versa such that a negative charge can be maintained; serine for threonine such that a free —OH can be maintained; and glutamine for asparagine such that a free —NH2 can be maintained.
The term “coding sequence” or “protein coding sequence” as used interchangeably herein refers to a segment of a polynucleotide that codes for a protein. The region or sequence is bounded nearer the 5′ end by a start codon and nearer the 3′ end with a stop codon. Coding sequences can also be referred to as open reading frames.
By “codon optimization” is meant a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/ (visited Jul. 9, 2002), and these tables can be adapted in a number of ways. See, Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding an engineered nuclease correspond to the most frequently used codon for a particular amino acid.
By “cytidine deaminase” is meant a polypeptide or fragment thereof capable of catalyzing a deamination reaction that converts an amino group to a carbonyl group. In one embodiment, the cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine. PmCDA1, which is derived from Petromyzon marinus (Petromyzon marinus cytosine deaminase 1 (“PmCDA1”)), or AID (Activation-induced cytidine deaminase (“AICDA”)), which is derived from a mammal (e.g., human, swine, bovine, horse, monkey etc.), and APOBEC are exemplary cytidine deaminases.
The base sequence and amino acid sequence of PmCDA1 and the base sequence and amino acid sequence of human AID are shown below.








>tr|A5H718|A5H718_PETMA Cytosine deaminase OS = Petromyzon marinus OX = 7757 PE = 2



SV = 1


MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERG


 


IHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYY


 


EKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELS


 


IMIQVKILHTTKSPAV


 


>EF094822.1 Petromyzon marinus isolate PmCDA.21 cytosine deaminase mRNA, complete cds


TGACACGACACAGCCGTGTATATGAGGAAGGGTAGCTGGATGGGGGGGGGGGGAATACGTTCAG


 


AGAGGACATTAGCGAGCGTCTTGTTGGTGGCCTTGAGTCTAGACACCTGCAGACATGACCGACG


 


CTGAGTACGTGAGAATCCATGAGAAGTTGGACATCTACACGTTTAAGAAACAGTTTTTCAACAA


 


CAAAAAATCCGTGTCGCATAGATGCTACGTTCTCTTTGAATTAAAACGACGGGGTGAACGTAGA


 


GCGTGTTTTTGGGGCTATGCTGTGAATAAACCACAGAGCGGGACAGAACGTGGAATTCACGCCG


 


AAATCTTTAGCATTAGAAAAGTCGAAGAATACCTGCGCGACAACCCCGGACAATTCACGATAAA


 


TTGGTACTCATCCTGGAGTCCTTGTGCAGATTGCGCTGAAAAGATCTTAGAATGGTATAACCAG


 


GAGCTGCGGGGGAACGGCCACACTTTGAAAATCTGGGCTTGCAAACTCTATTACGAGAAAAATG


 


CGAGGAATCAAATTGGGCTGTGGAACCTCAGAGATAACGGGGTTGGGTTGAATGTAATGGTAAG


 


TGAACACTACCAATGTTGCAGGAAAATATTCATCCAATCGTCGCACAATCAATTGAATGAGAAT


 


AGATGGCTTGAGAAGACTTTGAAGCGAGCTGAAAAACGACGGAGCGAGTTGTCCATTATGATTC


 


AGGTAAAAATACTCCACACCACTAAGAGTCCTGCTGTTTAAGAGGCTATGCGGATGGTTTTC


 


>tr|Q6QJ80|Q6QJ80_HUMAN Activation-induced cytidine deaminase OS = Homo sapiens


OX = 9606 GN = AICDA PE = 2 SV = 1


MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRY


 


ISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRR


 


LHRAGVQIAIMTFKAPV


 


>NG_011588.1: 5001-15681 Homo sapiens activation induced cytidine deaminase (AICDA),


RefSeqGene (LRG_17) on chromosome 12


AGAGAACCATCATTAATTGAAGTGAGATTTTTCTGGCCTGAGACTTGCAGGGAGGCAAGAAGAC


 


ACTCTGGACACCACTATGGACAGGTAAAGAGGCAGTCTTCTCGTGGGTGATTGCACTGGCCTTC


 


CTCTCAGAGCAAATCTGAGTAATGAGACTGGTAGCTATCCCTTTCTCTCATGTAACTGTCTGAC


 


TGATAAGATCAGCTTGATCAATATGCATATATATTTTTTGATCTGTCTCCTTTTCTTCTATTCA


 


GATCTTATACGCTGTCAGCCCAATTCTTTCTGTTTCAGACTTCTCTTGATTTCCCTCTTTTTCA


 


TGTGGCAAAAGAAGTAGTGCGTACAATGTACTGATTCGTCCTGAGATTTGTACCATGGTTGAAA


 


CTAATTTATGGTAATAATATTAACATAGCAAATCTTTAGAGACTCAAATCATGAAAAGGTAATA


 


GCAGTACTGTACTAAAAACGGTAGTGCTAATTTTCGTAATAATTTTGTAAATATTCAACAGTAA


 


AACAACTTGAAGACACACTTTCCTAGGGAGGCGTTACTGAAATAATTTAGCTATAGTAAGAAAA


 


TTTGTAATTTTAGAAATGCCAAGCATTCTAAATTAATTGCTTGAAAGTCACTATGATTGTGTCC


 


ATTATAAGGAGACAAATTCATTCAAGCAAGTTATTTAATGTTAAAGGCCCAATTGTTAGGCAGT


 


TAATGGCACTTTTACTATTAACTAATCTTTCCATTTGTTCAGACGTAGCTTAACTTACCTCTTA


 


GGTGTGAATTTGGTTAAGGTCCTCATAATGTCTTTATGTGCAGTTTTTGATAGGTTATTGTCAT


 


AGAACTTATTCTATTCCTACATTTATGATTACTATGGATGTATGAGAATAACACCTAATCCTTA


 


TACTTTACCTCAATTTAACTCCTTTATAAAGAACTTACATTACAGAATAAAGATTTTTTAAAAA


 


TATATTTTTTTGTAGAGACAGGGTCTTAGCCCAGCCGAGGCTGGTCTCTAAGTCCTGGCCCAAG


 


CGATCCTCCTGCCTGGGCCTCCTAAAGTGCTGGAATTATAGACATGAGCCATCACATCCAATAT


 


ACAGAATAAAGATTTTTAATGGAGGATTTAATGTTCTTCAGAAAATTTTCTTGAGGTCAGACAA


 


TGTCAAATGTCTCCTCAGTTTACACTGAGATTTTGAAAACAAGTCTGAGCTATAGGTCCTTGTG


 


AAGGGTCCATTGGAAATACTTGTTCAAAGTAAAATGGAAAGCAAAGGTAAAATCAGCAGTTGAA


 


ATTCAGAGAAAGACAGAAAAGGAGAAAAGATGAAATTCAACAGGACAGAAGGGAAATATATTAT


 


CATTAAGGAGGACAGTATCTGTAGAGCTCATTAGTGATGGCAAAATGACTTGGTCAGGATTATT


 


TTTAACCCGCTTGTTTCTGGTTTGCACGGCTGGGGATGCAGCTAGGGTTCTGCCTCAGGGAGCA


 


CAGCTGTCCAGAGCAGCTGTCAGCCTGCAAGCCTGAAACACTCCCTCGGTAAAGTCCTTCCTAC


 


TCAGGACAGAAATGACGAGAACAGGGAGCTGGAAACAGGCCCCTAACCAGAGAAGGGAAGTAAT


 


GGATCAACAAAGTTAACTAGCAGGTCAGGATCACGCAATTCATTTCACTCTGACTGGTAACATG


 


TGACAGAAACAGTGTAGGCTTATTGTATTTTCATGTAGAGTAGGACCCAAAAATCCACCCAAAG


 


TCCTTTATCTATGCCACATCCTTCTTATCTATACTTCCAGGACACTTTTTCTTCCTTATGATAA


 


GGCTCTCTCTCTCTCCACACACACACACACACACACACACACACACACACACACACACACAAAC


 


ACACACCCCGCCAACCAAGGTGCATGTAAAAAGATGTAGATTCCTCTGCCTTTCTCATCTACAC


 


AGCCCAGGAGGGTAAGTTAATATAAGAGGGATTTATTGGTAAGAGATGATGCTTAATCTGTTTA


 


ACACTGGGCCTCAAAGAGAGAATTTCTTTTCTTCTGTACTTATTAAGCACCTATTATGTGTTGA


 


GCTTATATATACAAAGGGTTATTATATGCTAATATAGTAATAGTAATGGTGGTTGGTACTATGG


 


TAATTACCATAAAAATTATTATCCTTTTAAAATAAAGCTAATTATTATTGGATCTTTTTTAGTA


 


TTCATTTTATGTTTTTTATGTTTTTGATTTTTTAAAAGACAATCTCACCCTGTTACCCAGGCTG


 


GAGTGCAGTGGTGCAATCATAGCTTTCTGCAGTCTTGAACTCCTGGGCTCAAGCAATCCTCCTG


 


CCTTGGCCTCCCAAAGTGTTGGGATACAGTCATGAGCCACTGCATCTGGCCTAGGATCCATTTA


 


GATTAAAATATGCATTTTAAATTTTAAAATAATATGGCTAATTTTTACCTTATGTAATGTGTAT


 


ACTGGCAATAAATCTAGTTTGCTGCCTAAAGTTTAAAGTGCTTTCCAGTAAGCTTCATGTACGT


 


GAGGGGAGACATTTAAAGTGAAACAGACAGCCAGGTGTGGTGGCTCACGCCTGTAATCCCAGCA


 


CTCTGGGAGGCTGAGGTGGGTGGATCGCTTGAGCCCTGGAGTTCAAGACCAGCCTGAGCAACAT


 


GGCAAAACGCTGTTTCTATAACAAAAATTAGCCGGGCATGGTGGCATGTGCCTGTGGTCCCAGC


 


TACTAGGGGGCTGAGGCAGGAGAATCGTTGGAGCCCAGGAGGTCAAGGCTGCACTGAGCAGTGC


 


TTGCGCCACTGCACTCCAGCCTGGGTGACAGGACCAGACCTTGCCTCAAAAAAATAAGAAGAAA


 


AATTAAAAATAAATGGAAACAACTACAAAGAGCTGTTGTCCTAGATGAGCTACTTAGTTAGGCT


 


GATATTTTGGTATTTAACTTTTAAAGTCAGGGTCTGTCACCTGCACTACATTATTAAAATATCA


 


ATTCTCAATGTATATCCACACAAAGACTGGTACGTGAATGTTCATAGTACCTTTATTCACAAAA


 


CCCCAAAGTAGAGACTATCCAAATATCCATCAACAAGTGAACAAATAAACAAAATGTGCTATAT


 


CCATGCAATGGAATACCACCCTGCAGTACAAAGAAGCTACTTGGGGATGAATCCCAAAGTCATG


 


ACGCTAAATGAAAGAGTCAGACATGAAGGAGGAGATAATGTATGCCATACGAAATTCTAGAAAA


 


TGAAAGTAACTTATAGTTACAGAAAGCAAATCAGGGCAGGCATAGAGGCTCACACCTGTAATCC


 


CAGCACTTTGAGAGGCCACGTGGGAAGATTGCTAGAACTCAGGAGTTCAAGACCAGCCTGGGCA


 


ACACAGTGAAACTCCATTCTCCACAAAAATGGGAAAAAAAGAAAGCAAATCAGTGGTTGTCCTG


 


TGGGGAGGGGAAGGACTGCAAAGAGGGAAGAAGCTCTGGTGGGGTGAGGGTGGTGATTCAGGTT


 


CTGTATCCTGACTGTGGTAGCAGTTTGGGGTGTTTACATCCAAAAATATTCGTAGAATTATGCA


 


TCTTAAATGGGTGGAGTTTACTGTATGTAAATTATACCTCAATGTAAGAAAAAATAATGTGTAA


 


GAAAACTTTCAATTCTCTTGCCAGCAAACGTTATTCAAATTCCTGAGCCCTTTACTTCGCAAAT


 


TCTCTGCACTTCTGCCCCGTACCATTAGGTGACAGCACTAGCTCCACAAATTGGATAAATGCAT


 


TTCTGGAAAAGACTAGGGACAAAATCCAGGCATCACTTGTGCTTTCATATCAACCATGCTGTAC


 


AGCTTGTGTTGCTGTCTGCAGCTGCAATGGGGACTCTTGATTTCTTTAAGGAAACTTGGGTTAC


 


CAGAGTATTTCCACAAATGCTATTCAAATTAGTGCTTATGATATGCAAGACACTGTGCTAGGAG


 


CCAGAAAACAAAGAGGAGGAGAAATCAGTCATTATGTGGGAACAACATAGCAAGATATTTAGAT


 


CATTTTGACTAGTTAAAAAAGCAGCAGAGTACAAAATCACACATGCAATCAGTATAATCCAAAT


 


CATGTAAATATGTGCCTGTAGAAAGACTAGAGGAATAAACACAAGAATCTTAACAGTCATTGTC


 


ATTAGACACTAAGTCTAATTATTATTATTAGACACTATGATATTTGAGATTTAAAAAATCTTTA


 


ATATTTTAAAATTTAGAGCTCTTCTATTTTTCCATAGTATTCAAGTTTGACAATGATCAAGTAT


 


TACTCTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTGAGATGGAGTTTTGGCTTGTTGCCCATGC


 


TGGAGTGGAATGGCATGACCATAGCTCACTGCAACCTCCACCTCCTGGGTTCAAGCAAAGCTGT


 


CGCCTCAGCCTCCCGGGTAGATGGGATTACAGGCGCCCACCACCACACTCGGCTAATGTTTGTA


 


TTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTCAGAGG


 


ATCCACCTGCCTCAGCCTCCCAAAGTGCTGGGATTACAGATGTAGGCCACTGCGCCCGGCCAAG


 


TATTGCTCTTATACATTAAAAAACAGGTGTGAGCCACTGCGCCCAGCCAGGTATTGCTCTTATA


 


CATTAAAAAATAGGCCGGTGCAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAAGCCAAGGC


 


GGGCAGAACACCCGAGGTCAGGAGTCCAAGGCCAGCCTGGCCAAGATGGTGAAACCCCGTCTCT


 


ATTAAAAATACAAACATTACCTGGGCATGATGGTGGGCGCCTGTAATCCCAGCTACTCAGGAGG


 


CTGAGGCAGGAGGATCCGCGGAGCCTGGCAGATCTGCCTGAGCCTGGGAGGTTGAGGCTACAGT


 


AAGCCAAGATCATGCCAGTATACTTCAGCCTGGGCGACAAAGTGAGACCGTAACAAAAAAAAAA


 


AAATTTAAAAAAAGAAATTTAGATCAAGATCCAACTGTAAAAAGTGGCCTAAACACCACATTAA


 


AGAGTTTGGAGTTTATTCTGCAGGCAGAAGAGAACCATCAGGGGGTCTTCAGCATGGGAATGGC


 


ATGGTGCACCTGGTTTTTGTGAGATCATGGTGGTGACAGTGTGGGGAATGTTATTTTGGAGGGA


 


CTGGAGGCAGACAGACCGGTTAAAAGGCCAGCACAACAGATAAGGAGGAAGAAGATGAGGGCTT


 


GGACCGAAGCAGAGAAGAGCAAACAGGGAAGGTACAAATTCAAGAAATATTGGGGGGTTTGAAT


 


CAACACATTTAGATGATTAATTAAATATGAGGACTGAGGAATAAGAAATGAGTCAAGGATGGTT


 


CCAGGCTGCTAGGCTGCTTACCTGAGGTGGCAAAGTCGGGAGGAGTGGCAGTTTAGGACAGGGG


 


GCAGTTGAGGAATATTGTTTTGATCATTTTGAGTTTGAGGTACAAGTTGGACACTTAGGTAAAG


 


ACTGGAGGGGAAATCTGAATATACAATTATGGGACTGAGGAACAAGTTTATTTTATTTTTTGTT


 


TCGTTTTCTTGTTGAAGAACAAATTTAATTGTAATCCCAAGTCATCAGCATCTAGAAGACAGTG


 


GCAGGAGGTGACTGTCTTGTGGGTAAGGGTTTGGGGTCCTTGATGAGTATCTCTCAATTGGCCT


 


TAAATATAAGCAGGAAAAGGAGTTTATGATGGATTCCAGGCTCAGCAGGGCTCAGGAGGGCTCA


 


GGCAGCCAGCAGAGGAAGTCAGAGCATCTTCTTTGGTTTAGCCCAAGTAATGACTTCCTTAAAA


 


AGCTGAAGGAAAATCCAGAGTGACCAGATTATAAACTGTACTCTTGCATTTTCTCTCCCTCCTC


 


TCACCCACAGCCTCTTGATGAACCGGAGGAAGTTTCTTTACCAATTCAAAAATGTCCGCTGGGC


 


TAAGGGTCGGCGTGAGACCTACCTGTGCTACGTAGTGAAGAGGCGTGACAGTGCTACATCCTTT


 


TCACTGGACTTTGGTTATCTTCGCAATAAGGTATCAATTAAAGTCGGCTTTGCAAGCAGTTTAA


 


TGGTCAACTGTGAGTGCTTTTAGAGCCACCTGCTGATGGTATTACTTCCATCCTTTTTTGGCAT


 


TTGTGTCTCTATCACATTCCTCAAATCCTTTTTTTTATTTCTTTTTCCATGTCCATGCACCCAT


 


ATTAGACATGGCCCAAAATATGTGATTTAATTCCTCCCCAGTAATGCTGGGCACCCTAATACCA


 


CTCCTTCCTTCAGTGCCAAGAACAACTGCTCCCAAACTGTTTACCAGCTTTCCTCAGCATCTGA


 


ATTGCCTTTGAGATTAATTAAGCTAAAAGCATTTTTATATGGGAGAATATTATCAGCTTGTCCA


 


AGCAAAAATTTTAAATGTGAAAAACAAATTGTGTCTTAAGCATTTTTGAAAATTAAGGAAGAAG


 


AATTTGGGAAAAAATTAACGGTGGCTCAATTCTGTCTTCCAAATGATTTCTTTTCCCTCCTACT


 


CACATGGGTCGTAGGCCAGTGAATACATTCAACATGGTGATCCCCAGAAAACTCAGAGAAGCCT


 


CGGCTGATGATTAATTAAATTGATCTTTCGGCTACCCGAGAGAATTACATTTCCAAGAGACTTC


 


TTCACCAAAATCCAGATGGGTTTACATAAACTTCTGCCCACGGGTATCTCCTCTCTCCTAACAC


 


GCTGTGACGTCTGGGCTTGGTGGAATCTCAGGGAAGCATCCGTGGGGTGGAAGGTCATCGTCTG


 


GCTCGTTGTTTGATGGTTATATTACCATGCAATTTTCTTTGCCTACATTTGTATTGAATACATC


 


CCAATCTCCTTCCTATTCGGTGACATGACACATTCTATTTCAGAAGGCTTTGATTTTATCAAGC


 


ACTTTCATTTACTTCTCATGGCAGTGCCTATTACTTCTCTTACAATACCCATCTGTCTGCTTTA


 


CCAAAATCTATTTCCCCTTTTCAGATCCTCCCAAATGGTCCTCATAAACTGTCCTGCCTCCACC


 


TAGTGGTCCAGGTATATTTCCACAATGTTACATCAACAGGCACTTCTAGCCATTTTCCTTCTCA


 


AAAGGTGCAAAAAGCAACTTCATAAACACAAATTAAATCTTCGGTGAGGTAGTGTGATGCTGCT


 


TCCTCCCAACTCAGCGCACTTCGTCTTCCTCATTCCACAAAAACCCATAGCCTTCCTTCACTCT


 


GCAGGACTAGTGCTGCCAAGGGTTCAGCTCTACCTACTGGTGTGCTCTTTTGAGCAAGTTGCTT


 


AGCCTCTCTGTAACACAAGGACAATAGCTGCAAGCATCCCCAAAGATCATTGCAGGAGACAATG


 


ACTAAGGCTACCAGAGCCGCAATAAAAGTCAGTGAATTTTAGCGTGGTCCTCTCTGTCTCTCCA


 


GAACGGCTGCCACGTGGAATTGCTCTTCCTCCGCTACATCTCGGACTGGGACCTAGACCCTGGC


 


CGCTGCTACCGCGTCACCTGGTTCACCTCCTGGAGCCCCTGCTACGACTGTGCCCGACATGTGG


 


CCGACTTTCTGCGAGGGAACCCCAACCTCAGTCTGAGGATCTTCACCGCGCGCCTCTACTTCTG


 


TGAGGACCGCAAGGCTGAGCCCGAGGGGCTGCGGCGGCTGCACCGCGCCGGGGTGCAAATAGCC


 


ATCATGACCTTCAAAGGTGCGAAAGGGCCTTCCGCGCAGGCGCAGTGCAGCAGCCCGCATTCGG


 


GATTGCGATGCGGAATGAATGAGTTAGTGGGGAAGCTCGAGGGGAAGAAGTGGGCGGGGATTCT


 


GGTTCACCTCTGGAGCCGAAATTAAAGATTAGAAGCAGAGAAAAGAGTGAATGGCTCAGAGACA


 


AGGCCCCGAGGAAATGAGAAAATGGGGCCAGGGTTGCTTCTTTCCCCTCGATTTGGAACCTGAA


 


CTGTCTTCTACCCCCATATCCCCGCCTTTTTTTCCTTTTTTTTTTTTTGAAGATTATTTTTACT


 


GCTGGAATACTTTTGTAGAAAACCACGAAAGAACTTTCAAAGCCTGGGAAGGGCTGCATGAAAA


 


TTCAGTTCGTCTCTCCAGACAGCTTCGGCGCATCCTTTTGGTAAGGGGCTTCCTCGCTTTTTAA


 


ATTTTCTTTCTTTCTCTACAGTCTTTTTTGGAGTTTCGTATATTTCTTATATTTTCTTATTGTT


 


CAATCACTCTCAGTTTTCATCTGATGAAAACTTTATTTCTCCTCCACATCAGCTTTTTCTTCTG


 


CTGTTTCACCATTCAGAGCCCTCTGCTAAGGTTCCTTTTCCCTCCCTTTTCTTTCTTTTGTTGT


 


TTCACATCTTTAAATTTCTGTCTCTCCCCAGGGTTGCGTTTCCTTCCTGGTCAGAATTCTTTTC


 


TCCTTTTTTTTTTTTTTTTTTTTTTTTTTTAAACAAACAAACAAAAAACCCAAAAAAACTCTTT


 


CCCAATTTACTTTCTTCCAACATGTTACAAAGCCATCCACTCAGTTTAGAAGACTCTCCGGCCC


 


CACCGACCCCCAACCTCGTTTTGAAGCCATTCACTCAATTTGCTTCTCTCTTTCTCTACAGCCC


 


CTGTATGAGGTTGATGACTTACGAGACGCATTTCGTACTTTGGGACTTTGATAGCAACTTCCAG


 


GAATGTCACACACGATGAAATATCTCTGCTGAAGACAGTGGATAAAAAACAGTCCTTCAAGTCT


 


TCTCTGTTTTTATTCTTCAACTCTCACTTTCTTAGAGTTTACAGAAAAAATATTTATATACGAC


 


TCTTTAAAAAGATCTATGTCTTGAAAATAGAGAAGGAACACAGGTCTGGCCAGGGACGTGCTGC


 


AATTGGTGCAGTTTTGAATGCAACATTGTCCCCTACTGGGAATAACAGAACTGCAGGACCTGGG


 


AGCATCCTAAAGTGTCAACGTTTTTCTATGACTTTTAGGTAGGATGAGAGCAGAAGGTAGATCC


 


TAAAAAGCATGGTGAGAGGATCAAATGTTTTTATATCAACATCCTTTATTATTTGATTCATTTG


 


AGTTAACAGTGGTGTTAGTGATAGATTTTTCTATTCTTTTCCCTTGACGTTTACTTTCAAGTAA


 


CACAAACTCTTCCATCAGGCCATGATCTATAGGACCTCCTAATGAGAGTATCTGGGTGATTGTG


 


ACCCCAAACCATCTCTCCAAAGCATTAATATCCAATCATGCGCTGTATGTTTTAATCAGCAGAA


 


GCATGTTTTTATGTTTGTACAAAAGAAGATTGTTATGGGTGGGGATGGAGGTATAGACCATGCA


 


TGGTCACCTTCAAGCTACTTTAATAAAGGATCTTAAAATGGGCAGGAGGACTGTGAACAAGACA


 


CCCTAATAATGGGTTGATGTCTGAAGTAGCAAATCTTCTGGAAACGCAAACTCTTTTAAGGAAG


 


TCCCTAATTTAGAAACACCCACAAACTTCACATATCATAATTAGCAAACAATTGGAAGGAAGTT


 


GCTTGAATGTTGGGGAGAGGAAAATCTATTGGCTCTCGTGGGTCTCTTCATCTCAGAAATGCCA


 


ATCAGGTCAAGGTTTGCTACATTTTGTATGTGTGTGATGCTTCTCCCAAAGGTATATTAACTAT


 


ATAAGAGAGTTGTGACAAAACAGAATGATAAAGCTGCGAACCGTGGCACACGCTCATAGTTCTA


 


GCTGCTTGGGAGGTTGAGGAGGGAGGATGGCTTGAACACAGGTGTTCAAGGCCAGCCTGGGCAA


 


CATAACAAGATCCTGTCTCTCAAAAAAAAAAAAAAAAAAAAGAAAGAGAGAGGGCCGGGCGTGG


 


TGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGCCGGGCGGATCACCTGTGGTCAGGA


 


GTTTGAGACCAGCCTGGCCAACATGGCAAAACCCCGTCTGTACTCAAAATGCAAAAATTAGCCA


 


GGCGTGGTAGCAGGCACCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGCTTGAA


 


CCCAGGAGGTGGAGGTTGCAGTAAGCTGAGATCGTGCCGTTGCACTCCAGCCTGGGCGACAAGA


 


GCAAGACTCTGTCTCAGAAAAAAAAAAAAAAAAGAGAGAGAGAGAGAAAGAGAACAATATTTGG


 


GAGAGAAGGATGGGGAAGCATTGCAAGGAAATTGTGCTTTATCCAACAAAATGTAAGGAGCCAA


 


TAAGGGATCCCTATTTGTCTCTTTTGGTGTCTATTTGTCCCTAACAACTGTCTTTGACAGTGAG


 


AAAAATATTCAGAATAACCATATCCCTGTGCCGTTATTACCTAGCAACCCTTGCAATGAAGATG


 


AGCAGATCCACAGGAAAACTTGAATGCACAACTGTCTTATTTTAATCTTATTGTACATAAGTTT


 


GTAAAAGAGTTAAAAATTGTTACTTCATGTATTCATTTATATTTTATATTATTTTGCGTCTAAT


 


GATTTTTTATTAACATGATTTCCTTTTCTGATATATTGAAATGGAGTCTCAAAGCTTCATAAAT


 


TTATAACTTTAGAAATGATTCTAATAACAACGTATGTAATTGTAACATTGCAGTAATGGTGCTA


 


CGAAGCCATTTCTCTTGATTTTTAGTAAACTTTTATGACAGCAAATTTGCTTCTGGCTCACTTT


 


CAATCAGTTAAATAAATGATAAATAATTTTGGAAGCTGTGAAGATAAAATACCAAATAAAATAA


 


TATAAAAGTGATTTATATGAAGTTAAAATAAAAAATCAGTATGATGGAATAAACTTG






Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) is a family of evolutionarily conserved cytidine deaminases. Members of this family are C-to-U editing enzymes. The N-terminal domain of APOBEC like proteins is the catalytic domain, while the C-terminal domain is a pseudocatalytic domain. More specifically, the catalytic domain is a zinc dependent cytidine deaminase domain and is important for cytidine deamination. APOBEC family members include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D (“APOBEC3E” now refers to this), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and Activation-induced (cytidine) deaminase. Many modified cytidine deaminases are commercially available, including but not limited to SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, VRER-BE3, YE1-BE3, EE-BE3, YE2-BE3, and YEE-BE3, which are available from Addgene (plasmids 85169, 85170, 85171, 85172, 85173, 85174, 85175, 85176, 85177).
Other exemplary deaminases that can be fused to Cas9 according to aspects of this disclosure are provided herein. It should be understood that, in some embodiments, the active domain of the respective sequence can be used, e.g., the domain without a localizing signal (nuclear localization sequence, without nuclear export signal, cytoplasmic localizing signal).
The term “deaminase” or “deaminase domain” as used herein, refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase, catalyzing the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain is a cytosine deaminase, catalyzing the hydrolytic deamination of cytosine to uracil. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenine to hypoxanthine. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine or adenine (A) to inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase catalyzing the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases (e.g., engineered adenosine deaminases, evolved adenosine deaminases) provided herein can be from any organism, such as a bacterium. In some embodiments, the adenosine deaminase is from a bacterium, such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, or Caulobacter crescentus. 
In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to a naturally occurring deaminase.
For example, deaminase domains are described in International PCT Application Nos. PCT/2017/045381 (WO 2018/027078) and PCT/US2016/058344 (WO 2017/070632), each of which is incorporated herein by reference for its entirety. Also, see Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017)), and Rees, H. A., et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 December; 19(12):770-788. doi: 10.1038/s41576-018-0059-1, the entire contents of which are hereby incorporated by reference.
“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.
By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an enzyme-linked immunosorbent assay (ELISA)), biotin, digoxigenin, or haptens.
By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. In one embodiment, the disease is a neoplasia or cancer. In some embodiments, the disease is a hematological cancer. By “hematological cancer” is meant a malignancy of immune system cells. In some embodiments, the hematological cancer is leukemia, myeloma, and/or lymphoma. Lymphomas and Leukemias are examples of “liquid cancers” or cancers present in the blood and are derived from the transformation of either a hematopoietic precursor in the bone marrow or a mature hematopoietic cell in the blood. Leukemias can be lymphoid or myeloid, and acute or chronic. In the case of myelomas, the transformed cell is a fully differentiated plasma cell that may be present as a dispersed collection of malignant cells or as a solid mass in the bone marrow. In the case of lymphomas, a transformed lymphocyte in a secondary lymphoid tissue generates a solid mass. Lymphomas are classified either Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL).
In some embodiments, the hematological cancer is a B cell cancer. In some embodiments, the B cell cancer is a lymphoma or a leukemia. In some cases, the leukemia comprises a pre-leukemia. In some cases, the leukemia is an acute leukemia. Acute leukemias include, for example, an acute myeloid leukemia (AML). Acute leukemias also include, for example, an acute lymphoid leukemia or an acute lymphocytic leukemia (ALL); ALL includes B-lineage ALL; T-lineage ALL; and T-cell acute lymphocytic leukemia (T-ALL).
Nonlimiting examples of diseases include T-cell acute lymphoblastic leukemia (T-ALL), mycosis fungoides (MF), Sézary syndrome (SS), Peripheral T/NK-cell lymphoma, Anaplastic large cell lymphoma ALK+, Primary cutaneous T-cell lymphoma, T-cell large granular lymphocytic leukemia, Angioimmunoblastic T/NK-cell lymphoma, Hepatosplenic T-cell lymphoma, Primary cutaneous CD30+ lymphoproliferative disorders, Extranodal NK/T-cell lymphoma, Adult T-cell leukemia/lymphoma, T-cell prolymphocytic leukemia, Subcutaneous panniculitis-like T-cell lymphoma, Primary cutaneous gamma-delta T-cell lymphoma, Aggressive NK-cell leukemia, and Enteropathy-associated T-cell lymphoma. In some embodiments, the disease is a liquid tumor. In some embodiments, the disease is T-cell acute lymphoblastic leukemia (T-ALL). In some embodiments, the disease is acute myelogenous leukemia (AML).
The term “effective amount,” as used herein, refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. In some embodiments, an effect amount is an amount required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of an active agent(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount. In one embodiment, an effective amount is the amount of a base editor of the invention (e.g., a fusion protein comprising a programable DNA binding protein, a nucleobase editor and gRNA) sufficient to introduce an alteration in a gene of interest in a cell (e.g., a cell in vitro or in vivo). In one embodiment, an effective amount is the amount of a base editor required to achieve a therapeutic effect (e.g., to reduce or control a disease or a symptom or condition thereof). Such therapeutic effect need not be sufficient to alter a gene of interest in all cells of a subject, tissue or organ, but only to alter a gene of interest in about 1%, 5%, 10%, 25%, 50%, 75% or more of the cells present in a subject, tissue or organ.
In some embodiments, an effective amount of a fusion protein provided herein, e.g., of a nucleobase editor comprising a nCas9 domain and a deaminase domain (e.g., adenosine deaminase or cytidine deaminase) refers to the amount of the fusion protein that is sufficient to induce editing of a target site specifically bound and edited by the nucleobase editors described herein. As will be appreciated by the skilled artisan, the effective amount of an agent, e.g., a fusion protein, a nuclease, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide, may vary depending on various factors as, for example, on the desired biological response, e.g., on the specific allele, genome, or target site to be edited, on the cell or tissue being targeted, and/or on the agent being used. In the context of a CAR-T cell, “an effective amount refers” to the quantity of cells necessary to administer to a patient to achieve a therapeutic response.
“Epitope,” as used herein, means an antigenic determinant. An epitope is the part of an antigen molecule that by its structure determines the specific antibody molecule that will recognize and bind it.
By “Fas Cell Surface Death Receptor (FAS) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession No. NP_000034.1 or fragment thereof. An exemplary amino acid sequence is provided below.







>NP_000034.1 tumor necrosis factor receptor super-


family member 6 isoform 1 precursor [Homo sapiens]








1
MLGIWTLLPL VLTSVARLSS KSVNAQVTDI NSKGLELRKT


 



VTTVETQNLE GLHHDGQFCH


 


61
KPCPPGERKA RDCTVNGDEP DCVPCQEGKE YTDKAHFSSK


 



CRRCRLCDEG HGLEVEINCT


 


121
RTQNTKCRCK PNFFCNSTVC EHCDPCTKCE HGIIKECTLT


 



SNTKCKEEGS RSNLGWLCLL


 


181
LLPIPLIVWV KRKEVQKTCR KHRKENQGSH ESPTLNPETV


 



AINLSDVDLS KYITTIAGVM


 


241
TLSQVKGFVR KNGVNEAKID EIKNDNVQDTAEQKVQLLRN


 



WHQLHGKKEA YDTLIKDLKK


 


301
ANLCTLAEKI QTIILKDITS DSENSNFRNE IQSLV






By “Fas Cell Surface Death Receptor (FAS) polynucleotide” is meant a nucleic acid encoding a FAS polypeptide. An exemplary FAS nucleic acid sequence is provided below.







>NM_000043.6 Homo sapiens Fas cell surface death


receptor (FAS), transcript variant 1, mRNA








1
ctcttctccc gcgggttggt ggacccgctc agtacggagt


 



tggggaagct ctttcacttc


 


61
ggaggattgc tcaacaacca tgctgggcat ctggaccctc


 



ctacctctgg ttcttacgtc


 


121
tgttgctaga ttatcgtcca aaagtgttaa tgcccaagtg


 



actgacatca actccaaggg


 


181
attggaattg aggaagactg ttactacagt tgagactcag


 



aacttggaag gcctgcatca


 


241
tgatggccaa ttctgccata agccctgtcc tccaggtgaa


 



aggaaagcta gggactgcac


 


301
agtcaatggg gatgaaccag actgcgtgcc ctgccaagaa


 



gggaaggagt acacagacaa


 


361
agcccatttt tcttccaaat gcagaagatg tagattgtgt


 



gatgaaggac atggcttaga


 


421
agtggaaata aactgcaccc ggacccagaa taccaagtgc


 



agatgtaaac caaacttttt


 


481
ttgtaactct actgtatgtg aacactgtga cccttgcacc


 



aaatgtgaac atggaatcat


 


541
caaggaatgc acactcacca gcaacaccaa gtgcaaagag


 



gaaggatcca gatctaactt


 


601
ggggtggctt tgtcttcttc ttttgccaat tccactaatt


 



gtttgggtga agagaaagga


 


661
agtacagaaa acatgcagaa agcacagaaa ggaaaaccaa


 



ggttctcatg aatctccaac


 


721
tttaaatcct gaaacagtgg caataaattt atctgatgtt


 



gacttgagta aatatatcac


 


781
cactattgct ggagtcatga cactaagtca agttaaaggc


 



tttgttcgaa agaatggtgt


 


841
caatgaagcc aaaatagatg agatcaagaa tgacaatgtc


 



caagacacag cagaacagaa


 


901
agttcaactg cttcgtaatt ggcatcaact tcatggaaag


 



aaagaagcgt atgacacatt


 


961
gattaaagat ctcaaaaaag ccaatctttg tactcttgca


 



gagaaaattc agactatcat


 


1021
cctcaaggac attactagtg actcagaaaa ttcaaacttc


 



agaaatgaaa tccaaagctt


 


1081
ggtctagagt gaaaaacaac aaattcagtt ctgagtatat


 



gcaattagtg tttgaaaaga


 


1141
ttcttaatag ctggctgtaa atactgcttg gttttttact


 



gggtacattt tatcatttat


 


1201
tagcgctgaa gagccaacat atttgtagat ttttaatatc


 



tcatgattct gcctccaagg


 


1261
atgtttaaaa tctagttggg aaaacaaact tcatcaagag


 



taaatgcagt ggcatgctaa


 


1321
gtacccaaat aggagtgtat gcagaggatg aaagattaag


 



attatgctct ggcatctaac


 


1381
atatgattct gtagtatgaa tgtaatcagt gtatgttagt


 



acaaatgtct atccacaggc


 


1441
taaccccact ctatgaatca atagaagaag ctatgacctt


 



ttgctgaaat atcagttact


 


1501
gaacaggcag gccactttgc ctctaaatta cctctgataa


 



ttctagagat tttaccatat


 


1561
ttctaaactt tgtttataac tctgagaaga tcatatttat


 



gtaaagtata tgtatttgag


 


1621
tgcagaattt aaataaggct ctacctcaaa gacctttgca


 



cagtttattg gtgtcatatt


 


1681
atacaatatt tcaattgtga attcacatag aaaacattaa


 



attataatgt ttgactatta


 


1741
tatatgtgta tgcattttac tggctcaaaa ctacctactt


 



ctttctcagg catcaaaagc


 


1801
attttgagca ggagagtatt actagagctt tgccacctct


 



ccatttttgc cttggtgctc


 


1861
atcttaatgg cctaatgcac ccccaaacat ggaaatatca


 



ccaaaaaata cttaatagtc


 


1921
caccaaaagg caagactgcc cttagaaatt ctagcctggt


 



ttggagatac taactgctct


 


1981
cagagaaagt agctttgtga catgtcatga acccatgttt


 



gcaatcaaag atgataaaat


 


2041
agattcttat ttttccccca cccccgaaaa tgttcaataa


 



tgtcccatgt aaaacctgct


 


2101
acaaatggca gcttatacat agcaatggca aaatcatcat


 



ctggatttag gaattgctct


 


2161
tgtcataccc ccaagtttct aagatttaag attctcctta


 



ctactatcct acgtttaaat


 


2221
atctttgaaa gtttgtatta aatgtgaatt ttaagaaata


 



atatttatat ttctgtaaat


 


2281
gtaaactgtg aagatagtta taaactgaag cagatacctg


 



gaaccaccta aagaacttcc


 


2341
atttatggag gatttttttg ccccttgtgt ttggaattat


 



aaaatatagg taaaagtacg


 


2401
taattaaata atgtttttgg tatttctggt tttctctttt


 



ttggtagggg cttgcttttt


 


2461
ggttttgtct tccttttctc taactgatgc taaatataac


 



ttgtctttaa tgcttcttgg


 


2521
atcccttaga aggtacttcc tttttaacct taaccctttt


 



agtagttaaa taattatttc


 


2581
cataggttgc tattgccaag aagacctctt ccaaacagca


 



catgattatt cgtcaaacag


 


2641
tttcgtattc cagatactgg aatgtggata agaaagtata


 



catttcaagg ggtaggtttt


 


2701
attattaaga aagccaaatg aggattttga aatattcttt


 



cctgcatatt atccattcta


 


2761
gctacatgct ggccagtggg ccacctttct tttctgcaat


 



ttaatgctag taatatattc


 


2821
tatttaaccc atgagtccca aagtattagc atttcaacat


 



gtaagcatgt cggtaagata


 


2881
gttgtgcttt gcttagggtt ccctcctgtg ttatggtctg


 



gaaagtgtct ttaggcagaa


 


2941
agtctgagtg atcacagggt tcactcatta atttctcttt


 



tctgagccat catagtctgt


 


3001
gctgtctgct ctccagtttt ctatttctag acagaagtag


 



ggcaagttag gtactagtta


 


3061
ttcttcatgg ccagaagtgc aagttctact ttgcaagaca


 



agattaagtt agagaacacc


 


3121
ctattccact ttggtgaact cagagcaaga actttgagtt


 



cctttgggag gaagacagtg


 


3181
gagaagtctt tgtacttggt gatgtggttt ttttcctcat


 



ggcttcacct agtggcccca


 


3241
agcatgactt ctcccatatc aatgagcaca gccacattcc


 



cgagttgagg tgaccccacg


 


3301
gtccagaatc atcctcattc tggtgaacct ggttctcttt


 



gtggtgggca tactgggtag


 


3361
gagaatcacc caaaggtcac ccatgagctg cagaaaaaaa


 



ggctatttgc agaaggagct


 


3421
cacagatcac attgaaagca ttgcatattc aaacatcttg


 



gtcttcttta ttggcatgcc


 


3481
cacagggtct tctgacctct gattagatca gacacttttt


 



agatattgaa tcatcagttt


 


3541
ctgtacaact atctgaataa ggtatataat caatgaaatt


 



tagaattttt ttctatactt


 


3601
actcctgatt ggtaatttgt ttgggtttag aattctatac


 



aaggccattt gtaattttcc


 


3661
tcagcacttt aaaaatatta aaccatgttt tcttaa






By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
By “fratricide” is meant the killing of immune cells by other immune cells, including self-antigen driven killing of immune cells. In certain embodiments, immune cells of the invention are genetically modified to prevent or reduce expression of antigens recognized by immune cells expressing a chimeric antigen receptor (CAR), thereby preventing or reducing fratricide. In various embodiments, fratricide may occur in vivo (e.g., in a subject) or ex vivo (e.g., in an immune cell preparation).
“Graft versus host disease” (GVHD) refers to a pathological condition where transplanted cells of a donor generate an immune response against cells of the host.
By “guide RNA” or “gRNA” is meant a polynucleotide which can be specific for a target sequence and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpf1). In an embodiment, the guide polynucleotide is a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs), though “gRNA” is used interchangeably to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules. Typically, gRNAs that exist as single RNA species comprise two domains: (1) a domain that shares homology to a target nucleic acid (e.g., and directs binding of a Cas9 complex to the target); and (2) a domain that binds a Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as a tracrRNA, and comprises a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., Science 337:816-821(2012), the entire contents of which is incorporated herein by reference. Other examples of gRNAs (e.g., those including domain 2) can be found in US20160208288, entitled “Switchable Cas9 Nucleases and Uses Thereof,” and U.S. Pat. No. 9,737,604, entitled “Delivery System For Functional Nucleases,” the entire contents of each are hereby incorporated by reference in their entirety. In some embodiments, a gRNA comprises two or more of domains (1) and (2), and may be referred to as an “extended gRNA.” An extended gRNA will bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions, as described herein. The gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to said target site, providing the sequence specificity of the nuclease:RNA complex. As will be appreciated by those skilled in the art, RNA polynucleotide sequences, e.g., gRNA sequences, include the nucleobase uracil (U), a pyrimidine derivative, rather than the nucleobase thymine (T), which is included in DNA polynucleotide sequences. In RNA, uracil base-pairs with adenine and replaces thymine during DNA transcription.
By “heterodimer” is meant a fusion protein comprising two domains, such as a wild type TadA domain and a variant of TadA domain (e.g., TadA*8) or two variant TadA domains (e.g., TadA*7.10 and TadA*8 or two TadA*8 domains).
“Host versus graft disease” (HVGD) refers to a pathological condition where the immune system of a host generates an immune response against transplanted cells of a donor.
“Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
By “immune cell” is meant a cell of the immune system capable of generating an immune response.
By “immune effector cell” is meant a lymphocyte, once activated, capable of effecting an immune response upon a target cell. In some embodiments, immune effector cells are effector T cells. In some embodiments, the effector T cell is a naïve CD8+ T cell, a cytotoxic T cell, a natural killer T (NKT) cell, a natural killer (NK) cell, or a regulatory T (Treg) cell. In some embodiments, immune effector cells are effector NK cells. In some embodiments, the effector T cells are thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. In some embodiments the immune effector cell is a CD4+ CD8+ T cell or a CD4− CD8− T cell. In some embodiments the immune effector cell is a T helper cell. In some embodiments the T helper cell is a T helper 1 (Th1), a T helper 2 (Th2) cell, or a helper T cell expressing CD4 (CD4+ T cell).
By “immune response regulation gene” or “immune response regulator” is meant a gene that encodes a polypeptide that is involved in the regulation of an immune response. An immune response regulation gene may regulate immune response in multiple mechanisms or on different levels. For example, an immune response regulation gene may inhibit or facilitate the activation of an immune cell, e.g. a T cell. An immune response regulation gene may increase or decrease the activation threshold of an immune cell. In some embodiments, the immune response regulation gene positively regulates an immune cell signal transduction pathway. In some embodiments, the immune response regulation gene negatively regulates an immune cell signal transduction pathway. In some embodiments, the immune response regulation gene encodes an antigen, an antibody, a cytokine, or a neuroendocrine.
By “immunogenic gene” is meant a gene that encodes a polypeptide that is able to elicit an immune response. For example, an immunogenic gene may encode an immunogen that elicits an immune response. In some embodiments, an immunogenic gene encodes a cell surface protein. In some embodiments, an immunogenic gene encodes a cell surface antigen or a cell surface marker. In some embodiments, the cell surface marker is a T cell marker or a B cell marker. In some embodiments, an immunogenic gene encodes a CD2, CD3e, CD3 delta, CD3 gamma, TRAC, TRBC1, TRBC2, CD4, CD5, CD7, CD8, CD19, CD23, CD27, CD28, CD30, CD33, CD52, CD70, CD127, CD122, CD130, CD132, CD38, CD69, CD11a, CD58, CD99, CD103, CCR4, CCR5, CCR6, CCR9, CCR10, CXCR3, CXCR4, CLA, CD161, B2M, or CIITA polypeptide.
The term “inhibitor of base repair” or “IBR” refers to a protein that is capable in inhibiting the activity of a nucleic acid repair enzyme, for example a base excision repair (BER) enzyme. In some embodiments, the IBR is an inhibitor of inosine base excision repair. Exemplary inhibitors of base repair include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGGl, hNEILl, T7 Endol, T4PDG, UDG, hSMUGl, and hAAG. In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is a catalytically inactive EndoV or a catalytically inactive hAAG. In some embodiments, the base repair inhibitor is an inhibitor of Endo V or hAAG. In some embodiments, the base repair inhibitor is a catalytically inactive EndoV or a catalytically inactive hAAG.
In some embodiments, the base repair inhibitor is uracil glycosylase inhibitor (UGI). UGI refers to a protein that is capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme. In some embodiments, a UGI domain comprises a wild-type UGI or a fragment of a wild-type UGI. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to a UGI or a UGI fragment. In some embodiments, the base repair inhibitor is an inhibitor of inosine base excision repair. In some embodiments, the base repair inhibitor is a “catalytically inactive inosine specific nuclease” or “dead inosine specific nuclease. Without wishing to be bound by any particular theory, catalytically inactive inosine glycosylases (e.g., alkyl adenine glycosylase (AAG)) can bind inosine, but cannot create an abasic site or remove the inosine, thereby sterically blocking the newly formed inosine moiety from DNA damage/repair mechanisms. In some embodiments, the catalytically inactive inosine specific nuclease can be capable of binding an inosine in a nucleic acid but does not cleave the nucleic acid. Non-limiting exemplary catalytically inactive inosine specific nucleases include catalytically inactive alkyl adenosine glycosylase (AAG nuclease), for example, from a human, and catalytically inactive endonuclease V (EndoV nuclease), for example, from E. coli. In some embodiments, the catalytically inactive AAG nuclease comprises an E125Q mutation or a corresponding mutation in another AAG nuclease.
By “increases” is meant a positive alteration of at least 10%, 25%, 50%, 75%, or 100%.
An “intein” is 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). For example, in cyanobacteria, DnaE, the catalytic subunit a of DNA polymerase III, is encoded by two separate genes, dnaE-n and dnaE-c. The intein encoded by the dnaE-n gene may be herein referred as “intein-N.” The intein encoded by the dnaE-c gene may be herein referred as “intein-C.”
Other intein systems may also be used. For example, 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, has been described (e.g., in Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5, incorporated herein by reference). 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.
Exemplary nucleotide and amino acid sequences of inteins are provided below.







DnaE Intein-N DNA:


TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATGGCCTTCTGCC


 


AATCGGGAAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCTGTCG


 


ATAACAATGGTAACATTTATACTCAGCCAGTTGCCCAGTGGCACGACCGG


 


GGAGAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAG


 


GGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAGATGCTGCCTA


 


TAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGAGTTGACAACCTT


 


CCTAAT


 


DnaE Intein-N Protein:


CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDR


 


GEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNL


 


PN


 


DnaE Intein-C DNA:


ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAAACGTTTATGA


 


TATTGGAGTCGAAAGAGATCACAACTTTGCTCTGAAGAACGGATTCATAG


 


CTTCTAAT


 


Intein-C:


MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN


 


Cfa-N DNA:


TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCC


 


TATTGGAAAGATTGTCGAAGAGAGAATTGAATGCACAGTATATACTGTAG


 


ACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGGCACAATCGC


 


GGCGAACAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACG


 


AGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAGATGTTGCCAA


 


TAGATGAGATATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGGATTG


 


CCA


 


Cfa-N Protein:


CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNR


 


GEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGL


 


P


 


Cfa-C DNA:


ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAGAG


 


GAAAGTAAAGATAATATCTCGAAAAAGTCTTGGTACCCAAAATGTCTATG


 


ATATTGGAGTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGTCTCGTA


 


GCCAGCAAC


 


Cfa-C Protein:


MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLV


 


ASN






Intein-N and intein-C may be fused to the N-terminal portion of the split Cas9 and the C-terminal portion of the 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 known in the art, e.g., as 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 WO2014004336, WO2017132580, US20150344549, and US20180127780, each of which is incorporated herein by reference in their entirety.
The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. By “leader peptide” is meant a short amino acid sequence (e.g., approximately 16-30 amino acids in length) that directs newly synthesized secretory or membrane proteins to and through membranes (e.g., the endoplasmic reticulum membrane). Leader peptides are typically located at the N-terminus of a polypeptide and can be removed by signal peptidases after the polypeptide has crossed the membrane. Leader peptide sequences typically contain three common structural features: N-terminal polar basic region (n-region), a hydrophobic core, and a hydrophilic c-region). In some embodiments, a CAR of the present invention includes a leader peptide sequence (e.g., N-terminal to the antigen binding domain). An exemplary leader peptide amino acid sequence is: METDTLLLWVLLLWVPGSTG (SEQ ID NO: 55).
The term “linker,” as used herein, refers to a bond (e.g., covalent bond), chemical group, or a molecule linking two molecules or moieties, e.g., two components of a protein complex or a ribonucleocomplex, or two domains of a fusion protein, such as, for example, a polynucleotide programmable DNA binding domain (e.g., dCas9) and a deaminase domain (e.g., an adenosine deaminase, a cytidine deaminase). A linker can join different components of, or different portions of components of, a base editor system. For example, in some embodiments, a linker can join a guide polynucleotide binding domain of a polynucleotide programmable nucleotide binding domain and a catalytic domain of a deaminase. In some embodiments, a linker can join a CRISPR polypeptide and a deaminase. In some embodiments, a linker can join a Cas9 and a deaminase. In some embodiments, a linker can join a dCas9 and a deaminase. In some embodiments, a linker can join a nCas9 and a deaminase. In some embodiments, a linker can join a guide polynucleotide and a deaminase. In some embodiments, a linker can join a deaminating component and a polynucleotide programmable nucleotide binding component of a base editor system. In some embodiments, a linker can join an RNA-binding portion of a deaminating component and a polynucleotide programmable nucleotide binding component of a base editor system. In some embodiments, a linker can join an RNA-binding portion of a deaminating component and an RNA-binding portion of a polynucleotide programmable nucleotide binding component of a base editor system. A linker can be positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond or non-covalent interaction, thus connecting the two. In some embodiments, the linker can be an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA linker. In some embodiments, the linker can be an RNA linker. In some embodiments, a linker can comprise an aptamer capable of binding to a ligand. In some embodiments, the ligand may be carbohydrate, a peptide, a protein, or a nucleic acid. In some embodiments, the linker may comprise an aptamer may be derived from a riboswitch. The riboswitch from which the aptamer is derived may be selected from a theophylline riboswitch, a thiamine pyrophosphate (TPP) riboswitch, an adenosine cobalamin (AdoCbl) riboswitch, an S-adenosyl methionine (SAM) riboswitch, an SAH riboswitch, a flavin mononucleotide (FMN) riboswitch, a tetrahydrofolate riboswitch, a lysine riboswitch, a glycine riboswitch, a purine riboswitch, a GlmS riboswitch, or a pre-queosine1 (PreQ1) riboswitch. In some embodiments, a linker may comprise an aptamer bound to a polypeptide or a protein domain, such as a polypeptide ligand. In some embodiments, the polypeptide ligand may be a K Homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif In some embodiments, the polypeptide ligand may be a portion of a base editor system component. For example, a nucleobase editing component may comprise a deaminase domain and an RNA recognition motif.
In some embodiments, the linker can be an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker can be about 5-100 amino acids in length, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acids in length. In some embodiments, the linker can be about 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500 amino acids in length. Longer or shorter linkers can be also contemplated.
In some embodiments, a linker joins a gRNA binding domain of an RNA-programmable nuclease, including a Cas9 nuclease domain, and the catalytic domain of a nucleic-acid editing protein (e.g., cytidine or adenosine deaminase). In some embodiments, a linker joins a dCas9 and a nucleic-acid editing protein. For example, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-200 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 70, 75, 80, 85, 90, 90, 95, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 175, 180, 190, or 200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, a linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 56), which may also be referred to as the XTEN linker. In some embodiments, a linker comprises the amino acid sequence SGGS (SEQ ID NO: 57). In some embodiments, a linker comprises (SGGS)n (SEQ ID NO: 58), (GGGS)n (SEQ ID NO: 59), (GGGGS)n (SEQ ID NO: 60), (G)n (SEQ ID NO: 61), (EAAAK)n (SEQ ID NO: 62), (GGS)n (SEQ ID NO: 63), SGSETPGTSESATPES (SEQ ID NO: 56), or (XP)n (SEQ ID NO: 64) motif, or a combination of any of these, where n is independently an integer between 1 and 30, and where X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, a linker comprises a plurality of proline residues and is 5-21, 5-14, 5-9, 5-7 amino acids in length, e.g., PAPAP (SEQ ID NO: 65), PAPAPA (SEQ ID NO: 66), PAPAPAP (SEQ ID NO: 67), PAPAPAPA (SEQ ID NO: 68), P(AP)4 (SEQ ID NO: 69), P(AP)7 (SEQ ID NO: 70), P(AP)10 (SEQ ID NO: 71). Such proline-rich linkers are also termed “rigid” linkers.
In some embodiments, the chimeric antigen receptor comprises at least one linker. The at least one linker joins, or links, a variable heavy (VH) region to a constant heavy (CH) region of the extracellular binding domain of the chimeric antigen receptor. Linkers can also link a variable light (VL) region to a variable constant (VC) region of the extracellular binding domain.
In some embodiments, the domains of the base editor are fused via a linker that comprises the amino acid sequence of: IDC-59 DNA







SGGSSGSETPGTSESATPESSGGS,


 


SGGSSGGSSGSETPGTSESATPESSGGSSGGS,


or


 


GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP


 


TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGG


 


SGGS.






In some embodiments, domains of the base editor are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 56), which may also be referred to as the XTEN linker. In some embodiments, a linker comprises the amino acid sequence SGGS (SEQ ID NO: 57). In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 75). In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 76).In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 77) SGGS. In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence







PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG


 


TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS.






The term “liquid cancer” as used herein refers to cancer cells that are present in body fluids, such as, for example, blood, lymph, and bone marrow. Liquid cancers include, but are not limited to, leukemias, myelomas, and liquid lymphomas. Liquid cancers as used herein do not include solid tumors, such as sarcomas and carcinomas, or solid lymphomas that do not contain cysts or liquid areas. A “liquid cancer” can be relapsed, refractory, or metastatic. A liquid cancer to be treated with the methods described herein can be, for example, a liquid lymphoma; liquid lymphomas include lymphomas that contain cysts or liquid areas.
By “Lymphocyte-activation gene 3 (LAG-3) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession No. NP_002277.4 or fragment thereof and having immunomodulatory activity. An exemplary amino acid sequence is provided below.







>NP_002277.4 lymphocyte activation gene 3 protein


precursor [Homo sapiens]








1
MWEAQFLGLL FLQPLWVAPV KPLQPGAEVP VVWAQEGAPA


 



QLPCSPTIPL QDLSLLRRAG


 


61
VTWQHQPDSG PPAAAPGHPL APGPHPAAPS SWGPRPRRYT


 



VLSVGPGGLR SGRLPLQPRV


 


121
QLDERGRQRG DFSLWLRPAR RADAGEYRAA VHLRDRALSC


 



RLRLRLGQAS MTASPPGSLR


 


181
ASDWVILNCS FSRPDRPASV HWFRNRGQGR VPVRESPHHH


 



LAESFLFLPQ VSPMDSGPWG


 


241
CILTYRDGFN VSIMYNLTVL GLEPPTPLTV YAGAGSRVGL


 



PCRLPAGVGT RSFLTAKWTP


 


301
PGGGPDLLVTGDNGDFTLRL EDVSQAQAGT YTCHIHLQEQ


 



QLNATVTLAI ITVTPKSFGS


 


361
PGSLGKLLCE VTPVSGQERF VWSSLDTPSQ RSFSGPWLEA


 



QEAQLLSQPW QCQLYQGERL


 


421
LGAAVYFTEL SSPGAQRSGR APGALPAGHL LLFLILGVLS


 



LLLLVTGAFG FHLWRRQWRP


 


481
RRFSALEQGI HPPQAQSKIE ELEQEPEPEP EPEPEPEPEP


 



EPEQL






By “Lymphocyte-activation gene 3 (LAG-3) polynucleotide” is meant a nucleic acid encoding a LAG-3 polypeptide. An exemplary LAG-3 nucleic acid sequence is provided below.







>NM_002286.6 Homo sapiens lymphocyte activating


3 (LAG3), mRNA








1
agagaccagc agaacggcat cccagccacg acggccactt


 



tgctctgtct gctctccgcc


 


61
acggccctgc tctgttccct gggacacccc cgcccccacc


 



tcctcaggct gcctgatctg


 


121
cccagctttc cagctttcct ctggattccg gcctctggtc


 



atccctcccc accctctctc


 


181
caaggccctc tcctggtctc ccttcttcta gaaccccttc


 



ctccacctcc ctctctgcag


 


241
aacttctcct ttacccccca ccccccacca ctgccccctt


 



tccttttctg acctcctttt


 


301
ggagggctca gcgctgccca gaccatagga gagatgtggg


 



aggctcagtt cctgggcttg


 


361
ctgtttctgc agccgctttg ggtggctcca gtgaagcctc


 



tccagccagg ggctgaggtc


 


421
ccggtggtgt gggcccagga gggggctcct gcccagctcc


 



cctgcagccc cacaatcccc


 


481
ctccaggatc tcagccttct gcgaagagca ggggtcactt


 



ggcagcatca gccagacagt


 


541
ggcccgcccg ctgccgcccc cggccatccc ctggcccccg


 



gccctcaccc ggcggcgccc


 


601
tcctcctggg ggcccaggcc ccgccgctac acggtgctga


 



gcgtgggtcc cggaggcctg


 


661
cgcagcggga ggctgcccct gcagccccgc gtccagctgg


 



atgagcgcgg ccggcagcgc


 


721
ggggacttct cgctatggct gcgcccagcc cggagcgcgg


 



acgccggcga gtaccgcgcc


 


781
gcggtgcacc tcagggaccg cgccctctcc tgccgcctcc


 



gtctgcgcct gggccaggcc


 


841
tcgatgactg ccagcccccc aggatctctc agagcctccg


 



actgggtcat tttgaactgc


 


901
tccttcagcc gccctgaccg cccagcctct gtgcattggt


 



tccggaaccg gggccagggc


 


961
cgagtccctg tccgggagtc cccccatcac cacttagcgg


 



aaagcttcct cttcctgccc


 


1021
caagtcagcc ccatggactc tgggccctgg ggctgcatcc


 



tcacctacag agatggcttc


 


1081
aacgtctcca tcatgtataa cctcactgtt ctgggtctgg


 



agcccccaac tcccttgaca


 


1141
gtgtacgctg gagcaggttc cagggtgggg ctgccctgcc


 



gcctgcctgc tggtgtgggg


 


1201
acccggtctt tcctcactgc caagtggact cctcctgggg


 



gaggccctga cctcctgggg


 


1261
actggagaca atggcgactt tacccttcga ctagaggatg


 



tgagccaggc ccaggctggg


 


1321
acctacacct gccatatcca tctgcaggaa cagcagctca


 



atgccactgt cacattggca


 


1381
atcatcacag tgactcccaa atcctttggg tcacctggat


 



ccctggqgaa gctgctttgt


 


1441
gaggtgactc cagtatctgg acaagaacgc tttgtgtgga


 



gctctctgga caccccatcc


 


1501
cagaggagtt tctcaggacc ttggctggag gcacaggagg


 



cccagctcct ttcccagcct


 


1561
tggcaatgcc agctgtacca gggggagagg cttcttggag


 



cagcagtgta cttcacagag


 


1621
ctgtctagcc caggtgccca acgctctggg agagccccag


 



gtgccctccc agcaggccac


 


1681
ctcctgctgt ttctcatcct tggtgtcctt tctctgctcc


 



ttttggtgac tggagccttt


 


1741
ggctttcacc tttggagaag acagtggcga ccaagacgat


 



tttctgcctt agagcaaggg


 


1801
attcaccctc cgcaggctca gagcaagata gaggagctgg


 



agcaagaacc ggagccggag


 


1861
ccggagccgg aaccggagcc cgagcccgag ccagagccgg


 



agcagctctg acctggagct


 


1921
gaggcagcca gcagatctca gcagcccagt ccaaataaac


 



tccctgtcag cagcaa






By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
The term “mutation,” as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). In some embodiments, the presently disclosed base editors can efficiently generate an “intended mutation,” such as a point mutation, in a nucleic acid (e.g., a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations. In some embodiments, an intended mutation is a mutation that is generated by a specific base editor (e.g., cytidine base editor or adenosine base editor) bound to a guide polynucleotide (e.g., gRNA), specifically designed to generate the intended mutation.
In general, mutations made or identified in a sequence (e.g., an amino acid sequence as described herein) are numbered in relation to a reference (or wild-type) sequence, i.e., a sequence that does not contain the mutations. The skilled practitioner in the art would readily understand how to determine the position of mutations in amino acid and nucleic acid sequences relative to a reference sequence.
“Neoplasia” refers to cells or tissues exhibiting abnormal growth or proliferation. The term neoplasia encompasses cancer, liquid and solid tumors. In some embodiments, the neoplasia is a solid tumor. In other embodiments, the neoplasia is a liquid tumor. In some embodiments, the neoplasia is a hematological cancer. In some embodiments, the hematological cancer is leukemia, myeloma, and/or lymphoma. In some embodiments, the hematological cancer is a B cell cancer. In some embodiments, the B cell cancer is a lymphoma or a leukemia. In some cases, the leukemia comprises a pre-leukemia. In some cases, the leukemia is an acute leukemia. Acute leukemias include, for example, an acute myeloid leukemia (AML). Acute leukemias also include, for example, an acute lymphoid leukemia or an acute lymphocytic leukemia (ALL); ALL includes B-lineage ALL; T-lineage ALL; and T-cell acute lymphocytic leukemia (T-ALL).
Nonlimiting examples of neoplasia include T-cell acute lymphoblastic leukemia (T-ALL), mycosis fungoides (MF), Sézary syndrome (SS), Peripheral T/NK-cell lymphoma, Anaplastic large cell lymphoma ALK+, Primary cutaneous T-cell lymphoma, T-cell large granular lymphocytic leukemia, Angioimmunoblastic T/NK-cell lymphoma, Hepatosplenic T-cell lymphoma, Primary cutaneous CD30+ lymphoproliferative disorders, Extranodal NK/T-cell lymphoma, Adult T-cell leukemia/lymphoma, T-cell prolymphocytic leukemia, Subcutaneous panniculitis-like T-cell lymphoma, Primary cutaneous gamma-delta T-cell lymphoma, Aggressive NK-cell leukemia, and Enteropathy-associated T-cell lymphoma. In some embodiments, the neoplasia is T-cell acute lymphoblastic leukemia (T-ALL). In some embodiments, the neoplasia is acute myelogenous leukemia (AML).
By “nuclear factor of activated T cells 1 (NFATc1) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession No. NM_172390.2 or a fragment thereof and is a component of the activated T cell DNA-binding transcription complex. An exemplary amino acid sequence is provided below.







>NP_765978.1 nuclear factor of activated T-cells, 


cytoplasmic 1 isoform A [Homo sapiens]


MPSTSFPVPSKFPLGPAAAVFGRGETLGPAPRAGGTMKSAEEEHYGYASS


 


NVSPALPLPTAHSTLPAPCHNLQTSTPGIIPPADHPSGYGAALDGGPAGY


 


FLSSGHTRPDGAPALESPRIEITSCLGLYHNNNQFFHDVEVEDVLPSSKR


 


SPSTATLSLPSLEAYRDPSCLSPASSLSSRSCNSEASSYESNYSYPYASP


 


QTSPWQSPCVSPKTTDPEEGFPRGLGACTLLGSPRHSPSTSPRASVTEES


 


WIGARSSRPASPCNKRKYSLNGRQPPYSPHHSPTPSPHGSPRVSVTDDSW


 


LGNTTQYTSSAIVAAINALTTDSSLDLGDGVPVKSRKTTLEQPPSVALKV


 


EPVGEDLGSPPPPADFAPEDYSSFQHIRKGGFCDQYLAVPQHPYQWAKPK


 


PLSPTSYMSPTLPALDWQLPSHSGPYELRIEVQPKSHHRAHYETEGSRGA


 


VKASAGGHPIVQLHGYLENEPLMLQLFIGTADDRLLRPHAFYQVHRITGK


 


TVSTTSHEAILSNTKVLEIPLLPENSMRAVIDCAGILKLRNSDIELRKGE


 


TDIGRKNTRVRLVFRVHVPQPSGRTLSLQVASNPIECSQRSAQELPLVEK


 


QSTDSYPVVGGKKMVLSGHNFLQDSKVIFVEKAPDGHHVWEMEAKTDRDL


 


CKPNSLVVEIPPFRNQRITSPVHVSFYVCNGKRKRSQYQRFTYLPANGNA


 


IFLTVSREHERVGCFF






By “nuclear factor of activated T cells 1 (NFATc1) polynucleotide” is meant a nucleic acid molecule encoding a NFATcl polypeptide. The NFATcl gene encodes a protein that is involved in in the inducible expression of cytokine genes, especially IL-2 and IL-4, in T-cells. An exemplary nucleic acid sequenced is provided below.







>NM_172390.2 Homo sapiens nuclear factor of 


activated T cells 1 (NFATC1), transcript 


variant 1, mRNA


GGCGGGCGCTCGGCGACTCGTCCCCGGGGCCCCGCGCGGGCCCGGGCAGC


 


AGGGGCGTGATGTCACGGCAGGGAGGGGGCGCGGGAGCCGCCGGGCCGGC


 


GGGGAGGCGGGGGAGGTGTTTTCCAGCTTTAAAAAGGCAGGAGGCAGAGC


 


GCGGCCCTGCGTCAGAGCGAGACTCAGAGGCTCCGAACTCGCCGGCGGAG


 


TCGCCGCGCCAGATCCCAGCAGCAGGGCGCGGGCACCGGGGCGCGGGCAG


 


GGCTCGGAGCCACCGCGCAGGTCCTAGGGCCGCGGCCGGGCCCCGCCACG


 


CGCGCACACGCCCCTCGATGACTTTCCTCCGGGGCGCGCGGCGCTGAGCC


 


CGGGGCGAGGGCTGTCTTCCCGGAGACCCGACCCCGGCAGCGCGGGGCGG


 


CCGCTTCTCCTGTGCCTCCGCCCGCCGCTCCACTCCCCGCCGCCGCCGCG


 


CGGATGCCAAGCACCAGCTTTCCAGTCCCTTCCAAGTTTCCACTTGGCCC


 


TGCGGCTGCGGTCTTCGGGAGAGGAGAAACTTTGGGGCCCGCGCCGCGCG


 


CCGGCGGCACCATGAAGTCAGCGGAGGAAGAACACTATGGCTATGCATCC


 


TCCAACGTCAGCCCCGCCCTGCCGCTCCCCACGGCGCACTCCACCCTGCC


 


GGCCCCGTGCCACAACCTTCAGACCTCCACACCGGGCATCATCCCGCCGG


 


CGGATCACCCCTCGGGGTACGGAGCAGCTTTGGACGGTGGGCCCGCGGGC


 


TACTTCCTCTCCTCCGGCCACACCAGGCCTGATGGGGCCCCTGCCCTGGA


 


GAGTCCTCGCATCGAGATAACCTCGTGCTTGGGCCTGTACCACAACAATA


 


ACCAGTTTTTCCACGATGTGGAGGTGGAAGACGTCCTCCCTAGCTCCAAA


 


CGGTCCCCCTCCACGGCCACGCTGAGTCTGCCCAGCCTGGAGGCCTACAG


 


AGACCCCTCGTGCCTGAGCCCGGCCAGCAGCCTGTCCTCCCGGAGCTGCA


 


ACTCAGAGGCCTCCTCCTACGAGTCCAACTACTCGTACCCGTACGCGTCC


 


CCCCAGACGTCGCCATGGCAGTCTCCCTGCGTGTCTCCCAAGACCACGGA


 


CCCCGAGGAGGGCTTTCCCCGCGGGCTGGGGGCCTGCACACTGCTGGGTT


 


CCCCGCGGCACTCCCCCTCCACCTCGCCCCGCGCCAGCGTCACTGAGGAG


 


AGCTGGCTGGGTGCCCGCTCCTCCAGACCCGCGTCCCCTTGCAACAAGAG


 


GAAGTACAGCCTCAACGGCCGGCAGCCGCCcTACTCACCCCACCACTCGC


 


CCACGCCGTCCCCGCACGGcTCCCCGCGGGTCAGCGTGACCGACGACTCG


 


TGGTTGGGCAACACCACCCAGTACACCAGCTCGGCCATCGTGGCCGCCAT


 


CAACGCGCTGACCACCGACAGCAGCCTGGACCTGGGAGATGGCGTCCCTG


 


TCAAGTCCCGCAAGACCACCCTGGAGCAGCCGCCCTCAGTGGCGCTCAAG


 


GTGGAGCCCGTCGGGGAGGACCTGGGCAGCCCCCCGCCCCCGGCCGACTT


 


CGCGCCCGAAGACTACTCCTCTTTCCAGCACATCAGGAAGGGCGGCTTCT


 


GCGACCAGTACCTGGCGGTGCCGCAGCACCCCTACCAGTGGGCGAAGCCC


 


AAGCCCCTGTCCCCTACGTCCTACATGAGCCCGACCCTGCCCGCCCTGGA


 


CTGGCAGCTGCCGTCCCACTCAGGCCCGTATGAGCTTCGGATTGAGGTGC


 


AGCCCAAGTCCCACCACCGAGCCCACTACGAGACGGAGGGCAGCCGGGGG


 


GCCGTGAAGGCGTCGGCCGGAGGACACCCCATCGTGCAGCTGCATGGCTA


 


CTTGGAGAATGAGCCGCTGATGCTGCAGCTTTTCATTGGGACGGCGGACG


 


ACCGCCTGCTGCGCCCGCACGCCTTCTACCAGGTGCACCGCATCACAGGG


 


AAGACCGTGTCCACCACCAGCCACGAGGCCATCCTCTCCAACACCAAAGT


 


CCTGGAGATCCCACTCCTGCCGGAGAACAGCATGCGAGCCGTCATTGACT


 


GTGCCGGAATCCTGAAACTCAGAAACTCCGACATTGAACTTCGGAAAGGA


 


GAGACGGACATCGGGAGGAAGAACACACGGGTACGGCTGGTGTTCCGCGT


 


TCACGTCCCGCAACCCAGCGGCCGCACGCTGTCCCTGCAGGTGGCCTCCA


 


ACCCCATCGAATGCTCCCAGCGCTCAGCTCAGGAGCTGCCTCTGGTGGAG


 


AAGCAGAGCACGGACAGCTATCCGGTCGTGGGCGGGAAGAAGATGGTCCT


 


GTCTGGCCACAACTTCCTGCAGGACTCCAAGGTCATTTTCGTGGAGAAAG


 


CCCCAGATGGCCACCATGTCTGGGAGATGGAAGCGAAAACTGACCGGGAC


 


CTGTGCAAGCCGAATTCTCTGGTGGTTGAGATCCCGCCATTTCGGAATCA


 


GAGGATAACCAGCCCCGTTCACGTCAGTTTCTACGTCTGCAACGGGAAGA


 


GAAAGCGAAGCCAGTACCAGCGTTTCACCTACCTTCCCGCCAACGGTAAC


 


GCCATCTTTCTAACCGTAAGCCGTGAACATGAGCGCGTGGGGTGCTTTTT


 


CTAAAGACGCAGAAACGACGTCGCCGTAAAGCAGCGTGGCGTGTTGCACA


 


TTTAACTGTGTGATGTCCCGTTAGTGAGACCGAGCCATCGATGCCCTGAA


 


AAGGAAAGGAAAAGGGAAGCTTCGGATGCATTTTCCTTGATCCCTGTTGG


 


GGGTGGGGGGCGGGGGTTGCATACTCAGATAGTCACGGTTATTTTGCTTC


 


TTGCGAATGTATAACAGCCAAGGGGAAAACATGGCTCTTCTGCTCCAAAA


 


AACTGAGGGGGTCCTGGTGTGCATTTGCACCCTAAAGCTGCTTACGGTGA


 


AAAGGCAAATAGGTATAGCTATTTTGCAGGCACCTTTAGGAATAAACTTT


 


GCTTTTAAGCCTGTAAAAAAAAAAAAAA






The term “non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with, or inhibit the biological activity of, the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the wild-type protein.
The term “nuclear localization sequence,” “nuclear localization signal,” or “NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus. Nuclear localization sequences are known in the art and described, for example, in Plank et al., International PCT application, PCT/EP2000/011690, filed Nov. 23, 2000, published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS described, for example, by Koblan et al., Nature Biotech. 2018 doi:10.1038/nbt.4172. In some embodiments, an NLS comprises the amino acid sequence









PKKKRKVEGADKRTADGSEFESPKKKRKV,



 



KRTADGSEFESPKKKRKV,



 



KRPAATKKAGQAKKKK, 



 



KKTELQTTNAENKTKKL,



 



KRGINDRNFWRGENGRKTR,



 



RKSGKIAAIVVKRPRK, 



 



PKKKRKV,



or



 



MDSLLMNRRKFLYQFKNVRWAKGRRETYLC.






The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g. nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5′ to 3′ direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages). The term “nucleic acid programmable DNA binding protein” or “napDNAbp” may be used interchangeably with “polynucleotide programmable nucleotide binding domain” to refer to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA), that guides the napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 protein. A Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, for example a nuclease active Cas9, a Cas9 nickase (nCas9), or a nuclease inactive Cas9 (dCas9). Non-limiting examples of nucleic acid programmable DNA binding proteins include, Cas9 (e.g., dCas9 and nCas9), Cas12a/Cpf1, Cas12b/C2cl, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i, and Cas12j/CasΦ. Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a/Cpf1, Cas12b/C2cl, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i, Cas12j/CasΦ, 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, homologues thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, although they may not be specifically listed in this disclosure. See, e.g., Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 October; 1:325-336. doi: 10.1089/crispr.2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan. 4; 363(6422):88-91. doi: 10.1126/science.aav7271, the entire contents of each are hereby incorporated by reference.
The term “nucleobase,” “nitrogenous base,” or “base,” used interchangeably herein, refers to a nitrogen-containing biological compound that forms a nucleoside, which in turn is a component of a nucleotide. The ability of nucleobases to form base pairs and to stack one upon another leads directly to long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Five nucleobases—adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U)—are called primary or canonical. Adenine and guanine are derived from purine, and cytosine, uracil, and thymine are derived from pyrimidine. DNA and RNA can also contain other (non-primary) bases that are modified. Non-limiting exemplary modified nucleobases can include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydromethylcytosine. Hypoxanthine and xanthine can be created through mutagen presence, both of them through deamination (replacement of the amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can result from deamination of cytosine. A “nucleoside” consists of a nucleobase and a five carbon sugar (either ribose or deoxyribose). Examples of a nucleoside include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of a nucleoside with a modified nucleobase includes inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). A “nucleotide” consists of a nucleobase, a five carbon sugar (either ribose or deoxyribose), and at least one phosphate group.
The terms “nucleobase editing domain” or “nucleobase editing protein,” as used herein, refers to a protein or enzyme that can catalyze a nucleobase modification in RNA or DNA, such as cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine), and adenine (or adenosine) to hypoxanthine (or inosine) deaminations, as well as non-templated nucleotide additions and insertions. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., an adenine deaminase or an adenosine deaminase; or a cytidine deaminase or a cytosine deaminase). In some embodiments, the nucleobase editing domain is more than one deaminase domain (e.g., an adenine deaminase or an adenosine deaminase and a cytidine or a cytosine deaminase). In some embodiments, the nucleobase editing domain can be a naturally occurring nucleobase editing domain. In some embodiments, the nucleobase editing domain can be an engineered or evolved nucleobase editing domain from the naturally occurring nucleobase editing domain. The nucleobase editing domain can be from any organism, such as a bacterium, human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse.
As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent. A “patient” or “subject” as used herein refers to a mammalian subject or individual diagnosed with, at risk of having or developing, or suspected of having or developing a disease or a disorder. In some embodiments, the term “patient” refers to a mammalian subject with a higher than average likelihood of developing a disease or a disorder. Exemplary patients can be humans, non-human primates, cats, dogs, pigs, cattle, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs) and other mammalians that can benefit from the therapies disclosed herein. Exemplary human patients can be male and/or female.
“Patient in need thereof” or “subject in need thereof” is referred to herein as a patient diagnosed with, at risk or having, predetermined to have, or suspected of having a disease or disorder (e.g., T- or NK-cell malignancy).
The terms “pathogenic mutation,” “pathogenic variant,” “disease casing mutation,” “disease causing variant,” “deleterious mutation,” or “predisposing mutation” refers to a genetic alteration or mutation that increases an individual's susceptibility or predisposition to a certain disease or disorder. In some embodiments, the pathogenic mutation comprises at least one wild-type amino acid substituted by at least one pathogenic amino acid in a protein encoded by a gene.
The term “pharmaceutically-acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g. the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.). The terms such as “excipient,” “carrier,” “pharmaceutically acceptable carrier,” “vehicle,” or the like are used interchangeably herein.
The term “pharmaceutical composition” means a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g., for specific delivery, increasing half-life, or other therapeutic compounds).
By “Programmed cell death 1 (PDCD1 or PD-1) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession No. AJS10360.1 or a fragment thereof. The PD-1 protein is thought to be involved in T cell function regulation during immune reactions and in tolerance conditions. An exemplary B2M polypeptide sequence is provided below. IDC-66 DNA







>AJS10360.1 programmed cell death 1 protein 


[Homo sapiens]


MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNA


 


TFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQL


 


PNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAE


 


VPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTI


 


GARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYAT


 


IVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL






By “Programmed cell death 1 (PDCD1 or PD-1) polynucleotide” is meant a nucleic acid molecule encoding a PD-1 polypeptide. The PDCD1 gene encodes an inhibitory cell surface receptor that inhibits T-cell effector functions in an antigen-specific manner. An exemplary PDCD1 nucleic acid sequence is provided below.







>AY238517.1 Homo sapiens programmed cell death


1 (PDCD1) mRNA, complete cds


ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACT


 


GGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACC


 


CCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACCCC


 


ACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTG


 


GTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCG


 


AGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTG


 


CCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGA


 


CAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGA


 


TCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAA


 


GTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCA


 


AACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGC


 


TAGTCTGGGTCCTGGCCGTCATCTGCTCCCGGGCCGCACGAGGGACAATA


 


GGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCCTCAGCCGTGCC


 


TGTGTTCTCTGTGGACTATGGGGAGCTGGATTTCCAGTGGCGAGAGAAGA


 


CCCCGGAGCCCCCCGTGCCCTGTGTCCCTGAGCAGACGGAGTATGCCACC


 


ATTGTCTTTCCTAGCGGAATGGGCACCTCATCCCCCGCCCGCAGGGGCTC


 


AGCTGACGGCCCTCGGAGTGCCCAGCCACTGAGGCCTGAGGATGGACACT


 


GCTCTTGGCCCCTCTGA






The terms “protein,” “peptide,” “polypeptide,” and their grammatical equivalents are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide can refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide can be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modifications, etc. A protein, peptide, or polypeptide can also be a single molecule or can be a multi-molecular complex. A protein, peptide, or polypeptide can be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide can be naturally occurring, recombinant, or synthetic, or any combination thereof. The term “fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein can be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an amino-terminal fusion protein or a carboxy-terminal fusion protein, respectively. A protein can comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain, or a catalytic domain of a nucleic acid editing protein. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent. In some embodiments, a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA or DNA. Any of the proteins provided herein can be produced by any method known in the art. For example, the proteins provided herein can be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.
Polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) can comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine, N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptane carboxylic acid, a-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. The polypeptides and proteins can be associated with post-translational modifications of one or more amino acids of the polypeptide constructs. Non-limiting examples of post-translational modifications include phosphorylation, acylation including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation including methylation and ethylation, ubiquitylation, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation and iodination.
By “promoter” is meant an array of nucleic acid control sequences, which direct transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor sequence elements. A “constitutive promoter” is a promoter that is continuously active and is not subject to regulation by external signals or molecules. In contrast, the activity of an “inducible promoter” is regulated by an external signal or molecule (for example, a transcription factor). By way of example, a promoter may be a CMV promoter.
The term “recombinant” as used herein in the context of proteins or nucleic acids refers to proteins or nucleic acids that do not occur in nature, but are the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations as compared to any naturally occurring sequence.
By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
By “reference” is meant a standard or control condition. In one embodiment, the reference is a wild-type or healthy cell. In other embodiments and without limitation, a reference is an untreated cell that is not subjected to a test condition, or is subjected to placebo or normal saline, medium, buffer, and/or a control vector that does not harbor a polynucleotide of interest.
A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, and about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween. In some embodiments, a reference sequence is a wild-type sequence of a protein of interest. In other embodiments, a reference sequence is a polynucleotide sequence encoding a wild-type protein.
The term “RNA-programmable nuclease,” and “RNA-guided nuclease” are used with (e.g., binds or associates with) one or more RNA(s) that is not a target for cleavage. In some embodiments, an RNA-programmable nuclease, when in a complex with an RNA, may be referred to as a nuclease:RNA complex. Typically, the bound RNA(s) is referred to as a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs), though “gRNA” is used interchangeably to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules. Typically, gRNAs that exist as single RNA species comprise two domains: (1) a domain that shares homology to a target nucleic acid (e.g., and directs binding of a Cas9 complex to the target); and (2) a domain that binds a Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as a tracrRNA, and comprises a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., Science 337:816-821(2012), the entire contents of which is incorporated herein by reference. Other examples of gRNAs (e.g., those including domain 2) can be found in U.S. Provisional Patent Application No. 61/874,682, filed Sep. 6, 2013, entitled “Switchable Cas9 Nucleases and Uses Thereof,” and U.S. Provisional Patent Application, No. 61/874,746, filed Sep. 6, 2013, entitled “Delivery System For Functional Nucleases,” the entire contents of each are hereby incorporated by reference in their entirety. In some embodiments, a gRNA comprises two or more of domains (1) and (2), and may be referred to as an “extended gRNA.” For example, an extended gRNA will, e.g., bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions, as described herein. The gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to said target site, providing the sequence specificity of the nuclease:RNA complex.
In some embodiments, the RNA-programmable nuclease is the (CRISPR-associated system) Cas9 endonuclease, for example, Cas9 (Csnl) from Streptococcus pyogenes (see, e.g., “Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti J. J., et al., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., et al., Nature 471:602-607(2011).Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to target DNA cleavage sites, these proteins are able to be targeted, in principle, to any sequence specified by the guide RNA. Methods of using RNA-programmable nucleases, such as Cas9, for site-specific cleavage (e.g., to modify a genome) are known in the art (see e.g., Cong, L. et al., Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 (2013); Mali, P. et al., RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, W. Y. et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al., RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, J. E. et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013); the entire contents of each of which are incorporated herein by reference).
By “signaling domain” is meant an intracellular portion of a protein expressed in a T cell that transduces a T cell effector function signal (e.g., an activation signal) and directs the T cell to perform a specialized function. T cell activation can be induced by a number of factors, including binding of cognate antigen to the T cell receptor on the surface of T cells and binding of cognate ligand to costimulatory molecules on the surface of the T cell. A T cell co-stimulatory molecule is a cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule. In some embodiments, the co-stimulatory domain is a CD2 cytoplasmic domain. Activation of a T cell leads to immune response, Such as T cell proliferation and differentiation (see, e.g., Smith-Garvin et al., Annu. Rev. Immunol., 27:591-619, 2009). Exemplary T cell signaling domains are known in the art. Non-limiting examples include the CD2, CD3ζ, CD8, CD28, CD27, CD154, GITR (TNFRSF18), CD134 (OX40), and CD137 (4-1BB) signaling domains.
By “single-chain antibody” or “scFv” is meant a genetically engineered molecule containing the VH and VL domains of one or more antibodies linked by a suitable polypeptide linker as a genetically fused single chain molecule (see, e.g., Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988: Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155/2012/980250: Marbry, IDrugs, 13:543-549, 2010). In some embodiments, the intramolecular orientation of the VH-domain and the VL-domain in an scFv is VH-domain-linker domain-VL-domain. In some embodiments, the intramolecular orientation of the VH-domain and the VL-domain in an scFv is VL-domain-linker domain-VH-domain.
The term “single nucleotide polymorphism (SNP)” is a variation in a single nucleotide that occurs at a specific position in the genome, where each variation is present to some appreciable degree within a population (e.g., >1%). For example, at a specific base position in the human genome, the C nucleotide can appear in most individuals, but in a minority of individuals, the position is occupied by an A. This means that there is a SNP at this specific position, and the two possible nucleotide variations, C or A, are said to be alleles for this position. SNPs underlie differences in susceptibility to disease. The severity of illness and the way our body responds to treatments are also manifestations of genetic variations. SNPs can fall within coding regions of genes, non-coding regions of genes, or in the intergenic regions (regions between genes). In some embodiments, SNPs within a coding sequence do not necessarily change the amino acid sequence of the protein that is produced, due to degeneracy of the genetic code. SNPs in the coding region are of two types: synonymous and nonsynonymous SNPs. Synonymous SNPs do not affect the protein sequence, while nonsynonymous SNPs change the amino acid sequence of protein. The nonsynonymous SNPs are of two types: missense and nonsense. SNPs that are not in protein-coding regions can still affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of noncoding RNA. Gene expression affected by this type of SNP is referred to as an eSNP (expression SNP) and can be upstream or downstream from the gene. A single nucleotide variant (SNV) is a variation in a single nucleotide without any limitations of frequency and can arise in somatic cells. A somatic single nucleotide variation (e.g., associated with cancer) can also be called a single-nucleotide alteration.
By “specifically binds” is meant a nucleic acid molecule, polypeptide, or complex thereof (e.g., a nucleic acid programmable DNA binding protein, a guide nucleic acid, and a chimeric antigen receptor), compound, or molecule that recognizes and binds a polypeptide and/or nucleic acid molecule of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample. For example, a chimeric antigen receptor specifically binds to a particular marker expressed on the surface of a cell, but does not bind to other polypeptides, carbohydrates, lipids, or any other compound on the surface of the cell.
Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 370 C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a one: embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In another embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be apparent to those skilled in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., more preferably of at least about 42° C., and even more preferably of at least about 68° C. In an embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
By “split” is meant divided into two or more fragments.
A “split Cas9 protein” or “split Cas9” refers to 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 particular 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. PDB file: 5F9R, each of which is incorporated herein by reference. In some embodiments, the protein is divided into two fragments at any C, T, A, or S within a region of SpCas9 between about 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 other embodiments, the N-terminal portion of the Cas9 protein comprises amino acids 1-573 or 1-637 S. pyogenes Cas9 wild-type (SpCas9) (NCBI Reference Sequence: NC_002737.2, Uniprot Reference Sequence: Q99ZW2) and the C-terminal portion of the Cas9 protein comprises a portion of amino acids 574-1368 or 638-1368 of SpCas9 wild-type, or a corresponding position thereof.
The C-terminal portion of the split Cas9 can be joined with the N-terminal portion of the split Cas9 to form a complete Cas9 protein. In some embodiments, the C-terminal portion of the Cas9 protein starts from where the N-terminal portion of the Cas9 protein ends. As such, in some embodiments, the C-terminal portion of the split Cas9 comprises a portion of amino acids (551-651)-1368 of spCas9. “(551-651)-1368” means starting at an amino acid between amino acids 551-651 (inclusive) and ending at amino acid 1368. For example, the C-terminal portion of the split Cas9 may comprise a portion of any one of amino acid 551-1368, 552-1368, 553-1368, 554-1368, 555-1368, 556-1368, 557-1368, 558-1368, 559-1368, 560-1368, 561-1368, 562-1368, 563-1368, 564-1368, 565-1368, 566-1368, 567-1368, 568-1368, 569-1368, 570-1368, 571-1368, 572-1368, 573-1368, 574-1368, 575-1368, 576-1368, 577-1368, 578-1368, 579-1368, 580-1368, 581-1368, 582-1368, 583-1368, 584-1368, 585-1368, 586-1368, 587-1368, 588-1368, 589-1368, 590-1368, 591-1368, 592-1368, 593-1368, 594-1368, 595-1368, 596-1368, 597-1368, 598-1368, 599-1368, 600-1368, 601-1368, 602-1368, 603-1368, 604-1368, 605-1368, 606-1368, 607-1368, 608-1368, 609-1368, 610-1368, 611-1368, 612-1368, 613-1368, 614-1368, 615-1368, 616-1368, 617-1368, 618-1368, 619-1368, 620-1368, 621-1368, 622-1368, 623-1368, 624-1368, 625-1368, 626-1368, 627-1368, 628-1368, 629-1368, 630-1368, 631-1368, 632-1368, 633-1368, 634-1368, 635-1368, 636-1368, 637-1368, 638-1368, 639-1368, 640-1368, 641-1368, 642-1368, 643-1368, 644-1368, 645-1368, 646-1368, 647-1368, 648-1368, 649-1368, 650-1368, or 651-1368 of spCas9. In some embodiments, the C-terminal portion of the split Cas9 protein comprises a portion of amino acids 574-1368 or 638-1368 of SpCas9.
By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline. Subjects include livestock, domesticated animals raised to produce labor and to provide commodities, such as food, including without limitation, cattle, goats, chickens, horses, pigs, rabbits, and sheep.
By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). In one embodiment, such a sequence is at least 60%, 80% or 85%, 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, COBALT, EMBOSS Needle, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e−3 and e−100 indicating a closely related sequence.
COBALT is used, for example, with the following parameters:

    
    
        a) alignment parameters: Gap penalties—11, −1 and End-Gap penalties—5, −1,
        b) CDD Parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conserved columns and Recompute on, and
        c) Query Clustering Parameters: Use query clusters on; Word Size 4; Max cluster distance 0.8; Alphabet Regular.
    
    


EMBOSS Needle is used, for example, with the following parameters:

    
    
        a) Matrix: BLOSUM62;
        b) GAP OPEN: 10;
        c) GAP EXTEND: 0.5;
        d) OUTPUT FORMAT: pair;
        e) END GAP PENALTY: false;
        f) END GAP OPEN: 10; and
        g) END GAP EXTEND: 0.5.
    
    


The term “target site” refers to a sequence within a nucleic acid molecule that is modified by a nucleobase editor. In one embodiment, the target site is deaminated by a deaminase or a fusion protein comprising a deaminase (e.g., cytidine or adenine deaminase).
By “T Cell Receptor Alpha Constant (TRAC) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession No. P01848.2 or fragment thereof and having immunomodulatory activity. An exemplary amino acid sequence is provided below.







>sp|P01848.2|TRAC_HUMAN RecName: Full = T cell 


receptor alpha constant


IQNPDPAVYQLRDSKSSDKSVCLETDFDSQTNVSQSKDSDVYITDKTVLD


 


MRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVE


 


KSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS






By “T Cell Receptor Alpha Constant (TRAC) polynucleotide” is meant a nucleic acid encoding a TRAC polypeptide. Exemplary TRAC nucleic acid sequences are provided below.








UCSC human genome database. Gene ENSG00000277734.8 Human T-cell 



receptor alpha chain (TCR-alpha)


catgctaatcctccggcaaacctctgtttcctcctcaaaaggcaggaggtcggaaagaataaac


 


aatgagagtcacattaaaaacacaaaatcctacggaaatactgaagaatgagtctcagcactaa


 


ggaaaagcctccagcagctcctgctttctgagggtgaaggatagacgctgtggctctgcatgac


 


tcactagcactctatcacggccatattctggcagggtcagtggctccaactaacatttgtttgg


 


tactttacagtttattaaatagatgtttatatggagaagctctcatttctttctcagaagagcc


 


tggctaggaaggtggatgaggcaccatattcattttgcaggtgaaattcctgagatgtaaggag


 


ctgctgtgacttgctcaaggccttatatcgagtaaacggtagtgctggggcttagacgcaggtg


 


ttctgatttatagttcaaaacctctatcaatgagagagcaatctcctggtaatgtgatagattt


 


cccaacttaatgccaacataccataaacctcccattctgctaatgcccagcctaagttggggag


 


accactccagattccaagatgtacagtttgctttgctgggcctttttcccatgcctgcctttac


 


tctgccagagttatattgctggggttttgaagaagatcctattaaataaaagaataagcagtat


 


tattaagtagccctgcatttcaggtttccttgagtggcaggccaggcctggccgtgaacgttca


 


ctgaaatcatggcctcttggccaagattgatagcttgtgcctgtccctgagtcccagtccatca


 


cgagcagctggtttctaagatgctatttcccgtataaagcatgagaccgtgacttgccagcccc


 


acagagccccgcccttgtccatcactggcatctggactccagcctgggttggggcaaagaggga


 


aatgagatcatgtcctaaccctgatcctcttgtcccacagATATCCAGAACCCTGACCCTGCCG


 


TGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTC


 


TCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGAC


 


ATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCAT


 


GTGCAAAGGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGgtaaggg


 


cagctttggtgccttcgcaggctgtttccttgcttcaggaatggccaggttctgcccagagctc


 


tggtcaatgatgtctaaaactcctctgattggtggtctcggccttatccattgccaccaaaacc


 


ctctttttactaagaaacagtgagccttgttctggcagtccagagaatgacacgggaaaaaagc


 


agatgaagagaaggtggcaggagagggcacgtggcccagcctcagtctctccaactgagttcct


 


gcctgcctgcctttgctcagactgtttgccccttactgctcttctaggcctcattctaagcccc


 


ttctccaagttgcctctccttatttctccctgtctgccaaaaaatctttcccagctcactaagt


 


cagtctcacgcagtcactcattaacccaccaatcactgattgtgccggcacatgaatgcaccag


 


gtgttgaagtggaggaattaaaaagtcagatgaggggtgtgcccagaggaagcaccattctagt


 


tgggggagcccatctgtcagctgggaaaagtccaaataacttcagattggaatgtgttttaact


 


cagggttgagaaaacagctaccttcaggacaaaagtcagggaagggctctctgaagaaatgcta


 


cttgaagataccagccctaccaagggcagggagaggaccctatagaggcctgggacaggagctc


 


aatgagaaaggagaagagcagcaggcatgagttgaatgaaggaggcagggccgggtcacagggc


 


cttctaggccatgagagggtagacagtattctaaggacgccagaaagctgttgatcggcttcaa


 


gcaggggagggacacctaatttgcttttcttttttttttttttttttttttttttttttgagat


 


ggagttttgctcttgttgcccaggctggagtgcaatggtgcatcttggctcactgcaacctccg


 


cctcccaggttcaagtgattctcctgcctcagcctcccgagtagctgagattacaggcacccgc


 


caccatgcctggctaattttttgtatttttagtagagacagggtttcactatgttggccaggct


 


ggtctcgaactcctgacctcaggtgatccacccgcttcagcctcccaaagtgctgggattacag


 


gcgtgagccaccacacccggcctgcttttcttaaagatcaatctgagtgctgtacggagagtgg


 


gttgtaagccaagagtagaagcagaaagggagcagttgcagcagagagatgatggaggcctggg


 


cagggtggtggcagggaggtaaccaacaccattcaggtttcaaaggtagaaccatgcagggatg


 


agaaagcaaagaggggatcaaggaaggcagctggattttggcctgagcagctgagtcaatgata


 


gtgccgtttactaagaagaaaccaaggaaaaaatttggggtgcagggatcaaaactttttggaa


 


catatgaaagtacgtgtttatactctttatggcccttgtcactatgtatgcctcgctgcctcca


 


ttggactctagaatgaagccaggcaagagcagggtctatgtgtgatggcacatgtggccagggt


 


catgcaacatgtactttgtacaaacagtgtatattgagtaaatagaaatggtgtccaggagccg


 


aggtatcggtcctgccagggccaggggctctccctagcaggtgctcatatgctgtaagttccct


 


ccagatctctccacaaggaggcatggaaaggctgtagttgttcacctgcccaagaactaggagg


 


tctggggtgggagagtcagcctgctctggatgctgaaagaatgtctgtttttccttttagAAAG


 


TTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGgtaagacaggggtctagcctggg


 


tttgcacaggattgcggaagtgatgaacccgcaataaccctgcctggatgagggagtgggaaga


 


aattagtagatgtgggaatgaatgatgaggaatggaaacagcggttcaagacctgcccagagct


 


gggtggggtctctcctgaatccctctcaccatctctgactttccattctaagcactttgaggat


 


gagtttctagcttcaatagaccaaggactctctcctaggcctctgtattcctttcaacagctcc


 


actgtcaagagagccagagagagcttctgggtggcccagctgtgaaatttctgagtcccttagg


 


gatagccctaaacgaaccagatcatcctgaggacagccaagaggttttgccttctttcaagaca


 


agcaacagtactcacataggctgtgggcaatggtcctgtctctcaagaatcccctgccactcct


 


cacacccaccctgggcccatattcatttccatttgagttgttcttattgagtcatccttcctgt


 


ggtagcggaactcactaaggggcccatctggacccgaggtattgtgatgataaattctgagcac


 


ctaccccatccccagaagggctcagaaataaaataagagccaagtctagtcggtgtttcctgtc


 


ttgaaacacaatactgttggccctggaagaatgcacagaatctgtttgtaaggggatatgcaca


 


gaagctgcaagggacaggaggtgcaggagctgcaggcctcccccacccagcctgctctgccttg


 


gggaaaaccgtgggtgtgtcctgcaggccatgcaggcctgggacatgcaagcccataaccgctg


 


tggcctcttggttttacagATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAA


 


TCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGAGg


 


tgaggggccttgaagctgggagtggggtttagggacgcgggtctctgggtgcatcctaagctct


 


gagagcaaacctccctgcagggtcttgcttttaagtccaaagcctgagcccaccaaactctcct


 


acttcttcctgttacaaattcctcttgtgcaataataatggcctgaaacgctgtaaaatatcct


 


catttcagccgcctcagttgcacttctcccctatgaggtaggaagaacagttgtttagaaacga


 


agaaactgaggccccacagctaatgagtggaggaagagagacacttgtgtacaccacatgcctt


 


gtgttgtacttctctcaccgtgtaacctcctcatgtcctctctccccagtacggctctcttagc


 


tcagtagaaagaagacattacactcatattacaccccaatcctggctagagtctccgcaccctc


 


ctcccccagggtccccagtcgtcttgctgacaactgcatcctgttccatcaccatcaaaaaaaa


 


actccaggctgggtgcgggggctcacacctgtaatcccagcactttgggaggcagaggcaggag


 


gagcacaggagctggagaccagcctgggcaacacagggagaccccgcctctacaaaaagtgaaa


 


aaattaaccaggtgtggtgctgcacacctgtagtcccagctacttaagaggctgagatgggagg


 


atcgcttgagccctggaatgttgaggctacaatgagctgtgattgcgtcactgcactccagcct


 


ggaagacaaagcaagatcctgtctcaaataataaaaaaaataagaactccagggtacatttgct


 


cctagaactctaccacatagccccaaacagagccatcaccatcacatccctaacagtcctgggt


 


cttcctcagtgtccagcctgacttctgttcttcctcattccagATCTGCAAGATTGTAAGACAG


 


CCTGTGCTCCCTCGCTCCTTCCTCTGCATTGCCCCTCTTCTCCCTCTCCAAACAGAGGGAACTC


 


TCCTACCCCCAAGGAGGTGAAAGCTGCTACCACCTCTGTGCCCCCCCGGCAATGCCACCAACTG


 


GATCCTACCCGAATTTATGATTAAGATTGCTGAAGAGCTGCCAAACACTGCTGCCACCCCCTCT


 


GTTCCCTTATTGCTGCTTGTCACTGCCTGACATTCACGGCAGAGGCAAGGCTGCTGCAGCCTCC


 


CCTGGCTGTGCACATTCCCTCCTGCTCCCCAGAGACTGCCTCCGCCATCCCACAGATGATGGAT


 


CTTCAGTGGGTTCTCTTGGGCTCTAGGTCCTGCAGAATGTTGTGAGGGGTTTATTTTTTTTTAA


 


TAGTGTTCATAAAGAAATACATAGTATTCTTCTTCTCAAGACGTGGGGGGAAATTATCTCATTA


 


TCGAGGCCCTGCTATGCTGTGTATCTGGGCGTGTTGTATGTCCTGCTGCCGATGCCTTCATTAA


 


AATGATTTGGAAGAGCAGA






Nucleotides in lower cases above are untranslated regions or introns, and nucleotides in upper cases are exons.







>X02592.1 Human mRNA for T-cell receptor alpha 


chain (TCR-alpha)


TTTTGAAACCCTTCAAAGGCAGAGACTTGTCCAGCCTAACCTGCCTGCTG


 


CTCCTAGCTCCTGAGGCTCAGGGCCCTTGGCTTCTGTCCGCTCTGCTCAG


 


GGCCCTCCAGCGTGGCCACTGCTCAGCCATGCTCCTGCTGCTCGTCCCAG


 


TGCTCGAGGTGATTTTTACCCTGGGAGGAACCAGAGCCCAGTCGGTGACC


 


CAGCTTGGCAGCCACGTCTCTGTCTCTGAAGGAGCCCTGGTTCTGCTGAG


 


GTGCAACTACTCATCGTCTGTTCCACCATATCTCTTCTGGTATGTGCAAT


 


ACCCCAACCAAGGACTCCAGCTTCTCCTGAAGTACACATCAGCGGCCACC


 


CTGGTTAAAGGCATCAACGGTTTTGAGGCTGAATTTAAGAAGAGTGAAAC


 


CTCCTTCCACCTGACGAAACCCTCAGCCCATATGAGCGACGCGGCTGAGT


 


ACTTCTGTGCTGTGAGTGATCTCGAACCGAACAGCAGTGCTTCCAAGATA


 


ATCTTTGGATCAGGGACCAGACTCAGCATCCGGCCAAATATCCAGAACCC


 


TGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTG


 


TCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAG


 


GATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTAT


 


GGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTG


 


CATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTC


 


CCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGA


 


AACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAA


 


TCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTG


 


TGGTCCAGCTGAGATCTGCAAGATTGTAAGACAGCCTGTGCTCCCTCGCT


 


CCTTCCTCTGCATTGCCCCTCTTCTCCCTCTCCAAACAGAGGGAACTCTC


 


CTACCCCCAAGGAGGTGAAAGCTGCTACCACCTCTGTGCCCCCCCGGTAA


 


TGCCACCAACTGGATCCTACCCGAATTTATGATTAAGATTGCTGAAGAGC


 


TGCCAAACACTGCTGCCACCCCCTCTGTTCCCTTATTGCTGCTTGTCACT


 


GCCTGACATTCACGGCAGAGGCAAGGCTGCTGCAGCCTCCCCTGGCTGTG


 


CACATTCCCTCCTGCTCCCCAGAGACTGCCTCCGCCATCCCACAGATGAC


 


GGATCTTCAGTGGGTTCTCTTGGGCTCTAGGTCCTGGAGAATGTTGTGAG


 


GGGTTTATTTTTTTTTAATAGTGTTCATAAAGAAATACATAGTATTCTTC


 


TTCTCAAGACGTGGGGGGAAATTATCTCATTATCGAGGCCCTGCTATGCT


 


GTGTGTCTGGGCGTGTTGTATGTCCTGCTGCCGATGCCTTCATTAAAATG


 


ATTTGGAA






By “T cell receptor beta constant 1 polypeptide (TRBC1)” is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession No. P01850 or fragment thereof and having immunomodulatory activity. An exemplary amino acid sequence is provided below.







>sp|P01850|TRBC1_HUMAN T cell receptor beta 


constant 1 OS = Homo sapiens OX = 9606


GN = RBC1 PE = 1 SV = 4


DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKE


 


VHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY


 


GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEI


 


LLGKATLYAVLVSALVLMAMVKRKDF






By “T cell receptor beta constant 1 polynucleotide (TRBC1)” is meant a nucleic acid encoding a TRBC1 polypeptide. An exemplary TRBC1 nucleic acid sequence is provided below.







>X00437.1


CTGGTCTAGAATATTCCACATCTGCTCTCACTCTGCCATGGACTCCTGGA


 


CCTTCTGCTGTGTGTCCCTTTGCATCCTGGTAGCGAAGCATACAGATGCT


 


GGAGTTATCCAGTCACCCCGCCATGAGGTGACAGAGATGGGACAAGAAGT


 


GACTCTGAGATGTAAACCAATTTCAGGCCACAACTCCCTTTTCTGGTACA


 


GACAGACCATGATGCGGGGACTGGAGTTGCTCATTTACTTTAACAACAAC


 


GTTCCGATAGATGATTCAGGGATGCCCGAGGATCGATTCTCAGCTAAGAT


 


GCCTAATGCATCATTCTCCACTCTGAAGATCCAGCCCTCAGAACCCAGGG


 


ACTCAGCTGTGTACTTCTGTGCCAGCAGTTTCTCGACCTGTTCGGCTAAC


 


TATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGACCT


 


GAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAG


 


AGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTC


 


TTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCA


 


CAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCA


 


ATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTC


 


TGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCT


 


CTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGA


 


TCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTG


 


TCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCT


 


AGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGG


 


CCATGGTCAAGAGAAAGGATTTCTGAAGGCAGCCCTGGAAGTGGAGTTAG


 


GAGCTTCTAACCCGTCATGGTTCAATACACATTCTTCTTTTGCCAGCGCT


 


TCTGAAGAGCTGCTCTCACCTCTCTGCATCCCAATAGATATCCCCCTATG


 


TGCATGCACACCTGCACACTCACGGCTGAAATCTCCCTAACCCAGGGGGA


 


C






By “T cell receptor beta constant 2 polypeptide (TRBC2)” is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession No. AOA5B9 or fragment thereof and having immunomodulatory activity. An exemplary amino acid sequence is provided below.







>sp|A0A5B9|TRBC2_HUMAN T cell receptor beta 


constant 2 OS = Homo sapiens OX = 9606


GN = TRBC2 PE = 1 SV = 2


DLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKE


 


VHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY


 


GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEI


 


LLGKATLYAVLVSALVLMAMVKRKDSRG






By “T cell receptor beta constant 2 polynucleotide (TRBC2)” is meant a nucleic acid encoding a TRAC polypeptide. An exemplary TRBC2 nucleic acid sequence is provided below.







>NG_001333.2:655095-656583 Homo sapiens T cell 


receptor beta locus (TRB) on chromosome7


AGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCA


 


GAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTATGCCTGGCCAC


 


AGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGG


 


AGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCC


 


GCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGC


 


CACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCT


 


ACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTC


 


ACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGGTGAGTGGGGCCT


 


GGGGAGATGCCTGGAGGAGATTAGGTGAGACCAGCTACCAGGGAAAATGG


 


AAAGATCCAGGTAGCGGACAAGACTAGATCCAGAAGAAAGCCAGAGTGGA


 


CAAGGTGGGATGATCAAGGTTCACAGGGTCAGCAAAGCACGGTGTGCACT


 


TCCCCCACCAAGAAGCATAGAGGCTGAATGGAGCACCTCAAGCTCATTCT


 


TCCTTCAGATCCTGACACCTTAGAGCTAAGCTTTCAAGTCTCCCTGAGGA


 


CCAGCCATACAGCTCAGCATCTGAGTGGTGTGCATCCCATTCTCTTCTGG


 


GGTCCTGGTTTCCTAAGATCATAGTGACCACTTCGCTGGCACTGGAGCAG


 


CATGAGGGAGACAGAACCAGGGCTATCAAAGGAGGCTGACTTTGTACTAT


 


CTGATATGCATGTGTTTGTGGCCTGTGAGTCTGTGATGTAAGGCTCAATG


 


TCCTTACAAAGCAGCATTCTCTCATCCATTTTTCTTCCCCTGTTTTCTTT


 


CAGACTGTGGCTTCACCTCCGGTAAGTGAGTCTCTCCTTTTTCTCTCTAT


 


CTTTCGCCGTCTCTGCTCTCGAACCAGGGCATGGAGAATCCACGGACACA


 


GGGGCGTGAGGGAGGCCAGAGCCACCTGTGCACAGGTGCCTACATGCTCT


 


GTTCTTGTCAACAGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCC


 


TCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGT


 


GCCCTCGTGCTGATGGCCATGGTAAGGAGGAGGGTGGGATAGGGCAGATG


 


ATGGGGGCAGGGGATGGAACATCACACATGGGCATAAAGGAATCTCAGAG


 


CCAGAGCACAGCCTAATATATCCTATCACCTCAATGAAACCATAATGAAG


 


CCAGACTGGGGAGAAAATGCAGGGAATATCACAGAATGCATCATGGGAGG


 


ATGGAGACAACCAGCGAGCCCTACTCAAATTAGGCCTCAGAGCCCGCCTC


 


CCCTGCCCTACTCCTGCTGTGCCATAGCCCCTGAAACCCTCAAAATGTTC


 


TCTCTTCCACAGGTCAAGAGAAAGGATTCCAGAGGCTAG






By “tet methylcytosine dioxygenase 2 (TET2) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession No. FM992369.1 or a fragment thereof and having catalytic activity to convert methylcytosine to 5-hydroxymethylcytosine. Defects in the gene have been associated with myeloproliferative disorders, and the enzyme's ability to methylate cytosine contributes to transcriptional regulation. An exemplary TET2 amino acid sequence is provided below.







>CAX30492.1 tet oncogene family member 2 


[Homo sapiens]


MEQDRTNHVEGNRLSPFLIPSPPICQTEPLATKLQNGSPLPERAHPEVNG


 


DTKWHSFKSYYGIPCMKGSQNSRVSPDFTQESRGYSKCLQNGGIKRTVSE


 


PSLSGLLQIKKLKQDQKANGERRNFGVSQERNPGESSQPNVSDLSDKKES


 


VSSVAQENAVKDFTSFSTHNCSGPENPELQILNEQEGKSANYHDKNIVLL


 


KNKAVLMPNGATVSASSVEHTHGELLEKTLSQYYPDCVSIAVQKTTSHIN


 


AINSQATNELSCEITHPSHTSGQINSAQTSNSELPPKPAAVVSEACDADD


 


ADNASKLAAMLNTCSFQKPEQLQQQKSVFEICPSPAENNIQGTTKLASGE


 


EFCSGSSSNLQAPGGSSERYLKQNEMNGAYFKQSSVFTKDSFSATTTPPP


 


PSQLLLSPPPPLPQVPQLPSEGKSTLNGGVLEEHHHYPNQSNTTLLREVK


 


IEGKPEAPPSQSPNPSTHVCSPSPMLSERPQNNCVNRNDIQTAGTMTVPL


 


CSEKTRPMSEHLKHNPPIFGSSGELQDNCQQLMRNKEQEILKGRDKEQTR


 


DLVPPTQHYLKPGWIELKAPRFHQAESHLKRNEASLPSILQYQPNLSNQM


 


TSKQYTGNSNMPGGLPRQAYTQKTTQLEHKSQMYQVEMNQGQSQGTVDQH


 


LQFQKPSHQVHFSKTDHLPKAHVQSLCGTRFHFQQRADSQTEKLMSPVLK


 


QHLNQQASETEPFSNSHLLQHKPHKQAAQTQPSQSSHLPQNQQQQQKLQI


 


KNKEEILQTFPHPQSNNDQQREGSFFGQTKVEECFHGENQYSKSSEFETH


 


NVQMGLEEVQNINRRNSPYSQTMKSSACKIQVSCSNNTHLVSENKEQTTH


 


PELFAGNKTQNLHHMQYFPNNVIPKQDLLHRCFQEQEQKSQQASVIQGYK


 


NRNQDMSGQQAAQLAQQRYLIHNHANVFPVPDQGGSHTQTPPQKDTQKHA


 


ALRWHLLQKQEQQQTQQPQTESCHSQMHRPIKVEPGCKPHACMHTAPPEN


 


KTWKKVTKQENPPASCDNVQQKSIIETMEQHLKQFHAKSLFDHKALTLKS


 


QKQVKVEMSGPVTVLTRQTTAAELDSHTPALEQQTTSSEKTPTKRTAASV


 


LNNFIESPSKLLDTPIKNLLDTPVKTQYDFPSCRCVEQIIEKDEGPFYTH


 


LGAGPNVAAIREIMEERFGQKGKAIRIERVIYTGKEGKSSQGCPIAKWVV


 


RRSSSEEKLLCLVRERAGHTCEAAVIVILILVWEGIPLSLADKLYSELTE


 


TLRKYGTLTNRRCALNEERTCACQGLDPETCGASFSFGCSWSMYYNGCKF


 


ARSKIPRKFKLLGDDPKEEEKLESHLQNLSTLMAPTYKKLAPDAYNNQIE


 


YEHRAPECRLGLKEGRPFSGVTACLDFCAHAHRDLHNMQNGSTLVCTLTR


 


EDNREFGGKPEDEQLHVLPLYKVSDVDEFGSVEAQEEKKRSGAIQVLSSF


 


RRKVRMLAEPVKTCRQRKLEAKKAAAEKLSSLENSSNKNEKEKSAPSRTK


 


QTENASQAKQLAELLRLSGPVMQQSQQPQPLQKQPPQPQQQQRPQQQQPH


 


HPQTESVNSYSASGSTNPYMRRPNPVSPYPNSSHTSDIYGSTSPMNFYST


 


SSQAAGSYLNSSNPMNPYPGLLNQNTQYPSYQCNGNLSVDNCSPYLGSYS


 


PQSQPMDLYRYPSQDPLSKLSLPPIHTLYQPRFGNSQSFTSKYLGYGNQN


 


MQGDGFSSCTIRPNVHHVGKLPPYPTHEMDGHFMGATSRLPPNLSNPNMD


 


YKNGEHHSPSHIIHNYSAAPGMFNSSLHALHLQNKENDMLSHTANGLSKM


 


LPALNHDRTACVQGGLHKLSDANGQEKQPLALVQGVASGAEDNDEVWSDS


 


EQSFLDPDIGGVAVAPTHGSILIECAKRELHATTPLKNPNRNHPTRISLV


 


FYQHKSMNEPKHGLALWEAKMAEKAREKEEECEKYGPDYVPQKSHGKKVK


 


REPAEPHETSEPTYLRFIKSLAERTMSVTTDSTVTTSPYAFTRVTGPYNR


 


YI






By “tet methylcytosine dioxygenase 2 (TET2) polynucleotide” is meant a nucleic acid molecule encoding a TET2 polypeptide. The TETs polypeptide encodes a methylcytosine dioxygenase and has transcription regulatory activity. An exemplary TET2 nucleic acid is presented below.








>FM992369.1 Homo sapiens mRNA for tet oncogene family 



member 2 (TET2 gene)


CCGTGCCATCCCAACCTCCCACCTCGCCCCCAACCTTCGCGCTTGCTCTGCTTCTTCTCCCAGG


 


GGTGGAGACCCGCCGAGGTCCCCGGGGTTCCCGAGGGCTGCACCCTTCCCCGCGCTCGCCAGCC


 


CTGGCCCCTACTCCGCGCTGGTCCGGGCGCACCACTCCCCCCGCGCCACTGCACGGCGTGAGGG


 


CAGCCCAGGTCTCCACTGCGCGCCCCGCTGTACGGCCCCAGGTGCCGCCGGCCTTTGTGCTGGA


 


CGCCCGGTGCGGGGGGCTAATTCCCTGGGAGCCGGGGCTGAGGGCCCCAGGGCGGCGGCGCAGG


 


CCGGGGCGGAGCGGGAGGAGGCCGGGGCGGAGCAGGAGGAGGCCCGGGCGGAGGAGGAGAGCCG


 


GCGGTAGCGGCAGTGGCAGCGGCGAGAGCTTGGGCGGCCGCCGCCGCCTCCTCGCGAGCGCCGC


 


GCGCCCGGGTCCCGCTCGCATGCAAGTCACGTCCGCCCCCTCGGCGCGGCCGCCCCGAGACGCC


 


GGCCCCGCTGAGTGATGAGAACAGACGTCAAACTGCCTTATGAATATTGATGCGGAGGCTAGGC


 


TGCTTTCGTAGAGAAGCAGAAGGAAGCAAGATGGCTGCCCTTTAGGATTTGTTAGAAAGGAGAC


 


CCGACTGCAACTGCTGGATTGCTGCAAGGCTGAGGGACGAGAACGAGGCTGGCAAACATTCAGC


 


AGCACACCCTCTCAAGATTGTTTACTTGCCTTTGCTCCTGTTGAGTTACAACGCTTGGAAGCAG


 


GAGATGGGCTCAGCAGCAGCCAATAGGACATGATCCAGGAAGAGCAAATTCAACTAGAGGGCAG


 


CCTTGTGGATGGCCCCGAAGCAAGCCTGATGGAACAGGATAGAACCAACCATGTTGAGGGCAAC


 


AGACTAAGTCCATTCCTGATACCATCACCTCCCATTTGCCAGACAGAACCTCTGGCTACAAAGC


 


TCCAGAATGGAAGCCCACTGCCTGAGAGAGCTCATCCAGAAGTAAATGGAGACACCAAGTGGCA


 


CTCTTTCAAAAGTTATTATGGAATACCCTGTATGAAGGGAAGCCAGAATAGTCGTGTGAGTCCT


 


GACTTTACACAAGAAAGTAGAGGGTATTCCAAGTGTTTGCAAAATGGAGGAATAAAACGCACAG


 


TTAGTGAACCTTCTCTCTCTGGGCTCCTTCAGATCAAGAAATTGAAACAAGACCAAAAGGCTAA


 


TGGAGAAAGACGTAACTTCGGGGTAAGCCAAGAAAGAAATCCAGGTGAAAGCAGTCAACCAAAT


 


GTCTCCGATTTGAGTGATAAGAAAGAATCTGTGAGTTCTGTAGCCCAAGAAAATGCAGTTAAAG


 


ATTTCACCAGTTTTTCAACACATAACTGCAGTGGGCCTGAAAATCCAGAGCTTCAGATTCTGAA


 


TGAGCAGGAGGGGAAAAGTGCTAATTACCATGACAAGAACATTGTATTACTTAAAAACAAGGCA


 


GTGCTAATGCCTAATGGTGCTACAGTTTCTGCCTCTTCCGTGGAACACACACATGGTGAACTCC


 


TGGAAAAAACACTGTCTCAATATTATCCAGATTGTGTTTCCATTGCGGTGCAGAAAACCACATC


 


TCACATAAATGCCATTAACAGTCAGGCTACTAATGAGTTGTCCTGTGAGATCACTCACCCATCG


 


CATACCTCAGGGCAGATCAATTCCGCACAGACCTCTAACTCTGAGCTGCCTCCAAAGCCAGCTG


 


CAGTGGTGAGTGAGGCCTGTGATGCTGATGATGCTGATAATGCCAGTAAACTAGCTGCAATGCT


 


AAATACCTGTTCCTTTCAGAAACCAGAACAACTACAACAACAAAAATCAGTTTTTGAGATATGC


 


CCATCTCCTGCAGAAAATAACATCCAGGGAACCACAAAGCTAGCGTCTGGTGAAGAATTCTGTT


 


CAGGTTCCAGCAGCAATTTGCAAGCTCCTGGTGGCAGCTCTGAACGGTATTTAAAACAAAATGA


 


AATGAATGGTGCTTACTTCAAGCAAAGCTCAGTGTTCACTAAGGATTCCTTTTCTGCCACTACC


 


ACACCACCACCACCATCACAATTGCTTCTTTCTCCCCCTCCTCCTCTTCCACAGGTTCCTCAGC


 


TTCCTTCAGAAGGAAAAAGCACTCTGAATGGTGGAGTTTTAGAAGAACACCACCACTACCCCAA


 


CCAAAGTAACACAACACTTTTAAGGGAAGTGAAAATAGAGGGTAAACCTGAGGCACCACCTTCC


 


CAGAGTCCTAATCCATCTACACATGTATGCAGCCCTTCTCCGATGCTTTCTGAAAGGCCTCAGA


 


ATAATTGTGTGAACAGGAATGACATACAGACTGCAGGGACAATGACTGTTCCATTGTGTTCTGA


 


GAAAACAAGACCAATGTCAGAACACCTCAAGCATAACCCACCAATTTTTGGTAGCAGTGGAGAG


 


CTACAGGACAACTGCCAGCAGTTGATGAGAAACAAAGAGCAAGAGATTCTGAAGGGTCGAGACA


 


AGGAGCAAACACGAGATCTTGTGCCCCCAACACAGCACTATCTGAAACCAGGATGGATTGAATT


 


GAAGGCCCCTCGTTTTCACCAAGCGGAATCCCATCTAAAACGTAATGAGGCATCACTGCCATCA


 


ATTCTTCAGTATCAACCCAATCTCTCCAATCAAATGACCTCCAAACAATACACTGGAAATTCCA


 


ACATGCCTGGGGGGCTCCCAAGGCAAGCTTACACCCAGAAAACAACACAGCTGGAGCACAAGTC


 


ACAAATGTACCAAGTTGAAATGAATCAAGGGCAGTCCCAAGGTACAGTGGACCAACATCTCCAG


 


TTCCAAAAACCCTCACACCAGGTGCACTTCTCCAAAACAGACCATTTACCAAAAGCTCATGTGC


 


AGTCACTGTGTGGCACTAGATTTCATTTTCAACAAAGAGCAGATTCCCAAACTGAAAAACTTAT


 


GTCCCCAGTGTTGAAACAGCACTTGAATCAACAGGCTTCAGAGACTGAGCCATTTTCAAACTCA


 


CACCTTTTGCAACATAAGCCTCATAAACAGGCAGCACAAACACAACCATCCCAGAGTTCACATC


 


TCCCTCAAAACCAGCAACAGCAGCAAAAATTACAAATAAAGAATAAAGAGGAAATACTCCAGAC


 


TTTTCCTCACCCCCAAAGCAACAATGATCAGCAAAGAGAAGGATCATTCTTTGGCCAGACTAAA


 


GTGGAAGAATGTTTTCATGGTGAAAATCAGTATTCAAAATCAAGCGAGTTCGAGACTCATAATG


 


TCCAAATGGGACTGGAGGAAGTACAGAATATAAATCGTAGAAATTCCCCTTATAGTCAGACCAT


 


GAAATCAAGTGCATGCAAAATACAGGTTTCTTGTTCAAACAATACACACCTAGTTTCAGAGAAT


 


AAAGAACAGACTACACATCCTGAACTTTTTGCAGGAAACAAGACCCAAAACTTGCATCACATGC


 


AATATTTTCCAAATAATGTGATCCCAAAGCAAGATCTTCTTCACAGGTGCTTTCAAGAACAGGA


 


GCAGAAGTCACAACAAGCTTCAGTTCTACAGGGATATAAAAATAGAAACCAAGATATGTCTGGT


 


CAACAAGCTGCGCAACTTGCTCAGCAAAGGTACTTGATACATAACCATGCAAATGTTTTTCCTG


 


TGCCTGACCAGGGAGGAAGTCACACTCAGACCCCTCCCCAGAAGGACACTCAAAAGCATGCTGC


 


TCTAAGGTGGCATCTCTTACAGAAGCAAGAACAGCAGCAAACACAGCAACCCCAAACTGAGTCT


 


TGCCATAGTCAGATGCACAGGCCAATTAAGGTGGAACCTGGATGCAAGCCACATGCCTGTATGC


 


ACACAGCACCACCAGAAAACAAAACATGGAAAAAGGTAACTAAGCAAGAGAATCCACCTGCAAG


 


CTGTGATAATGTGCAGCAAAAGAGCATCATTGAGACCATGGAGCAGCATCTGAAGCAGTTTCAC


 


GCCAAGTCGTTATTTGACCATAAGGCTCTTACTCTCAAATCACAGAAGCAAGTAAAAGTTGAAA


 


TGTCAGGGCCAGTCACAGTTTTGACTAGACAAACCACTGCTGCAGAACTTGATAGCCACACCCC


 


AGCTTTAGAGCAGCAAACAACTTCTTCAGAAAAGACACCAACCAAAAGAACAGCTGCTTCTGTT


 


CTCAATAATTTTATAGAGTCACCTTCCAAATTACTAGATACTCCTATAAAAAATTTATTGGATA


 


CACCTGTCAAGACTCAATATGATTTCCCATCTTGCAGATGTGTAGAGCAAATTATTGAAAAAGA


 


TGAAGGTCCTTTTTATACCCATCTAGGAGCAGGTCCTAATGTGGCAGCTATTAGAGAAATCATG


 


GAAGAAAGGTTTGGACAGAAGGGTAAAGCTATTAGGATTGAAAGAGTCATCTATACTGGTAAAG


 


AAGGCAAAAGTTCTCAGGGATGTCCTATTGCTAAGTGGGTGGTTCGCAGAAGCAGCAGTGAAGA


 


GAAGCTACTGTGTTTGGTGCGGGAGCGAGCTGGCCACACCTGTGAGGCTGCAGTGATTGTGATT


 


CTCATCCTGGTGTGGGAAGGAATCCCGCTGTCTCTGGCTGACAAACTCTACTCGGAGCTTACCG


 


AGACGCTGAGGAAATACGGCACGCTCACCAATCGCCGGTGTGCCTTGAATGAAGAGAGAACTTG


 


CGCCTGTCAGGGGCTGGATCCAGAAACCTGTGGTGCCTCCTTCTCTTTTGGTTGTTCATGGAGC


 


ATGTACTACAATGGATGTAAGTTTGCCAGAAGCAAGATCCCAAGGAAGTTTAAGCTGCTTGGGG


 


ATGACCCAAAAGAGGAAGAGAAACTGGAGTCTCATTTGCAAAACCTGTCCACTCTTATGGCACC


 


AACATATAAGAAACTTGCACCTGATGCATATAATAATCAGATTGAATATGAACACAGAGCACCA


 


GAGTGCCGTCTGGGTCTGAAGGAAGGCCGTCCATTCTCAGGGGTCACTGCATGTTTGGACTTCT


 


GTGCTCATGCCCACAGAGACTTGCACAACATGCAGAATGGCAGCACATTGGTATGCACTCTCAC


 


TAGAGAAGACAATCGAGAATTTGGAGGAAAACCTGAGGATGAGCAGCTTCACGTTCTGCCTTTA


 


TACAAAGTCTCTGACGTGGATGAGTTTGGGAGTGTGGAAGCTCAGGAGGAGAAAAAACGGAGTG


 


GTGCCATTCAGGTACTGAGTTCTTTTCGGCGAAAAGTCAGGATGTTAGCAGAGCCAGTCAAGAC


 


TTGCCGACAAAGGAAACTAGAAGCCAAGAAAGCTGCAGCTGAAAAGCTTTCCTCCCTGGAGAAC


 


AGCTCAAATAAAAATGAAAAGGAAAAGTCAGCCCCATCACGTACAAAACAAACTGAAAACGCAA


 


GCCAGGCTAAACAGTTGGCAGAACTTTTGCGACTTTCAGGACCAGTCATGCAGCAGTCCCAGCA


 


GCCCCAGCCTCTACAGAAGCAGCCACCACAGCCCCAGCAGCAGCAGAGACCCCAGCAGCAGCAG


 


CCACATCACCCTCAGACAGAGTCTGTCAACTCTTATTCTGCTTCTGGATCCACCAATCCATACA


 


TGAGACGGCCCAATCCAGTTAGTCCTTATCCAAACTCTTCACACACTTCAGATATCTATGGAAG


 


CACCAGCCCTATGAACTTCTATTCCACCTCATCTCAAGCTGCAGGTTCATATTTGAATTCTTCT


 


AATCCCATGAACCCTTACCCTGGGCTTTTGAATCAGAATACCCAATATCCATCATATCAATGCA


 


ATGGAAACCTATCAGTGGACAACTGCTCCCCATATCTGGGTTCCTATTCTCCCCAGTCTCAGCC


 


GATGGATCTGTATAGGTATCCAAGCCAAGACCCTCTGTCTAAGCTCAGTCTACCACCCATCCAT


 


ACACTTTACCAGCCAAGGTTTGGAAATAGCCAGAGTTTTACATCTAAATACTTAGGTTATGGAA


 


ACCAAAATATGCAGGGAGATGGTTTCAGCAGTTGTACCATTAGACCAAATGTACATCATGTAGG


 


GAAATTGCCTCCTTATCCCACTCATGAGATGGATGGCCACTTCATGGGAGCCACCTCTAGATTA


 


CCACCCAATCTGAGCAATCCAAACATGGACTATAAAAATGGTGAACATCATTCACCTTCTCACA


 


TAATCCATAACTACAGTGCAGCTCCGGGCATGTTCAACAGCTCTCTTCATGCCCTGCATCTCCA


 


AAACAAGGAGAATGACATGCTTTCCCACACAGCTAATGGGTTATCAAAGATGCTTCCAGCTCTT


 


AACCATGATAGAACTGCTTGTGTCCAAGGAGGCTTACACAAATTAAGTGATGCTAATGGTCAGG


 


AAAAGCAGCCATTGGCACTAGTCCAGGGTGTGGCTTCTGGTGCAGAGGACAACGATGAGGTCTG


 


GTCAGACAGCGAGCAGAGCTTTCTGGATCCTGACATTGGGGGAGTGGCCGTGGCTCCAACTCAT


 


GGGTCAATTCTCATTGAGTGTGCAAAGCGTGAGCTGCATGCCACAACCCCTTTAAAGAATCCCA


 


ATAGGAATCACCCCACCAGGATCTCCCTCGTCTTTTACCAGCATAAGAGCATGAATGAGCCAAA


 


ACATGGCTTGGCTCTTTGGGAAGCCAAAATGGCTGAAAAAGCCCGTGAGAAAGAGGAAGAGTGT


 


GAAAAGTATGGCCCAGACTATGTGCCTCAGAAATCCCATGGCAAAAAAGTGAAACGGGAGCCTG


 


CTGAGCCACATGAAACTTCAGAGCCCACTTACCTGCGTTTCATCAAGTCTCTTGCCGAAAGGAC


 


CATGTCCGTGACCACAGACTCCACAGTAACTACATCTCCATATGCCTTCACTCGGGTCACAGGG


 


CCTTACAACAGATATATATGAAGATATATATGATATCACCCCCTTTTGTTGGTTACCTCACTTG


 


AAAAGACCACAACCAACCTGTCAGTAGTATAGTTCTCATGACGTGGGCAGTGGGGAAAGGTCAC


 


AGTATTCATGACAAATGTGGTGGGAAAAACCTCAGCTCACCAGCAACAAAAGAGGTTATCTTAC


 


CATAGCACTTAATTTTCACTGGCTCCCAAGTGGTCACAGATGGCATCTAGGAAAAGACCAAAGC


 


ATTCTATGCAAAAAGAAGGTGGGGAAGAAAGTGTTCCGCAATTTACATTTTTAAACACTGGTTC


 


TATTATTGGACGAGATGATATGTAAATGTGATCCCCCCCCCCCGCTTACAACTCTACACATCTG


 


TGACCACTTTTAATAATATCAAGTTTGCATAGTCATGGAACACAAATCAAACAAGTACTGTAGT


 


ATTACAGTGACAGGAATCTTAAAATACCATCTGGTGCTGAATATATGATGTACTGAAATACTGG


 


AATTATGGCTTTTTGAAATGCAGTTTTTACTGTAATCTTAACTTTTATTTATCAAAATAGCTAC


 


AGGAAACATGAATAGCAGGAAAACACTGAATTTGTTTGGATGTTCTAAGAAATGGTGCTAAGAA


 


AATGGTGTCTTTAATAGCTAAAAATTTAATGCCTTTATATCATCAAGATGCTATCAGTGTACTC


 


CAGTGCCCTTGAATAATAGGGGTACCTTTTCATTCAAGTTTTTATCATAATTACCTATTCTTAC


 


ACAAGCTTAGTTTTTAAAATGTGGACATTTTAAAGGCCTCTGGATTTTGCTCATCCAGTGAAGT


 


CCTTGTAGGACAATAAACGTATATATGTACATATATACACAAACATGTATATGTGCACACACAT


 


GTATATGTATAAATATTTTAAATGGTGTTTTAGAAGCACTTTGTCTACCTAAGCTTTGACAACT


 


TGAACAATGCTAAGGTACTGAGATGTTTAAAAAACAAGTTTACTTTCATTTTAGAATGCAAAGT


 


TGATTTTTTTAAGGAAACAAAGAAAGCTTTTAAAATATTTTTGCTTTTAGCCATGCATCTGCTG


 


ATGAGCAATTGTGTCCATTTTTAACACAGCCAGTTAAATCCACCATGGGGCTTACTGGATTCAA


 


GGGAATACGTTAGTCCACAAAACATGTTTTCTGGTGCTCATCTCACATGCTATACTGTAAAACA


 


GTTTTATACAAAATTGTATGACAAGTTCATTGCTCAAAAATGTACAGTTTTAAGAATTTTCTAT


 


TAACTGCAGGTAATAATTAGCTGCATGCTGCAGACTCAACAAAGCTAGTTCACTGAAGCCTATG


 


CTATTTTATGGATCATAGGCTCTTCAGAGAACTGAATGGCAGTCTGCCTTTGTGTTGATAATTA


 


TGTACATTGTGACGTTGTCATTTCTTAGCTTAAGTGTCCTCTTTAACAAGAGGATTGAGCAGAC


 


TGATGCCTGCATAAGATGAATAAACAGGGTTAGTTCCATGTGAATCTGTCAGTTAAAAAGAAAC


 


AAAAACAGGCAGCTGGTTTGCTGTGGTGGTTTTAAATCATTAATTTGTATAAAGAAGTGAAAGA


 


GTTGTATAGTAAATTAAATTGTAAACAAAACTTTTTTAATGCAATGCTTTAGTATTTTAGTACT


 


GTAAAAAAATTAAATATATACATATATATATATATATATATATATATATATATGAGTTTGAAGC


 


AGAATTCACATCATGATGGTGCTACTCAGCCTGCTACAAATATATCATAATGTGAGCTAAGAAT


 


TCATTAAATGTTTGAGTGATGTTCCTACTTGTCATATACCTCAACACTAGTTTGGCAATAGGAT


 


ATTGAACTGAGAGTGAAAGCATTGTGTACCATCATTTTTTTCCAAGTCCTTTTTTTTATTGTTA


 


AAAAAAAAAGCATACCTTTTTTCAATACTTGATTTCTTAGCAAGTATAACTTGAACTTCAACCT


 


TTTTGTTCTAAAAATTCAGGGATATTTCAGCTCATGCTCTCCCTATGCCAACATGTCACCTGTG


 


TTTATGTAAAATTGTTGTAGGTTAATAAATATATTCTTTGTCAGGGATTTAACCCTTTTATTTT


 


GAATCCCTTCTATTTTACTTGTACATGTGCTGATGTAACTAAAACTAATTTTGTAAATCTGTTG


 


GCTCTTTTTATTGTAAAGAAAAGCATTTTAAAAGTTTGAGGAATCTTTTGACTGTTTCAAGCAG


 


GAAAAAAAAATTACATGAAAATAGAATGCACTGAGTTGATAAAGGGAAAAATTGTAAGGCAGGA


 


GTTTGGCAAGTGGCTGTTGGCCAGAGACTTACTTGTAACTCTCTAAATGAAGTTTTTTTGATCC


 


TGTAATCACTGAAGGTACATACTCCATGTGGACTTCCCTTAAACAGGCAAACACCTACAGGTAT


 


GGTGTGCAACAGATTGTACAATTACATTTTGGCCTAAATACATTTTTGCTTACTAGTATTTAAA


 


ATAAATTCTTAATCAGAGGAGGCCTTTGGGTTTTATTGGTCAAATCTTTGTAAGCTGGCTTTTG


 


TCTTTTTAAAAAATTTCTTGAATTTGTGGTTGTGTCCAATTTGCAAACATTTCCAAAAATGTTT


 


GCTTTGCTTACAAACCACATGATTTTAATGTTTTTTGTATACCATAATATCTAGCCCCAAACAT


 


TTGATTACTACATGTGCATTGGTGATTTTGATCATCCATTCTTAATATTTGATTTCTGTGTCAC


 


CTACTGTCATTTGTTAAACTGCTGGCCAACAAGAACAGGAAGTATAGTTTGGGGGGTTGGGGAG


 


AGTTTACATAAGGAAGAGAAGAAATTGAGTGGCATATTGTAAATATCAGATCTATAATTGTAAA


 


TATAAAACCTGCCTCAGTTAGAATGAATGGAAAGCAGATCTACAATTTGCTAATATAGGAATAT


 


CAGGTTGACTATATAGCCATACTTGAAAATGCTTCTGAGTGGTGTCAACTTTACTTGAATGAAT


 


TTTTCATCTTGATTGACGCACAGTGATGTACAGTTCACTTCTGAAGCTAGTGGTTAACTTGTGT


 


AGGAAACTTTTGCAGTTTGACACTAAGATAACTTCTGTGTGCATTTTTCTATGCTTTTTTAAAA


 


ACTAGTTTCATTTCATTTTCATGAGATGTTTGGTTTATAAGATCTGAGGATGGTTATAAATACT


 


GTAAGTATTGTAATGTTATGAATGCAGGTTATTTGAAAGCTGTTTATTATTATATCATTCCTGA


 


TAATGCTATGTGAGTGTTTTTAATAAAATTTATATTTATTTAATGCACTCTAAGTGTTGTCTTC


 


CT






By “transforming growth factor receptor 2 (TGFBRII) polypeptide” is meant a protein having at least about 85% sequence identity to NCBI Accession No. ABG65632.1 or a fragment thereof and having immunosuppressive activity. An exemplary amino acid sequence is provided below.







>ABG65632.1 transforming growth factor beta 


receptor II [Homo sapiens]


MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQL


 


CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETV


 


CHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNLLFS


 


EEYNTSNPDLLLVIFQVTGISLLPPLGVAISVLILFYCYRVNRQQKLSST


 


WETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLV


 


GKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLK


 


HENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKL


 


GSSLARGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGL


 


SLRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSM


 


ALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEI


 


PSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGR


 


SCSEEKIPEDGSLNTTK






By “transforming growth factor receptor 2 (TGFBRII) polynucleotide” is meant a nucleic acid that encodes a TGFBRII polypeptide. The TGFBRII gene encodes a transmembrane protein having serine/threonine kinase activity. An exemplary TGFBRII nucleic acid is provided below.







>M85079.1 Human TGF-beta type II receptor


mRNA, complete cds


GTTGGCGAGGAGTTTCCTGTTTCCCCCGCAGCGCTGAGTTGAAGTTGAGT


 


GAGTCACTCGCGCGCACGGAGCGACGACACCCCCGCGCGTGCACCCGCTC


 


GGGACAGGAGCCGGACTCCTGTGCAGCTTCCCTCGGCCGCCGGGGGCCTC


 


CCCGCGCCTCGCCGGCCTCCAGGCCCCTCCTGGCTGGCGAGCGGGCGCCA


 


CATCTGGCCCGCACATCTGCGCTGCCGGCCCGGCGCGGGGTCCGGAGAGG


 


GCGCGGCGCGGAGCGCAGCCAGGGGTCCGGGAAGGCGCCGTCCGTGCGCT


 


GGGGGCTCGGTCTATGACGAGCAGCGGGGTCTGCCATGGGTCGGGGGCTG


 


CTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGTGGACGCGTATCGCCAG


 


CACGATCCCACCGCACGTTCAGAAGTCGGTTAATAACGACATGATAGTCA


 


CTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGAT


 


GTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAG


 


CATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGA


 


GAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAG


 


CTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCAT


 


TATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTA


 


GCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACC


 


AGCAATCCTGACTTGTTGCTAGTCATATTTCAAGTGACAGGCATCAGCCT


 


CCTGCCACCACTGGGAGTTGCCATATCTGTCATCATCATCTTCTACTGCT


 


ACCGCGTTAACCGGCAGCAGAAGCTGAGTTCAACCTGGGAAACCGGCAAG


 


ACGCGGAAGCTCATGGAGTTCAGCGAGCACTGTGCCATCATCCTGGAAGA


 


TGACCGCTCTGACATCAGCTCCACGTGTGCCAACAACATCAACCACAACA


 


CAGAGCTGCTGCCCATTGAGCTGGACACCCTGGTGGGGAAAGGTCGCTTT


 


GCTGAGGTCTATAAGGCCAAGCTGAAGCAGAACACTTCAGAGCAGTTTGA


 


GACAGTGGCAGTCAAGATCTTTCCCTATGAGGAGTATGCCTCTTGGAAGA


 


CAGAGAAGGACATCTTCTCAGACATCAATCTGAAGCATGAGAACATACTC


 


CAGTTCCTGACGGCTGAGGAGCGGAAGACGGAGTTGGGGAAACAATACTG


 


GCTGATCACCGCCTTCCACGCCAAGGGCAACCTACAGGAGTACCTGACGC


 


GGCATGTCATCAGCTGGGAGGACCTGCGCAAGCTGGGCAGCTCCCTCGCC


 


CGGGGGATTGCTCACCTCCACAGTGATCACACTCCATGTGGGAGGCCCAA


 


GATGCCCATCGTGCACAGGGACCTCAAGAGCTCCAATATCCTCGTGAAGA


 


ACGACCTAACCTGCTGCCTGTGTGACTTTGGGCTTTCCCTGCGTCTGGAC


 


CCTACTCTGTCTGTGGATGACCTGGCTAACAGTGGGCAGGTGGGAACTGC


 


AAGATACATGGCTCCAGAAGTCCTAGAATCCAGGATGAATTTGGAGAATG


 


CTGAGTCCTTCAAGCAGACCGATGTCTACTCCATGGCTCTGGTGCTCTGG


 


GAAATGACATCTCGCTGTAATGCAGTGGGAGAAGTAAAAGATTATGAGCC


 


TCCATTTGGTTCCAAGGTGCGGGAGCACCCCTGTGTCGAAAGCATGAAGG


 


ACAACGTGTTGAGAGATCGAGGGCGACCAGAAATTCCCAGCTTCTGGCTC


 


AACCACCAGGGCATCCAGATGGTGTGTGAGACGTTGACTGAGTGCTGGGA


 


CCACGACCCAGAGGCCCGTCTCACAGCCCAGTGTGTGGCAGAACGCTTCA


 


GTGAGCTGGAGCATCTGGACAGGCTCTCGGGGAGGAGCTGCTCGGAGGAG


 


AAGATTCCTGAAGACGGCTCCCTAAACACTACCAAATAGCTCTTATGGGG


 


CAGGCTGGGCATGTCCAAAGAGGCTGCCCCTCTCACCAAA






By “T Cell Immunoreceptor with Ig and ITIM Domains (TIGIT) polypeptide” is meant a protein having at least about 85% sequence identity to NCBI Accession No. ACD74757.1 or a fragment thereof and having immunomodulatory activity. An exemplary TIGIT amino acid sequence is provided below.







>ACD74757.1 T cell immunoreceptor with


Ig and ITIM domains [Homo sapiens]


MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSST


 


TAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTV


 


NDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATL


 


VVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSC


 


VQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG






By “T Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) polynucleotide” is meant a nucleic acid encoding a TIGIT polypeptide. The TIGIT gene encodes an inhibitory immune receptor that is associated with neoplasia and T cell exhaustion. An exemplary nucleic acid sequence is provided below.







>EU675310.1 Homo sapiens T cell immunoreceptor


with Ig and ITIM domains (TIGIT) mRNA,


complete cds


CGTCCTATCTGCAGTCGGCTACTTTCAGTGGCAGAAGAGGCCACATCTGC


 


TTCCTGTAGGCCCTCTGGGCAGAAGCATGCGCTGGTGTCTCCTCCTGATC


 


TGGGCCCAGGGGCTGAGGCAGGCTCCCCTCGCCTCAGGAATGATGACAGG


 


CACAATAGAAACAACGGGGAACATTTCTGCAGAGAAAGGTGGCTCTATCA


 


TCTTACAATGTCACCTCTCCTCCACCACGGCACAAGTGACCCAGGTCAAC


 


TGGGAGCAGCAGGACCAGCTTCTGGCCATTTGTAATGCTGACTTGGGGTG


 


GCACATCTCCCCATCCTTCAAGGATCGAGTGGCCCCAGGTCCCGGCCTGG


 


GCCTCACCCTCCAGTCGCTGACCGTGAACGATACAGGGGAGTACTTCTGC


 


ATCTATCACACCTACCCTGATGGGACGTACACTGGGAGAATCTTCCTGGA


 


GGTCCTAGAAAGCTCAGTGGCTGAGCACGGTGCCAGGTTCCAGATTCCAT


 


TGCTTGGAGCCATGGCCGCGACGCTGGTGGTCATCTGCACAGCAGTCATC


 


GTGGTGGTCGCGTTGACTAGAAAGAAGAAAGCCCTCAGAATCCATTCTGT


 


GGAAGGTGACCTCAGGAGAAAATCAGCTGGACAGGAGGAATGGAGCCCCA


 


GTGCTCCCTCACCCCCAGGAAGCTGTGTCCAGGCAGAAGCTGCACCTGCT


 


GGGCTCTGTGGAGAGCAGCGGGGAGAGGACTGTGCCGAGCTGCATGACTA


 


CTTCAATGTCCTGAGTTACAGAAGCCTGGGTAACTGCAGCTTCTTCACAG


 


AGACTGGTTAGCAACCAGAGGCATCTTCTGG






As used herein “transduction” means to transfer a gene or genetic material to a cell via a viral vector.
“Transformation,” as used herein refers to the process of introducing a genetic change in a cell produced by the introduction of exogenous nucleic acid.
“Transfection” refers to the transfer of a gene or genetical material to a cell via a chemical or physical means.
By “translocation” is meant the rearrangement of nucleic acid segments between non-homologous chromosomes.
By “transmembrane domain” is meant an amino acid sequence that inserts into a lipid bilayer, such as the lipid bilayer of a cell or virus or virus-like particle. A transmembrane domain can be used to anchor a protein of interest (e.g., a CAR) to a membrane. The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane domains for use in the disclosed CARs can include at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane domain is derived from CD4, CD8α, CD28 and CD3ζ. In some embodiments, the transmembrane domain is a CD8α hinge and transmembrane domain.
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or a symptom associated therewith or obtaining a desired pharmacologic and/or physiologic effect. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely reduces, diminishes, abrogates, abates, alleviates, decreases the intensity of, or cures a disease and/or adverse symptom attributable to the disease. In some embodiments, the effect is preventative, i.e., the effect protects or prevents an occurrence or reoccurrence of a disease or condition. To this end, the presently disclosed methods comprise administering a therapeutically effective amount of a compositions as described herein.
The term “uracil glycosylase inhibitor” or “UGI” is meant an agent that inhibits the uracil-excision repair system. In one embodiment, the agent is a protein or fragment thereof that binds a host uracil-DNA glycosylase and prevents removal of uracil residues from DNA. In an embodiment, a UGI is a protein, a fragment thereof, or a domain that is capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme. In some embodiments, a UGI domain comprises a wild-type UGI or a modified version thereof. In some embodiments, a UGI domain comprises a fragment of the exemplary amino acid sequence set forth below. In some embodiments, a UGI fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the exemplary UGI sequence provided below. In some embodiments, a UGI comprises an amino acid sequence that is homologous to the exemplary UGI amino acid sequence or fragment thereof, as set forth below. In some embodiments, the UGI, or a portion thereof, is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a wild-type UGI or a UGI sequence, or portion thereof, as set forth below. An exemplary UGI comprises an amino acid sequence as follows: >splP14739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitor
MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLT SD APEYKPWALVIQDSNGENKIKML (SEQ ID NO: 106). The term “vector” refers to a means of introducing a nucleic acid sequence into a cell, resulting in a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, and episome. “Expression vectors” are nucleic acid sequences comprising the nucleotide sequence to be expressed in the recipient cell. Expression vectors may include additional nucleic acid sequences to promote and/or facilitate the expression of the of the introduced sequence such as start, stop, enhancer, promoter, and secretion sequences.
By “zeta chain of T cell receptor associated protein kinase 70 (ZAP70) polypeptide” is meant a protein having at least about 85% amino acid sequence identity to NCBI Accession No. AAH53878.1 and having kinase activity. An exemplary amino acid sequence is provided below.







>AAH53878.1 Zeta-chain (TCR) associated


protein kinase 70kDa [Homo sapiens]


MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSL


 


VHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRK


 


PCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVE


 


KLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYAL


 


SLIYGKTVYHYLTSQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCL


 


KEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARIT


 


SPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFG


 


SVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVR


 


LIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMK


 


YLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGK


 


WPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMA


 


FTEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSL


 


ASKVEGPPGSTQKAEAACA






By “zeta chain of T cell receptor associated protein kinase 70 (ZAP70) polynucleotide” is meant a nucleic acid encoding a ZAP70 polypeptide. The ZAP70 gene encodes a tyrosine kinase that is involved in T cell development and lymphocyte activation. Absence of functional ZAP10 can lead to a severe combined immunodeficiency characterized by the lack of CD8+ T cells. An exemplary ZAP70 nucleic acid sequence is provided below.







>BC053878.1 Homo sapiens zeta-chain


(TCR) associated protein kinase 70kDa, mRNA


(cDNA clone MGC:61743 IMAGE:5757161),


complete cds


GCTTGCCGGAGCTCAGCAGACACCAGGCCTTCCGGGCAGGCCTGGCCCAC


 


CGTGGGCCTCAGAGCTGCTGCTGGGGCATTCAGAACCGGCTCTCCATTGG


 


CATTGGGACCAGAGACCCCGCAAGTGGCCTGTTTGCCTGGACATCCACCT


 


GTACGTCCCCAGGTTTCGGGAGGCCCAGGGGCGATGCCAGACCCCGCGGC


 


GCACCTGCCCTTCTTCTACGGCAGCATCTCGCGTGCCGAGGCCGAGGAGC


 


ACCTGAAGCTGGCGGGCATGGCGGACGGGCTCTTCCTGCTGCGCCAGTGC


 


CTGCGCTCGCTGGGCGGCTATGTGCTGTCGCTCGTGCACGATGTGCGCTT


 


CCACCACTTTCCCATCGAGCGCCAGCTCAACGGCACCTACGCCATTGCCG


 


GCGGCAAAGCGCACTGTGGACCGGCAGAGCTCTGCGAGTTCTACTCGCGC


 


GACCCCGACGGGCTGCCCTGCAACCTGCGCAAGCCGTGCAACCGGCCGTC


 


GGGCCTCGAGCCGCAGCCGGGGGTCTTCGACTGCCTGCGAGACGCCATGG


 


TGCGTGACTACGTGCGCCAGACGTGGAAGCTGGAGGGCGAGGCCCTGGAG


 


CAGGCCATCATCAGCCAGGCCCCGCAGGTGGAGAAGCTCATTGCTACGAC


 


GGCCCACGAGCGGATGCCCTGGTACCACAGCAGCCTGACGCGTGAGGAGG


 


CCGAGCGCAAACTTTACTCTGGGGCGCAGACCGACGGCAAGTTCCTGCTG


 


AGGCCGCGGAAGGAGCAGGGCACATACGCCCTGTCCCTCATCTATGGGAA


 


GACGGTGTACCACTACCTCATCAGCCAAGACAAGGCGGGCAAGTACTGCA


 


TTCCCGAGGGCACCAAGTTTGACACGCTCTGGCAGCTGGTGGAGTATCTG


 


AAGCTGAAGGCGGACGGGCTCATCTACTGCCTGAAGGAGGCCTGCCCCAA


 


CAGCAGTGCCAGCAACGCCTCAGGGGCTGCTGCTCCCACACTCCCAGCCC


 


ACCCATCCACGTTGACTCATCCTCAGAGACGAATCGACACCCTCAACTCA


 


GATGGATACACCCCTGAGCCAGCACGCATAACGTCCCCAGACAAACCGCG


 


GCCGATGCCCATGGACACGAGCGTGTATGAGAGCCCCTACAGCGACCCAG


 


AGGAGCTCAAGGACAAGAAGCTCTTCCTGAAGCGCGATAACCTCCTCATA


 


GCTGACATTGAACTTGGCTGCGGCAACTTTGGCTCAGTGCGCCAGGGCGT


 


GTACCGCATGCGCAAGAAGCAGATCGACGTGGCCATCAAGGTGCTGAAGC


 


AGGGCACGGAGAAGGCAGACACGGAAGAGATGATGCGCGAGGCGCAGATC


 


ATGCACCAGCTGGACAACCCCTACATCGTGCGGCTCATTGGCGTCTGCCA


 


GGCCGAGGCCCTCATGCTGGTCATGGAGATGGCTGGGGGCGGGCCGCTGC


 


ACAAGTTCCTGGTCGGCAAGAGGGAGGAGATCCCTGTGAGCAATGTGGCC


 


GAGCTGCTGCACCAGGTGTCCATGGGGATGAAGTACCTGGAGGAGAAGAA


 


CTTTGTGCACCGTGACCTGGCGGCCCGCAACGTCCTGCTGGTTAACCGGC


 


ACTACGCCAAGATCAGCGACTTTGGCCTCTCCAAAGCACTGGGTGCCGAC


 


GACAGCTACTACACTGCCCGCTCAGCAGGGAAGTGGCCGCTCAAGTGGTA


 


CGCACCCGAATGCATCAACTTCCGCAAGTTCTCCAGCCGCAGCGATGTCT


 


GGAGCTATGGGGTCACCATGTGGGAGGCCTTGTCCTACGGCCAGAAGCCC


 


TACAAGAAGATGAAAGGGCCGGAGGTCATGGCCTTCATCGAGCAGGGCAA


 


GCGGATGGAATGCCCACCAGAGTGTCCACCCGAACTGTACGCACTCATGA


 


GTGACTGCTGGATCTACAAGTGGGAGGATCGCCCCGACTTCCTGACCGTG


 


GAGCAGCGCATGCGAGCCTGTTACTACAGCCTGGCCAGCAAGGTGGAAGG


 


GCCCCCAGGCAGCACACAGAAGGCTGAGGCTGCCTGTGCCTGAGCTCCCG


 


CTGCCCAGGGGAGCCCTCCACACCGGCTCTTCCCCACCCTCAGCCCCACC


 


CCAGGTCCTGCAGTCTGGCTGAGCCCTGCTTGGTTGTCTCCACACACAGC


 


TGGGCTGTGGTAGGGGGTGTCTCAGGCCACACCGGCCTTGCATTGCCTGC


 


CTGGCCCCCTGTCCTCTCTGGCTGGGGAGCAGGGAGGTCCGGGAGGGTGC


 


GGCTGTGCAGCCTGTCCTGGGCTGGTGGCTCCCGGAGGGCCCTGAGCTGA


 


GGGCATTGCTTACACGGATGCCTTCCCCTGGGCCCTGACATTGGAGCCTG


 


GGCATCCTCAGGTGGTCAGGCGTAGATCACCAGAATAAACCCAGCTTCCC


 


TCTTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA


 


AAAAAAAAAAAAAAAAAA






The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
DNA editing has emerged as a viable means to modify disease states by correcting pathogenic mutations at the genetic level. Until recently, all DNA editing platforms have functioned by inducing a DNA double strand break (DSB) at a specified genomic site and relying on endogenous DNA repair pathways to determine the product outcome in a semi-stochastic manner, resulting in complex populations of genetic products. Though precise, user-defined repair outcomes can be achieved through the homology directed repair (HDR) pathway, a number of challenges have prevented high efficiency repair using HDR in therapeutically-relevant cell types. In practice, this pathway is inefficient relative to the competing, error-prone non-homologous end joining pathway. Further, HDR is tightly restricted to the Gi and S phases of the cell cycle, preventing precise repair of DSBs in post-mitotic cells. As a result, it has proven difficult or impossible to alter genomic sequences in a user-defined, programmable manner with high efficiencies in these populations.
INCORPORATION BY REFERENCE
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.



BRIEF DESCRIPTION OF THE DRAWINGS
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
FIGS. 1A-1B are illustrations of three proteins that impact T cell function. FIG. 1A is an illustration of the TRAC protein, which is a key component in graft versus host disease. FIG. 1B is an illustration of the B2M protein, a component of the MHC class 1 antigen presenting complex present on nucleated cells that can be recognized by a host's CD8+ T cells. FIG. 1C is an illustration of T cell signaling that leads to expression of the PDCD1 gene, and the resulting PD-1 protein acts to inhibit the T cell signaling.
FIG. 2 is a graph of the percentage of cells with knocked down expression of target genes after base editing. “EP” denotes electroporation.
FIG. 3 is a graph of the percentages of the observed types of genetic modification in untransduced cells or in cells transduced with a BE4 base editing system or a Cas9 nuclease.
FIG. 4 is a graph depicting target nucleotide modification percentage as measured by percentage of cells that are negative for target protein expression as determined by flow cytometry (FC) in cells transduced with BE4 and sgRNAs directing BE4 to splice site acceptors (SA) or donors (SD) or that generate a STOP codon. Control cells were mock electroporated (EP).
FIG. 5 is a diagram of the BE4 system disrupting splice site acceptors (SA), splice donors (SD), or generate STOP codons. Figure discloses SEQ ID NOS 1497-1499 and 1501-1503, respectively, in order of appearance.
FIG. 6 is a chart summarizing off-target binding sites of sgRNAs employed to disrupt target genes.
FIG. 7 is a graph summarizing flow cytometry (FC) data of the percentage of cells edited with BE4 or Cas9 that exhibit reduced protein expression. Cells were either gated to B2M or CD3, the latter being a proxy for TRAC expression.
FIG. 8A is a scatter plot of FACS data of unedited control cells. FIG. 8B is a scatter plot of FACS data of cells that have been edited at the B2M, TRAC, and PD1 loci.
FIG. 9 is a graph illustration the effectiveness of the base editing techniques described herein to modify specific genes that can negatively impact CAR-T immunotherapy.
FIG. 10 is a diagram depicting a droplet digital PCR (ddPCR) protocol to detect and quantify gene modifications and translocations.
FIG. 11 presents two graphs showing the data generated from next generation sequencing (NGS) analysis or ddPCR of cells edited using either the BE4 system or the Cas9 system.
FIG. 12 is a schematic diagram that illustrates the role Cbl-b plays in suppressing T cell activation.
FIG. 13 is a graph depicting the efficiency of Cbl-b knockdown by disruption of splice sites. SA=Splice Acceptor; SD=Splice Donor; STOP—generated STOP codon; 2° Only=secondary antibody only; C373 refers to a loss of function variant (C373R); RL1-A::APC-A=laser; ICS=intracellular staining.
FIG. 14 is a graph illustrating the rate of Cas12b-mediated indels in the GRIN2B and DNMT1 genes in T cells. EP denotes electroporation.
FIG. 15 is a graph summarizing fluorescence assisted cell sorting (FACS) data of cells transduced via electroporation (EP) with by Cas12b and guide RNAs specific for TRAC, GRIN2B, and DNMT1 and gated for CD3.
FIG. 16 is a scatter plot of fluorescence assisted cell sorting data of cells transduced CAR-P2A-mCherry lentivirus demonstrating CAR expression.
FIG. 17 is a scatter plot of fluorescence assisted cell sorting data demonstrating CAR expression in cells transduced with a poly(1,8-octanediol citrate) (POC) lentiviral vector.
FIG. 18 is graph showing that BE4 produced efficient, durable gene knockout with high product purity.
FIG. 19A is a representative FACS analysis showing loss of surface expression of a protein due to gene knockout by BE4 or spCas9. FIG. 19B is a graph show that gene knockout by BE4 or spCas9 produces loss of B2M surface expression.
FIG. 20 is a schematic depicting the locations of B2M, TRAC, and PD-1 target sites. Translocations can be detected when B2M, TRAC, and PD-1 sequences recombine.
FIG. 21 is a graph showing that multiplexed base editing does not significantly impair cell expansion.
FIG. 22 is a graph showing that BE4 generated triple-edited T cells with similar on-target editing efficiency and cellular phenotype as spCas9.
FIG. 23 depicts flow cytometry analysis showing the generation of triple-edited CD3-, B2M−, PD1− T cells.
FIG. 24 depicts flow cytometry analysis showing the CAR expression in BE4 and Cas9 edited cells.
FIG. 25 is a graph showing CAR-T cell killing or antigen positive cells.
FIG. 26 are graphs showing that Cas12b and BE4 can be paired for efficient multiplex editing in T cells.
FIG. 27 is a graph showing that Cas12b can direct insertion of a chimeric antigen receptor (CAR) into a locus by introducing into a cell a double-stranded DNA template encoding the CAR in the presence of a Cas12 nuclease and a sgRNA targeting the locus.
FIGS. 28A-B are depictions showing base editing approaches to silence genes for engineering CAR-T cells. FIG. 28A illustrates targets for CAR-T editing. FIG. 28B shows two strategies for silencing with base editors: creating a stop codon with CBE and splice disruption with CBE.
FIG. 29 is an illustrative cartoon representation that, in some cases, Multiple CAR-Ts required to address clonality of disease in AML.
FIG. 30 is an illustrative cartoon representation that, in some cases, multiple edits may be required to eliminate fratricide for a T-ALL CAR-T combination.
FIGS. 31A-B illustrate the results of experiments testing four simultaneous base edits for T-ALL; CAR-T does not impact yield compared to nuclease. FIG. 31A illustrates the theoretical yield in contest of quad T-ALL edits for no electroporation (no EP; dark circles), EP only (dark squares), CBE Variant 1 (light, upright triangles), CBE Variant 2 (light, upside down triangles), and Cas9 (light diamonds). FIG. 31B illustrates viability of cells post electroporation. For each set of results at 24 hours (hr), 48 hr, 72 hr, 96 hr, and 168 hr, results are shown from right to left as no EP, EP only, CBE variant 1, CBE variant 2, and Cas9.
FIG. 32 illustrates that greater than 90% quad knockout by BE4. Electroporation was conducted at a 5M cell scale and the quad edit was performed in a single electroporation (EP) step. Greater than 90% edit efficiency was achieved for all 4 targets with rBE4. For each result group, data is from left to right: PD1, CD7, TRAC, and CD52.
FIG. 33 illustrates that base editing causes no difference in cell yields. Data is: no EP (circles), EP only (squares), rBE4 (triangles), ppBE4 (upside down triangles), and Cas9 (diamonds).
FIG. 34 illustrates that edited CAR-Ts target CD3 and CD7 on model tumor cells. Data is: UTD 1:1 (circles), 7CAR8 1:1 (squares), and 3CAR2 1:1 (triangles).
FIG. 35 is a schematic drawing of an exemplary CD7 CAR-T cell for targeting T-ALL Tumor cells.
FIG. 36 is a flow chart depicting a clinical protocol for treating patients with CD7 CAR-T cells.
FIGS. 37A-37C depict CD7 CAR-T cell production. FIG. 37A is a flow chart depicting a protocol for producing TALL017 heterogenous thawed CD7 CAR-T cells. FIG. 37B is a flow chart depicting a protocol for producing TALL083 CD7 CAR-T cells. FIG. 37C is a scatter plot of fluorescence assisted cell sorting data demonstrating that TALL017 CAR-T cells are highly activated post-thaw.
FIG. 38 is a graph depicting total editing in TALL017 and TALL038 CD7 CAR-T cells by next generation sequencing (NGS).
FIGS. 39A-39B depict 24 hr post thaw protein expression of TCRα/β, CD7, and CD52 in TALL017 and TALL038 CD7 CAR-T cells. FIG. 39A is a scatter plot of fluorescence assisted cell sorting data for TALL017 and TALL038 CD7 CAR-T cells. FIG. 39B is a graph depicting residual protein expression via FACS.
FIG. 40 depicts a gating scheme for an identity panel.
FIG. 41 is a graph depicting the identity of final cell product as a mix of CD2+/− and CD56+/− cells.
FIG. 42 is a graph depicting 24 h and 48h post-thaw CD7 CAR-T cell expression.
FIG. 43 includes graphs depicting that CD25 expression post thaw is lower in TALL038 CD7 CAR-T cells compared to TALL017 CD7 CAR-T cells.
FIG. 44 is a flow chart depicting a CD7 CAR-T bead based potency protocol for in vitro characterization.
FIGS. 45A-45B depict that TALL038 CD7 CAR-T cells release IFNγ, TNFα, and IL-2 compared in response to CD7 antigen. FIG. 45A is a graph depicting the release of IFNγ by TALL038 CD7 CAR-T cells. FIG. 45B is a graph depicting the release of TNFα, IL-10 and IL-2 by TALL038 CD7 CAR-T cells.
FIG. 46 is a schematic depicting a co-culture protocol for measurement of CAR directed T-cell killing of tumor cells.
FIGS. 47A-47B depict that TALL038 CD7 CAR-T cells exhibit increased CCRF when re-challenged compared to TALL017 CAR T cells. FIG. 47A is a graph depicting a primary challenge. FIG. 47B is a graph depicting a secondary challenge.
FIG. 48 is a graph depicting Bioluminescent Radiance Data at Day 10 Post CCRF Implant/Day −1 to CD7 CAR-T Treatment (mean, SEM). Total flux is measured on a linear scale.
FIGS. 49A-49B depict Bioluminescent Radiance Data at Day 19 Post CCRF Implant/Day 8 Post CD7 CAR-T Treatment (mean, SEM). FIG. 49A is a graph depicting mean tumor burden. Total flux is measured on a linear scale. FIG. 49B is a graph depicting mean mouse weights.
FIGS. 50A-50B depict Bioluminescent Radiance Data at Day 38 Post CCRF Implant/Day 27 Post CD7 CAR-T Treatment (mean, SEM). FIG. 50A is a graph depicting mean tumor burden. Total flux is measured on a linear scale. FIG. 50B is a graph depicting mean tumor burden. Total flux is measured on a logarithmic scale.
FIGS. 51A-51B depict Bioluminescent Radiance Data at Day 38 Post CCRF Implant/Day 27 Post CD7 CAR-T Treatment (individual mice). FIG. 51A is a graph depicting mean tumor burden. Total flux is measured on a linear scale. FIG. 51B is a graph depicting mean tumor burden. Total flux is measured on a logarithmic scale.
FIGS. 52A-52B depict Bioluminescent Radiance Data at Day 38 Post CCRF Implant/Day 27 Post CD7 CAR-T Treatment (individual mice). FIG. 52A includes graphs depicting mean tumor burden. Total flux is measured on a linear scale (Top) and on a logarithmic scale (bottom). FIG. 52B includes graphs depicting mean tumor burden. Total flux is measured on a linear scale (Top) and on a logarithmic scale (bottom).
FIG. 53 is a graph depicting editing efficiency of CD5 gRNA candidates.
FIG. 54 is a graph depicting editing efficiency via NGS of CD5 gRNA candidates g103 and g104 in combination with BE4.
FIG. 55 is a schematic depicting production of an exemplary CD5 CAR construct.
FIG. 56 is a graph depicting CAR expression from a lentiviral vector (LVV) screen using CD5 gRNA candidates g103 and g104.
FIG. 57 is a graph depicting CAR LVV expression based on hinge sequence.
FIGS. 58A-58D are graphs depicting edited and unedited constructs are fully cytotoxic against CD5+ CCRF in live imaging cell killing assay.
FIGS. 59A-59B depict cytokine (IFNγ) production by CD5 CAR LVV constructs using sgRNA 103 or sgRNA 104 in the presence of CD5+ CCRF-CEM leukemia cell lines. FIG. 59A (left) is a graph depicting IFNγ production with CD5 CAR LVV (LV63-69) transduced T cells alone or with CCRF cells edited using sgRNA 103. FIG. 59A (right) is a graph depicting IFNγ production with CD5 CAR LVV (LV63-69) transduced T cells alone or with CCRF cells edited using sgRNA 104. FIG. 59A (bottom) is a graph depicting IFNγ production using unedited CD5 CAR LVV (LV63-69) transduced T cells or CCRF cells. FIG. 59B (top) is a graph depicting IFNγ production with CD5 CAR LVV (LV63-69) transduced T cells alone either unedited or edited using sgRNA 103 or 104. FIG. 59B (bottom) is a graph depicting IFNγ production with CD5 CAR LVV (LV63-69) transduced CCRF cells either unedited or edited using sgRNA 103 or 104.



DETAILED DESCRIPTION OF THE INVENTION
The present invention features genetically modified immune cells having enhanced anti-neoplasia activity, resistance to immune suppression, and decreased risk of eliciting a graft versus host reaction or a host versus graft reaction, or a combination thereof. The present invention also features methods for producing and using these modified immune cells (e.g., immune effector cells, such as T cells).
In one embodiment, a subject having or having a propensity to develop graft versus host disease (GVHD) is administered a CAR-T cell that lacks or has reduced levels of functional TRAC. In one embodiment, a subject having or having a propensity to develop host versus graft disease (HVGD) is administered a CAR-T cell that lacks or has reduced levels of functional beta2 microglobulin (B2M).
The modification of immune effector cells to express chimeric antigen receptors and to knockout or knockdown specific genes to diminish the negative impact that their expression can have on immune cell function is accomplished using a base editor system comprising a cytidine deaminase or adenosine deaminase as described herein.
Autologous, patient-derived chimeric antigen receptor-T cell (CAR-T) therapies have demonstrated remarkable efficacy in treating some hematologic cancers. While these products have led to significant clinical benefit for patients, the need to generate individualized therapies creates substantial manufacturing challenges and financial burdens. Allogeneic CAR-T therapies were developed as a potential solution to these challenges, having similar clinical efficacy profiles to autologous products while treating many patients with cells derived from a single healthy donor, thereby substantially reducing cost of goods and lot-to-lot variability.
Most first-generation allogeneic CAR-Ts use nucleases to introduce two or more targeted genomic DNA double strand breaks (DSBs) in a target T cell population, relying on error-prone DNA repair to generate mutations that knock out target genes in a semi-stochastic manner. Such nuclease-based gene knockout strategies aim to reduce the risk of graft-versus-host-disease and host rejection of CAR-Ts. However, the simultaneous induction of multiple DSBs results in a final cell product containing large-scale genomic rearrangements such as balanced and unbalanced translocations, and a relatively high abundance of local rearrangements including inversions and large deletions. Furthermore, as increasing numbers of simultaneous genetic modifications are made by induced DSBs, considerable genotoxicity is observed in the treated cell population. This has the potential to significantly reduce the cell expansion potential from each manufacturing run, thereby decreasing the number of patients that can be treated per healthy donor.
Base editors (BEs) are a class of emerging gene editing reagents that enable highly efficient, user-defined modification of target genomic DNA without the creation of DSBs. Here, an alternative means of producing allogeneic CAR-T cells is proposed by using base editing technology to reduce or eliminate detectable genomic rearrangements while also improving cell expansion. As shown herein, in contrast to a nuclease-only editing strategy, concurrent modification of one or more, for example, one, two, three, four, five, six, seven, eight, night, ten, or more, genetic loci by base editing produces highly efficient gene knockouts with no detectable translocation events.
In some embodiments, at least one or more genes or regulatory elements thereof are modified in an immune cell with the base editing compositions and methods provided herein. In some embodiments, the at least one or more genes or regulatory elements thereof are selected from ACAT1, ACLY, ADORA2A, AXL, B2M, BATF, BCL2L11, BTLA, CAMK2D, cAMP, CASP8, CBLB, CCR5, CD2, CD3D, CD3E, CD3G, CD4, CD5, CD7, CD8A, CD33, CD38, CD52, CD70, CD82, CD86, CD96, CD123, CD160, CD244, CD276, CDK8, CDKN1B, Chi3l1, CIITA, CISH, CSF2CSK, CTLA-4, CUL3, Cypl1a1, DCK, DGKA, DGKZ, DHX37, ELOB(TCEB2), ENTPD1 (CD39), FADD, FAS, GATA3, IL6, IL6R, IL10, IL10RA, IRF4, IRF8, JUNB, Lag3, LAIR-1 (CD305), LDHA, LIF, LYN, MAP4K4, MAPK14, MCJ, MEF2D, MGAT5, NR4A1, NR4A2, NR4A3, NT5E (CD73), ODC1, OTULINL (FAM105A), PAGI, PDCD1, PDIA3, PHD1 (EGLN2), PHD2 (EGLN1), PHD3 (EGLN3), PIK3CD, PIKFYVE, PPARa, PPARd, PRDMI1, PRKACA, PTEN, PTPN2, PTPN6, PTPN11, PVRIG (CDI12R), RASA2, RFXANK, SELPG/PSGL1, SIGLEC15, SLA, SLAMF7, SOCS1, Spry1, Spry2, STK4, SUV39, HITET2, TGFbRII, TIGIT, Tim-3, TMEM222, TNFAIP3, TNFRSF8 (CD30), TNFRSF10B, TOX, TOX2, TRAC, TRBC1, TRBC2, UBASH3A, VHL, VISTA, XBP1, YAP1, and ZC3H12A. In some embodiments, the at least one or more genes or regulatory elements thereof are selected from CD3, CD5, CD7, CD33, CD123, TRAC, LAG-3, FAS, CD52, TRBC1, TRBC2, B2M, and CIITA and PD-1. In some embodiments, the modified immune cell comprises a modification in CD5 and at least one or more genes or regulatory elements thereof selected from TRAC, LAG-3, FAS, CD52, TRBC1, TRBC2, B2M, and CIITA and PD-1. In some embodiments, the modified immune cell comprises a modification in CD7 and at least one or more genes or regulatory elements thereof selected from TRAC, LAG-3, FAS, CD52, TRBC1, TRBC2, B2M, and CIITA and PD-1. In some embodiments, the modified immune cell comprises a modification in CD33 and at least one or more genes or regulatory elements thereof selected from TRAC, LAG-3, FAS, CD52, TRBC1, TRBC2, B2M, and CIITA and PD-1. In some embodiments, the modified immune cell comprises a modification in CD3 and at least one or more genes or regulatory elements thereof selected from TRAC, LAG-3, FAS, CD52, TRBC1, TRBC2, B2M, and CIITA and PD-1. In some embodiments, the modified immune cell comprises a modification in CD123 and at least one or more genes or regulatory elements thereof selected from TRAC, LAG-3, FAS, CD52, TRBC1, TRBC2, B2M, and CIITA and PD-1. Multiplex editing of genes may be useful in the creation of CAR-T cell therapies with improved therapeutic properties. This method addresses known limitations of multiplex-edited T cell products and are a promising development towards the next generation of precision cell-based therapies.
In one aspect, provided herein is a universal CAR-T cell. In some embodiments, the CAR-T cell described herein is an allogeneic cell. In some embodiments, the universal CAR-T cell is an allogeneic T cell that can be used to express a desired CAR, and can be universally applicable, irrespective of the donor and the recipient's immunogenic compatibility. An allogenic immune cell may be derived from one or more donors. In certain embodiments, the allogenic immune cell is derived from a single human donor. For example, the allogenic T cell may be derived from PBMCs of a single healthy human donor. In certain embodiments, the allogenic immune cell is derived from multiple human donors. In some embodiments, an universal CAR-T cell may be generated, as described herein by using gene modification to introduce concurrent edits at multiple gene loci, for example, three, four, five, six, seven, eight, nine, ten or more genetic loci. A modification, or concurrent modifications as described herein may be a genetic editing, such as a base editing, generated by a base editor. The base editor may be a C base editor or A base editor. As is discussed herein, base editing may be used to achieve a gene disruption, such that the gene is not expressed. A modification by base editing may be used to achieve a reduction in gene expression. In some embodiments base editor may be used to introduce a genetic modification such that the edited gene does not generate a structurally or functionally viable protein product. In some embodiments, a modification, such as the concurrent modifications described herein may comprise a genetic editing, such as base editing, such that the expression or functionality of the gene product is altered in any way. For example, the expression of the gene product may be enhanced or upregulated as compared to baseline expression levels. In some embodiments the activity or functionality of the gene product may be upregulated as a result of the base editing, or multiple base editing events acting in concert.
In some embodiments, generation of universal CAR-T cell may be advantageous over autologous T cell (CAR-T), which may be difficult to generate for an urgent use. Allogeneic approaches are preferred over autologous cell preparation for a number of situations related to uncertainty of engineering autologous T cells to express a CAR and finally achieving the desired cellular products for a transplant at the time of medical emergency. However, for allogeneic T cells, or “off-the-shelf” T cells, it is important to carefully negotiate the host's reactivity to the CAR-T cells (HVGD) as well as the allogeneic T cell's potential hostility towards a host cell (GVHD). Given the scenario, base editing can be successfully used to generate multiple simultaneous gene editing events, such that (a) it is possible to reduce or down regulate expression of antigens to generate a fratricide resistant immune cell; (b) it is possible to generate a platform cell type that is devoid of or expresses low amounts of an endogenous T cell receptor, for example, a TCR alpha chain (such a via base editing of TRAC), or a TCR beta chain (such a through base editing of TRBC1/TRBC2); and/or (c) it is possible to reduce or down regulate expression of antigens that may be incompatible to a host tissue system and vice versa.
In some embodiments, the methods described herein can be used to generate an autologous T cell expressing a CAR-T. In some embodiments, multiple base editing events can be accomplished in a single electroporation event, thereby reducing electroporation event associated toxicity. Any known methods for incorporation of exogenous genetic material into a cell may be used to replace electroporation, and such methods known in the art are hereby contemplated for use in any of the methods described herein.
In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD2 and expresses a CD2 chimeric antigen receptor containing a CD2 co-stimulatory domain. In some embodiments, the CD2 modified immune cell administered to a subject is further modified in one or more genes or regulatory elements (e.g., CD52, TRAC, PD-1) with the base editing compositions and methods provided herein.
As shown herein, base editing in combination with a CAR insertion is a useful strategy for generating fratricide resistant allogeneic T cells with minimal genomic rearrangements. Multiplex editing of genes may also be useful in the creation of CAR-T cell therapies with improved therapeutic properties. This method addresses known limitations of CAR-T therapy and is a promising development towards the next generation of precision cell based therapies.
In one experiment, the base editor BE4 demonstrated high efficiency multiplex base editing of three cell surface targets in T cells (TRAC, B2M, and PD-1), knocking out gene expression by 95%, 95% and 88%, respectively, in a single electroporation to generate cell populations with high percentages of cells with reduced protein expression of B2M and CD3. Editing each of these genes may be useful in the creation of CAR-T cell therapies with improved therapeutic properties. Each of the genes was silenced by a single targeted base change (C to T) without the creation of double strand breaks. As a result, the BE4-treated cells also did not show any measurable translocations (large-scale genomic rearrangements), whereas cells receiving the same three edits with a nuclease did show detectable genomic rearrangements.
Thus, coupling nuclease-based knockout of the TRAC gene with simultaneous BE-mediated knockout of two additional genes yields a homogeneous allogeneic T cell population with minimal genomic rearrangements, enabling the targeted insertion of a CAR transgene at the TRAC locus. Taken together, this demonstrates that base editing alone or in combination with a single nuclease knockout and CAR insertion is a useful strategy for generating allogeneic T cells with minimal genomic rearrangements compared to nuclease-alone approaches. This method addresses known limitations of multiplex-edited T cell products and are a promising development towards the next generation of precision cell based therapies.
Chimeric Antigen Receptor and CAR-T Cells
The invention provides immune cells modified using nucleobase editors described herein that express chimeric antigen receptors (CARs). Modification of immune cells to express a chimeric antigen receptor can enhance an immune cell's immunoreactive activity, wherein the chimeric antigen receptor has an affinity for an epitope on an antigen, wherein the antigen is associated with an altered fitness of an organism. For example, the chimeric antigen receptor can have an affinity for an epitope on a protein expressed in a neoplastic cell. Because the CAR-T cells can act independently of major histocompatibility complex (MHC), activated CAR-T cells can kill the neoplastic cell expressing the antigen. The direct action of the CAR-T cell evades neoplastic cell defensive mechanisms that have evolved in response to MHC presentation of antigens to immune cells.
In some embodiments, the invention provides immune effector cells that express chimeric antigen receptors that target B cells involved in an autoimmune response (e.g., B cells of a subject that express antibodies generated against the subject's own tissues).
Some embodiments comprise autologous immune cell immunotherapy, wherein immune cells are obtained from a subject having a disease or altered fitness characterized by cancerous or otherwise altered cells expressing a surface marker. The obtained immune cells are genetically modified to express a chimeric antigen receptor and are effectively redirected against specific antigens. Thus, in some embodiments, immune cells are obtained from a subject in need of CAR-T immunotherapy. In some embodiments, these autologous immune cells are cultured and modified shortly after they are obtained from the subject. In other embodiments, the autologous cells are obtained and then stored for future use. This practice may be advisable for individuals who may be undergoing parallel treatment that will diminish immune cell counts in the future. In allogeneic immune cell immunotherapy, immune cells can be obtained from a donor other than the subject who will be receiving treatment. In some embodiments, immune cells are obtained from a healthy subject or donor and are genetically modified to express a chimeric antigen receptor and are effectively redirected against specific antigens. The immune cells, after modification to express a chimeric antigen receptor, are administered to a subject for treating a neoplasia (e.g., leukemia). In some embodiments, immune cells to be modified to express a chimeric antigen receptor can be obtained from pre-existing stock cultures of immune cells.
Immune cells and/or immune effector cells can be isolated or purified from a sample collected from a subject or a donor using standard techniques known in the art. For example, immune effector cells can be isolated or purified from a whole blood sample by lysing red blood cells and removing peripheral mononuclear blood cells by centrifugation. The immune effector cells can be further isolated or purified using a selective purification method that isolates the immune effector cells based on cell-specific markers such as CD25, CD3, CD4, CD8, CD28, CD45RA, or CD45RO. In one embodiment, CD25+ is used as a marker to select regulatory T cells. In one embodiment, CD4+ is used as a marker to select T cells. In one embodiment, CD8+ is used as a marker to select T cells. In one embodiment, CD4+ and CD8+ are used as a marker to select T cells. In one embodiment, CD4+ and CD25+ are used as a marker to select T cells.
In another embodiment, the invention provides T cells that have targeted gene knockouts at the TCR constant region (TRAC), which is responsible for TCRαβ surface expression. TCRαβ-deficient CAR T cells are compatible with allogeneic immunotherapy (Qasim et al., Sci. Transl. Med. 9, eaaj2013 (2017); Valton et al., Mol Ther. 2015 September; 23(9): 1507-1518). If desired, residual TCRαβ T cells are removed using CliniMACS magnetic bead depletion to minimize the risk of GVHD. In another embodiment, the invention provides donor T cells selected ex vivo to recognize minor histocompatibility antigens expressed on recipient hematopoietic cells, thereby minimizing the risk of graft-versus-host disease (GVHD), which is the main cause of morbidity and mortality after transplantation (Warren et al., Blood 2010; 115(19):3869-3878). Another technique for isolating or purifying immune effector cells is flow cytometry. In fluorescence activated cell sorting a fluorescently labelled antibody with affinity for an immune effector cell marker is used to label immune effector cells in a sample. A gating strategy appropriate for the cells expressing the marker is used to segregate the cells. For example, T lymphocytes can be separated from other cells in a sample by using, for example, a fluorescently labeled antibody specific for an immune effector cell marker (e.g., CD4, CD8, CD28, CD45) and corresponding gating strategy. In one embodiment, a CD45 gating strategy is employed. In some embodiments, a gating strategy for other markers specific to an immune effector cell is employed instead of, or in combination with, the CD45 gating strategy. In one embodiment, a CD4 gating strategy is employed. In one embodiment, a CD8 gating strategy is employed. In one embodiment, a CD25 gating strategy is employed. In one embodiment, a CD4 and CD8 gating strategy is employed. In one embodiment, a CD4 and CD25 gating strategy is employed. In some embodiments, a gating strategy for other markers specific to an immune effector cell is employed instead of, or in combination with, the CD4, CD25 and/or CD8 gating strategy. In some embodiments, the gating strategy as provided in FIG. 40 is employed.
The immune effector cells contemplated in the invention are effector T cells. In some embodiments, the effector T cell is a naïve CD8+ T cell, a cytotoxic T cell, a natural killer T (NKT) cell, a natural killer (NK) cell, or a regulatory T (Treg) cell. In some embodiments, the effector T cells are thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. In some embodiments the immune effector cell is a CD4+ CD8+ T cell or a CD4− CD8− T cell. In some embodiments the immune effector cell is a T helper cell. In some embodiments the T helper cell is a T helper 1 (Th1), a T helper 2 (Th2) cell, or a helper T cell expressing CD4 (CD4+ T cell). In some embodiments, immune effector cells are effector NK cells. In some embodiments, the immune effector cell is any other subset of T cells. The modified immune effector cell may express, in addition to the chimeric antigen receptor, an exogenous cytokine, a different chimeric receptor, or any other agent that would enhance immune effector cell signaling or function. For example, co-expression of the chimeric antigen receptor and a cytokine may enhance the CAR-T cell's ability to lyse a target cell.
Chimeric antigen receptors as contemplated in the present invention comprise an extracellular binding domain, a transmembrane domain, and an intracellular domain. Binding of an antigen to the extracellular binding domain can activate the CAR-T cell and generate an effector response, which includes CAR-T cell proliferation, cytokine production, and other processes that lead to the death of the antigen expressing cell. In some embodiments of the present invention, the chimeric antigen receptor further comprises a linker. In some embodiments, the linker is a (GGGGS)n linker (SEQ ID NO: 109). In some embodiments, the linker is a (GGGGS)3 linker (SEQ ID NO: 110). In some embodiments, a CAR of the present invention includes a leader peptide sequence (e.g., N-terminal to the antigen binding domain). An exemplary leader peptide amino acid sequence is: METDTLLLWVLLLWVPGSTG (SEQ ID NO: 55).
The extracellular binding domain of a chimeric antigen receptor contemplated herein comprises an amino acid sequence of an antibody, or an antigen binding fragment thereof, that has an affinity for a specific antigen. In various embodiments, the CAR specifically binds 5T4. Exemplary anti-5T4 CARs include, without limitation, CART-5T4 (Oxford BioMedica plc) and UCART-5T4 (Cellectis SA).
In various embodiments, the CAR specifically binds Alpha-fetoprotein. Exemplary anti-Alpha-fetoprotein CARs include, without limitation, ET-1402 (Eureka Therapeutics Inc).
In various embodiments, the CAR specifically binds Axl. Exemplary anti-Axl CARs include, without limitation, CCT-301-38 (F1 Oncology Inc).
In various embodiments, the CAR specifically binds B7H6. Exemplary anti-B7H6 CARs include, without limitation, CYAD-04 (Celyad SA).
In various embodiments, the CAR specifically binds BCMA. Exemplary anti-BCMA CARs include, without limitation, ACTR-087+SEA-BCMA (Seattle Genetics Inc), ALLO-715 (Cellectis SA), ARI-0002 (Institut d'Investigacions Biomediques August Pi I Sunyer), bb-2121 (bluebird bio Inc), bb-21217 (bluebird bio Inc), CART-BCMA (University of Pennsylvania), CT-053 (Carsgen Therapeutics Ltd), Descartes-08 (Cartesian Therapeutics), FCARH-143 (Juno Therapeutics Inc), ICTCAR-032 (Innovative Cellular Therapeutics Co Ltd), IM21 CART (Beijing Immunochina Medical Science & Technology Co Ltd), JCARH-125 (Memorial Sloan-Kettering Cancer Center), KITE-585 (Kite Pharma Inc), LCAR-B38M (Nanjing Legend Biotech Co Ltd), LCAR-B4822M (Nanjing Legend Biotech Co Ltd), MCARH-171 (Memorial Sloan-Kettering Cancer Center), P-BCMA-101 (Poseida Therapeutics Inc), P-BCMA-ALLO1 (Poseida Therapeutics Inc), spCART-269 (Shanghai Unicar-Therapy Bio-medicine Technology Co Ltd), and BCMA02/bb2121 (bluebird bio Inc). The polypeptide sequence of the BCMA02/bb2121 CAR is provided below:







MALPVTALLLPLALLLHAARPDIVLTQSPPSLAMSLGKRATISCRASESV


 


TILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTI


 


DPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKG


 


QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGW


 


INTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDY


 


SYAMDYWGQGTSVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPA


 


AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF


 


KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQ


 


LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE


 


AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR






In various embodiments, the CAR specifically binds CCK2R. Exemplary anti-CCK2R CARs include, without limitation, anti-CCK2R CAR-T adaptor molecule (CAM)+anti-FITC CAR T-cell therapy (cancer), Endocyte/Purdue (Purdue University),
In various embodiments, the CAR specifically binds a CD antigen. Exemplary anti-CD antigen CARs include, without limitation, VM-802 (ViroMed Co Ltd).
In various embodiments, the CAR specifically binds CD123. Exemplary anti-CD123 CARs include, without limitation, MB-102 (Fortress Biotech Inc), RNA CART123 (University of Pennsylvania), SFG-iMC-CD123.zeta (Bellicum Pharmaceuticals Inc), and UCART-123 (Cellectis SA).
In various embodiments, the CAR specifically binds CD133. Exemplary anti-CD133 CARs include, without limitation, KD-030 (Nanjing Kaedi Biotech Inc).
In various embodiments, the CAR specifically binds CD138. Exemplary anti-CD138 CARs include, without limitation, ATLCAR.CD138 (UNC Lineberger Comprehensive Cancer Center) and CART-138 (Chinese PLA General Hospital).
In various embodiments, the CAR specifically binds CD171. Exemplary anti-CD171 CARs include, without limitation, JCAR-023 (Juno Therapeutics Inc).
In various embodiments, the CAR specifically binds CD19. Exemplary anti-CD19 CARs include, without limitation, 1928z-41BBL (Memorial Sloan-Kettering Cancer Center), 1928z-E27 (Memorial Sloan-Kettering Cancer Center), 19-28z-T2 (Guangzhou Institutes of Biomedicine and Health), 4G7-CARD (University College London), 4SCAR19 (Shenzhen Geno-Immune Medical Institute), ALLO-501 (Pfizer Inc), ATA-190 (QIMR Berghofer Medical Research Institute), AUTO-1 (University College London), AVA-008 (Avacta Ltd), axicabtagene ciloleucel (Kite Pharma Inc), BG-T19 (Guangzhou Bio-gene Technology Co Ltd), BinD-19 (Shenzhen BinDeBio Ltd.), BPX-401 (Bellicum Pharmaceuticals Inc), CAR19h28TM41BBz (Westmead Institute for Medical Research), C-CAR-011 (Chinese PLA General Hospital), CDI9CART (Innovative Cellular Therapeutics Co Ltd), CIK-CAR.CD19 (Formula Pharmaceuticals Inc), CLIC-1901 (Ottawa Hospital Research Institute), CSG-CD19 (Carsgen Therapeutics Ltd), CTL-119 (University of Pennsylvania), CTX-101 (CRISPR Therapeutics AG), DSCAR-01 (Shanghai Hrain Biotechnology), ET-190 (Eureka Therapeutics Inc), FT-819 (Memorial Sloan-Kettering Cancer Center), ICAR-19 (Immune Cell Therapy Inc), IM19 CAR-T (Beijing Immunochina Medical Science & Technology Co Ltd), JCAR-014 (Juno Therapeutics Inc), JWCAR-029 (MingJu Therapeutics (Shanghai) Co., Ltd), KD-C-19 (Nanjing Kaedi Biotech Inc), LinCART19 (iCell Gene Therapeutics), lisocabtagene maraleucel (Juno Therapeutics Inc), MatchCART (Shanghai Hrain Biotechnology), MB-CART19.1 (Shanghai Children's Medical Center), PBCAR-0191 (Precision BioSciences Inc), PCAR-019 (PersonGen Biomedicine (Suzhou) Co Ltd), pCAR-19B (Chongqing Precision Biotech Co Ltd), PZ-01 (Pinze Lifetechnology Co Ltd), RB-1916 (Refuge Biotechnologies Inc), SKLB-083019 (Chengdu Yinhe Biomedical Co Ltd), spCART-19 (Shanghai Unicar-Therapy Bio-medicine Technology Co Ltd), TBI-1501 (Takara Bio Inc), TC-110 (TCR2 Therapeutics Inc), TI-1007 (Timmune Biotech Inc), tisagenlecleucel (Abramson Cancer Center of the University of Pennsylvania), U-CART (Shanghai Bioray Laboratory Inc), UCART-19 (Wugen Inc), UCART-19 (Cellectis SA), vadacabtagene leraleucel (Memorial Sloan-Kettering Cancer Center), XLCART-001 (Nanjing Medical University), and yinnuokati-19 (Shenzhen Innovation Immunotechnology Co Ltd).
In various embodiments, the CAR specifically binds CD2. Exemplary anti-CD2 CARs include, without limitation, UCART-2 (Wugen Inc).
In various embodiments, the CAR specifically binds CD20. Exemplary anti-CD20 CARs include, without limitation, ACTR-087 (National University of Singapore), ACTR-707 (Unum Therapeutics Inc), CBM-C20.1 (Chinese PLA General Hospital), MB-106 (Fred Hutchinson Cancer Research Center), and MB-CART20.1 (Miltenyi Biotec GmbH).
In various embodiments, the CAR specifically binds CD22. Exemplary anti-CD22 CARs include, without limitation, anti-CD22 CAR T-cell therapy (B-cell acute lymphoblastic leukemia), University of Pennsylvania (University of Pennsylvania), CD22-CART (Shanghai Unicar-Therapy Bio-medicine Technology Co Ltd), JCAR-018 (Opus Bio Inc), MendCART (Shanghai Hrain Biotechnology), and UCART-22 (Cellectis SA).
In various embodiments, the CAR specifically binds CD30. Exemplary anti-CD30 CARs include, without limitation, ATLCAR.CD30 (UNC Lineberger Comprehensive Cancer Center), CBM-C30.1 (Chinese PLA General Hospital), and Hu30-CD28zeta (National Cancer Institute).
In various embodiments, the CAR specifically binds CD33. Exemplary anti-CD33 CARs include, without limitation, anti-CD33 CAR gamma delta T-cell therapy (acute myeloid leukemia), TC BioPharm/University College London (University College London), CAR33VH (Opus Bio Inc), CART-33 (Chinese PLA General Hospital), CIK-CAR.CD33 (Formula Pharmaceuticals Inc), UCART-33 (Cellectis SA), and VOR-33 (Columbia University).
In various embodiments, the CAR specifically binds CD38. Exemplary anti-CD38 CARs include, without limitation, UCART-38 (Cellectis SA).
In various embodiments, the CAR specifically binds CD38 A2. Exemplary anti-CD38 A2 CARs include, without limitation, T-007 (TNK Therapeutics Inc).
In various embodiments, the CAR specifically binds CD4. Exemplary anti-CD4 CARs include, without limitation, CD4CAR (iCell Gene Therapeutics).
In various embodiments, the CAR specifically binds CD44. Exemplary anti-CD44 CARs include, without limitation, CAR-CD44v6 (Istituto Scientifico H San Raffaele).
In various embodiments, the CAR specifically binds CD5. Exemplary anti-CD5 CARs include, without limitation, CD5CAR (iCell Gene Therapeutics). Exemplary CD5 CAR amino acid sequences are provided below:








>5CAR-CH3-CD28TM-CD28-CD3Z











1
MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW
60



 


61
VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC
120


 


121
TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC
180


 


181
KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM
240


 


241
GIYYCQQYDESPWTFGGGTKLEMKGSGDPAEPKSPDKTHTCPGQPREPQVYTLPPSRDEL
300


 


301
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
360


 


361
QGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWV
420


 


421
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQ
480


 


481
LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE
540


 


541
RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
573


 








>5CAR-CD8aH-CD28TM-CD28-CD3Z











1
MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW
60



 


61
VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC
120


 


121
TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC
180


 


181
KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM
240


 


241
GIYYCQQYDESPWTFGGGTKLEMKGSGDPATTTPAPRPPTPAPTIASQPLSLRPEACRPA
300


 


301
AGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFTIFWVRSKRSRLLHSDYMNMTPR
360


 


361
RPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK
420


 


421
RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT
480


 


481
KDTYDALHMQALPPR
495


 








>5CAR-CD28H-CD28TM-CD28-CD3Z











1
MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW
60



 


61
VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC
120


 


121
TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC
180


 


181
KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM
240


 


241
GIYYCQQYDESPWTFGGGTKLEMKGSGDPAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPS
300


 


301
PLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTR
360


 


361
KHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP
420


 


421
EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA
480


 


481
LHMQALPPR
489


 








>5CAR-CH3-CD8aTM-41BB-CD3Z











1
MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW
60



 


61
VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC
120


 


121
TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC
180


 


181
KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM
240


 


241
GIYYCQQYDESPWTFGGGTKLEMKGSGDPAEPKSPDKTHTCPGQPREPQVYTLPPSRDEL
300


 


301
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
360


 


361
QGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKPTTTPAPRPPTPAPTIASQPLSLRPEA
420


 


421
CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMR
480


 


481
PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL
540


 


541
DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST
600


 


601
ATKDTYDALHMOALPPR
617


 








>5CAR-CD8aH-CD8aTM-41BB-CD3Z











1
MEFGLSWLFLVAILKGVQCIDAMGNIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNW
60



 


61
VKQAPGKGLRWMGWINTHTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFC
120


 


121
TRRGYDWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIKMTQSPSSMYASLGERVTITC
180


 


181
KASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLDYEDM
240


 


241
GIYYCQQYDESPWTFGGGTKLEMKGSGDPATTTPAPRPPTPAPTIASQPLSLRPEACRPA
300


 


301
AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQT
360


 


361
TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR
420


 


421
GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD
480


 


481
TYDALHMQALPPRX
494






In various embodiments, the CAR specifically binds CD7. Exemplary anti-CD7 CARs include, without limitation, CAR-pNK (PersonGen Biomedicine (Suzhou) Co Ltd), and CD7.CAR/28zeta CAR T cells (Baylor College of Medicine), UCART7 (Washington University in St Louis). An exemplary CD7 CAR amino acid sequence is as follows:








>7CAR8











1
MALPVTALLLPLALLLHAARPGSDIELTQSPAIMSASLGEEITLTCSASSSVSYMHWYQQ
60



 


61
KSGTSPKLLIYSTSNLASGVPSRFSGSGSGTFYSLTISSVEAEDAADYYCHQWSSYTFGG
120


 


121
GTKLEIKRGGGGSGGGGSGGGGSQVKLQESGGGLVKPGGSLKLSCAASGFTFSSYAMSWV
180


 


181
RQTPEKRLEWVATISSGGSYTYYPDSVKGRFTTSRDNAKNTLYLQMSSLRSEDTAMYYCA
240


 


241
RQDGYYPGWEANWGQGTTVTVSSAAATTTPAPRPPTPAPTLASQPLSLRPEACRPAAGGA
300


 


301
VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRK
360


 


361
HYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE
420


 


421
MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL
480


 


481
HMQALPPR
488






In various embodiments, the CAR specifically binds CDH17. Exemplary anti-CDH17 CARs include, without limitation, ARB-001.T (Arbele Ltd).
In various embodiments, the CAR specifically binds CEA. Exemplary anti-CEA CARs include, without limitation, HORC-020 (HumOrigin Inc).
In various embodiments, the CAR specifically binds Chimeric TGF-beta receptor (CTBR). Exemplary anti-Chimeric TGF-beta receptor (CTBR) CARs include, without limitation, CAR-CTBR T cells (bluebird bio Inc).
In various embodiments, the CAR specifically binds Claudin18.2. Exemplary anti-Claudin18.2 CARs include, without limitation, CAR-CLD18 T-cells (Carsgen Therapeutics Ltd) and KD-022 (Nanjing Kaedi Biotech Inc).
In various embodiments, the CAR specifically binds CLL1. Exemplary anti-CLL1 CARs include, without limitation, KITE-796 (Kite Pharma Inc).
In various embodiments, the CAR specifically binds DLL3. Exemplary anti-DLL3 CARs include, without limitation, AMG-119 (Amgen Inc).
In various embodiments, the CAR specifically binds Dual BCMA/TACI (APRIL). Exemplary anti-Dual BCMA/TACI (APRIL) CARs include, without limitation, AUTO-2 (Autolus Therapeutics Limited).
In various embodiments, the CAR specifically binds Dual CD19/CD22. Exemplary anti-Dual CD19/CD22 CARs include, without limitation, AUTO-3 (Autolus Therapeutics Limited) and LCAR-L10D (Nanjing Legend Biotech Co Ltd).
In various embodiments, the CAR specifically binds CD19.
In various embodiments, the CAR specifically binds Dual CLL1/CD33. Exemplary anti-Dual CLL1/CD33 CARs include, without limitation, ICG-136 (iCell Gene Therapeutics).
In various embodiments, the CAR specifically binds Dual EpCAM/CD3. Exemplary anti-Dual EpCAM/CD3 CARs include, without limitation, IKT-701 (Icell Kealex Therapeutics).
In various embodiments, the CAR specifically binds Dual ErbB/4ab. Exemplary anti-Dual ErbB/4ab CARs include, without limitation, LEU-001 (King's College London).
In various embodiments, the CAR specifically binds Dual FAP/CD3. Exemplary anti-Dual FAP/CD3 CARs include, without limitation, IKT-702 (Icell Kealex Therapeutics).
In various embodiments, the CAR specifically binds EBV. Exemplary anti-EBV CARs include, without limitation, TT-18 (Tessa Therapeutics Pte Ltd).
In various embodiments, the CAR specifically binds EGFR. Exemplary anti-EGFR CARs include, without limitation, anti-EGFR CAR T-cell therapy (CBLB MegaTAL, cancer), bluebird bio (bluebird bio Inc), anti-EGFR CAR T-cell therapy expressing CTLA-4 checkpoint inhibitor+PD-1 checkpoint inhibitor mAbs (EGFR-positive advanced solid tumors), Shanghai Cell Therapy Research Institute (Shanghai Cell Therapy Research Institute), CSG-EGFR (Carsgen Therapeutics Ltd), and EGFR-IL12-CART (Pregene (Shenzhen) Biotechnology Co Ltd).
In various embodiments, the CAR specifically binds EGFRvIII. Exemplary anti-EGFRvIII CARs include, without limitation, KD-035 (Nanjing Kaedi Biotech Inc) and UCART-EgfrVIII (Cellectis SA).
In various embodiments, the CAR specifically binds Flt3. Exemplary anti-Flt3 CARs include, without limitation, ALLO-819 (Pfizer Inc) and AMG-553 (Amgen Inc).
In various embodiments, the CAR specifically binds Folate receptor. Exemplary anti-Folate receptor CARs include, without limitation, EC17/CAR T (Endocyte Inc).
In various embodiments, the CAR specifically binds G250. Exemplary anti-G250 CARs include, without limitation, autologous T-lymphocyte cell therapy (G250-scFV-transduced, renal cell carcinoma), Erasmus Medical Center (Daniel den Hoed Cancer Center).
In various embodiments, the CAR specifically binds GD2. Exemplary anti-GD2 CARs include, without limitation, 1RG-CART (University College London), 4SCAR-GD2 (Shenzhen Geno-Immune Medical Institute), C7R-GD2.CART cells (Baylor College of Medicine), CMD-501 (Baylor College of Medicine), CSG-GD2 (Carsgen Therapeutics Ltd), GD2-CART01 (Bambino Gesu Hospital and Research Institute), GINAKIT cells (Baylor College of Medicine), iC9-GD2-CAR-IL-15 T-cells (UNC Lineberger Comprehensive Cancer Center), and IKT-703 (Icell Kealex Therapeutics).
In various embodiments, the CAR specifically binds GD2 and MUC1. Exemplary anti-GD2/MUC1 CARs include, without limitation, PSMA CAR-T (University of Pennsylvania).
In various embodiments, the CAR specifically binds GPC3. Exemplary anti-GPC3 CARs include, without limitation, ARB-002.T (Arbele Ltd), CSG-GPC3 (Carsgen Therapeutics Ltd), GLYCAR (Baylor College of Medicine), and TT-14 (Tessa Therapeutics Pte Ltd).
In various embodiments, the CAR specifically binds Her2. Exemplary anti-Her2 CARs include, without limitation, ACTR-087+trastuzumab (Unum Therapeutics Inc), ACTR-707+ trastuzumab (Unum Therapeutics Inc), CIDeCAR (Bellicum Pharmaceuticals Inc), MB-103 (Mustang Bio Inc), RB-H21 (Refuge Biotechnologies Inc), and TT-16 (Baylor College of Medicine).
In various embodiments, the CAR specifically binds IL13R. Exemplary anti-IL13R CARs include, without limitation, MB-101 (City of Hope) and YYB-103 (YooYoung Pharmaceuticals Co Ltd).
In various embodiments, the CAR specifically binds integrin beta-7. Exemplary anti-integrin beta-7 CARs include, without limitation, MMG49 CAR T-cell therapy (Osaka University).
In various embodiments, the CAR specifically binds LC antigen. Exemplary anti-LC antigen CARs include, without limitation, VM-803 (ViroMed Co Ltd) and VM-804 (ViroMed Co Ltd).
In various embodiments, the CAR specifically binds mesothelin. Exemplary anti-mesothelin CARs include, without limitation, CARMA-hMeso (Johns Hopkins University), CSG-MESO (Carsgen Therapeutics Ltd), iCasp9M28z (Memorial Sloan-Kettering Cancer Center), KD-021 (Nanjing Kaedi Biotech Inc), m-28z-T2 (Guangzhou Institutes of Biomedicine and Health), MesoCART (University of Pennsylvania), meso-CAR-T+PD-78 (MirImmune LLC), RB-M1 (Refuge Biotechnologies Inc), and TC-210 (TCR2 Therapeutics Inc).
In various embodiments, the CAR specifically binds MUC1. Exemplary anti-MUC1 CARs include, without limitation, anti-MUC1 CAR T-cell therapy+PD-1 knockout T cell therapy (esophageal cancer/NSCLC), Guangzhou Anjie Biomedical Technology/University of Technology Sydney (Guangzhou Anjie Biomedical Technology Co LTD), ICTCAR-043 (Innovative Cellular Therapeutics Co Ltd), ICTCAR-046 (Innovative Cellular Therapeutics Co Ltd), P-MUCIC-101 (Poseida Therapeutics Inc), and TAB-28z (OncoTab Inc).
In various embodiments, the CAR specifically binds MUC16. Exemplary anti-MUC16 CARs include, without limitation, 4H1128Z-E27 (Eureka Therapeutics Inc) and JCAR-020 (Memorial Sloan-Kettering Cancer Center).
In various embodiments, the CAR specifically binds nfP2X7. Exemplary anti-nfP2X7 CARs include, without limitation, BIL-022c (Biosceptre International Ltd).
In various embodiments, the CAR specifically binds PSCA. Exemplary anti-PSCA CARs include, without limitation, BPX-601 (Bellicum Pharmaceuticals Inc).
In various embodiments, the CAR specifically binds PSMA. CIK-CAR.PSMA (Formula Pharmaceuticals Inc), and P-PSMA-101 (Poseida Therapeutics Inc).
In various embodiments, the CAR specifically binds ROR1. Exemplary anti-ROR1 CARs include, without limitation, JCAR-024 (Fred Hutchinson Cancer Research Center).
In various embodiments, the CAR specifically binds ROR2. Exemplary anti-ROR2 CARs include, without limitation, CCT-301-59 (F1 Oncology Inc).
In various embodiments, the CAR specifically binds SLAMF7. Exemplary anti-SLAMF7 CARs include, without limitation, UCART-CS1 (Cellectis SA).
In various embodiments, the CAR specifically binds TRBC1. Exemplary anti-TRBC1 CARs include, without limitation, AUTO-4 (Autolus Therapeutics Limited).
In various embodiments, the CAR specifically binds TRBC2. Exemplary anti-TRBC2 CARs include, without limitation, AUTO-5 (Autolus Therapeutics Limited).
In various embodiments, the CAR specifically binds TSHR. Exemplary anti-TSHR CARs include, without limitation, ICTCAT-023 (Innovative Cellular Therapeutics Co Ltd).
In various embodiments, the CAR specifically binds VEGFR-1. Exemplary anti-VEGFR-1 CARs include, without limitation, SKLB-083017 (Sichuan University).
In various embodiments, the CAR is AT-101 (AbClon Inc); AU-101, AU-105, and AU-180 (Aurora Biopharma Inc); CARMA-0508 (Carisma Therapeutics); CAR-T (Fate Therapeutics Inc); CAR-T (Cell Design Labs Inc); CM-CX1 (Celdara Medical LLC); CMD-502, CMD-503, and CMD-504 (Baylor College of Medicine); CSG-002 and CSG-005 (Carsgen Therapeutics Ltd); ET-1501, ET-1502, and ET-1504 (Eureka Therapeutics Inc); FT-61314 (Fate Therapeutics Inc); GB-7001 (Shanghai GeneChem Co Ltd); IMA-201 (Immatics Biotechnologies GmbH); IMM-005 and IMM-039 (Immunome Inc); ImmuniCAR (TC BioPharm Ltd); NT-0004 and NT-0009 (BioNTech Cell and Gene Therapies GmbH), OGD-203 (OGD2 Pharma SAS), PMC-005B (PharmAbcine), and TI-7007 (Timmune Biotech Inc).
Provided herein are also nucleic acids that encode the chimeric antigen receptors described herein. In some embodiments, the nucleic acid is isolated or purified. Delivery of the nucleic acids ex vivo can be accomplished using methods known in the art. For example, immune cells obtained from a subject may be transformed with a nucleic acid vector encoding the chimeric antigen receptor. The vector may then be used to transform recipient immune cells so that these cells will then express the chimeric antigen receptor. Efficient means of transforming immune cells include transfection and transduction. Such methods are well known in the art. For example, applicable methods for delivery the nucleic acid molecule encoding the chimeric antigen receptor (and the nucleic acid(s) encoding the base editor) can be found in International Application No. PCT/US2009/040040 and U.S. Pat. Nos. 8,450,112; 9,132,153; and 9,669,058, each of which is incorporated herein in its entirety. Additionally, those methods and vectors described herein for delivering the nucleic acid encoding the base editor are applicable to delivering the nucleic acid encoding the chimeric antigen receptor.
Extracellular Binding Domain
The chimeric antigen receptors of the invention include an extracellular binding domain. The extracellular binding domain of a chimeric antigen receptor contemplated herein comprises an amino acid sequence of an antibody, or an antigen binding fragment thereof, that has an affinity for a specific antigen. In some embodiments, the antigen is CD3. In some embodiments, the antigen is CD5. In some embodiments, the antigen is CD7. In some embodiments, the antigen is CD33. In some embodiments, the antigen is CD123.
In some embodiments the chimeric antigen receptor comprises an amino acid sequence of an antibody. In some embodiments, the chimeric antigen receptor comprises the amino acid sequence of an antigen binding fragment of an antibody. The antibody (or fragment thereof) portion of the extracellular binding domain recognizes and binds to an epitope of an antigen. In some embodiments, the antibody fragment portion of a chimeric antigen receptor is a single chain variable fragment (scFv). A scFv comprises the light and variable fragments of a monoclonal antibody. In other embodiments, the antibody fragment portion of a chimeric antigen receptor is a multichain variable fragment, which can comprise more than one extracellular binding domains and therefore bind to more than one antigen simultaneously. In a multiple chain variable fragment embodiment, a hinge region may separate the different variable fragments, providing necessary spatial arrangement and flexibility.
In other embodiments, the antibody portion of a chimeric antigen receptor comprises at least one heavy chain and at least one light chain. In some embodiments, the antibody portion of a chimeric antigen receptor comprises two heavy chains, joined by disulfide bridges and two light chains, wherein the light chains are each joined to one of the heavy chains by disulfide bridges. In some embodiments, the light chain comprises a constant region and a variable region. Complementarity determining regions residing in the variable region of an antibody are responsible for the antibody's affinity for a particular antigen. Thus, antibodies that recognize different antigens comprise different complementarity determining regions. Complementarity determining regions reside in the variable domains of the extracellular binding domain, and variable domains (i.e., the variable heavy and variable light) can be linked with a linker or, in some embodiments, with disulfide bridges. In some embodiments, the variable heavy chain and variable light chain are linked by a (GGGGS)n linker, wherein the n is an integer from 1 to 10 (SEQ ID NO: 114). In some embodiments, the linker is a (GGGGS)3 linker (SEQ ID NO: 110).
In some embodiments, the antigen recognized and bound by the extracellular domain is a protein or peptide, a nucleic acid, a lipid, or a polysaccharide. Antigens can be heterologous, such as those expressed in a pathogenic bacteria or virus. Antigens can also be synthetic; for example, some individuals have extreme allergies to synthetic latex and exposure to this antigen can result in an extreme immune reaction. In some embodiments, the antigen is autologous, and is expressed on a diseased or otherwise altered cell. For example, in some embodiments, the antigen is expressed in a neoplastic cell. In some embodiments, the neoplastic cell is a solid tumor cell. In other embodiments, the neoplastic cell is a liquid tumor cell. In other embodiments, the neoplastic cell is a hematological cancer, such as a B cell cancer. In some embodiments, the B cell cancer is a lymphoma or a leukemia.
A liquid cancer to be treated with the methods described herein can be, for example, a leukemia. In some cases, the leukemia comprises a pre-leukemia. In some cases, the leukemia is an acute leukemia. Acute leukemias include, for example, an acute myeloid leukemia (AML). Acute leukemias also include, for example, an acute lymphoid leukemia or an acute lymphocytic leukemia (ALL); ALL includes B-lineage ALL; T-lineage ALL; and T-cell acute lymphocytic leukemia (T-ALL).
Nonlimiting examples of neoplasias include T-cell acute lymphoblastic leukemia (T-ALL), mycosis fungoides (MF), Sézary syndrome (SS), Peripheral T/NK-cell lymphoma, Anaplastic large cell lymphoma ALK+, Primary cutaneous T-cell lymphoma, T-cell large granular lymphocytic leukemia, Angioimmunoblastic T/NK-cell lymphoma, Hepatosplenic T-cell lymphoma, Primary cutaneous CD30+ T lymphoproliferative disorders, Extranodal NK/T-cell lymphoma, Adult T-cell leukemia/lymphoma, T-cell prolymphocytic leukemia, Subcutaneous panniculitis-like T-cell lymphoma, Primary cutaneous gamma-delta T-cell lymphoma, Aggressive NK-cell leukemia, and Enteropathy-associated T-cell lymphoma. In some embodiments, the neoplasia is T-cell acute lymphoblastic leukemia (T-ALL). In some embodiments, the neoplasia is acute myelogenous leukemia (AML).
Antibody-antigen interactions are noncovalent interactions resulting from hydrogen bonding, electrostatic or hydrophobic interactions, or from van der Waals forces. The affinity of extracellular binding domain of the chimeric antigen receptor for an antigen can be calculated with the following formula:

KA=[Antibody−Antigen]/[Antibody][Antigen], wherein

    
    
        [Ab]=molar concentration of unoccupied binding sites on the antibody;
        [Ag]=molar concentration of unoccupied binding sites on the antigen; and
        [Ab−Ag]=molar concentration of the antibody-antigen complex.
    
    


The antibody-antigen interaction can also be characterized based on the dissociation of the antigen from the antibody. The dissociation constant (KD) is the ratio of the association rate to the dissociation rate and is inversely proportional to the affinity constant. Thus, KD=1/KA. Those skilled in the art will be familiar with these concepts and will know that traditional methods, such as ELISA assays, can be used to calculate these constants.
Transmembrane Domain
The chimeric antigen receptors of the invention include a transmembrane domain. The transmembrane domain of the chimeric antigen receptors described herein spans the CAR-T cells lipid bilayer cellular membrane and separates the extracellular binding domain and the intracellular signaling domain. In some embodiments, this domain is derived from other receptors having a transmembrane domain, while in other embodiments, this domain is synthetic. In some embodiments, the transmembrane domain may be derived from a non-human transmembrane domain and, in some embodiments, humanized. By “humanized” is meant having the sequence of the nucleic acid encoding the transmembrane domain optimized such that it is more reliably or efficiently expressed in a human subject. In some embodiments, the transmembrane domain is derived from another transmembrane protein expressed in a human immune effector cell. Examples of such proteins include, but are not limited to, subunits of the T cell receptor (TCR) complex, PDT, or any of the Cluster of Differentiation proteins, or other proteins, that are expressed in the immune effector cell and that have a transmembrane domain. In some embodiments, the transmembrane domain will be synthetic, and such sequences will comprise many hydrophobic residues.
Transmembrane domains for use in the disclosed CARs can include at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane domain is derived from CD4, CD8α, CD28 or CD3ζ. In some embodiments, the transmembrane domain is a CD28 transmembrane domain. In some embodiments, the transmembrane domain is a CD8α transmembrane domain.
In some embodiments the transmembrane domain is a CD8α hinge and transmembrane domain. In some embodiments, the CD8α hinge and transmembrane domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:







SDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY


 


TWAPLAGTCGVLLLSLVITLYC






The chimeric antigen receptor is designed, in some embodiments, to comprise a spacer between the transmembrane domain and the extracellular domain, the intracellular domain, or both. Such spacers can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the spacer can be 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids in length. In still other embodiments the spacer can be between 100 and 500 amino acids in length. The spacer can be any polypeptide that links one domain to another and are used to position such linked domains to enhance or optimize chimeric antigen receptor function. In some embodiments the hinge/spacer is selected from CH3, CD8α, or CD28.
Intracellular Signaling Domain
The chimeric antigen receptors of the invention include an intracellular signaling domain. The intracellular signaling domain is the intracellular portion of a protein expressed in a T cell that transduces a T cell effector function signal (e.g., an activation signal) and directs the T cell to perform a specialized function. T cell activation can be induced by a number of factors, including binding of cognate antigen to the T cell receptor on the surface of T cells and binding of cognate ligand to costimulatory molecules on the surface of the T cell. A T cell co-stimulatory molecule is a cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule. Activation of a T cell leads to immune response, Such as T cell proliferation and differentiation (see, e.g., Smith-Garvin et al., Annu. Rev. Immunol., 27:591-619, 2009). Exemplary T cell signaling domains are known in the art. Non-limiting examples include the CD3ζ, CD8, CD28, CD27, CD154, GITR (TNFRSF18), CD134 (OX40), and CD137 (4-1BB) signaling domains.
The intracellular signaling domain of the chimeric antigen receptor contemplated herein comprises a primary signaling domain. In some embodiments, the chimeric antigen receptor comprises the primary signaling domain and a secondary, or co-stimulatory, signaling domain.
In some embodiments, the primary signaling domain comprises one or more immunoreceptor tyrosine-based activation motifs, or ITAMs. In some embodiments, the primary signaling domain comprises more than one ITAM. ITAMs incorporated into the chimeric antigen receptor may be derived from ITAMs from other cellular receptors. In some embodiments, the primary signaling domain comprising an ITAM may be derived from subunits of the TCR complex, such as CD3γ, CD3ε, CD3ζ, or CD3δ (see FIG. 1A). In some embodiments, the primary signaling domain comprising an ITAM may be derived from FcRγ, FcRβ, CD5, CD22, CD79a, CD79b, or CD66d.
In some embodiments, the primary signaling domain is selected from the group consisting of CD8, CD28, CD134 (OX40), CD137 (4-1BB), and CD3ζ. In some embodiments, the primary signaling domain is a CD3ζ signaling domain. In some embodiments, the CD3ζ signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:







RVKFSRSADAPAYQQGQNQLYNELLLGRREEYDVLDKRRGRDPEMGGKPR


 


RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGIYQGLSTATKDT


 


YDALHMQALPPR






In some embodiments, the primary signaling domain is a CD134 (OX40) signaling domain. In some embodiments, the CD134 (OX40) signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:










 
RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI






In some embodiments, the secondary, or co-stimulatory, signaling domain is derived from 4-1BB, CD2, CD4, CD28, CDS, CD8α, CD83, CD134, CD137, ICOS, or CD154. In some embodiments, the secondary signaling domain is a CD28 signaling domain. In some embodiments, the CD28 signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:










 
SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS






In some embodiments, the secondary signaling domain is a CD137 (4-1BB) signaling domain. In some embodiments, the CD137 (4-1BB) signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:










 
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL






In some embodiments, the CD137 (4-1BB) signaling domain is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an exemplary amino acid sequence as provided below:








 
RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.






In some embodiments, the CAR comprises one or more signaling domains. In some embodiments, the CAR comprises a 4-1BB signaling domain and a CD3(signaling domain. In some embodiments, the CAR comprises a CD28 signaling domain and a CD3(signaling domain.
Editing of Target Genes in Immune Cells
The present invention provides for immune cells comprising a chimeric antigen (CAR) and one or more edited genes, one or more regulatory elements thereof, or combinations thereof, wherein expression of the edited gene is either knocked out or knocked down. In some embodiments, the immune cell comprises a CAR and an altered endogenous gene that provides resistance to fratricide, enhances immune cell function, resistance to immunosuppression or inhibition, or a combination thereof. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a CAR-T cell. In some embodiments, the immune cell is a NK cell. In some embodiments, each edited gene may comprise a single base edit. In some embodiments, each edited gene may comprise multiple base edits at different regions of the gene.
In some embodiments, a single modification event (such as electroporation), may introduce one or more gene edits. In some embodiments at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more edits may be introduced in one or more genes simultaneously. In some embodiments, an immune cell, including but not limited to any immune cell comprising an edited gene selected from any of the aforementioned gene edits, can be edited to generate mutations in other genes that enhance the CAR-T's function or reduce immunosuppression or inhibition of the cell.
In some embodiments, the CAR-T cells have increased fratricide resistance as compared to a similar CAR-T cell but without further having the one or more edited genes as described herein. In some embodiments, the CAR-T cells have reduced immunogenicity as compared to a similar CAR-T cell but without further having the one or more edited genes as described herein. In some embodiments, the CAR-T cells have lower activation threshold as compared to a similar CAR-T but without further having the one or more edited genes as described herein. In some embodiments, the CAR-T cells have increased anti-neoplasia activity as compared to a similar CAR-T cell but without further having the one or more edited genes as described herein.
In some embodiments, provided herein is an immune cell with at least one modification in an endogenous gene or regulatory elements thereof. In some embodiments, the immune cell may comprise a further modification in at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more endogenous genes or regulatory elements thereof.
In some embodiments the one or more genes, or one or more regulatory elements thereof, or combinations thereof, may be selected from a group consisting of: CD3 antigen (CD3); CD5 antigen (CD5); CD7 antigen (CD7); CD33 antigen (CD33); CD52 antigen (CD52); CD123 antigen (CD123); T cell receptor alpha constant (TRAC); Programmed cell death 1 (PDCD1 or PD-1); Fas cell surface death receptor (FAS); Lymphocyte-activation gene 3 (LAG-3); Class II Major Histocompatibility Complex Transactivator (CIITA); T Cell Receptor Beta Constant 1 (TRBC1); T Cell Receptor Beta Constant 2 (TRBC2); and beta-2 microglobulin (B2M). In some embodiments, CD3, CD5, CD7, CD33 or CD123 is edited. In some embodiments, the immune cell comprises an edited CD3 gene, and additionally, at least one edited gene. In some embodiments, the immune cell comprises an edited CD5 gene, and additionally, at least one edited gene. In some embodiments, the immune cell comprises an edited CD7 gene, and additionally, at least one edited gene. In some embodiments, the immune cell comprises an edited CD33 gene, and additionally, at least one edited gene. In some embodiments, the immune cell comprises an edited CD123 gene, and additionally, at least one edited gene. The at least one edited gene may be selected from the list of genes mentioned in the preceding paragraphs. In some embodiments, CD3, CD5, CD7, CD33 or CD123 is edited in combination with one or more of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.
In various embodiments, the modified immune cell comprises mutations in one or more of CD5, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, or a combination thereof. In various embodiments, the modified immune cell comprises mutations in two, three, four, five, six, seven, eight, nine or ten of CD5, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In various embodiments, the modified immune cell comprises a mutation in CD5 and mutations in one or more of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, or a combination thereof. In one embodiment, the modified immune cell comprises mutations in CD5, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.
In various embodiments, the modified immune cell comprises mutations in one or more of CD7, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, or a combination thereof. In various embodiments, the modified immune cell comprises mutations in two, three, four, five, six, seven, eight, nine or ten of CD7, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In various embodiments, the modified immune cell comprises a mutation in CD7 and mutations in one or more of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, or a combination thereof. In one embodiment, the modified immune cell comprises mutations in CD7, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.
In various embodiments, the modified immune cell comprises mutations in one or more of CD3, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, or a combination thereof. In various embodiments, the modified immune cell comprises mutations in two, three, four, five, six, seven, eight, nine or ten of CD3, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In various embodiments, the modified immune cell comprises a mutation in CD3 and mutations in one or more of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, or a combination thereof. In one embodiment, the modified immune cell comprises mutations in CD3, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.
In various embodiments, the modified immune cell comprises mutations in one or more of CD33, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, or a combination thereof. In various embodiments, the modified immune cell comprises mutations in two, three, four, five, six, seven, eight, nine or ten of CD33, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In various embodiments, the modified immune cell comprises a mutation in CD33 and mutations in one or more of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, or a combination thereof. In one embodiment, the modified immune cell comprises mutations in CD33, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.
In various embodiments, the modified immune cell comprises mutations in one or more of CD123, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, or a combination thereof. In various embodiments, the modified immune cell comprises mutations in two, three, four, five, six, seven, eight, nine or ten of CD123, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1. In various embodiments, the modified immune cell comprises a mutation in CD123 and mutations in one or more of TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1, or a combination thereof. In one embodiment, the modified immune cell comprises mutations in CD123, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PD1.
In some embodiments, the at least one modification is a single nucleobase modification. In some embodiments, the altered endogenous gene may be created by base editing. In some embodiments, the base editing may reduce or attenuate the gene expression. In some embodiments, the base editing may reduce or attenuate the gene activation. In some embodiments, the base editing may reduce or attenuate the functionality of the gene product. In some other embodiments, the base editing may activate or enhance the gene expression. In some embodiments, the base editing may increase the functionality of the gene product.
Allogeneic immune cells expressing an endogenous immune cell receptor as well as a chimeric antigen receptor may recognize and attack host cells, a circumstance termed graft versus host disease (GVHD). The alpha component of the immune cell receptor complex is encoded by the TRAC gene, and in some embodiments, this gene is edited such that the alpha subunit of the TCR complex is nonfunctional or absent. Because this subunit is necessary for endogenous immune cell signaling, editing this gene can reduce the risk of graft versus host disease caused by allogeneic immune cells.
In some embodiments, editing of genes to provide fratricide resistance, enhance the function of the immune cell or to reduce immunosuppression or inhibition can occur in the immune cell before the cell is transformed to express a chimeric antigen receptor. In other aspects, editing of genes to enhance the function of the immune cell or to reduce immunosuppression or inhibition can occur in a CAR-T cell, i.e., after the immune cell has been transformed to express a chimeric antigen receptor.
In some embodiments of the present invention, the CD5 gene is edited in the CAR-T cell to knockout or knockdown expression. The CAR-T is then transformed to express a chimeric antigen receptor with a CD5 scFv. By knocking out or knocking down expression of the CD5 gene, the modified CAR-T cells are less likely to commit fratricide.
In some embodiments of the present invention, the CD7 gene is edited in the CAR-T cell to knockout or knockdown expression. The CAR-T is then transformed to express a chimeric antigen receptor with a CD7 scFv. By knocking out or knocking down expression of the CD7 gene, the modified CAR-T cells are less likely to commit fratricide.
In some embodiments of the present invention, the CD33 gene is edited in the CAR-T cell to knockout or knockdown expression. The CAR-T is then transformed to express a chimeric antigen receptor with a CD33 scFv. By knocking out or knocking down expression of the CD33 gene, the modified CAR-T cells are less likely to commit fratricide.
In some embodiments of the present invention, the CD3 gene is edited in the CAR-T cell to knockout or knockdown expression. The CAR-T is then transformed to express a chimeric antigen receptor with a CD3 scFv. By knocking out or knocking down expression of the CD3 gene, the modified CAR-T cells are less likely to commit fratricide.
In some embodiments of the present invention, the CD123 gene is edited in the CAR-T cell to knockout or knockdown expression. The CAR-T is then transformed to express a chimeric antigen receptor with a CD123 scFv. By knocking out or knocking down expression of the CD123 gene, the modified CAR-T cells are less likely to commit fratricide.
Host immune cells can potentially recognize allogeneic CAR-T cells as non-self and elicit an immune response to remove the non-self cells. B2M is expressed in nearly all nucleated cells and is associated with MHC class I complex (FIG. 1B). Circulating host CD8+ T cells can recognize this B2M protein as non-self and kill the allogeneic cells. To overcome this graft rejection, in some embodiments, the B2M gene is edited to either knockout or knockdown expression. In some embodiments, provided herein is an immune cell with an edited B2M gene, such that the immune cell does not express an endogenous functional B2M. In some embodiments, provided herein is a CAR-T cell with an edited B2M gene, such that the CAR-T cell exhibits reduced or negligible expression or no expression of endogenous B2M.
In some embodiments, an immune cell comprises a chimeric antigen receptor and one or more edited genes, a regulatory element thereof, or combinations thereof. An edited gene may be an immune response regulation gene, an immunogenic gene, a checkpoint inhibitor gene, a gene involved in immune responses, a cell surface marker, e.g. a T cell surface marker, or any combination thereof. In some embodiments, an immune cell comprises a chimeric antigen receptor and an edited gene that is associated with activated T cell proliferation, alpha-beta T cell activation, gamma-delta T cell activation, positive regulation of T cell proliferation, negative regulation of T-helper cell proliferation or differentiation, or their regulatory elements thereof, or combinations thereof. In some embodiments, the edited gene may be a checkpoint inhibitor gene, for example, such as a PD1 gene, a PDC1 gene, or a member related to or regulating the pathway of their formation or activation. In some embodiments, the edited gene is a TRAC gene. In some embodiments, the edited gene is a CD5 gene. In some embodiments, the edited gene is a CD7 gene. In some embodiments, the edited gene is a CD33 gene. In some embodiments, the edited gene is a CD3 gene. In some embodiments, the edited gene is a CD123 gene. In some embodiments, the edited gene is a B2M gene. In some embodiments, the edited gene is a CHTA gene. In some embodiments, the edited gene is a TRBC1/2 gene. In some embodiments, the edited gene is a CD5 gene. In some embodiments, the edited gene is a CD7 gene. In some embodiments, the edited gene is a CD52 gene. In some embodiments, at least one gene is edited selected from PD-1, CD2, CD3, CD5, CD7, CD52, B2M, TRBC12, CIITA, and TRAC, or combinations thereof. In some embodiments, the PD-1, CD2, CD52, and TRAC genes are edited. In some embodiments, the PD-1, CD2, CD52, B2M, TRBC1/2, CIITA and TRAC genes are edited. In some embodiments, the PD-1, CD5, CD52, and TRAC genes are edited. In some embodiments, the PD-1, CD3, CD7, and CD52 genes are edited.
In some embodiments, the editing of the endogenous gene reduces expression of the gene. In some embodiments, the editing of the endogenous gene reduces expression of the gene by at least 50% as compared to a control cell without the modification. In some embodiments, the editing of the endogenous gene reduces expression of the gene by at least 60% as compared to a control cell without the modification. In some embodiments, the editing of the endogenous gene reduces expression of the gene by at least 70% as compared to a control cell without the modification. In some embodiments, the editing of the endogenous gene reduces expression of the gene by at least 80% as compared to a control cell without the modification. In some embodiments, the editing of the endogenous gene reduces expression of the gene by at least 90% as compared to a control cell without the modification. In some embodiments, the editing of the endogenous gene reduces expression of the gene by at least 100% as compared to a control cell without the modification. In some embodiments, the editing of the endogenous gene eliminates gene expression.
In some embodiments of the present invention, the PDCD1 gene is edited in the CAR-T cell to knockout or knockdown expression. The PDCD1 gene encodes the cell surface receptor PD-1, an immune system checkpoint expressed in immune cells, and it is involved in reducing autoimmunity by promoting apoptosis of antigen specific immune cells. By knocking out or knocking down expression of the PDCD1 gene, the modified CAR-T cells are less likely to apoptose, are more likely to proliferate, and can escape the programmed cell death immune checkpoint.
The CBLB gene encodes an E3 ubiquitin ligase that plays a significant role in inhibiting immune effector cell activation. Referring to FIG. 1C, the CBLB protein favors the signaling pathway resulting in immune effector cell tolerance and actively inhibits signaling that leads to immune effector cell activation. Because immune effector cell activation is necessary for the CAR-T cells to proliferate in vivo post-transplant, in some embodiments of the present invention the CBLB gene is edited to knockout or knockdown expression.
In some embodiments, provided herein is an immune cell with an edited TRAC gene (wherein, the TRAC gene may comprise one, two, three, four, five, six, seven eight, nine, ten or more base edits), such that the immune cell does not express an endogenous functional T cell receptor alpha chain. In some embodiments, the immune cell is a T cell expressing a chimeric antigen receptor (a CAR-T cell). In some embodiments, provided herein is a CAR-T cell with base edits in TRAC gene, such that the CAR-T cell have reduced or negligible or no expression of endogenous T cell receptor alpha protein.
In some embodiments, provided herein is an immune cell with an edited CIITA gene, such that the immune cell does not express an endogenous functional class II, major histocompatibility complex, transactivator. In some embodiments, provided herein is a CAR-T cell with an edited CIITA gene, such that the CAR-T cell exhibits reduced or negligible expression or no expression of endogenous class II, major histocompatibility complex, transactivator.
In some embodiments, provided herein is an immune cell with an edited TRBC1 or TRBC2 gene, such that the immune cell does not express an endogenous functional T cell receptor beta chain. In some embodiments, provided herein is a CAR-T cell with an edited TRBC1/TRBC2 gene, such that the CAR-T cell exhibits reduced or negligible expression or no expression of endogenous T cell receptor beta chain.
In some embodiments, an immune cell comprises a chimeric antigen receptor and an edited TRAC, B2M, PDCD1, CBLB gene, or a combination thereof, wherein expression of the edited gene is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and an edited TRAC gene, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited TRAC and B2M genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited TRAC and PDCD1 genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited TRAC and CBLB genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited TRAC, B2M, and PDCD1 genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited TRAC, B2M, and CBLB genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell or immune effector cell comprises a chimeric antigen receptor and edited TRAC, PDCD1, and CBLB genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen and edited TRAC, B2M, PDCD1, and CBLB genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and an edited B2M gene, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited B2M and PDCD1 genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited B2M and CBLB genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited B2M, PDCD1, and CBLB genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and an edited PDCD gene, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited PDCD and CBLB genes, wherein expression of the edited genes is either knocked out or knocked down. And in some embodiments, an immune cell comprises a chimeric antigen receptor and an edited CBLB, expression of the edited gene is either knocked out or knocked down.
In some embodiments, an immune cell comprises a chimeric antigen receptor and edited CD5 gene, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited CD7 gene, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited CD33 gene, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited CD3 gene, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited CD123 gene, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited FAS gene, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited LAG-3 gene, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited CIITA gene, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited TRBC1 gene, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited TRBC2 gene, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited CD52 gene, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and one or more edited CD3, CD5, CD7, CD33, CD123, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and/or PD1 genes, wherein expression of the edited genes is either knocked out or knocked down.
The base editing may be positioned at any suitable position of the gene, or in a regulatory element of the gene. Thus, it may be appreciated that a single base editing at a start codon, for example, can completely abolish the expression of the gene. In some embodiments, the altered endogenous gene may be modified or edited in an exon, an intron, an exon-intron injunction, or a regulatory element thereof. The modification may be edit to a single nucleobase in a gene or a regulatory element thereof. The modification may be in a exon, more than one exons, an intron, or more than one introns, or a combination thereof. The modification may be in an open reading frame of a gene. The modification may be in an untranslated region of the gene, for example, a 3′-UTR or a 5′-UTR. In some embodiments, the modification is in a regulatory element of an endogenous gene. In some embodiments, the modification is in a promoter, an enhancer, an operator, a silencer, an insulator, a terminator, a transcription initiation sequence, a translation initiation sequence (e.g. a Kozak sequence), or any combination thereof. In some embodiments, base editing may introduce a premature STOP codon into an exon, resulting in either lack of a translated product or in a truncated that may be misfolded and thereby eliminated by degradation, or may produce an unstable mRNA that is readily degraded.
In some embodiments, base editing may be performed, for example on exon 1, exon 2, or exon 3, or exon 4, or exon 5 of human PDC1/PD-1 gene. In some embodiments, base editing in the human PDC1/PD-1 gene is performed at a site within exon 1. In some embodiments, base editing in the human PDC1/PD-1 gene is performed at a site within exon 2. In some embodiments, base editing in the human PDC1/PD-1 gene is performed at a site within exon 3. In some embodiments, base editing in the human PDC1/PD-1 gene is performed at a site within exon 4. In some embodiments, base editing in the human PDC1/PD-1 gene is performed at a site within exon 5. In some embodiments one or more base editing actions can be performed on the human PDC1/PD-1 gene, at exon 1, exon 2, exon 3, exon 4, exon 5, or any combination thereof.
In some embodiments, base editing in the human PDC1/PD-1A gene is performed by editing position 4, 6, 7, 8 or 9 of a guide RNA spacer sequence targeting exon 1. In some embodiments, base editing in the human PDC1/PD-1A gene is performed by editing position 4, 6, 7, 8 or 9 of a guide RNA spacer sequence targeting exon 1. In some embodiments, base editing in the human PDC1/PD-1A gene is performed by editing position 7, 8 or 9 of a guide RNA spacer sequence targeting exon 2. In some embodiments, base editing in the human PDC/PD-1A gene is performed by editing position 5, 7, or 8 of a guide RNA spacer sequence targeting exon 3. In some embodiments, base editing in the human PDC/PD-1A gene is performed by editing position 5 or 8 of a guide RNA spacer sequence targeting exon 5.
In some embodiments, base editing may be performed, for example on exon 1, exon 2, or exon 3 of human CD7 gene. In some embodiments, base editing in the human CD7 gene is performed at a site within exon 1. In some embodiments, base editing in the human CD7 gene is performed at a site within exon 2. In some embodiments, base editing in the human CD7 gene is performed at a site within exon 3. In some embodiments one or more base editing actions can be performed on the human CD7 gene, at exon 1, exon 2, exon 3, or any combination thereof. In some embodiments, base editing in the human CD7 gene is performed at position 4, 8, 9 within exon 1. In some embodiments, base editing in the human CD7 gene is performed by editing position 5, 6, 7, 8, or 9 of a guide RNA spacer sequence targeting exon 2. In some embodiments, base editing in the human CD7 gene is performed by editing position 4 or 9 of a guide RNA spacer sequence targeting exon 3.
In some embodiments, base editing may be performed, for example on exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or exon 8 of human LAG-3 gene. In some embodiments, base editing in the human LAG-3 gene is performed at a site within exon 1. In some embodiments, base editing in the human LAG-3 gene is performed at a site within exon 2. In some embodiments, base editing in the human LAG-3 gene is performed at a site within exon 3. In some embodiments, base editing in the human LAG-3 gene is performed at a site within exon 4. In some embodiments, base editing in the human LAG-3 gene is performed at a site within exon 5. In some embodiments, base editing in the human LAG-3 gene is performed at a site within exon 6. In some embodiments, base editing in the human LAG-3 gene is performed at a site within exon 7. In some embodiments, base editing in the human LAG-3 gene is performed at a site within exon 8. In some embodiments one or more base editing actions can be performed on the human LAG-3 gene, at exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or any combination thereof. In some embodiments, base editing in the human LAG-3 gene is performed by editing position 4 or 8 of a guide RNA spacer sequence targeting exon 1. In some embodiments, base editing in the human LAG-3 gene is performed by editing position 4, 6, or 8 of a guide RNA spacer sequence targeting exon 2. In some embodiments, base editing in the human LAG-3 gene is performed by editing position 4, 5, 6 or 7 of a guide RNA spacer sequence targeting exon 3. In some embodiments, base editing in the human LAG-3 gene is performed by editing position 4, 8 or 9 of a guide RNA spacer sequence targeting exon 4. In some embodiments, base editing in the human LAG-3 gene is performed by editing position 8 or 9 of a guide RNA spacer sequence targeting exon 5. In some embodiments, base editing in the human LAG-3 gene is performed by editing position 4, 6, 7 or 8 of a guide RNA spacer sequence targeting exon 6. In some embodiments, base editing in the human LAG-3 gene is performed by editing position 4, 6 or 7 of a guide RNA spacer sequence targeting exon 7. In some embodiments, base editing in the human LAG-3 gene is performed by editing position 8 of a guide RNA spacer sequence targeting exon 8.
In some embodiments, base editing may be performed, for example on exon 1, exon 2, exon 3, exon 4, exon 5, or exon 6 of human CD33 gene. In some embodiments, base editing in the human CD33 gene is performed at a site within exon 1. In some embodiments, base editing in the human CD33 gene is performed at a site within exon 2. In some embodiments, base editing in the human CD33 gene is performed at a site within exon 3. In some embodiments, base editing in the human CD33 gene is performed at a site within exon 4. In some embodiments, base editing in the human CD33 gene is performed at a site within exon 5. In some embodiments, base editing in the human CD33 gene is performed at a site within exon 6. In some embodiments one or more base editing actions can be performed on the human CD33 gene, at exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, or any combination thereof. In some embodiments, base editing in the human CD33 gene is performed by editing position 7, 8 or 9 of a guide RNA spacer sequence targeting exon 1. In some embodiments, base editing in the human CD33 gene is performed by editing position 4, 5, 6, or 8 of a guide RNA spacer sequence targeting exon 2. In some embodiments, base editing in the human CD33 gene is performed by editing position 4, 5, 6 or 7 of a guide RNA spacer sequence targeting exon 3. In some embodiments, base editing in the human CD33 gene is performed by editing position 6 or 7 of a guide RNA spacer sequence targeting exon 4. In some embodiments, base editing in the human CD33 gene is performed by editing position 7 or 8 of a guide RNA spacer sequence targeting exon 5. In some embodiments, base editing in the human CD33 gene is performed by editing position 4, 5, or 6 of a guide RNA spacer sequence targeting exon 6.
In some embodiments, base editing may be performed, for example on exon 1, exon 2, exon 3, exon 4, exon 5, exon 7, exon 8, exon 10, or exon 11 of human CD123 gene. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 1. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 2. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 3. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 4. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 5. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 7. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 8. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 10. In some embodiments, base editing in the human CD123 gene is performed at a site within exon 11. In some embodiments one or more base editing actions can be performed on the human CD123 gene, at exon 1, exon 2, exon 3, exon 4, exon 5, exon 7, exon 8, exon 10, exon 11, or any combination thereof. In some embodiments, base editing in the human CD123 gene is performed by editing position 6 of a guide RNA spacer sequence targeting exon 1. In some embodiments, base editing in the human CD123 gene is performed by editing position 4, 6, or 8 of a guide RNA spacer sequence targeting exon 2. In some embodiments, base editing in the human CD123 gene is performed by editing position 5, 6 or 8 of a guide RNA spacer sequence targeting exon 3. In some embodiments, base editing in the human CD123 gene is performed by editing position 5 or 6 of a guide RNA spacer sequence targeting exon 4. In some embodiments, base editing in the human CD123 gene is performed by editing position 4 or 5 of a guide RNA spacer sequence targeting exon 5. In some embodiments, base editing in the human CD123 gene is performed by editing position 5 of a guide RNA spacer sequence targeting exon 7. In some embodiments, base editing in the human CD123 gene is performed by editing position 5, 6, 7 or 8 of a guide RNA spacer sequence targeting exon 8. In some embodiments, base editing in the human CD123 gene is performed by editing position 4, 7 or 8 of a guide RNA spacer sequence targeting exon 10. In some embodiments, base editing in the human CD123 gene is performed by editing position 5 or 8 of a guide RNA spacer sequence targeting exon 11.
In some embodiments, base editing may be performed, for example on exon 1, or exon 3, or exon 4, or exon 5, or exon 6, or exon 7, or exon 8, or exon 9 of human FAS gene. In some embodiments, base editing in the human FAS gene is performed at a site within exon 1. In some embodiments, base editing in the human FAS gene is performed at a site within exon 3. In some embodiments, base editing in the human FAS gene is performed at a site within exon 4. In some embodiments, base editing in the human FAS gene is performed at a site within exon 5. In some embodiments, base editing in the human FAS is performed at a site within exon 6. In some embodiments, base editing in the human FAS is performed at a site within exon 7. In some embodiments, base editing in the human FAS is performed at a site within exon 8. In some embodiments, base editing in the human FAS is performed at a site within exon 9. In some embodiments one or more base editing actions can be performed on the human FAS gene, at exon 1, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, or any combination thereof.
In some embodiments, base editing in the human FAS gene is performed by editing position 9 of a guide RNA spacer sequence targeting exon 1. In some embodiments, base editing in the human FAS gene is performed by editing position 6 of a guide RNA spacer sequence targeting exon 3. In some embodiments, base editing in the human FAS gene is performed by editing position 7 of a guide RNA spacer sequence targeting exon 4. In some embodiments, base editing in the human FAS gene is performed by editing position 5 of a guide RNA spacer sequence targeting exon 5. In some embodiments, base editing in the human FAS gene is performed by editing position 4 or 7 of a guide RNA spacer sequence targeting exon 6. In some embodiments, base editing in the human FAS gene is performed by editing position 8 of a guide RNA spacer sequence targeting exon 7. In some embodiments, base editing in the human FAS gene is performed by editing position 8 of a guide RNA spacer sequence targeting exon 8. In some embodiments, base editing in the human FAS gene is performed by editing position 5 or 6 of a guide RNA spacer sequence targeting exon 9.
In some embodiments, base editing may be performed, for example on exon 1, exon 2, or exon 3 of human CD52 gene. In some embodiments, base editing in the human CD52 gene is performed at a site within exon 1. In some embodiments, base editing in the human CD52 gene is performed at a site within exon 2. In some embodiments, base editing in the human CD7 gene is performed at a site within exon 3. In some embodiments one or more base editing actions can be performed on the human CD52 gene, at exon 1, exon 2, exon 3, or any combination thereof. In some embodiments, base editing in the human CD52 gene is performed by editing position 4 or 7 of a guide RNA spacer sequence targeting exon 1. In some embodiments, base editing in the human CD52 gene is performed by editing position 5, 6, or 7 of a guide RNA spacer sequence targeting exon 2.
In some embodiments, base editing may be performed, for example on exon 1, exon 2, or exon 3, or exon 4, or exon 5, or exon 6, or exon 7, or exon 8, or exon 9, or exon 10 of human CD5 gene. In some embodiments, base editing in the human CD5 gene is performed at a site within exon 1. In some embodiments, base editing in the human CD5 gene is performed at a site within exon 2. In some embodiments, base editing in the human CD5 gene is performed at a site within exon 3. In some embodiments, base editing in the human CD5 gene is performed at a site within exon 4. In some embodiments, base editing in the human CD5 gene is performed at a site within exon 5. In some embodiments, base editing in the human CD5 gene is performed at a site within exon 6. In some embodiments, base editing in the human CD5 gene is performed at a site within exon 7. In some embodiments, base editing in the human CD5 gene is performed at a site within exon 8. In some embodiments, base editing in the human CD5 gene is performed at a site within exon 9. In some embodiments, base editing in the human CD5 gene is performed at a site within exon 10. In some embodiments one or more base editing actions can be performed on the human CD5 gene, at exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, or any combination thereof.
In some embodiments, base editing in the human CD5 gene is performed by editing position 6 of a guide RNA spacer sequence targeting exon 1. In some embodiments, base editing in the human CD5 gene is performed by editing position 6 of a guide RNA spacer sequence targeting exon 1. In some embodiments, base editing in the human CD5 gene is performed by editing position 5 and/or 6 of a guide RNA spacer sequence targeting exon 2. In some embodiments, base editing in the human CD5 gene is performed by editing position 5 and/or 6 of a guide RNA spacer sequence targeting exon 2. In some embodiments, base editing in the human CD5 gene is performed by editing position 5, 6, 8 and/or 9 of a guide RNA spacer sequence targeting exon 3. In some embodiments, base editing in the human CD5 gene is performed by editing position 4 or 5 of a guide RNA spacer sequence targeting exon 4. In some embodiments, base editing in the human CD5 gene is performed by editing position 4, 5, 7, 8 or 9 of a guide RNA spacer sequence targeting exon 5. In some embodiments, base editing in the human CD5 gene is performed by editing position 4, 5, 6, 7, 8 and/or 9 of a guide RNA spacer sequence targeting exon 6. In some embodiments, base editing in the human CD5 gene is performed by editing position 4 of a guide RNA spacer sequence targeting exon 7. In some embodiments, base editing in the human CD5 gene is performed by editing position 4, 5, or 7 of a guide RNA spacer sequence targeting exon 8. In some embodiments, base editing in the human CD5 gene is performed by editing position 6 or 8 of a guide RNA spacer sequence targeting exon 9. In some embodiments, base editing in the human CD5 gene is performed by editing position 9 of a guide RNA spacer sequence targeting exon 10.
In some embodiments, base editing may be performed, for example on exon 1, or exon 2, or exon 3 or exon 4 of human TRAC gene (UCSC genomic database ENSG00000277734.8). In some embodiments, base editing in human TRAC gene is performed at a site within exon 1. In some embodiments, base editing in human TRAC gene is performed at a site within exon 2. In some embodiments, base editing in human TRAC gene is performed at a site within exon 3. In some embodiments, base editing in human TRAC gene is performed at a site within exon 4. In some embodiments one or more base editing actions can be performed on human TRAC gene, at exon 1, exon 2, exon 3, exon 4 or any combination thereof. In some embodiments, base editing in the human TRAC gene is performed by editing at position 5, 6, or 9 of a guide RNA spacer sequence targeting within exon 1.
In some embodiments, base editing may be performed, for example, on exon 1, exon 2, exon 3, or exon 4 of human B2M gene (Chromosome 15, NC_000015.10, 44711492-44718877; exemplary mRNA sequence NM 004048). In some embodiments, base editing in human B2M gene is performed at a site within exon 1. In some embodiments, base editing in human B2M gene is performed at a site within exon 2. In some embodiments, base editing in human B2M gene is performed at a site within exon 3. In some embodiments, base editing in human B2M gene is performed at a site within exon 4. In some embodiments one or more base editing actions can be performed on human B2M gene, at exon 1, exon 2, exon 3, exon 4 or any combination thereof. In some embodiments, base editing in the human B2M gene is performed by editing position 5 of a guide RNA spacer sequence targeting exon 1. In some embodiments, base editing in the human B2M gene is performed by editing position 4, 6 or 9 of a guide RNA spacer sequence targeting exon 2.
In some embodiments, base editing may be performed, for example on exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, or exon 19 of human CIITA gene. In some embodiments, base editing in the human CD52 gene is performed at a site within exon 1. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 2. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 3. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 4. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 5. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 6. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 7. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 8. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 9. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 10. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 11. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 12. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 13. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 14. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 15. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 16. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 17. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 18. In some embodiments, base editing in the human CIITA gene is performed at a site within exon 19. In some embodiments one or more base editing actions can be performed on the human CIITA gene, at exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, or any combination thereof.
In some embodiments, base editing in the human CIITA gene is performed by editing position 6 or 7 of a guide RNA spacer sequence targeting exon 1. In some embodiments, base editing in the human CIITA gene is performed by editing position 7 or 8 of a guide RNA spacer sequence targeting exon 2. In some embodiments, base editing in the human CIITA gene is performed by editing position 8 of a guide RNA spacer sequence targeting exon 4. In some embodiments, base editing in the human CIITA gene is performed by editing position 4, 7 or 8 of a guide RNA spacer sequence targeting exon 7. In some embodiments, base editing in the human CIITA gene is performed by editing position 8 of a guide RNA spacer sequence targeting exon 8. In some embodiments, base editing in the human CIITA gene is performed by editing position 4, 6, or 7 of a guide RNA spacer sequence targeting exon 9. In some embodiments, base editing in the human CIITA gene is performed by editing position 4, 5, or 7 of a guide RNA spacer sequence targeting exon 10. In some embodiments, base editing in the human CIITA gene is performed by editing position 4, 5, 6, 7 or 8 of a guide RNA spacer sequence targeting exon 11. In some embodiments, base editing in the human CIITA gene is performed by editing position 6 of a guide RNA spacer sequence targeting exon 12. In some embodiments, base editing in the human CIITA gene is performed by editing position 4 or 5 of a guide RNA spacer sequence targeting exon 14. In some embodiments, base editing in the human CIITA gene is performed by editing position 4, 7 or 8 of a guide RNA spacer sequence targeting exon 15. In some embodiments, base editing in the human CIITA gene is performed by editing position 5, 7 or 8 of a guide RNA spacer sequence targeting exon 16. In some embodiments, base editing in the human CIITA gene is performed by editing position 7 or 8 of a guide RNA spacer sequence targeting exon 17. In some embodiments, base editing in the human CIITA gene is performed by editing position 5 of a guide RNA spacer sequence targeting exon 18. In some embodiments, base editing in the human CIITA gene is performed by editing position 5, 6, 7 or 8 of a guide RNA spacer sequence targeting exon 19.
In some embodiments, base editing may be performed, for example, on exon 1, exon 2, or exon 3 of human TRBC1 gene. In some embodiments, base editing in human TRBC1 gene is performed at a site within exon 1. In some embodiments, base editing in human TRBC1 gene is performed at a site within exon 2. In some embodiments, base editing in human TRBC1 gene is performed at a site within exon 3. In some embodiments one or more base editing actions can be performed on human TRBC1 gene, at exon 1, exon 2, exon 3, or any combination thereof. In some embodiments, base editing in the human TRBC1 gene is performed by editing position 5, 6, 7 or 8 of a guide RNA spacer sequence targeting exon 1. In some embodiments, base editing in the human TRBC1 gene is performed by editing position 8 of a guide RNA spacer sequence targeting exon 2. In some embodiments, base editing in the human TRBC1 gene is performed by editing position 4 or 5 of a guide RNA spacer sequence targeting exon 3.
In some embodiments, base editing may be performed, for example, on exon 1, exon 2, or exon 3 of human TRBC2 gene. In some embodiments, base editing in human TRBC2 gene is performed at a site within exon 1. In some embodiments, base editing in human TRBC2 gene is performed at a site within exon 2. In some embodiments, base editing in human TRBC2 gene is performed at a site within exon 3. In some embodiments one or more base editing actions can be performed on human TRBC2 gene, at exon 1, exon 2, exon 3, or any combination thereof. In some embodiments, base editing in the human TRBC2 gene is performed by editing position 5, 6, 7 or 8 of a guide RNA spacer sequence targeting exon 1. In some embodiments, base editing in the human TRBC2 gene is performed by editing position 7 or 8 of a guide RNA spacer sequence targeting exon 2. In some embodiments, base editing in the human TRBC2 gene is performed by editing position 4 of a guide RNA spacer sequence targeting exon 3.
In some embodiments, base editing may be performed on an intron. For example, base editing may be performed on an intron. In some embodiments, the base editing may be performed at a site within an intron. In some embodiments, the base editing may be performed at a site one or more introns. In some embodiments, the base editing may be performed at any exon of the multiple introns in a gene. In some embodiments, one or more base editing may be performed on an exon, an intron or any combination of exons and introns.
In some embodiments, the modification or base edit may be within a promoter site. In some embodiments, the base edit may be introduced within an alternative promoter site. In some embodiments, the base edit may be in a 5′ regulatory element, such as an enhancer. In some embodiment, base editing may be introduced to disrupt the binding site of a nucleic acid binding protein. Exemplary nucleic acid binding proteins may be a polymerase, nuclease, gyrase, topoisomerase, methylase or methyl transferase, transcription factors, enhancer, PABP, zinc finger proteins, among many others.
In some embodiments, base editing may be used for splice disruption to silence target protein expression. In some embodiments, base editing may generate a splice acceptor-splice donor (SA-SD) site. Targeted base editing generating a SA-SD, or at a SA-SD site can result in reduced expression of a gene. In some embodiments, base editors (e.g., ABE, CBE) are used to target dinucleotide motifs that constitute splice acceptor and splice donor sites, which are the first and last two nucleotides of each intron. For example, the exon 3 splice donor (SD) site of CD2 may be targeted for base editing. In some embodiments, splice disruption is achieved with an adenosine base editor (ABE). In some embodiments, splice disruption is achieved with a cytidine base editor (CBE). In some embodiments, base editors (e.g., ABE, CBE) are used to edit exons by creating STOP codons. The premature stop codon may be generated in an exon, an intron, or an untranslated region. In some embodiments, base editing may be used to introduce more than one STOP codon, in one or more alternative reading frames. For example, a premature STOP codon can be introduced at position 8 within exon 2, at position 4 within exon 3, at position 6 within exon 3, at position 9 within exon 3, at position 4 within exon 4, at position 5 within exon 4, or at position 4 within exon 5. In some embodiments, the stop codon is generated by a adenosine base editor (ABE). In some embodiments, the stop codon is generated by a cytidine base editor (CBE). In some embodiments, the CBE generates any one of the following edits (shown in underlined font) to generate a STOP codon: CAG→TAG; CAA→TAA; CGA→TGA; TGG→TGA; TGG→TAG; or TGG+TAA.
In some embodiments, modification/base edits may be introduced at a 3′-UTR, for example, in a poly adenylation (poly-A) site. In some embodiments, base editing may be performed on a 5′-UTR region.
In some embodiments, an immune cell, including by not limited to any immune cell comprising any of the aforementioned gene edits, can be edited to generate mutations in other genes that enhance the CAR-T's function or reduce immunosuppression or inhibition of the cell. For example, in some embodiments, an immune cell comprises a chimeric antigen receptor and an edited TGFBR2, ZAP70, NFATc1, TET2 gene, or a combination thereof, wherein expression of the edited gene is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and an edited TGFBR2 gene, wherein expression of the edited gene is knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited TGFBR2 and ZAP70 genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited TGFBR2 and ZAP70 genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited TGFBR2 and NFATC1 genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited TGFBR2 and TET2 genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited TGFBR2, ZAP70, and NFATC1 genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited TGFBR2, ZAP70, and TET2 genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited TGFBR2, NFATC1, and TET2 genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen and edited TGFBR2, ZAP70, NFATC1, and TET2 genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and an edited ZAP70 gene, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited ZAP70 and NFATC1 genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited ZAP70 and TET2 genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited ZAP70, PDCD1, and TET2 genes, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and an edited PDCD1 gene, wherein expression of the edited genes is either knocked out or knocked down. In some embodiments, an immune cell comprises a chimeric antigen receptor and edited PDCD1 and TET2 genes, wherein expression of the edited genes is either knocked out or knocked down. And in some embodiments, an immune cell comprises a chimeric antigen receptor and an edited TET2, expression of the edited gene is either knocked out or knocked down.
In some embodiments, a chimeric antigen receptor is inserted into the TRAC gene. This has advantages. First, because TRAC is highly expressed in immune cell, the chimeric antigen receptor will be similarly expressed when its construct is designed to insert the chimeric antigen receptor into the TRAC gene such that expression of the receptor is driven by the TRAC promoter. Second, inserting the chimeric antigen receptor into the TRAC gene will knockout TRAC expression. In some embodiments, the gene editing system described herein can be used to insert the chimeric antigen receptor into the TRAC locus. gRNAs specific for the TRAC locus can guide the gene editing system to the locus and initiate double-stranded DNA cleavage. In particular embodiments, the gRNA is used in conjunction with Cas12b. In various embodiments, the gene editing system is used in conjunction with a nucleic acid having a sequence encoding a CAR receptor. Exemplary guide RNAs are provided in the following Table 1A.







TABLE 1A





Guide RNAs


















GUUCUGUCUUUUGGUCAGGACAACCGUCUAGCUAUAAGUG
ATTN
BhCas12b
TRAC KO


CUGCAGGGUGUGAGAAACUCCUAUUGCUGGACGAUGUCUC

nuclease
gRNA 1


UUACGAGGCAUUAGCACAGAGUCUCUCAGCUGGUACAC


(Exon 2)


 


GUUCUGUCUUUUGGUCAGGACAACCGUCUAGCUAUAAGUG
ATTN
BhCas12b
TRAC KO


CUGCAGGGUGUGAGGAAACUCCUAUUGCUGGACGAUGUUU

nuclease
gRNA 2


CUUACGAGGCAUUAGCACACCGAUUUUGAUUCUCAAACA


(Exon 2)


 


GUUCUGUCUUUUGGUCAGGACAACCGUCUAGCUAUAAGUG
ATTN
BhCas12b
TRAC KO


CUGCAGGGUGUGAGAAACUCCUAUUGCUGGACGAUGUCUC

nuclease
gRNA 3


UUACGAGGCAUUAGCACUCAAACAAAUGUGCACAAAG


(Exon 2)


 


GUUCUGUCUUUUGGUCAGGACAACCGUCUAGCUAUAAGUG
ATTN
BhCas12b
TRAC KO


CUGCAGGGUGUGAGAAACUCCUAUUGCUGGACGAUGUCUC

nuclease
gRNA 4


UUACGAGGCAUUAGCACUCAAACAAAUGUGUCACAAAG


(Exon 2)


 


GUUCUGUCUUUUGGUCAGGACAACCGUCUAGCUAUAAGUG
ATTN
BhCas12b
TRAC KO


CUGCAGGGUGUGAGAAACUCCUAUUGCUGGACGAUGUCUC

nuclease
gRNA 5


UUACGAGGCAUUAGCACUUUGAGAAUCAAAAUCGGUA


(Exon 2)


 


GUUCUGUCUUUUGGUCAGGACAACCGUCUAGCUAUAAGUG
ATTN
BhCas12b
TRAC KO


CUGCAGGGUGUGAGAAACUCCUAUUGCUGGACGAUGUCUC

nuclease
gRNA 6


UUACGAGGCAUUAGCACUGAUGUGUAUAUCACAGACAA


(Exon 2)


 


GUUCUGUCUUUUGGUCAGGACAACCGUCUAGCUAUAAGUG
ATTN
BhCas12b
TRAC KO


CUGCAGGGUGUGAGAAACUCCUAUUGCUGGACGAUGUCUC

nuclease
gRNA 7


UUACGAGGCAUUAGCAGUUGCUCCAGGCCACAGCAU


(Exon 2)


 


GUUCUGUCUUUUGGUCAGGACAACCGUCUAGCUAUAAGUG
ATTN
BhCas12b
TRAC KO


CUGCAGGGUGUGAGAAACUCCUAUUGCUGGACGAUGUCUC

nuclease
gRNA 8


UUACGAGGCAUUAGCACUUCCAGAAGACACCUUCUUCC


(Exon 2)


 


GUUCUGUCUUUUGGUCAGGACAACCGUCUAGCUAUAAGUG
ATTN
BhCas12b
TRAC KO


CUGCAGGGUGUGAGAAACUCCUAUUGCUGGACGAUGUCUC

nuclease
gRNA 9


UUACGAGGCAUUAGCACCAGAAGACACCUUCUUCCCCA


(Exon 2)


 


GUUCUGUCUUUUGGUCAGGACAACCGUCUAGCUAUAAGUG
ATTN
BhCas12b
TRAC KO


CUGCAGGGUGUGAGAGAAACUCCUAUUGCUGGACGAUGUC

nuclease
gRNA 10


UCUUACGAGGCAUUAGCACGGUUCCGAAUCCUCCUGA


(Exon 4)


 


GUUCUGUCUUUUGGUCAGGACAACCGUCUAGCUAUAAGUG
ATTN
BhCas12b
TRAC KO


CUGCAGGGUGUGAGAAACUCCUAUUGCUGGACGAUGUCUC

nuclease
gRNA 11


UUACGAGGCAUUAGCACGGAACCCAAUCACUGACAGGU


(Exon 4)









A DNA construct encoding the chimeric antigen receptor and nucleic acid containing extended stretches of TRAC DNA that flank the gRNA targeting sequences. Without being bound by theory, the construct binds to the complementary TRAC sequences, and the chimeric antigen receptor DNA, residing in proximity to the TRAC sequences on the construct is then inserted at the site of the lesion, effectively knocking out the TRAC gene and knocking in the chimeric antigen receptor nucleic acid. Table 1B provides guide RNAs for the TRAC gene that can guide the base editing machinery to the TRAC locus, which enables insertion of the chimeric antigen receptor nucleic acid. The first 11 gRNAs are for BhCas12b nuclease. The second set of 11 are for the BvCas12b nuclease. These are all for inserting the CAR at TRAC by creating a double stranded break, and not for base editing.







TABLE 1B







TRAC guide RNAs









Guide RNA
SEQ ID NO:
Gene Exon





GTTCTGTCTTTTGGTCAGGACAACCGTCTAGCTATAAGTGCTGCA
132
TRAC KO


GGGTGTGAGAAACTCCTATTGCTGGACGATGTCTCTTACGAGGCA

gRNA 1


TTAGCACAGAGTCTCTCAGCTGGTACA




 


GTTCTGTCTTTTGGTCAGGACAACCGTCTAGCTATAAGTGCTGCA
133
TRAC KO


GGGTGTGAGAAACTCCTATTGCTGGACGATGTCTCTTACGAGGCA

gRNA 2


TTAGCACACCGATTTTGATTCTCAAAC




 


GTTCTGTCTTTTGGTCAGGACAACCGTCTAGCTATAAGTGCTGCA
134
TRAC KO


GGGTGTGAGAAACTCCTATTGCTGGACGATGTCTCTTACGAGGCA

gRNA 3


TTAGCACTGATTCTCAAACAAATGTGT




 


GTTCTGTCTTTTGGTCAGGACAACCGTCTAGCTATAAGTGCTGCA
135
TRAC KO


GGGTGTGAGAAACTCCTATTGCTGGACGATGTCTCTTACGAGGCA

gRNA 4


TTAGCACTCAAACAAATGTGTCACAAA




 


GTTCTGTCTTTTGGTCAGGACAACCGTCTAGCTATAAGTGCTGCA
136
TRAC KO


GGGTGTGAGAAACTCCTATTGCTGGACGATGTCTCTTACGAGGCA

gRNA 5


TTAGCACGTTTGAGAATCAAAATCGGT




 


GTTCTGTCTTTTGGTCAGGACAACCGTCTAGCTATAAGTGCTGCA
137
TRAC KO


GGGTGTGAGAAACTCCTATTGCTGGACGATGTCTCTTACGAGGCA

gRNA 6


TTAGCACTGATGTGTATATCACAGACA




 


GTTCTGTCTTTTGGTCAGGACAACCGTCTAGCTATAAGTGCTGCA
138
TRAC KO


GGGTGTGAGAAACTCCTATTGCTGGACGATGTCTCTTACGAGGCA

gRNA 7


TTAGCACGTTGCTCCAGGCCACAGCAC




 


GTTCTGTCTTTTGGTCAGGACAACCGTCTAGCTATAAGTGCTGCA
139
TRAC KO


GGGTGTGAGAAACTCCTATTGCTGGACGATGTCTCTTACGAGGCA

gRNA 8


TTAGCACTTCCAGAAGACACCTTCTTC




 


GTTCTGTCTTTTGGTCAGGACAACCGTCTAGCTATAAGTGCTGCA
140
TRAC KO


GGGTGTGAGAAACTCCTATTGCTGGACGATGTCTCTTACGAGGCA

gRNA 9


TTAGCACCAGAAGACACCTTCTTCCCC




 


GTTCTGTCTTTTGGTCAGGACAACCGTCTAGCTATAAGTGCTGCA
141
TRAC KO


GGGTGTGAGAAACTCCTATTGCTGGACGATGTCTCTTACGAGGCA

gRNA 10


TTAGCACGGTTCCGAATCCTCCTCCTG




 


GTTCTGTCTTTTGGTCAGGACAACCGTCTAGCTATAAGTGCTGCA
142
TRAC KO


GGGTGTGAGAAACTCCTATTGCTGGACGATGTCTCTTACGAGGCA

gRNA 11


TTAGCACGGAACCCAATCACTGACAGG




 


GACCTATAGGGTCAATGAATCTGTGCGTGTGCCATAAGTAATTAA
143
TRAC KO


AAATTACCCACCACAGGAGCACCTGAAAACAGGTGCTTGGCACAG

gRNA 1


AGTCTCTCAGCTGGTACA




 


GACCTATAGGGTCAATGAATCTGTGCGTGTGCCATAAGTAATTAA
144
TRAC KO


AAATTACCCACCACAGGAGCACCTGAAAACAGGTGCTTGGCACAC

gRNA 2


CGATTTTGATTCTCAAAC




 


GACCTATAGGGTCAATGAATCTGTGCGTGTGCCATAAGTAATTAA
145
TRAC KO


AAATTACCCACCACAGGAGCACCTGAAAACAGGTGCTTGGCACTG

gRNA 3


ATTCTCAAACAAATGTGT




 


GACCTATAGGGTCAATGAATCTGTGCGTGTGCCATAAGTAATTAAA
146
TRAC KO


AATTACCCACCACAGGAGCACCTGAAAACAGGTGCTTGGCACTCAA




ACAAATGTGTCACAAA

gRNA 4


 


GACCTATAGGGTCAATGAATCTGTGCGTGTGCCATAAGTAATTAAA
147
TRAC KO


AATTACCCACCACAGGAGCACCTGAAAACAGGTGCTTGGCACGTTT




GAGAATCAAAATCGGT

gRNA 5


 


GACCTATAGGGTCAATGAATCTGTGCGTGTGCCATAAGTAATTAAA
148
TRAC KO


AATTACCCACCACAGGAGCACCTGAAAACAGGTGCTTGGCACTGAT




GTGTATATCACAGACA

gRNA 6


 


GACCTATAGGGTCAATGAATCTGTGCGTGTGCCATAAGTAATTAAA
149
TRAC KO


AATTACCCACCACAGGAGCACCTGAAAACAGGTGCTTGGCACGTTG

gRNA 7


CTCCAGGCCACAGCAC




 


GACCTATAGGGTCAATGAATCTGTGCGTGTGCCATAAGTAATTAAA
150
TRAC KO


AATTACCCACCACAGGAGCACCTGAAAACAGGTGCTTGGCACTTCC

gRNA 8


AGAAGACACCTTCTTC




 


GACCTATAGGGTCAATGAATCTGTGCGTGTGCCATAAGTAATTAAA
151
TRAC KO


AATTACCCACCACAGGAGCACCTGAAAACAGGTGCTTGGCACCAGA

gRNA 9


AGACACCTTCTTCCCC




 


GACCTATAGGGTCAATGAATCTGTGCGTGTGCCATAAGTAATTAAA
152
TRAC KO


AATTACCCACCACAGGAGCACCTGAAAACAGGTGCTTGGCACGGTT

gRNA 10


CCGAATCCTCCTCCTG




 


GACCTATAGGGTCAATGAATCTGTGCGTGTGCCATAAGTAATTAAA
153
TRAC KO


AATTACCCACCACAGGAGCACCTGAAAACAGGTGCTTGGCACGGAA

gRNA 11


CCCAATCACTGACAGG









First 11 gRNAs are for BhCas112b nuclease. Second set of 11 gRNAs are for the BvCas12b nuclease. Scaffold sequence in bold, in first instance.
In some embodiments, a nucleic acid encoding a chimeric antigen receptor of the present invention can be targeted to the TRAC locus using the BE4 base editor. In some embodiments, the chimeric antigen receptor is targeted to the TRAC locus using a CRISPR/Cas9 base editing system.
To produce the gene edits described above, immune cells are collected from a subject and contacted with two or more guide RNAs and a nucleobase editor polypeptide comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a cytidine deaminase or adenosine deaminase. In some embodiments, the collected immune cells are contacted with at least one nucleic acid, wherein the at least one nucleic acid encodes two or more guide RNAs and a nucleobase editor polypeptide comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a cytidine deaminase. In some embodiments, the gRNA comprises nucleotide analogs. These nucleotide analogs can inhibit degradation of the gRNA from cellular processes. Table 2 provides target sequences to be used for gRNAs.







TABLE 2







Exemplary Target Sequences













Target
Target

SEQ





protein
residue
gRNA target
ID NO:
Base editor
Codon change
Residue function





NFATC1
R118
CTCGATGCGAGGACTCTCCA
154
BE
CGC>CAC
Calcineurin binding



I119
TCTCGATGCGAGGACTCTCC
155
ABE
ATC>ACC
Calcineurin binding



E120
CATCGAGATAACCTCGTGCT
156
ABE
GAG>GGG
Calcineurin binding



S172
TGGCCGGGCTCAGGCACGAG
157
BE
AGC>AAC
PHOSPHORYLATION



W396
GCCCACTGGTAGGGGTGCTG
158
ABE
TGG>CGG
Calcineurin binding



R439
TGGGCTCGGTGGTGGGACTT
159
BE
CGA>CAA
DNA BINDING



H441
CGAGCCCACTACGAGACGGA
160
ABE
CAC>CGC
DNA BINDING



Y442
CTCGTAGTGGGCTCGGTGGT
161
ABE
TAC>CAC
DNA BINDING



K452
GCCGTGAAGGCGTCGGCCGG
162
ABE
AAG>GGG
DNA BINDING



R540
GTTTCTGAGTTTCAGGATTC
163
BE
AGA>AAA
DNA BINDING



R555
CATCGGGAGGAAGAACACAC
164
ABE
AGG>GGG
DNA BINDING



K556
GGAGGAAGAACACACGGGTA
165
ABE
AAG>GGG
DNA BINDING



Q589
GAGCGCTGGGCTGCATCAGA
166
BE
CAG>CAT
DNA BINDING


 


NFATC2
E114
TGATCTCGATCCGAGGGCTC
167
BE
GAG>AAA
Calcineurin binding



I115
ACGGAGTGATCTCGATCCGA
168
ABE
ATC>ACC
Calcineurin binding



R253
GCGGAGGCATTCGTGCGCCG
169
ABE
AGG>GGG
NLS



S99
GCCGCGCTCAGAAACTTCTG
170
BE
AGC>AAC
PHOSPHORYLATION



S107
GGGCCTCGGGCCTGAGCCCT
171
BE
TCG>TTG
PHOSPHORYLATION



S148
CCTCGGGCTGGCGGCCACCC
172
BE
AGC>AAC
PHOSPHORYLATION



S236
CCACTCGCCCGTGCCCCGTC
173
BE
TCG>TTG
PHOSPHORYLATION



S255
GCATTCGTGCGCCGAGGCCT
174
BE
TCG>TTG
PHOSPHORYLATION



S268
GAGCCTCACCCCAGCGCTCC
175
BE
TCA>TTA
PHOSPHORYLATION



S274
GAGGGGCTCCGGGAGCGCTG
176
BE
AGC>AAC
PHOSPHORYLATION



S326
AGGGCTGGTCTTCCACATCT
177
BE
AGC>AAC
PHOSPHORYLATION


 


NFATC4
S213
GCGGGGAGCCCAGGCCAAAG
178
ABE
TCC>CCC
PHOSPHORYLATION


 


AKT1
T305
GCCACCATGAAGACCTTTTG
179
BE
ACC>ATT
PHOSPHORYLATION



T312
TTGCGGCACACCTGAGTACC
180
BE
ACA>ATA
PHOSPHORYLATION



S473
GTAGGAGAACTGGGGGAAGT
181
ABE
TCC>CCC
PHOSPHORYLATION



Y474
CTCCTACTCGGCCAGCGGCA
182
ABE
TAC>TGC
PHOSPHORYLATION


 


AKT2
T309
GAAAACCTTCTGTGGGACCC
183
BE
ACC>ATT
PHOSPHORYLATION



S474
AGTAGGAGAACTGGGGGAAG
184
ABE
TCC>CCC
PHOSPHORYLATION


 


BLIMP1
C608 (ZF2)
GTTGCAAGTCTGACATTTGA
185
ABE
TGC>CGC
DNA BINDING



C608 (ZF2)
GTTGCAAGTCTGACATTTGA
185
BE
TGC>TAC
DNA BINDING



H621 (ZF2)
GAAACACTACCTGGTACACA
186
BE
CAC>TAT
DNA BINDING



C636 (ZF3)
TGTGGCAGACCTACAGTGTA
187
BE
TGC>TAC
DNA BINDING



C664 (ZF4)
GGGCACACCTTGCATTGGTA
188
ABE
TGC>CGC
DNA BINDING



Splice site 1
CTGCGCACCTGGCATTCATG
189
BE




 


GCN2 kinase
Exon 1 SD
CCTACCGGTCCGCAAGCGTC
190
BE

KNOCKOUT


(IDO
Exon 2 SD
ACTCACACATCTGGATAGGT
191
BE

KNOCKOUT


pathway)
Exon 5 SD
GACTTACCTAGACCTTCCTG
192
BE

KNOCKOUT


 


CBL-B
C373
AATCTTACAGAGCTGAAAAG
193
BE
TGT>TAT
E3 UBIQUITIN LIGASE



Y665.1
CATCATATTCTTCACTTCCA
194
ABE
TAT>CAC




Y665.2
AAGAATATGATGTTCCTCCC
195
ABE
TAT>TGT




K907
CCCCTAAACCACGACCGCGC
196
ABE
AAA>GGG




R911
TCCTGCGCGGTCGTGGTTTA
197
BE
CGC>CAC



 


SHP1
Y377
CCCTACTCTGTGACCAACTG
198
ABE
TAC>TGC



 


IRF4
R96
CGCAGGCGCGTCTTCCAGGT
199
BE
CGC>CAC
DNA BINDING



R98
GCACCGCAGGCGCGTCTTCC
200
BE
CGG>CAG
DNA BINDING



K103
GAACAAGAGCAATGACTTTG
201
ABE
AAG>GGG
DNA BINDING


 


PD1
Exon 1 STOP
CACCTACCTAAGAACCATCC
202
BE

KNOCKOUT



Exon 2 STOP
GGGGTTCCAGGGCCTGTCTG
203
BE

KNOCKOUT


 


TET2
H1386
GACTTGCACAACATGCAGAA
204
BE
CAC>TAC
DNA BINDING



R1302
TTGCCAGAAGCAAGATCCCA
205
ABE
AGA>GGG
DNA BINDING



S1290
CCATGAACAACCAAAAGAGA
206
ABE
TCA>CCA
DNA BINDING


 


SMARCA4
T353
TCACCCCCATCCAGAAGCCG
207
BE
ACC>ATT
PHOSPHORYLATION



S610
ATCTGGCTGGTCTCGTCCAG
208
BE
AGC>ATC
PHOSPHORYLATION



S613
GATGAGCGACCTCCCGGTGA
209
ABE
AGC>GGC
PHOSPHORYLATION



S695
AGACAGCGATGACGTCTCTG
210
ABE
AGC>GGC
PHOSPHORYLATION



S699
ACGTCTCTGAGGTGGACGCG
211
BE
TCT>TTT
PHOSPHORYLATION



S1452
TTAGGGGAGAGTTTCTCGGC
212
ABE
TCC>CCC
PHOSPHORYLATION



S1575
GGAGAGTGAGGAGGAGGAAG
213
ABE
AGT>GGT
PHOSPHORYLATION



S1586
AAGGCTCCGAATCCGAATCT
214
BE
TCC>TTT
PHOSPHORYLATION



S1627
ATCGTCACTCACGACCGGCT
215
BE
AGT>AAT
PHOSPHORYLATION



S1631
TGACAGTGAGGAGGAACAAG
216
ABE
AGT>GGT
PHOSPHORYLATION


 


CDK4
P173
CACCCGTGGTTGTTACACTC
217
BE
CCC>CTT



 


ZAP70
S144
CATCAGCCAGGCCCCGCAGG
218
ABE
AGC>TGC
PHOSPHORYLATION



Y292
GGTGTATCCATCTGAGTTGA
219
ABE
TAC>CAC
PHOSPHORYLATION



Y292
GGGTGTATCCATCTGAGTTG
220
ABE
TAC>CAC
PHOSPHORYLATION



R360
GCGCAAGAAGCAGATCGACG
221
BE
CGC>TGC
Hypermorphic activity



Y598
TTACTACAGCCTGGCCAGCA
222
ABE
TAC>TGC
PHOSPHORYLATION









The cytidine and adenosine deaminase nucleobase editors used in this invention can act on DNA, including single stranded DNA. Methods of using them to generate modifications in target nucleobase sequences in immune cells are presented.
In certain embodiments, the fusion proteins provided herein comprise one or more features that improve the base editing activity of the fusion proteins. For example, any of the fusion proteins provided herein may comprise a Cas9 domain that has reduced nuclease activity. In some embodiments, any of the fusion proteins provided herein may have a Cas9 domain that does not have nuclease activity (dCas9), or a Cas9 domain that cuts one strand of a duplexed DNA molecule, referred to as a Cas9 nickase (nCas9). Without wishing to be bound by any particular theory, the presence of the catalytic residue (e.g., H840) maintains the activity of the Cas9 to cleave the non-edited (e.g., non-methylated) strand opposite the targeted nucleobase. Mutation of the catalytic residue (e.g., D10 to A10) prevents cleavage of the edited strand containing the targeted A residue. Such Cas9 variants can generate a single-strand DNA break (nick) at a specific location based on the gRNA-defined target sequence, leading to repair of the non-edited strand, ultimately resulting in a nucleobase change on the non-edited strand.
Nucleobase Editors
Disclosed herein is a base editor or a nucleobase editor for editing, modifying or altering a target nucleotide sequence of a polynucleotide. Described herein is a nucleobase editor or a base editor comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain (e.g., adenosine deaminase or cytidine deaminase). A polynucleotide programmable nucleotide binding domain, when in conjunction with a bound guide polynucleotide (e.g., gRNA), can specifically bind to a target polynucleotide sequence (i.e., via complementary base pairing between bases of the bound guide nucleic acid and bases of the target polynucleotide sequence) and thereby localize the base editor to the target nucleic acid sequence desired to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded DNA or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA hybrid.
Polynucleotide Programmable Nucleotide Binding Domain
It should be appreciated that polynucleotide programmable nucleotide binding domains can also include nucleic acid programmable proteins that bind RNA. For example, the polynucleotide programmable nucleotide binding domain can be associated with a nucleic acid that guides the polynucleotide programmable nucleotide binding domain to an RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, though they are not specifically listed in this disclosure.
A polynucleotide programmable nucleotide binding domain of a base editor can itself comprise one or more domains. For example, a polynucleotide programmable nucleotide binding domain can comprise one or more nuclease domains. In some embodiments, the nuclease domain of a polynucleotide programmable nucleotide binding domain can comprise an endonuclease or an exonuclease. Herein the term “exonuclease” refers to a protein or polypeptide capable of digesting a nucleic acid (e.g., RNA or DNA) from free ends, and the term “endonuclease” refers to a protein or polypeptide capable of catalyzing (e.g., cleaving) internal regions in a nucleic acid (e.g., DNA or RNA). In some embodiments, an endonuclease can cleave a single strand of a double-stranded nucleic acid. In some embodiments, an endonuclease can cleave both strands of a double-stranded nucleic acid molecule. In some embodiments a polynucleotide programmable nucleotide binding domain can be a deoxyribonuclease. In some embodiments a polynucleotide programmable nucleotide binding domain can be a ribonuclease.
In some embodiments, a nuclease domain of a polynucleotide programmable nucleotide binding domain can cut zero, one, or two strands of a target polynucleotide. In some embodiments, the polynucleotide programmable nucleotide binding domain can comprise a nickase domain. Herein the term “nickase” refers to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain that is capable of cleaving only one strand of the two strands in a duplexed nucleic acid molecule (e.g., DNA). In some embodiments, a nickase can be derived from a fully catalytically active (e.g., natural) form of a polynucleotide programmable nucleotide binding domain by introducing one or more mutations into the active polynucleotide programmable nucleotide binding domain. For example, where a polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain can include a D10A mutation and a histidine at position 840. In such embodiments, the residue H840 retains catalytic activity and can thereby cleave a single strand of the nucleic acid duplex. In another example, a Cas9-derived nickase domain can comprise an H840A mutation, while the amino acid residue at position 10 remains a D. In some embodiments, a nickase can be derived from a fully catalytically active (e.g., natural) form of a polynucleotide programmable nucleotide binding domain by removing all or a portion of a nuclease domain that is not required for the nickase activity. For example, where a polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain can comprise a deletion of all or a portion of the RuvC domain or the HNH domain.
The amino acid sequence of an exemplary catalytically active Cas9 is as follows:








MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL



 


KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY


 


HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY


 


NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF


 


DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS


 


MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD


 


GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI


 


PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS


 


LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD


 


SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA


 


HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF


 


KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ


 


TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR


 


LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK


 


FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS


 


KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK


 


SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS


 


MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG


 


KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS


 


AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV


 


ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD


 


ATLIHQSITGLYETRIDLSQLGGD.






A base editor comprising a polynucleotide programmable nucleotide binding domain comprising a nickase domain is thus able to generate a single-strand DNA break (nick) at a specific polynucleotide target sequence (e.g., determined by the complementary sequence of a bound guide nucleic acid). In some embodiments, the strand of a nucleic acid duplex target polynucleotide sequence that is cleaved by a base editor comprising a nickase domain (e.g., Cas9-derived nickase domain) is the strand that is not edited by the base editor (i.e., the strand that is cleaved by the base editor is opposite to a strand comprising a base to be edited). In other embodiments, a base editor comprising a nickase domain (e.g., Cas9-derived nickase domain) can cleave the strand of a DNA molecule which is being targeted for editing. In such embodiments, the non-targeted strand is not cleaved.
Also provided herein are base editors comprising a polynucleotide programmable nucleotide binding domain which is catalytically dead (i.e., incapable of cleaving a target polynucleotide sequence). Herein the terms “catalytically dead” and “nuclease dead” are used interchangeably to refer to a polynucleotide programmable nucleotide binding domain which has one or more mutations and/or deletions resulting in its inability to cleave a strand of a nucleic acid. In some embodiments, a catalytically dead polynucleotide programmable nucleotide binding domain base editor can lack nuclease activity as a result of specific point mutations in one or more nuclease domains. For example, in the case of a base editor comprising a Cas9 domain, the Cas9 can comprise both a D10A mutation and an H840A mutation. Such mutations inactivate both nuclease domains, thereby resulting in the loss of nuclease activity. In other embodiments, a catalytically dead polynucleotide programmable nucleotide binding domain can comprise one or more deletions of all or a portion of a catalytic domain (e.g., RuvC1 and/or HNH domains). In further embodiments, a catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (e.g., D10A or H840A) as well as a deletion of all or a portion of a nuclease domain.
Also contemplated herein are mutations capable of generating a catalytically dead polynucleotide programmable nucleotide binding domain from a previously functional version of the polynucleotide programmable nucleotide binding domain. For example, in the case of catalytically dead Cas9 (“dCas9”), variants having mutations other than D10A and H840A are provided, which result in nuclease inactivated Cas9. Such mutations, by way of example, include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvC1 subdomain). Additional suitable nuclease-inactive dCas9 domains can be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A/H840A, D10A/D839A/H840A, and D10A/D839A/H840A/N863A mutant domains (See, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference).
Non-limiting examples of a polynucleotide programmable nucleotide binding domain which can be incorporated into a base editor include a CRISPR protein-derived domain, a restriction nuclease, a meganuclease, TAL nuclease (TALEN), and a zinc finger nuclease (ZFN). In some embodiments, a base editor comprises a polynucleotide programmable nucleotide binding domain comprising a natural or modified protein or portion thereof which via a bound guide nucleic acid is capable of binding to a nucleic acid sequence during CRISPR (i.e., Clustered Regularly Interspaced Short Palindromic Repeats)-mediated modification of a nucleic acid. Such a protein is referred to herein as a “CRISPR protein.” Accordingly, disclosed herein is a base editor comprising a polynucleotide programmable nucleotide binding domain comprising all or a portion of a CRISPR protein (i.e. a base editor comprising as a domain all or a portion of a CRISPR protein, also referred to as a “CRISPR protein-derived domain” of the base editor). A CRISPR protein-derived domain incorporated into a base editor can be modified compared to a wild-type or natural version of the CRISPR protein. For example, as described below a CRISPR protein-derived domain can comprise one or more mutations, insertions, deletions, rearrangements and/or recombinations relative to a wild-type or natural version of the CRISPR protein.
CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, and then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA,” or simply “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., et al. Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self.
In some embodiments, the methods described herein can utilize an engineered Cas protein. A guide RNA (gRNA) is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user-defined ˜20 nucleotide spacer that defines the genomic target to be modified. Thus, a skilled artisan can change the genomic target of the Cas protein specificity is partially determined by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome.
In some embodiments, the gRNA scaffold sequence is as follows:







GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA


CUUGAAAAAGUGGCACCGAGUCGGUGCUUUU.






In an embodiment, the RNA scaffold comprises a stem loop. In an embodiment, the RNA scaffold comprises the nucleic acid sequence:







GUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAACGAAACUUACACAG


UUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUC


AACACCCUGUCAUUUUAUGGCAGGGUG.






In an embodiment, the RNA scaffold comprises a canonical stem loop. In an embodiment, the RNA scaffold comprises the nucleic acid sequence:







GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA


CUUGAAAAAGUGGCACCGAGUCGGUGCU*mU*mU*mU






where m=2′-O-methyl modification and *=3′ phosphorothioate internucleotide linkages (i.e., at the first 3′ terminal RNA residues as shown here).

In an embodiment, the RNA scaffold comprises the nucleic acid sequence:







GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA


CUUGAAAAAGUGGCACCGAGUCGGUGCUUUU






where m=2′-O-methyl modification and *=3′ phosphorothioate internucleotide linkages (i.e., at the first 3′ terminal RNA residues as shown here).

In an embodiment, an S. pyogenes sgRNA scaffold polynucleotide sequence is as follows:







GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA


CUUGAAAAAGUGGCACCGAGUCGGUGC.






In an embodiment, an S. aureus sgRNA scaffold polynucleotide sequence is as follows:







GUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACUAAAACAAGGCAA


AAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGA.






In an embodiment, a BhCas12b sgRNA scaffold has the following polynucleotide sequence:







GUUCUGTCUUUUGGUCAGGACAACCGUCUAGCUAUAAGUGCUGCAGGGU


GUGAGAAACUCCUAUUGCUGGACGAUGUCUCUUACGAGGCAUUAGCAC.






In an embodiment, a BvCas12b sgRNA scaffold has the following polynucleotide sequence:







GACCUAUAGGGUCAAUGAAUCUGUGCGUGUGCCAUAAGUAAUUAAAAAU


UACCCACCACAGGAGCACCUGAAAACAGGUGCUUGGCAC.






In an embodiment, the RNA scaffold comprises a non-canonical sequence. In an embodiment, the RNA scaffold comprises the nucleic acid sequence:







GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA


CUUGAAAAAGUGGGACCGAGUCGGUGCU*mU*mU*mU






where m=2′-O-methyl modification and *=3′ phosphorothioate internucleotide linkages (i.e., at the first 3′ terminal RNA residues as shown here).

In some embodiments, a CRISPR protein-derived domain incorporated into a base editor is an endonuclease (e.g., deoxyribonuclease or ribonuclease) capable of binding a target polynucleotide when in conjunction with a bound guide nucleic acid. In some embodiments, a CRISPR protein-derived domain incorporated into a base editor is a nickase capable of binding a target polynucleotide when in conjunction with a bound guide nucleic acid. In some embodiments, a CRISPR protein-derived domain incorporated into a base editor is a catalytically dead domain capable of binding a target polynucleotide when in conjunction with a bound guide nucleic acid. In some embodiments, a target polynucleotide bound by a CRISPR protein derived domain of a base editor is DNA. In some embodiments, a target polynucleotide bound by a CRISPR protein-derived domain of a base editor is RNA.
Cas proteins that can be used herein include class 1 and class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, 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, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i, and Cas12j/CasΦ, CARF, DinG, homologues thereof, or modified versions thereof. An unmodified CRISPR enzyme can have DNA cleavage activity, such as Cas9, which has two functional endonuclease domains: RuvC and HNH. A CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within a target sequence and/or within a complement of a target sequence. For example, a CRISPR enzyme can direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.
A vector that encodes a CRISPR enzyme that is mutated to with respect, to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence can be used. Cas9 can refer to a polypeptide with at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and/or sequence homology to a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from S. pyogenes). Cas9 can refer to a polypeptide with at most or at most about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and/or sequence homology to a wild-type exemplary Cas9 polypeptide (e.g., from S. pyogenes). Cas9 can refer to the wild-type or a modified form of the Cas9 protein that can comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof.
In some embodiments, a CRISPR protein-derived domain of a base editor can include all or a portion of Cas9 from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquis (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); Neisseria meningitidis (NCBI Ref: YP_002342100.1), Streptococcus pyogenes, or Staphylococcus aureus. 
Cas9 Domains of Nucleobase Editors
Cas9 nuclease sequences and structures are well known to those of skill in the art (See, e.g., “Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti et al., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., et al., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., et al., Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.
In some embodiments, a nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-limiting, exemplary Cas9 domains are provided herein. The Cas9 domain may be a nuclease active Cas9 domain, a nuclease inactive Cas9 domain (dCas9), or a Cas9 nickase (nCas9). In some embodiments, the Cas9 domain is a nuclease active domain. For example, the Cas9 domain may be a Cas9 domain that cuts both strands of a duplexed nucleic acid (e.g., both strands of a duplexed DNA molecule). In some embodiments, the Cas9 domain comprises any one of the amino acid sequences as set forth herein. In some embodiments the Cas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that has 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any one of the amino acid sequences set forth herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that has at least 10, at least 15, 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 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth herein.
In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as “Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9. In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild-type Cas9. In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.
In some embodiments, Cas9 fusion proteins as provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas9 sequence, but only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments will be apparent to those of skill in the art.
A Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence that has complementary to the guide RNA. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 domain, for example a nuclease active Cas9, a Cas9 nickase (nCas9), or a nuclease inactive Cas9 (dCas9). Examples of nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), Cas12a/Cpf1, Cas12b/C2cl, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i, and Cas12j/CasΦ. Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a/Cpf1, Cas12b/C2cl, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i, Cas12j/CasΦ, 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, homologues thereof, or modified or engineered versions thereof.
In some embodiments, wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1). An exemplary Streptococcus pyogenes Cas9 (spCas9) nucleic acid sequence is provided below:








ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCA



 


CTGATGATTATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTAT


 


CAAAAAAAATCTTATAGGGGCTCTTTTATTTGGCAGTGGAGAGACAGCGGAAGCGACTCGTCTC


 


AAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTT


 


TTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGT


 


GGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTAT


 


CATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGCAGATTCTACTGATAAAGCGG


 


ATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGA


 


GGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAATCTAC


 


AATCAATTATTTGAAGAAAACCCTATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTG


 


CACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTGAGCTCCCCGGTGAGAAGAGAAA


 


TGGCTTGTTTGGGAATCTCATTGCTTTGTCATTGGGATTGACCCCTAATTTTAAATCAAATTTT


 


GATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATT


 


TATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGC


 


TATTTTACTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCTATCAGCTTCA


 


ATGATTAAGCGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAAC


 


AACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATAT


 


TGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGAT


 


GGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTG


 


ACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGA


 


AGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATT


 


CCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTG


 


AAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATT


 


TATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGT


 


TTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAGGGAA


 


TGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAAC


 


AAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGAT


 


AGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGCGCCTACCATGATTTGC


 


TAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATAT


 


TGTTTTAACATTGACCTTATTTGAAGATAGGGGGATGATTGAGGAAAGACTTAAAACATATGCT


 


CACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTT


 


TGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTT


 


GAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTT


 


AAAGAAGATATTCAAAAAGCACAGGTGTCTGGACAAGGCCATAGTTTACATGAACAGATTGCTA


 


ACTTAGCTGGCAGTCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAATTGTTGATGAACT


 


GGTCAAAGTAATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACA


 


ACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAAT


 


TAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTA


 


TCTCTATTATCTACAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTA


 


AGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAGACGATTCAATAGACAATA


 


AGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGT


 


CAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTT


 


GATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAAC


 


GCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAA


 


TACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAA


 


TTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATC


 


ATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACT


 


TGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCT


 


GAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCA


 


AAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGA


 


AACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATG


 


CCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTT


 


TACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGG


 


TGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAA


 


TCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTG


 


AAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCAT


 


TAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCC


 


GGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAG


 


CTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGA


 


GCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATT


 


TTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATAC


 


GTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTT


 


TAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCC


 


ACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAG


 


GTGACTGA






An exemplary Streptococcus pyogenes Cas9 (spCas9) amino acid sequence is provided below:








MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGALLFGSGETAEATRL



 


KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY


 


HEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIY


 


NQLFEENPINASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSNE


 


DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSAS


 


MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD


 


GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI


 


PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS


 


LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD


 


SVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYA


 


HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF


 


KEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQT


 


TQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRL


 


SDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF


 


DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK


 


LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKS


 


EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM


 


PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGK


 


SKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASA


 


GELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVI


 


LADANLDKVLSAYNKHRDKPIREQAENIIHLETLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDA


 


TLIHQSITGLYETRIDLSQLGGD






(single underline: HNH domain; double underline: RuvC domain)

In some embodiments, wild-type Cas9 corresponds to, or comprises the following nucleotide sequences:








ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCATAA



 


CCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGAT


 


TAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTG


 


AAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTT


 


TTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGT


 


CGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATAT


 


CATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGG


 


ACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGA


 


GGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTAT


 


AATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCG


 


CCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAA


 


TGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTC


 


GACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATC


 


TACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGC


 


AATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCA


 


ATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGC


 


AACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATAT


 


TGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGAT


 


GGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCG


 


ACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGA


 


GGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATA


 


CCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCG


 


AAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTT


 


CATCGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGT


 


TTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCA


 


TGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGAC


 


CAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGAT


 


TCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCC


 


TAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATAT


 


AGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCT


 


CACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGAT


 


TGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCT


 


AAAGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGACTCTTTAACCTTC


 


AAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGA


 


ATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCT


 


AGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAA


 


ACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAG


 


AACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGAAACT


 


TTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGT


 


TTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACA


 


ATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGT


 


CGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAG


 


TTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTA


 


AACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTAGATTCCCGAAT


 


GAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCA


 


AAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACC


 


ACCATGCGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAA


 


GCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAA


 


AGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCT


 


TTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGG


 


GGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCC


 


ATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGA


 


TTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTA


 


CGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGA


 


AAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTT


 


TTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCAT


 


AATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGC


 


GCCGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATT


 


TAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGT


 


TGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTC


 


ATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCA


 


TACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGC


 


ATTCAAGTATTTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTAGAC


 


GCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTG


 


GGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGA


 


TTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCTGCAGGA






In some embodiments, wild-type Cas9 corresponds to, or comprises the following amino acid sequence:








MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL



 


KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY


 


HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY


 


NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF


 


DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS


 


MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD


 


GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI


 


PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS


 


LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD


 


SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA


 


HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTE


 


KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ


 


TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR


 


LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK


 


FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS


 


KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK


 


SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS


 


MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG


 


KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS


 


AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV


 


ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD


 


ATLIHQSITGLYETRIDLSQLGGD






(single underline: HNH domain; double underline: RuvC domain).

In some embodiments, wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_002737.2 (nucleotide sequence as follows)):








ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCA



 


CTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTAT


 


CAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTC


 


AAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTT


 


TTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGT


 


GGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTAT


 


CATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGG


 


ATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGA


 


GGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTAC


 


AATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTG


 


CACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAA


 


TGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTT


 


GATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATT


 


TATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGC


 


TATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCA


 


ATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAAC


 


AACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATAT


 


TGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGAT


 


GGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTG


 


ACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGA


 


AGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATT


 


CCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTG


 


AAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATT


 


TATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGT


 


TTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAA


 


TGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAAC


 


AAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGAT


 


AGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGC


 


TAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATAT


 


TGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCT


 


CACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTT


 


TGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTT


 


GAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTT


 


AAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCAA


 


ATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATT


 


GGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAG


 


ACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAG


 


AATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCT


 


CTATCTCTATTATCTCCAAAATGGAAGAGACAAGTATGTGGACCAAGAAATAGATATTAATCGT


 


TTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACA


 


ATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGT


 


AGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAG


 


TTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCA


 


AACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCAT


 


GAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCT


 


AAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACC


 


ATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAA


 


ACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAG


 


TCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCT


 


TCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGG


 


GGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCC


 


ATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAA


 


TTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATA


 


TGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGG


 


AAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCT


 


TTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAAT


 


CATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGT


 


GCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATT


 


TAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGT


 


GGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTT


 


ATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAA


 


TACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGC


 


TTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGAT


 


GCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAG


 


GAGGTGACTGA






In some embodiments, wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (Uniprot Reference Sequence: Q99ZW2 (amino acid sequence as follows)):








MDKKYSIGLDIGTNSVGWAVITEDYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL



 


KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY


 


HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY


 


NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF


 


DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS


 


MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD


 


GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI


 


PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS


 


LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD


 


SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA


 


HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF


 


KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ


 


TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR


 


LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK


 


FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS


 


KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK


 


SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS


 


MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG


 


KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS


 


AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV


 


ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD


 


ATLIHQSITGLYETRIDLSQLGGD






(single underline: HNH domain; double underline: RuvC domain)

In some embodiments, Cas9 refers to Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1) or Neisseria meningitidis (NCBI Ref: YP_002342100.1) or to a Cas9 from any other organism.
It should be appreciated that additional Cas9 proteins (e.g., a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9), including variants and homologs thereof, are within the scope of this disclosure. Exemplary Cas9 proteins include, without limitation, those provided below. In some embodiments, the Cas9 protein is a nuclease dead Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease active Cas9.
In some embodiments, the Cas9 domain is a nuclease-inactive Cas9 domain (dCas9). For example, the dCas9 domain may bind to a duplexed nucleic acid molecule (e.g., via a gRNA molecule) without cleaving either strand of the duplexed nucleic acid molecule. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10X mutation and a H840X mutation of the amino acid sequence set forth herein, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid change. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10A mutation and a H840A mutation of the amino acid sequence set forth herein, or a corresponding mutation in any of the amino acid sequences provided herein. As one example, a nuclease-inactive Cas9 domain comprises the amino acid sequence set forth in Cloning vector pPlatTET-gRNA2 (Accession No. BAV54124).
The amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows: MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 236) (see, e.g., Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.” Cell. 2013; 152(5):1173-83, the entire contents of which are incorporated herein by reference).
Additional suitable nuclease-inactive dCas9 domains will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A/H840A, D10A/D839A/H840A, and D10A/D839A/H840A/N863A mutant domains (See, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference).
In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase, referred to as an “nCas9” protein (for “nickase” Cas9). A nuclease-inactivated Cas9 protein may interchangeably be referred to as a “dCas9” protein (for nuclease-“dead” Cas9) or catalytically inactive Cas9. Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell. 28; 152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28; 152(5):1173-83 (2013)).
In some embodiments, the dCas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the dCas9 domains provided herein. In some embodiments, the Cas9 domain comprises an amino acid sequences that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more or more mutations compared to any one of the amino acid sequences set forth herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that has at least 10, at least 15, 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 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth herein.
In some embodiments, dCas9 corresponds to, or comprises in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10X mutation and a H840X mutation of the amino acid sequence set forth herein, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid change. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10A mutation and a H840A mutation of the amino acid sequence set forth herein, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, a nuclease-inactive Cas9 domain comprises the amino acid sequence set forth in Cloning vector pPlatTET-gRNA2 (Accession No. BAV54124).
In some embodiments, the dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A):








MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL



 


KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY


 


HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY


 


NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNE


 


DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS


 


MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD


 


GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI


 


PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS


 


LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD


 


SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA


 


HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF


 


KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ


 


TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR


 


LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK


 


FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS


 


KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK


 


SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS


 


MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG


 


KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS


 


AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV


 


ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD


 


ATLIHQSITGLYETRIDLSQLGGD






(single underline: HNH domain; double underline: RuvC domain).

In some embodiments, the amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows:








MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL



 


KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY


 


HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY


 


NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNE


 


DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS


 


MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD


 


GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI


 


PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS


 


LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD


 


SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA


 


HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTE


 


KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ


 


TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR


 


LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK


 


FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS


 


KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK


 


SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS


 


MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG


 


KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS


 


AGELQKGNELALPSKIVNFLYLASHIEKLKGSPEDNEQKQLHVEQHKHYDDEIIEQISEFSKRV


 


ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD


 


ATLIHQSITGLYETRIDLSQLGGD






(see, e.g., Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.” Cell. 2013; 152(5):1173-83, the entire contents of which are incorporated herein by reference).

In some embodiments, the amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows:








MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL



 


KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY


 


HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY


 


NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF


 


DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS


 


MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD


 


GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI


 


PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS


 


LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD


 


SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA


 


HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF


 


KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ


 


TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR


 


LSDYDVDAIVPQSFLKDDS1DNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK


 


FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS


 


KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK


 


SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS


 


MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG


 


KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS


 


AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLHVEQHKHYLDEIIEQISEFSKRV


 


ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD


 


ATLIHQSITGLYETRIDLSQLGGD






In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 remains a histidine in the amino acid sequence provided above, or at corresponding positions in any of the amino acid sequences provided herein.
In other embodiments, dCas9 variants having mutations other than D10A and H840A are provided, which, e.g., result in nuclease inactivated Cas9 (dCas9). Such mutations, by way of example, include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvC1 subdomain). In some embodiments, variants or homologues of dCas9 are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, variants of dCas9 are provided having amino acid sequences which are shorter, or longer, by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more.
Additional suitable nuclease-inactive dCas9 domains will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A/H840A, D10A/D839A/H840A, and D10A/D839A/H840A/N863A mutant domains (See, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference).
In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase, referred to as an “nCas9” protein (for “nickase” Cas9). The Cas9 nickase may be a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule (e.g., a duplexed DNA molecule). In some embodiments the Cas9 nickase cleaves the target strand of a duplexed nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is base paired to (complementary to) a gRNA (e.g., an sgRNA) that is bound to the Cas9. In some embodiments, a Cas9 nickase comprises a D10A mutation and has a histidine at position 840. In some embodiments the Cas9 nickase cleaves the non-target, non-base-edited strand of a duplexed nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is not base paired to a gRNA (e.g., an sgRNA) that is bound to the Cas9. In some embodiments, a Cas9 nickase comprises an H840A mutation and has an aspartic acid residue at position 10, or a corresponding mutation. In some embodiments the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 nickases provided herein. Additional suitable Cas9 nickases will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure.
The amino acid sequence of an exemplary catalytically Cas9 nickase (nCas9) is as follows:








MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL



 


KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY


 


HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY


 


NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNE


 


DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS


 


MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD


 


GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI


 


PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS


 


LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD


 


SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA


 


HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF


 


KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ


 


TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR


 


LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK


 


FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS


 


KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK


 


SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKYLS


 


MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG


 


KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS


 


AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV


 


ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD


 


ATLIHQSITGLYETRIDLSQLGGD






In some embodiments, Cas9 refers to a Cas9 from archaea (e.g., nanoarchaea), which constitute a domain and kingdom of single-celled prokaryotic microbes. In some embodiments, the nucleic acid programmable DNA binding protein may be a CasX or CasY protein, which have been described in, for example, Burstein et al., “New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017 Feb. 21. doi: 10.1038/cr.2017.21, the entire contents of which is hereby incorporated by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems were discovered, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. In some embodiments, in a base editor system described herein Cas9 is replaced by CasX, or a variant of CasX. In some embodiments, in a base editor system described herein Cas9 is replaced by CasY, or a variant of CasY. It should be appreciated that other RNA-guided DNA binding proteins may be used as a nucleic acid programmable DNA binding protein (napDNAbp), and are within the scope of this disclosure.
In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein may be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the programmable nucleotide binding protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to any CasX or CasY protein described herein. In some embodiments, the programmable nucleotide binding protein is a naturally-occurring CasX or CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to a naturally-occurring CasX or CasY protein. It should be appreciated that CasX and CasY from other bacterial species may also be used in accordance with the present disclosure.
An exemplary CasX ((uniprot.org/uniprot/F0NN87; uniprot.org/uniprot/F0NH53) tr|F0NN87|F0NN87_SULIHCRISPR-associatedCasx protein OS=Sulfolobus islandicus (strain HVE10/4) GN=SiH_0402 PE=4 SV=1) amino acid sequence is as follows:








MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAK



 


KKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVK


 


PEFYEFGRSPGMVERTRRVKLEVEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILY


 


SLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLT


 


KLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNI


 


NSDDGKVRDLKLISAYVNGELIRGEG.






An exemplary CasX (>tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx OS=Sulfolobus islandicus (strain REY15A) GN=SiRe_0771 PE=4 SV=1) amino acid sequence is as follows:








MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAK



 


KKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVK


 


PEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILY


 


SLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLT


 


KLLEKRDLLSERLEAIARNALSISSNMRERYTVLANYIYEYLTGSKRLEDLLYFANRDLIMNLN


 


SDDGKVRDLKLISAYVNGELIRGEG.


 


Deltaproteobacteria CasX


MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANN


 


LRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFAC


 


SQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRA


 


LDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKG


 


NQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDfAYNEVIARVRMWVNLNLWQKLKLSRDDA


 


KPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYL


 


PNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIERE


 


EARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQKWYGDLRGNPFAVEAENRV


 


VDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLY


 


GGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKI


 


GRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTD


 


ILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLV


 


FANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTIT


 


YADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDI


 


SKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSY


 


KSGKQPFVGAWQAFYKRRLKEVWKPNA






An exemplary CasY ((ncbi.nlm.nih.gov/protein/APG80656.1)>APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacterium]) amino acid sequence is as follows:








MSKRHPRISGVKGYRLHAQRLEYTGKSGAMRTIKYPLYSSPSGGRTVPREIVSAINDDYVGLYG



 


LSNFDDLYNAEKRNEEKVYSVLDFWYDCVQYGAVFSYTAPGLLKNVAEVRGGSYELTKTLKGSH


 


LYDELQIDKVIKFLNKKEISRANGSLDKLKKDIIDCFKAEYRERHKDQCNKLADDIKNAKKDAG


 


ASLGERQKKLFRDFFGISEQSENDKPSFTNPLNLTCCLLPFDTVNNNRNRGEVLFNKLKEYAQK


 


LDKNEGSLEMWEYIGIGNSGTAFSNFLGEGFLGRLRENKITELKKAMMDITDAWRGQEQEEELE


 


KRLRILAALTIKLREPKFDNHWGGYRSDINGKLSSWLQNYINQTVKIKEDLKGHKKDLKKAKEM


 


INRFGESDTKEEAVVSSLLESIEKIVPDDSADDEKPDIPAIAIYRRFLSDGRLTLNRFVQREDV


 


QEALLKERLEAEKKKKPKKRKKKSDAEDEKETIDEKELFPHLAKPLKLVPNEYGDSKRELYKKY


 


KNAAIYTDALWKAVEKIYKSAFSSSLKNSFFDTDFDKDFFIKRLQKIFSVYRRFNTDKWKPIVK


 


NSFAPYCDIVSLAENEVLYKPKQSRSRKSAAIDKNRVRLPSTENIAKAGIALARELSVAGFDWK


 


DLLKKEEHEEYIDLIELHKTALALLLAVTETQLDISALDFVENGTVKDFMKTRDGNLVLEGRFL


 


EMFSQSIVFSELRGLAGLMSRKEFITRSAIQTMNGKQAELLYIPHEFQSAKITTPKEMSRAFLD


 


LAPAEFATSLEPESLSEKSLLKLKQMRYYPHYFGYELTRTGQGIDGGVAENALRLEKSPVKKRE


 


IKCKQYKTLGRGQNKIVLYVRSSYYQTQFLEWFLHRPKNVQTDVAVSGSFLIDEKKVKTRWNYD


 


ALTVALEPVSGSERVFVSQPFTIFPEKSAEEEGQRYLGIDIGEYGIAYTALEITGDSAKILDQN


 


FISDPQLKTLREEVKGLKLDQRRGTFAMPSTKIARIRESLVHSLRNRIHHLALKHKAKIVYELE


 


VSRFEEGKQKIKKVYATLKKADVYSEIDADKNLQTTVWGKLAVASEISASYTSQFCGACKKLWR


 


AEMQVDETITTQELTGTVRVIKGGTLIDAIKDFMRPPIFDENDTPFPKYRDFCDKHHISKKMRG


 


NSCLFICPFCRANADADIQASQTIALLRYVKEEKKVEDYFERFRKLKNIKVLGQMKKI.






The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Cas9 undergoes a conformational change upon target binding that positions the nuclease domains to cleave opposite strands of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (˜3-4 nucleotides upstream of the PAM sequence). The resulting DSB is then repaired by one of two general repair pathways: (1) the efficient but error-prone non-homologous end joining (NHEJ) pathway; or (2) the less efficient but high-fidelity homology directed repair (HDR) pathway.
The “efficiency” of non-homologous end joining (NHEJ) and/or homology directed repair (HDR) can be calculated by any convenient method. For example, in some embodiments, efficiency can be expressed in terms of percentage of successful HDR. For example, a surveyor nuclease assay can be used to generate cleavage products and the ratio of products to substrate can be used to calculate the percentage. For example, a surveyor nuclease enzyme can be used that directly cleaves DNA containing a newly integrated restriction sequence as the result of successful HDR. More cleaved substrate indicates a greater percent HDR (a greater efficiency of HDR). As an illustrative example, a fraction (percentage) of HDR can be calculated using the following equation [(cleavage products)/(substrate plus cleavage products)](e.g., (b+c)/(a+b+c), where “a” is the band intensity of DNA substrate and “b” and “c” are the cleavage products).
In some embodiments, efficiency can be expressed in terms of percentage of successful NHEJ. For example, a T7 endonuclease I assay can be used to generate cleavage products and the ratio of products to substrate can be used to calculate the percentage NHEJ. T7 endonuclease I cleaves mismatched heteroduplex DNA which arises from hybridization of wild-type and mutant DNA strands (NHEJ generates small random insertions or deletions (indels) at the site of the original break). More cleavage indicates a greater percent NHEJ (a greater efficiency of NHEJ). As an illustrative example, a fraction (percentage) of NHEJ can be calculated using the following equation: (1−(1−(b+c)/(a+b+c))1/2)×100, where “a” is the band intensity of DNA substrate and “b” and “c” are the cleavage products (Ran et. al., Cell. 2013 Sep. 12; 154(6):1380-9; and Ran et al., Nat Protoc. 2013 November; 8(11): 2281-2308).
The NHEJ repair pathway is the most active repair mechanism, and it frequently causes small nucleotide insertions or deletions (indels) at the DSB site. The randomness of NHEJ-mediated DSB repair has important practical implications, because a population of cells expressing Cas9 and a gRNA or a guide polynucleotide can result in a diverse array of mutations. In most embodiments, NHEJ gives rise to small indels in the target DNA that result in amino acid deletions, insertions, or frameshift mutations leading to premature stop codons within the open reading frame (ORF) of the targeted gene. The ideal end result is a loss-of-function mutation within the targeted gene.
While NHEJ-mediated DSB repair often disrupts the open reading frame of the gene, homology directed repair (HDR) can be used to generate specific nucleotide changes ranging from a single nucleotide change to large insertions like the addition of a fluorophore or tag.
In order to utilize HDR for gene editing, a DNA repair template containing the desired sequence can be delivered into the cell type of interest with the gRNA(s) and Cas9 or Cas9 nickase. The repair template can contain the desired edit as well as additional homologous sequence immediately upstream and downstream of the target (termed left & right homology arms). The length of each homology arm can be dependent on the size of the change being introduced, with larger insertions requiring longer homology arms. The repair template can be a single-stranded oligonucleotide, double-stranded oligonucleotide, or a double-stranded DNA plasmid. The efficiency of HDR is generally low (<10% of modified alleles) even in cells that express Cas9, gRNA and an exogenous repair template. The efficiency of HDR can be enhanced by synchronizing the cells, since HDR takes place during the S and G2 phases of the cell cycle. Chemically or genetically inhibiting genes involved in NHEJ can also increase HDR frequency.
In some embodiments, Cas9 is a modified Cas9. A given gRNA targeting sequence can have additional sites throughout the genome where partial homology exists. These sites are called off-targets and need to be considered when designing a gRNA. In addition to optimizing gRNA design, CRISPR specificity can also be increased through modifications to Cas9. Cas9 generates double-strand breaks (DSBs) through the combined activity of two nuclease domains, RuvC and HNH. Cas9 nickase, a D10A mutant of SpCas9, retains one nuclease domain and generates a DNA nick rather than a DSB. The nickase system can also be combined with HDR-mediated gene editing for specific gene edits.
In some embodiments, Cas9 is a variant Cas9 protein. A variant Cas9 polypeptide has an amino acid sequence that is different by one amino acid (e.g., has a deletion, insertion, substitution, fusion) when compared to the amino acid sequence of a wild-type Cas9 protein. In some instances, the variant Cas9 polypeptide has an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nuclease activity of the Cas9 polypeptide. For example, in some instances, the variant Cas9 polypeptide has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 protein. In some embodiments, the variant Cas9 protein has no substantial nuclease activity. When a subject Cas9 protein is a variant Cas9 protein that has no substantial nuclease activity, it can be referred to as “dCas9.”
In some embodiments, a variant Cas9 protein has reduced nuclease activity. For example, a variant Cas9 protein exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1%, of the endonuclease activity of a wild-type Cas9 protein, e.g., a wild-type Cas9 protein.
In some embodiments, a variant Cas9 protein can cleave the complementary strand of a guide target sequence but has reduced ability to cleave the non-complementary strand of a double stranded guide target sequence. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, a variant Cas9 protein has a D10A (aspartate to alanine at amino acid position 10) and can therefore cleave the complementary strand of a double stranded guide target sequence but has reduced ability to cleave the non-complementary strand of a double stranded guide target sequence (thus resulting in a single strand break (SSB) instead of a double strand break (DSB) when the variant Cas9 protein cleaves a double stranded target nucleic acid) (see, for example, Jinek et al., Science. 2012 Aug. 17; 337(6096):816-21).
In some embodiments, a variant Cas9 protein can cleave the non-complementary strand of a double stranded guide target sequence but has reduced ability to cleave the complementary strand of the guide target sequence. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC/HNH/RuvC domain motifs). As a non-limiting example, in some embodiments, the variant Cas9 protein has an H840A (histidine to alanine at amino acid position 840) mutation and can therefore cleave the non-complementary strand of the guide target sequence but has reduced ability to cleave the complementary strand of the guide target sequence (thus resulting in a SSB instead of a DSB when the variant Cas9 protein cleaves a double stranded guide target sequence). Such a Cas9 protein has a reduced ability to cleave a guide target sequence (e.g., a single stranded guide target sequence) but retains the ability to bind a guide target sequence (e.g., a single stranded guide target sequence).
In some embodiments, a variant Cas9 protein has a reduced ability to cleave both the complementary and the non-complementary strands of a double stranded target DNA. As a non-limiting example, in some embodiments, the variant Cas9 protein harbors both the D10A and the H840A mutations such that the polypeptide has a reduced ability to cleave both the complementary and the non-complementary strands of a double stranded target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA).
As another non-limiting example, in some embodiments, the variant Cas9 protein harbors W476A and WI 126A mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA).
As another non-limiting example, in some embodiments, the variant Cas9 protein harbors P475A, W476A, N477A, D1125A, WI126A, and D1127A mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA).
As another non-limiting example, in some embodiments, the variant Cas9 protein harbors H840A, W476A, and WI 126A, mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA). As another non-limiting example, in some embodiments, the variant Cas9 protein harbors H840A, D10A, W476A, and WI 126A, mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA). In some embodiments, the variant Cas9 has restored catalytic His residue at position 840 in the Cas9 HNH domain (A840H).
As another non-limiting example, in some embodiments, the variant Cas9 protein harbors, H840A, P475A, W476A, N477A, D1125A, WI 126A, and D1127A mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA). As another non-limiting example, in some embodiments, the variant Cas9 protein harbors D10A, H840A, P475A, W476A, N477A, D1125A, W1126A, and D1127A mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA). In some embodiments, when a variant Cas9 protein harbors W476A and WI126A mutations or when the variant Cas9 protein harbors P475A, W476A, N477A, D1125A, W1126A, and D1127A mutations, the variant Cas9 protein does not bind efficiently to a PAM sequence. Thus, in some such embodiments, when such a variant Cas9 protein is used in a method of binding, the method does not require a PAM sequence. In other words, in some embodiments, when such a variant Cas9 protein is used in a method of binding, the method can include a guide RNA, but the method can be performed in the absence of a PAM sequence (and the specificity of binding is therefore provided by the targeting segment of the guide RNA). Other residues can be mutated to achieve the above effects (i.e., inactivate one or the other nuclease portions). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987 can be altered (i.e., substituted). Also, mutations other than alanine substitutions are suitable.
In some embodiments, a variant Cas9 protein that has reduced catalytic activity (e.g., when a Cas9 protein has a D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or a A987 mutation, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and/or D986A), the variant Cas9 protein can still bind to target DNA in a site-specific manner (because it is still guided to a target DNA sequence by a guide RNA) as long as it retains the ability to interact with the guide RNA.
In some embodiments, the variant Cas protein can be spCas9, spCas9-VRQR, spCas9-VRER, xCas9 (sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL.
In some embodiments, a modified SpCas9 including amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and having specificity for the altered PAM 5′-NGC-3′ was used.
Alternatives to S. pyogenes Cas9 can include RNA-guided endonucleases from the Cpf1 family that display cleavage activity in mammalian cells. CRISPR from Prevotella and Francisella 1 (CRISPR/Cpf1) is a DNA-editing technology analogous to the CRISPR/Cas9 system. Cpf1 is an RNA-guided endonuclease of a class II CRISPR/Cas system. This acquired immune mechanism is found in Prevotella and Francisella bacteria. Cpf1 genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations. Unlike Cas9 nucleases, the result of Cpf1-mediated DNA cleavage is a double-strand break with a short 3′ overhang. Cpf1's staggered cleavage pattern can open up the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning, which can increase the efficiency of gene editing. Like the Cas9 variants and orthologues described above, Cpf1 can also expand the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM sites favored by SpCas9. The Cpf1 locus contains a mixed alpha/beta domain, a RuvC-I followed by a helical region, a RuvC-II and a zinc finger-like domain. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9.
Furthermore, Cpf1, unlike Cas9, does not have a HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alpha-helical recognition lobe of Cas9. Cpf1 CRISPR-Cas domain architecture shows that Cpf1 is functionally unique, being classified as Class 2, type V CRISPR system. The Cpf1 loci encode Cas1, Cas2 and Cas4 proteins that are more similar to types I and III than type II systems. Functional Cpf1 does not require the trans-activating CRISPR RNA (tracrRNA), therefore, only CRISPR (crRNA) is required. This benefits genome editing because Cpf1 is not only smaller than Cas9, but also it has a smaller sgRNA molecule (approximately half as many nucleotides as Cas9). The Cpf1-crRNA complex cleaves target DNA or RNA by identification of a protospacer adjacent motif 5′-YTN-3′ or 5′-TTN-3′ in contrast to the G-rich PAM targeted by Cas9. After identification of PAM, Cpf1 introduces a sticky-end-like DNA double-stranded break having an overhang of 4 or 5 nucleotides.
In some embodiments, the Cas9 is a Cas9 variant having specificity for an altered PAM sequence. In some embodiments, the Additional Cas9 variants and PAM sequences are described in Miller, S. M., et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs, Nat. Biotechnol. (2020), the entirety of which is incorporated herein by reference. in some embodiments, a Cas9 variate have no specific PAM requirements. In some embodiments, a Cas9 variant, e.g. a SpCas9 variant has specificity for a NRNH PAM, wherein R is A or G and H is A, C, or T. In some embodiments, the SpCas9 variant has specificity for a PAM sequence AAA, TAA, CAA, GAA, TAT, GAT, or CAC. In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1218, 1219, 1221, 1249, 1256, 1264, 1290, 1318, 1317, 1320, 1321, 1323, 1332, 1333, 1335, 1337, or 1339 or a corresponding position thereof. In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1135, 1218, 1219, 1221, 1249, 1320, 1321, 1323, 1332, 1333, 1335, or 1337 or a corresponding position thereof. In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1219, 1221, 1256, 1264, 1290, 1318, 1317, 1320, 1323, 1333 or a corresponding position thereof. In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1131, 1135, 1150, 1156, 1180, 1191, 1218, 1219, 1221, 1227, 1249, 1253, 1286, 1293, 1320, 1321, 1332, 1335, 1339 or a corresponding position thereof. In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1127, 1135, 1180, 1207, 1219, 1234, 1286, 1301, 1332, 1335, 1337, 1338, 1349 or a corresponding position thereof. Exemplary amino acid substitutions and PAM specificity of SpCas9 variants are shown in Tables 3A-3D.









TABLE 3A









SpCas9 amino acid position





















1114
1135
1218
1219
1221
1249
1320
1321
1323
1332
1333
1335
1337


SpCas9
R
D
G
E
Q
P
A
P
A
D
R
R
T





AAA

N

V
H





G




AAA

N

V
H





G


AAA



V






G


TAA
G
N

V






I


TAA

N

V






I

A


TAA
G
N

V






I

A


CAA



V






K


CAA

N

V






K


CAA

N

V






K


GAA



V
H

V



K


GAA

N

V


V



K


GAA



V
H

V



K


TAT


S
V
H
S

S



L


TAT


S
V
H
S

S



L


TAT


S
V
H
S

S



L


GAT



V






I


GAT



V




D


Q


GAT



V




D


Q


CAC



V





N

Q
N


CAC

N

V







Q
N


CAC



V





N

Q
N


















TABLE 3B









SpCas9 amino acid position



























1114
1134
1135
1137
1139
1151
1180
1188
1211
1219
1221
1256
1264
1290
1318
1317
1320
1323
1333


SpCas9
R
F
D
P
V
K
D
K
K
E
Q
Q
H
V
L
N
A
A
R





GAA









V
H





V

K


GAA


N
S





V






V
D
K


GAA


N






V
H

Y



V

K


CAA


N






V
H

Y



V

K


CAA
G

N
S





V
H

Y



V

K


CAA


N




R

V
H





V

K


CAA


N



G

R
V
H

Y



V

K


CAA


N






V
H

Y



V

K


AAA


N



G


V
H
R
Y



V
D
K


CAA
G

N



G


V
H

Y



V
D
K


CAA

L
N



G


V
H

Y


T
V
D
K


TAA
G

N



G


V
H

Y
G
S

V
D
K


TAA
G

N


E
G


V
H

Y

S

V

K


TAA
G

N



G


V
H

Y

S

V
D
K


TAA
G

N



G

R
V
H





V

K


TAA


N



G

R
V
H

Y



V

K


TAA
G

N

A

G


V
H





V

K


TAA
G

N






V
H





V

K


















TABLE 3C









SpCas9 amino acid position


















1114
1131
1135
1150
1156
1180
1191
1218
1219
1221


SpCas9
R
Y
D
E
K
D
K
G
E
Q





SacB.TAT


N



N

V
H


SacB.TAT


N




S
V
H


AAT


N




S
V
H


TAT
G

N


G

S
V
H


TAT
G

N


G

S
V
H


TAT
G
C
N


G

S
V
H


TAT
G
C
N


G

S
V
H


TAT
G
C
N


G

S
V
H


TAT
G
C
N

E
G

S
V
H


TAT
G
C
N
V

G

S
V
H


TAT

C
N


G

S
V
H


TAT
G
C
N


G

S
V
H












SpCas9 amino acid position




















1227
1249
1253
1286
1293
1320
1321
1332
1335
1339



SpCas9
A
P
E
N
A
A
P
D
R
T







SacB.TAT





V
S

L



SacB.TAT

S




S
G
L



AAT
V
S

K
T

S
G
L
I



TAT

S
K



S
G
L



TAT

S




S
G
L



TAT

S




S
G
L



TAT

S




S
G
L



TAT

S




S
G
L



TAT

S




S
G
L



TAT

S




S
G
L



TAT

S




S
G
L



TAT

S




S
G
L



















TABLE 3D









SpCas9 amino acid position






















1114
1127
1135
1180
1207
1219
1234
1286
1301
1332
1335
1337
1338
1349


SpCas9
R
D
D
D
E
E
N
N
P
D
R
T
S
H























SacB.CAC


N


V


N
Q
N




AAC
G

N


V


N
Q
N


AAC
G

N


V


N
Q
N


TAC
G

N


V


N
Q
N


TAC
G

N


V

H
N
Q
N


TAC
G

N

G
V
D
H
N
Q
N


TAC
G

N


V


N
Q
N


TAC
G
G
N
E

V

H
N
Q
N


TAC
G

N


V

H
N
Q
N


TAC
G

N


V


N
Q
N
T
R









In some embodiments, the Cas9 is a Neisseria meningitidis Cas9 (NmeCas9) or a variant thereof. In some embodiments, the NmeCas9 has specificity for a NNNNGAYW PAM, wherein Y is C or T and W is A or T. In some embodiments, the NmeCas9 has specificity for a NNNNGYTT PAM, wherein Y is C or T. In some embodiments, the NmeCas9 has specificity for a NNNNGTCT PAM. In some embodiments, the NmeCas9 is a Nme1 Cas9. In some embodiments, the NmeCas9 has specificity for a NNNNGATT PAM, a NNNNCCTA PAM, a NNNNCCTC PAM, a NNNNCCTT PAM, a NNNNCCTG PAM, a NNNNCCGT PAM, a NNNNCCGGPAM, a NNNNCCCA PAM, a NNNNCCCT PAM, a NNNNCCCC PAM, a NNNNCCAT PAM, a NNNNCCAG PAM, a NNNNCCAT PAM, or a NNNGATT PAM. In some embodiments, the Nme1Cas9 has specificity for a NNNNGATT PAM, a NNNNCCTA PAM, a NNNNCCTC PAM, a NNNNCCTT PAM, or a NNNNCCTG PAM. In some embodiments, the NmeCas9 has specificity for a CAA PAM, a CAAA PAM, or a CCA PAM. In some embodiments, the NmeCas9 is a Nme2 Cas9. In some embodiments, the NmeCas9 has specificity for a NNNNCC (N4CC) PAM, wherein N is any one of A, G, C, or T. in some embodiments, the NmeCas9 has specificity for a NNNNCCGT PAM, a NNNNCCGGPAM, a NNNNCCCA PAM, a NNNNCCCT PAM, a NNNNCCCC PAM, a NNNNCCAT PAM, a NNNNCCAG PAM, a NNNNCCAT PAM, or a NNNGATT PAM. In some embodiments, the NmeCas9 is a Nme3Cas9. In some embodiments, the NmeCas9 has specificity for a NNNNCAAA PAM, a NNNNCC PAM, or a NNNNCNNN PAM. Additional NmeCas9 features and PAM sequences as described in Edraki et al. Mol. Cell. (2019) 73(4): 714-726 is incorporated herein by reference in its entirety.
An exemplary Neisseria meningitidis Cas9 protein, Nme1Cas9, (NCBI Reference: WP_002235162.1; type II CRISPR RNA-guided endonuclease Cas9) has the following amino acid sequence:









1
MAAFKPNPIN YILGLDIGIA SVGWAMVEID EDENPICLID LGVRVFERAE VPKTGDSLAM



 


61
ARRLARSVRR LTRRRAHRLL RARRLLKREG VLQAADFDEN GLIKSLPNTP WQLRAAALDR


 


121
KLTPLEWSAV LLHLIKHRGY LSQRKNEGET ADKELGALLK GVADNAHALQ TGDFRTPAEL


 


181 
ALNKFEKESG HIRNQRGDYS HTFSRKDLQA ELILLFEKQK EFGNPHVSGG LKEGIETLLM



 


241
TQRPALSGDA VQKMLGHCTF EPAEPKAAKN TYTAERFIWL TKLNNLRILE QGSERPLTDT



 


301
ERATLMDEPY RKSKLTYAQA RKLLGLEDTA FFKGLRYGKD NAEASTLMEM KAYHAISEAL


 


361
EKEGLKDKKS PLNLSPELQD EIGTAFSLFK TDEDITGRLK DRIQPEILEA LLKHISFDKF


 


421
VQISLKALRR IVPLMEQGKR YDEACAEIYG DHYGKKNTEE KIYLPPIPAD EIRNPVVLRA


 


481 
LSQARKVING VVRRYGSPAR IHIETAREVG KSFKDRKEIE KRQEENRKDR EKAAAKFREY



 


541 
FPNFVGEPKS KDILKLRLYE QQHGKCLYSG KEINLGRLNE KGYVEIDHAL PFSRTWDDSF


 


601 
NNKVLVIGSE NQNKGNQTPY EYFNGKDNSR EWQEFKARVE TSRFPRSKKQ RILLQKFDED


 


661 
GFKERNLNDT RYVNRFLCQF VADRMRLTGK GKKRVFASNG QITNLLRGFW GLRKVRAEND


 


721 
RHHALDAVVV ACSTVAMQQK ITRFVRYKEM NAFDGKTIDK ETGEVLHQKT HFPQPWEFFA


 


781 
QEVMIRVFGK PDGKPEFEEA DTPEKLRTLL AEKLSSRPEA VHEYVTPLFV SRAPNRKMSG


 


841 
QGHMETVKSA KRLDEGVSVL RVPLTQLKLK DLEKMVNRER EPKLYEALKA RLEAHKDDPA


 


901 
KAFAEPFYKY DKAGNRTQQV KAVRVEQVQK TGVWVRNHNG IADNATMVRV DVFEKGDKYY


 


961 
LVPIYSWQVA KGILPDRAVV QGKDEEDWQL IDDSFNFKFS LHPNDLVEVI TKKARMFGYF


 


1021 
ASCHRGTGNI NIRIHDLDHK IGKNGILEGI GVKTALSFQK YQIDELGKEI RPCRLKKRPP


 


1081
VR







An exemplary Neisseria meningitidis Cas9 protein, Nme2Cas9, (NCBI Reference: WP_002230835; type II CRISPR RNA-guided endonuclease Cas9) has the following amino acid sequence:









1
MAAFKPNPIN YILGLDIGIA SVGWAMVEID EEENPIRLID LGVRVFERAE VPKTGDSLAM



 


61
ARRLARSVRR LTRRRAHRLL RARRLLKREG VLQAADFDEN GLIKSLPNTP WQLRAAALDR


 


121
KLTPLEWSAV LLHLIKHRGY LSQRKNEGET ADKELGALLK GVANNAHALQ TGDFRTPAEL


 


181
ALNKFEKESG HIRNQRGDYS HTFSRKDLQA ELILLFEKQK EFGNPHVSGG LKEGIETLLM


 


241
TQRPALSGDA VQKMLGHCTF EPAEPKAAKN TYTAERFIWL TKLNNLRILE QGSERPLTDT


 


301
ERATLMDEPY RKSKLTYAQA RKLLGLEDTA FFKGLRYGKD NAEASTLMEM KAYHAISRAL


 


361
EKEGLKDKKS PLNLSSELQD EIGTAFSLFK TDEDITGRLK DRVQPEILEA LLKHISFDKF


 


421
VQISLKALRR IVPLMEQGKR YDEACAEIYG DHYGKKNTEE KIYLPPIPAD EIRNPVVLRA


 


481
LSQARKVING VVRRYGSPAR IHIETAREVG KSFKDRKEIE KRQEENRKDR EKAAAKFREY


 


541
FPNFVGEPKS KDILKLRLYE QQHGKCLYSG KEINLVRLNE KGYVEIDHAL PFSRTWDDSF


 


601
NNKVLVLGSE NQNKGNQTPY EYFNGKDNSR EWQEFKARVE TSRFPPSKKQ RILLQKFDED


 


661
GFKECNLDNT RYVNRFLCQF VADHILLTGK GKRRVFASNG QITNLLRGFW GLRKVRAEND


 


721
RHHALDAVVV ACSTVAMQQK ITRFVRYKEM NAFDGKTIDK ETGKVLHQKT HFPQPWEFFA


 


781
QEVMIRVFGK PDGKPEFEEA DTPEKLRTLL AEKLSSRPEA VHEYVTPLFV SRAPNRKMSG


 


841
AHKDTLRSAK RFVKHNEKIS VKRVWLTEIK LADLENMVNY KNGREIELYE ALKARLEAYG


 


901
GNAKQAFDPK DNPFYKKGGQ LVKAVRVEKT QESGVLLNKK NAYTIADNGD MVRVDVFCKV


 


961
DKKGKNQYFI VPIYAWQVAE NILPDIDCKG YRIDDSYTFC FSLHKYDLIA FQKDEKSKVE


 


1021
FAYYINCDSS NGRFYLAWHD KGSKEQQFRI STQNVLVIQK YQVNELGKEI RPCRLKKRPP


 


1081
VR






Cas12 Domains of Nucleobase Editors

Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multisubunit effector complexes, while Class 2 systems have a single protein effector. For example, Cas9 and Cpf1 are Class 2 effectors, albeit different types (Type II and Type V, respectively). In addition to Cpf1, Class 2, Type V CRISPR-Cas systems also comprise Cas12a/Cpf1, Cas12b/C2cl, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i and Cas12j/CasΦ). See, e.g., Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems,” Mol. Cell, 2015 Nov. 5; 60(3): 385-397; Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR Journal, 2018, 1(5): 325-336; and Yan et al., “Functionally Diverse Type V CRISPR-Cas Systems,” Science, 2019 Jan. 4; 363: 88-91; the entire contents of each is hereby incorporated by reference. Type V Cas proteins contain a RuvC (or RuvC-like) endonuclease domain. While production of mature CRISPR RNA (crRNA) is generally tracrRNA-independent, Cas12b/C2c1, for example, requires tracrRNA for production of crRNA. Cas12b/C2c1 depends on both crRNA and tracrRNA for DNA cleavage.
Nucleic acid programmable DNA binding proteins contemplated in the present invention include Cas proteins that are classified as Class 2, Type V (Cas12 proteins). Non-limiting examples of Cas Class 2, Type V proteins include Cas12a/Cpf1, Cas12b/C2cl, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i, and Cas12j/CasΦ homologues thereof, or modified versions thereof. As used herein, a Cas12 protein can also be referred to as a Cas12 nuclease, a Cas12 domain, or a Cas12 protein domain. In some embodiments, the Cas12 proteins of the present invention comprise an amino acid sequence interrupted by an internally fused protein domain such as a deaminase domain.
In some embodiments, the Cas12 domain is a nuclease inactive Cas12 domain or a Cas12 nickase. In some embodiments, the Cas12 domain is a nuclease active domain. For example, the Cas12 domain may be a Cas12 domain that nicks one strand of a duplexed nucleic acid (e.g., duplexed DNA molecule). In some embodiments, the Cas12 domain comprises any one of the amino acid sequences as set forth herein. In some embodiments the Cas12 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth herein. In some embodiments, the Cas12 domain comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any one of the amino acid sequences set forth herein. In some embodiments, the Cas12 domain comprises an amino acid sequence that has at least 10, at least 15, 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 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth herein.
In some embodiments, proteins comprising fragments of Cas12 are provided. For example, in some embodiments, a protein comprises one of two Cas12 domains: (1) the gRNA binding domain of Cas12; or (2) the DNA cleavage domain of Cas12. In some embodiments, proteins comprising Cas12 or fragments thereof are referred to as “Cas12 variants.” A Cas12 variant shares homology to Cas12, or a fragment thereof. For example, a Cas12 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild type Cas12. In some embodiments, the Cas12 variant may have 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild type Cas12. In some embodiments, the Cas12 variant comprises a fragment of Cas12 (e.g., a gRNA binding domain or a DNA cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas12. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas12. In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.
In some embodiments, Cas12 corresponds to, or comprises in part or in whole, a Cas12 amino acid sequence having one or more mutations that alter the Cas12 nuclease activity. Such mutations, by way of example, include amino acid substitutions within the RuvC nuclease domain of Cas12. In some embodiments, variants or homologues of Cas12 are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to a wild type Cas12. In some embodiments, variants of Cas12 are provided having amino acid sequences which are shorter, or longer, by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more.
In some embodiments, Cas12 fusion proteins as provided herein comprise the full-length amino acid sequence of a Cas12 protein, e.g., one of the Cas12 sequences provided herein. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas12 sequence, but only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas12 domains are provided herein, and additional suitable sequences of Cas12 domains and fragments will be apparent to those of skill in the art.
Generally, the class 2, Type V Cas proteins have a single functional RuvC endonuclease domain (See, e.g., Chen et al., “CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity,” Science 360:436-439 (2018)). In some cases, the Cas12 protein is a variant Cas12b protein. (See Strecker et al., Nature Communications, 2019, 10(1): Art. No.: 212). In one embodiment, a variant Cas12 polypeptide has an amino acid sequence that is different by 1, 2, 3, 4, 5 or more amino acids (e.g., has a deletion, insertion, substitution, fusion) when compared to the amino acid sequence of a wild type Cas12 protein. In some instances, the variant Cas12 polypeptide has an amino acid change (e.g., deletion, insertion, or substitution) that reduces the activity of the Cas12 polypeptide. For example, in some instances, the variant Cas12 is a Cas12b polypeptide that has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nickase activity of the corresponding wild-type Cas12b protein. In some cases, the variant Cas12b protein has no substantial nickase activity.
In some cases, a variant Cas12b protein has reduced nickase activity. For example, a variant Cas12b protein exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1%, of the nickase activity of a wild-type Cas12b protein.
In some embodiments, the Cas12 protein includes RNA-guided endonucleases from the Cas12a/Cpf1 family that displays activity in mammalian cells. CRISPR from Prevotella and Francisella 1 (CRISPR/Cpf1) is a DNA editing technology analogous to the CRISPR/Cas9 system. Cpf1 is an RNA-guided endonuclease of a class II CRISPR/Cas system. This acquired immune mechanism is found in Prevotella and Francisella bacteria. Cpf1 genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations. Unlike Cas9 nucleases, the result of Cpf1-mediated DNA cleavage is a double-strand break with a short 3′ overhang. Cpf1's staggered cleavage pattern can open up the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning, which can increase the efficiency of gene editing. Like the Cas9 variants and orthologues described above, Cpf1 can also expand the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM sites favored by SpCas9. The Cpf1 locus contains a mixed alpha/beta domain, a RuvC-I followed by a helical region, a RuvC-II and a zinc finger-like domain. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. Furthermore, Cpf1, unlike Cas9, does not have a HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alpha-helical recognition lobe of Cas9. Cpf1 CRISPR-Cas domain architecture shows that Cpf1 is functionally unique, being classified as Class 2, type V CRISPR system. The Cpf1 loci encode Cas1, Cas2, and Cas4 proteins are more similar to types I and III than type II systems. Functional Cpf1 does not require the trans-activating CRISPR RNA (tracrRNA), therefore, only CRISPR (crRNA) is required. This benefits genome editing because Cpf1 is not only smaller than Cas9, but also it has a smaller sgRNA molecule (approximately half as many nucleotides as Cas9). The Cpf1-crRNA complex cleaves target DNA or RNA by identification of a protospacer adjacent motif 5′-YTN-3′ or 5′-TTTN-3′ in contrast to the G-rich PAM targeted by Cas9. After identification of PAM, Cpf1 introduces a sticky-end-like DNA double-stranded break having an overhang of 4 or 5 nucleotides.
In some aspects of the present invention, a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence can be used. Cas12 can refer to a polypeptide with at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and/or sequence homology to a wild type exemplary Cas12 polypeptide (e.g., Cas12 from Bacillus hisashii). Cas12 can refer to a polypeptide with at most or at most about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and/or sequence homology to a wild type exemplary Cas12 polypeptide (e.g., from Bacillus hisashii (BhCas12b), Bacillus sp. V3-13 (BvCas12b), and Alicyclobacillus acidiphilus (AaCas12b)). Cas12 can refer to the wild type or a modified form of the Cas12 protein that can comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof.
In some embodiments, BhCas12b guide polynucleotide has the following sequence:







BhCas12b sgRNA scaffold (underlined) + 20 nt to


23 nt guide sequence (denoted by Nn)


5′GUUCUGTCUUUUGGUCAGGACAACCGUCUAGCUAUAAGUGCUGCAGG


GUGUGAGAAACUCCUAUUGCUGGACGAUGUCUCUUACGAGGCAUUAGCA


CNNNNNNNNNNNNNNNNNNNN 3′






In some embodiments, BvCas12b and AaCas12b guide polynucleotides have the following sequences:







BvCas12b sgRNA scaffold (underlined) + 20 nt to


23 nt guide sequence (denoted by Nn)


5′GACCUAUAGGGUCAAUGAAUCUGUGCGUGUGCCAUAAGUAAUUAAAA


AUUACCCACCACAGGAGCACCUGAAAACAGGUGCUUGGCACNNNNNNNN


NNNNNNNNNNNN-3′


 


AaCasl2b sgRNA scaffold (underlined) + 20 nt to


23 nt guide sequence (denoted by Nn)


5′GUCUAAAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCC


AGGUGGCAAAGCCCGUUGAACUUCUCAAAAAGAACGAUCUGAGAAGUGG


CACNNNNNNNNNNNNNNNNNNNN-3′






Nucleic Acid Programmable DNA Binding Proteins

Some aspects of the disclosure provide fusion proteins comprising domains that act as nucleic acid programmable DNA binding proteins, which may be used to guide a protein, such as a base editor, to a specific nucleic acid (e.g., DNA or RNA) sequence. In particular embodiments, a fusion protein comprises a nucleic acid programmable DNA binding protein domain and a deaminase domain. Non-limiting examples of nucleic acid programmable DNA binding proteins include, Cas9 (e.g., dCas9 and nCas9), Cas12a/Cpf1, Cas12b/C2cl, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i and Cas12j/CasΦ. Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a/Cpf1, Cas12b/C2cl, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i, Cas12j/CasΦ, 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, homologues thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, although they may not be specifically listed in this disclosure. See, e.g., Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 October; 1:325-336. doi: 10.1089/crispr.2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan. 4; 363(6422):88-91. doi: 10.1126/science.aav7271, the entire contents of each are hereby incorporated by reference.
One example of a nucleic acid programmable DNA-binding protein that has different PAM specificity than Cas9 is Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (Cpf1). Similar to Cas9, Cpf1 is also a class 2 CRISPR effector. It has been shown that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif (TTN, TTTN, or YTN). Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells. Cpf1 proteins are known in the art and have been described previously, for example Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962; the entire contents of which is hereby incorporated by reference.
Useful in the present compositions and methods are nuclease-inactive Cpf1 (dCpf1) variants that may be used as a guide nucleotide sequence-programmable DNA-binding protein domain. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9 but does not have a HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alfa-helical recognition lobe of Cas9. It was shown in Zetsche et al., Cell, 163, 759-771, 2015 (which is incorporated herein by reference) that, the RuvC-like domain of Cpf1 is responsible for cleaving both DNA strands and inactivation of the RuvC-like domain inactivates Cpf1 nuclease activity. For example, mutations corresponding to D917A, E1006A, or D1255A in Francisella novicida Cpf1 inactivate Cpf1 nuclease activity. In some embodiments, the dCpf1 of the present disclosure comprises mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, or D917A/E1006A/D1255A. It is to be understood that any mutations, e.g., substitution mutations, deletions, or insertions that inactivate the RuvC domain of Cpf1, may be used in accordance with the present disclosure.
In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein may be a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase (nCpf1). In some embodiments, the Cpf1 protein is a nuclease inactive Cpf1 (dCpf1). In some embodiments, the Cpf1, the nCpf1, or the dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a Cpf1 sequence disclosed herein. In some embodiments, the dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to a Cpf1 sequence disclosed herein, and comprises mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, or D917A/E1006A/D1255A. It should be appreciated that Cpf1 from other bacterial species may also be used in accordance with the present disclosure.








Wild-type Francisella novicida Cpf1 (D917, E1006, and D1255 are bolded and



underlined)


MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEE


 


ILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLI


 


DAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSS


 


NDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQR


 


VFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK


 


MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQ


 


KLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKA


 


KYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKK


 


DLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIV


 


PLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI


 


FDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQ


 


KGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFEN


 


ISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFY


 


RKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKF


 


NDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI


 


EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVY


 


QKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPV


 


TGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLI


 


NFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQ


 


MRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGK


 


KLNLVIKNEEYFEFVQNRNN


 


Francisella novicida Cpf1 D917A (A917, E1006, and D1255 are bolded and underlined)


MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEE


 


ILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLI


 


DAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSS


 


NDIPTS11YRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQR


 


VFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK


 


MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQ


 


KLDLSKIYFKNDKSITDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKA


 


KYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKK


 


DLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIV


 


PLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI


 


FDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQ


 


KGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFEN


 


ISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFY


 


RKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKF


 


NDEINLLLKEKANDVHILSIARGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI


 


EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVY


 


QKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPV


 


TGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLI


 


NFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQ


 


MRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGK


 


KLNLVIKNEEYFEFVQNRNN


 


Francisella novicida Cpf1 E1006A (D917, A1006, and D1255 are bolded and


underlined)


MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEE


 


ILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLI


 


DAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSS


 


NDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQR


 


VFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK


 


MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQ


 


KLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKA


 


KYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKK


 


DLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIV


 


PLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI


 


FDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQ


 


KGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFEN


 


ISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFY


 


RKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKF


 


NDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI


 


EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFADLNFGFKRGRFKVEKQVY


 


QKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPV


 


TGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTTASFGSRDI


 


NFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQ


 


MRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGK


 


KLNLVIKNEEYFEFVQNRNN


 


Francisella novicida Cpf1 D1255A (D917, E1006, and A1255 are bolded and


underlined)


MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEE


 


ILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLI


 


DAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSS


 


NDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQR


 


VFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK


 


MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQ


 


KLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKA


 


KYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKK


 


DLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIV


 


PLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI


 


FDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQ


 


KGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFEN


 


ISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFY


 


RKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCP1T1NFKSSGANKF


 


NDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI


 


EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVY


 


QKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPV


 


TGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLI


 


NFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQ


 


MRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDAAANGAYHIGLKGLMLLGRIKNNQEGK


 


KLNLVIKNEEYFEFVQNRNN


 


Francisella novicida Cpf1 D917A/E1006A (A917, A1006, and D1255 are bolded and


underlined)


MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEE


 


ILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLI


 


DAKKGQESDTTTWTKQSKDNGTELFKANSDITDTDEALETTKSFKGWTTYFKGFHENRKNVYSS


 


NDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQR


 


VFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK


 


MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQ


 


KLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKA


 


KYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKK


 


DLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIV


 


PLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI


 


FDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQ


 


KGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFEN


 


ISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFY


 


RKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKF


 


NDEINLLLKEKANDVHILSIARGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI


 


EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFADLNFGFKRGRFKVEKQVY


 


QKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPV


 


TGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLI


 


NFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQ


 


MRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGK


 


KLNLVIKNEEYFEFVQNRNN


 


Francisella novicida Cpf1 D917A/D1255A (A917, E1006, and A1255 are bolded and


underlined)


MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEE


 


ILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLI


 


DAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSS


 


NDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDlDYKTSEVNQR


 


VFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK


 


MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQ


 


KLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKA


 


KYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKK


 


DLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIV


 


PLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI


 


FDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQ


 


KGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFEN


 


ISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFY


 


RKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKF


 


NDEINLLLKEKANDVHILSIARGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI


 


EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVY


 


QKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPV


 


TGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLI


 


NFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQ


 


MRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDAAANGAYHIGLKGLMLLGRIKNNQEGK


 


KLNLVIKNEEYFEFVQNRNN


 


Francisella novicida Cpf1 E1006A/D1255A (D917, A1006, and A1255 are bolded and


underlined)


MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEE


 


ILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLI


 


DAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSS


 


NDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQR


 


VFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK


 


MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQ


 


KLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKA


 


KYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKK


 


DLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIV


 


PLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI


 


FDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQ


 


KGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFEN


 


ISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFY


 


RKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCP1T1NFKSSGANKF


 


NDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI


 


EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFADLNFGFKRGRFKVEKQVY


 


QKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPV


 


TGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLI


 


NFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQ


 


MRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDAAANGAYHIGLKGLMLLGRIKNNQEGK


 


KLNLVIKNEEYFEFVQNRNN


 


Francisella novicida Cpf1 D917A/E1006A/D1255A (A917, A1006, and A1255 are


bolded and underlined)


MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEE


 


ILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLI


 


DAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSS


 


NDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEATNYEQIKKDLAEELTFDIDYKTSEVNQR


 


VFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYK


 


MSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQ


 


KLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKA


 


KYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKK


 


DLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIV


 


PLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKI


 


FDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQ


 


KGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFEN


 


ISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFY


 


RKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKF


 


NDEINLLLKEKANDVHILSIARGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAI


 


EKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFADLNFGFKRGRFKVEKQVY


 


QKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPV


 


TGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLI


 


NFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQ


 


MRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDAAANGAYHIGLKGLMLLGRIKNNQEGK


 


KLNLVIKNEEYFEFVQNRNN






In some embodiments, one of the Cas9 domains present in the fusion protein may be replaced with a guide nucleotide sequence-programmable DNA-binding protein domain that has no requirements for a PAM sequence.
In some embodiments, the Cas9 domain is a Cas9 domain from Staphylococcus aureus (SaCas9). In some embodiments, the SaCas9 domain is a nuclease active SaCas9, a nuclease inactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, the SaCas9 comprises a N579A mutation, or a corresponding mutation in any of the amino acid sequences provided herein.
In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a NNGRRT or a NNGRRT PAM sequence. In some embodiments, the SaCas9 domain comprises one or more of a E781X, a N967X, and a R1014X mutation, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more of a E781K, a N967K, and a R1014H mutation, or one or more corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SaCas9 domain comprises a E781K, a N967K, or a R1014H mutation, or corresponding mutations in any of the amino acid sequences provided herein.








Exemplary SaCas9 sequence



KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQ


 


RVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTG


 


NELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQL


 


DQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYN


 


ALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKP


 


EFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYOSSEDIQEELTNLNSELTQEEIEQISN


 


LKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPV


 


VKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEE11RTTG


 


KENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ


 


EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDF


 


INRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDA


 


L11ANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDY


 


KYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHD


 


PQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDY


 


PNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAE


 


FIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQ


 


SIKKYSTDILGNLYEVKSKKHPQIIKKG






Residue N579 above, which is underlined and in bold, may be mutated (e.g., to a A579) to yield a SaCas9 nickase.









Exemplary SaCas9n sequence



KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQ


 


RVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTG


 


NELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQL


 


DQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYN


 


ALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKP


 


EFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISN


 


LKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPV


 


VKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTG


 


KENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ


 


EEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDF


 


INRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDA


 


LCIANADFIEKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDY


 


KYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHD


 


PQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDY


 


PNSRNKVVKISLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAE


 


FIASFYNNDIIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQ


 


SIKKYSTDILGNLYEVKSKKHPQIIKKG






Residue A579 above, which can be mutated from N579 to yield a SaCas9 nickase, is underlined and in bold.









Exemplary SaKKH Cas9



KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQ


 


RVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTG


 


NELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQL


 


DQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYN


 


ALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKP


 


EFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYOSSEDIQEELTNLNSELTQEEIEQISN


 


LKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPV


 


VKRSFIQSIKVINAIIKKYGLPNDIIIEDAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTG


 


KENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ


 


EEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDF


 


INRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDA


 


LIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDY


 


KYSHBVDKKPNRKLINDTLYSTRKDDKGNTLTVNNLNGLYDKDNDKLKKLTNKSPEKLLMYHHD


 


PQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDY


 


PNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAE


 


FIASFYKNDLIKTNGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQ


 


SIKKYSTDILGNLYEVKSKKHPQIIKKG.






Residue A579 above, which can be mutated from N579 to yield a SaCas9 nickase, is underlined and in bold. Residues K781, K967, and H1014 above, which can be mutated from E781, N967, and R1014 to yield a SaKKH Cas9 are underlined and in italics.

In some embodiments, the napDNAbp is a circular permutant. In the following sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, and the underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations. The asterisk (*) denotes a STOP codon.








CP5 (with MSP “NGC = Pam Variant with mutations Regular Cas9 likes NGG”



PID = Protein Interacting Domain and “D10A” nickase):


EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQ


 


VNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFMQPTVAYSVLVVAKVEKGKSK


 


KLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKF


 


LQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILA


 


DANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIARKEYRSTKEVLDATL


 


IHQSITGLYETRIDLSQLGGDGGSGGSGGSGGSGGSGGSGGMDKKYSIGLAIGTNSVGWAVITD


 


EYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFS


 


NEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL


 


RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSAR


 


LSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLL


 


AQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL


 


PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDN


 


GSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEE


 


TITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMR


 


KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLK


 


IIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS


 


RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL


 


AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKEL


 


GSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK


 


VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR


 


QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHH


 


AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEGADKRTADGSEFESPKKKR


 


KV*






In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a single effector of a microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, without limitation, Cas9, Cpf1, Cas12b/C2c1, and Cas12c/C2c3. Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multisubunit effector complexes, while Class 2 systems have a single protein effector. For example, Cas9 and Cpf1 are Class 2 effectors. In addition to Cas9 and Cpf1, three distinct Class 2 CRISPR-Cas systems (Cas12b/C2c1, and Cas12c/C2c3) have been described by Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems”, Mol. Cell, 2015 Nov. 5; 60(3): 385-397, the entire contents of which is hereby incorporated by reference. Effectors of two of the systems, Cas12b/C2c1, and Cas12c/C2c3, contain RuvC-like endonuclease domains related to Cpf1. A third system contains an effector with two predicated HEPN RNase domains. Production of mature CRISPR RNA is tracrRNA-independent, unlike production of CRISPR RNA by Cas12b/C2c1. Cas12b/C2c1 depends on both CRISPR RNA and tracrRNA for DNA cleavage.
The crystal structure of Alicyclobaccillus acidoterrastris Cas12b/C2c1 (AacC2c1) has been reported in complex with a chimeric single-molecule guide RNA (sgRNA). See e.g., Liu et al., “C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism”, Mol. Cell, 2017 Jan. 19; 65(2):310-322, the entire contents of which are hereby incorporated by reference. The crystal structure has also been reported in Alicyclobacillus acidoterrestris C2c1 bound to target DNAs as ternary complexes. See e.g., Yang et al., “PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease”, Cell, 2016 Dec. 15; 167(7):1814-1828, the entire contents of which are hereby incorporated by reference. Catalytically competent conformations of AacC2c1, both with target and non-target DNA strands, have been captured independently positioned within a single RuvC catalytic pocket, with Cas12b/C2c1-mediated cleavage resulting in a staggered seven-nucleotide break of target DNA. Structural comparisons between Cas12b/C2c1 ternary complexes and previously identified Cas9 and Cpf1 counterparts demonstrate the diversity of mechanisms used by CRISPR-Cas9 systems.
In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein may be a Cas12b/C2c1, or a Cas12c/C2c3 protein. In some embodiments, the napDNAbp is a Cas12b/C2c1 protein. In some embodiments, the napDNAbp is a Cas12c/C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to a naturally-occurring Cas12b/C2c1 or Cas12c/C2c3 protein. In some embodiments, the napDNAbp is a naturally-occurring Cas12b/C2c1 or Cas12c/C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to any one of the napDNAbp sequences provided herein. It should be appreciated that Cas12b/C2c1 or Cas12c/C2c3 from other bacterial species may also be used in accordance with the present disclosure.
A Cas12b/C2c1 ((uniprot.org/uniprot/TOD7A2#2) sp|T0D7A2|C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS=Alicyclobacillus acido-terrestris (strain ATCC 49025/DSM 3922/CIP 106132/NCIMB 13137/GD3B) GN=c2c1 PE=1 SV=1) amino acid sequence is as follows:








MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECDKT



 


AEECKAELLERLRARQVENGHRGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPL


 


ADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKEKAETRKSADRTADVLRALADFGLKPLMR


 


VYTDSEMSSVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGQEYAKLVEQKNRFEQKN


 


FVGQEHLVHLVNQLQQDMKEASPGLESKEQTAHYVTGRALRGSDKVFEKWGKLAPDAPFDLYDA


 


EIKNVQRRNTRRFGSHDLFAKLAEPEYQALWREDASFLTRYAVYNSILRKLNHAKMFATFTLPD


 


ATAHPIWTRFDKLGGNLHQYTFLFNEFGERRHAIRFHKLLKVENGVAREVDDVTVPISMSEQLD


 


NLLPRDPNEPIALYFRDYGAEQHFTGEFGGAKIQCRRDQLAHMHRRRGARDVYLNVSVRVQSQS


 


EARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRWISVDLGLRTS


 


ASISVFRVARKDELKPNSKGRVPFFFPIKGNDNLVAVHERSQLLKLPGETESKDLRAIREERQR


 


TLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPVDAANHMTPDWREAFENELQKLKSLHG


 


ICSDKEWMDAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYAKDVVGGNSIEQIEYLERQY


 


KFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDERGK


 


GKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELINQAQVHDLLVGTMYAA


 


FSSRFDARTGAPGIRCRRVPARCTQEHNPEPFPWWLNKFVVEHTLDACPLRADDLIPTGEGEIF


 


VSPFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQIRLRCDWGEVDGELVLIPRLTGKRTADS


 


YSNKVFYTNTGVTYYERERGKKRRKVFAQEKLSEEEAELLVEADEAREKSVVLMRDPSGIINRG


 


NWTRQKEFWSMVNQRIEGYLVKQIRSRVPLQDSACENTGDI


 


AacCasl2b (Alicyclobacillus acidiphilus)-WP_067623834


MAVKSMKVKLRLDNMPEIRAGLWKLHTEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECYKT


 


AEECKAELLERLRARQVENGHCGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKFLSPL


 


ADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKAKAEARKSTDRTADVLRALADFGLKPLMR


 


VYTDSDMSSVQWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGEAYAKLVEQKSRFEQKN


 


FVGQEHLVQLVNQLQQDMKEASHGLESKEQTAHYLTGRALRGSDKVFEKWEKLDPDAPFDLYDT


 


EIKNVQRRNTRRFGSHDLFAKLAEPKYQALWREDASFLTRYAVYNSIVRKLNHAKMFATFTLPD


 


ATAHPIWTRFDKLGGNLHQYTFLFNEFGEGRHAIRFQKLLTVEDGVAKEVDDVTVPISMSAQLD


 


DLLPRDPHELVALYFQDYGAEQHLAGEFGGAKIQYRRDQLNHLHARRGARDVYLNLSVRVQSQS


 


EARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGLLSGLRVMSVDLGLRTS


 


ASISVFRVARKDELKPNSEGRVPFCFPIEGNENLVAVHERSQLLKLPGETESKDLRAIREERQR


 


TLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPMDANQMTPDWREAFEDELQKLKSLYGI


 


CGDREWTEAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYQKDVVGGNSIEQIEYLERQYK


 


FLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVYALDDERGK


 


GKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGVFQELLNQAQVHDLLVGTMYAA


 


FSSRFDARTGAPGIRCRRVPARCAREQNPEPFPWWLNKFVAEHKLDGCPLRADDLIPTGEGEFF


 


VSPFSAEEGDFHQIHADLNAAQNLQRRLWSDFDISQIRLRCDWGEVDGEPVLIPRTTGKRTADS


 


YGNKVFYTKTGVTYYERERGKKRRKVFAQEELSEEEAELLVEADEAREKSVVLMRDPSGIINRG


 


DWTRQKEFWSMVNQRIEGYLVKQIRSRVRLQESACENTGDI


 


BhCas12b (Bacillus hisashii) NCBI Reference Sequence: WP_095142515


MAPKKKRKVGIHGVPAAATRSFTLKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYE


 


HHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVEKKGE


 


ANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILG


 


KLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEE


 


YEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWRE11QKWL


 


KMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKK


 


KDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESG


 


GWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRY


 


PHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKELTEWIKDSKGKKLKSG


 


IESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGET


 


LVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDE


 


LIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRK


 


FLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQ


 


AKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSR


 


FHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDR


 


KCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFIL


 


KDGVYEWVNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKW


 


MAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSMKRPAATKKAGQAKKKK






In some embodiments, the Cas12b is BvCas12b (V4), which is a variant of BhCas12b and comprises the following changes relative to BhCas12b: S893R, K846R, and E837G. BhCas12b (V4) is expressed as follows: 5′ mRNA Cap-5′UTR-bhCas12b-STOP sequence-3′UTR-120polyA tail.








5′UTR:



GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC


 


3′ UTR (TriLink standard UTR)


GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCC


 


TGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGA


 


Nucleic acid sequence of bhCas12b (V4)


ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGCCACCAGATCCT


 


TCATCCTGAAGATCGAGCCCAACGAGGAAGTGAAGAAAGGCCTCTGGAAAACCCACGAGGTGCT


 


GAACCACGGAATCGCCTACTACATGAATATCCTGAAGCTGATCCGGCAAGAGGCCATCTACGAG


 


CACCACGAGCAGGACCCCAAGAATCCCAAGAAGGTGTCCAAGGCCGAGATCCAGGCCGAGCTGT


 


GGGATTTCGTGCTGAAGATGCAGAAGTGCAACAGCTTCACACACGAGGTGGACAAGGACGAGGT


 


GTTCAACATCCTGAGAGAGCTGTACGAGGAACTGGTGCCCAGCAGCGTGGAAAAGAAGGGCGAA


 


GCCAACCAGCTGAGCAACAAGTTTCTGTACCCTCTGGTGGACCCCAACAGCCAGTCTGGAAAGG


 


GAACAGCCAGCAGCGGCAGAAAGCCCAGATGGTACAACCTGAAGATTGCCGGCGATCCCTCCTG


 


GGAAGAAGAGAAGAAGAAGTGGGAAGAAGATAAGAAAAAGGACCCGCTGGCCAAGATCCTGGGC


 


AAGCTGGCTGAGTACGGACTGATCCCTCTGTTCATCCCCTACACCGACAGCAACGAGCCCATCG


 


TGAAAGAAATCAAGTGGATGGAAAAGTCCCGGAACCAGAGCGTGCGGCGGCTGGATAAGGACAT


 


GTTCATTCAGGCCCTGGAACGGTTCCTGAGCTGGGAGAGCTGGAACCTGAAAGTGAAAGAGGAA


 


TACGAGAAGGTCGAGAAAGAGTACAAGACCCTGGAAGAGAGGATCAAAGAGGACATCCAGGCTC


 


TGAAGGCTCTGGAACAGTATGAGAAAGAGCGGCAAGAACAGCTGCTGCGGGACACCCTGAACAC


 


CAACGAGTACCGGCTGAGCAAGAGAGGCCTTAGAGGCTGGCGGGAAATCATCCAGAAATGGCTG


 


AAAATGGACGAGAACGAGCCCTCCGAGAAGTACCTGGAAGTGTTCAAGGACTACCAGCGGAAGC


 


ACCCTAGAGAGGCCGGCGATTACAGCGTGTACGAGTTCCTGTCCAAGAAAGAGAACCACTTCAT


 


CTGGCGGAATCACCCTGAGTACCCCTACCTGTACGCCACCTTCTGCGAGATCGACAAGAAAAAG


 


AAGGACGCCAAGCAGCAGGCCACCTTCACACTGGCCGATCCTATCAATCACCCTCTGTGGGTCC


 


GATTCGAGGAAAGAAGCGGCAGCAACCTGAACAAGTACAGAATCCTGACCGAGCAGCTGCACAC


 


CGAGAAGCTGAAGAAAAAGCTGACAGTGCAGCTGGACCGGCTGATCTACCCTACAGAATCTGGC


 


GGCTGGGAAGAGAAGGGCAAAGTGGACATTGTGCTGCTGCCCAGCCGGCAGTTCTACAACCAGA


 


TCTTCCTGGACATCGAGGAAAAGGGCAAGCACGCCTTCACCTACAAGGATGAGAGCATCAAGTT


 


CCCTCTGAAGGGCACACTCGGCGGAGCCAGAGTGCAGTTCGACAGAGATCACCTGAGAAGATAC


 


CCTCACAAGGTGGAAAGCGGCAACGTGGGCAGAATCTACTTCAACATGACCGTGAACATCGAGC


 


CTACAGAGTCCCCAGTGTCCAAGTCTCTGAAGATCCACCGGGACGACTTCCCCAAGGTGGTCAA


 


CTTCAAGCCCAAAGAACTGACCGAGTGGATCAAGGACAGCAAGGGCAAGAAACTGAAGTCCGGC


 


ATCGAGTCCCTGGAAATCGGCCTGAGAGTGATGAGCATCGACCTGGGACAGAGACAGGCCGCTG


 


CCGCCTCTATTTTCGAGGTGGTGGATCAGAAGCCCGACATCGAAGGCAAGCTGTTTTTCCCAAT


 


CAAGGGCACCGAGCTGTATGCCGTGCACAGAGCCAGCTTCAACATCAAGCTGCCCGGCGAGACA


 


CTGGTCAAGAGCAGAGAAGTGCTGCGGAAGGCCAGAGAGGACAATCTGAAACTGATGAACCAGA


 


AGCTCAACTTCCTGCGGAACGTGCTGCACTTCCAGCAGTTCGAGGACATCACCGAGAGAGAGAA


 


GCGGGTCACCAAGTGGATCAGCAGACAAGAGAACAGCGACGTGCCCCTGGTGTACCAGGATGAG


 


CTGATCCAGATCCGCGAGCTGATGTACAAGCCTTACAAGGACTGGGTCGCCTTCCTGAAGCAGC


 


TCCACAAGAGACTGGAAGTCGAGATCGGCAAAGAAGTGAAGCACTGGCGGAAGTCCCTGAGCGA


 


CGGAAGAAAGGGCCTGTACGGCATCTCCCTGAAGAACATCGACGAGATCGATCGGACCCGGAAG


 


TTCCTGCTGAGATGGTCCCTGAGGCCTACCGAACCTGGCGAAGTGCGTAGACTGGAACCCGGCC


 


AGAGATTCGCCATCGACCAGCTGAATCACCTGAACGCCCTGAAAGAAGATCGGCTGAAGAAGAT


 


GGCCAACACCATCATCATGCACGCCCTGGGCTACTGCTACGACGTGCGGAAGAAGAAATGGCAG


 


GCTAAGAACCCCGCCTGCCAGATCATCCTGTTCGAGGATCTGAGCAACTACAACCCCTACGAGG


 


AAAGGTCCCGCTTCGAGAACAGCAAGCTCATGAAGTGGTCCAGACGCGAGATCCCCAGACAGGT


 


TGCACTGCAGGGCGAGATCTATGGCCTGCAAGTGGGAGAAGTGGGCGCTCAGTTCAGCAGCAGA


 


TTCCACGCCAAGACAGGCAGCCCTGGCATCAGATGTAGCGTCGTGACCAAAGAGAAGCTGCAGG


 


ACAATCGGTTCTTCAAGAATCTGCAGAGAGAGGGCAGACTGACCCTGGACAAAATCGCCGTGCT


 


GAAAGAGGGCGATCTGTACCCAGACAAAGGCGGCGAGAAGTTCATCAGCCTGAGCAAGGATCGG


 


AAGTGCGTGACCACACACGCCGACATCAACGCCGCTCAGAACCTGCAGAAGCGGTTCTGGACAA


 


GAACCCACGGCTTCTACAAGGTGTACTGCAAGGCCTACCAGGTGGACGGCCAGACCGTGTACAT


 


CCCTGAGAGCAAGGACCAGAAGCAGAAGATCATCGAAGAGTTCGGCGAGGGCTACTTCATTCTG


 


AAGGACGGGGTGTACGAATGGGTCAACGCCGGCAAGCTGAAAATCAAGAAGGGCAGCTCCAAGC


 


AGAGCAGCAGCGAGCTGGTGGATAGCGACATCCTGAAAGACAGCTTCGACCTGGCCTCCGAGCT


 


GAAAGGCGAAAAGCTGATGCTGTACAGGGACCCCAGCGGCAATGTGTTCCCCAGCGACAAATGG


 


ATGGCCGCTGGCGTGTTCTTCGGAAAGCTGGAACGCATCCTGATCAGCAAGCTGACCAACCAGT


 


ACTCCATCAGCACCATCGAGGACGACAGCAGCAAGCAGTCTATGAAAAGGCCGGCGGCCACGAA


 


AAAGGCCGGCCAGGCAAAAAAGAAAAAG






In some embodiments, the Cas12b is BvCas12B. In some embodiments, the Cas12b comprises amino acid substitutions S893R, K846R, and E837G as numbered in the BvCas12b exemplary sequence provided below.








ByCas12b (Bacillus sp. V3-13) NCBI Reference Sequence: WP_101661451.1



MAIRSIKLKMKTNSGTDSIYLRKALWRTHQLINEGIAYYMNLLTLYRQEAIGDKTKEAYQAELI


 


NIIRNQQRNNGSSEEHGSDQEILALLRQLYELIIPSSIGESGDANQLGNKFLYPLVDPNSQSGK


 


GTSNAGRKPRWKRLKEEGNPDWELEKKKDEERKAKDPTVKIFDNLNKYGLLPLFPLFTNIQKDI


 


EWLPLGKRQSVRKWDKDMFIQAIERLLSWESWNRRVADEYKQLKEKTESYYKEHLTGGEEWIEK


 


IRKFEKERNMELEKNAFAPNDGYFITSRQIRGWDRVYEKWSKLPESASPEELWKVVAEQQNKMS


 


EGFGDPKVFSFLANRENRDIWRGHSERIYHIAAYNGLQKKLSRTKEQATFTLPDAIEHPLWIRY


 


ESPGGTNLNLFKLEEKQKKNYYVTLSKIIWPSEEKWIEKENIEIPLAPSIQFNRQIKLKQHVKG


 


KQEISFSDYSSRISLDGVLGGSRIQFNRKYIKNHKELLGEGDIGPVFFNLVVDVAPLQETRNGR


 


LQSPIGKALKVISSDFSKVIDYKPKELMDWMNTGSASNSFGVASLLEGMRVMSIDMGQRTSASV


 


SIFEVVKELPKDQEQKLFYSINDTELFAIHKRSFLLNLPGEVVTKNNKQQRQERRKKRQFVRSQ


 


TRMLANVLRLETKKTPDERKKAIHKLMEIVQSYDSWTASQKEVWEKELNLLTNMAAFNDEIWKE


 


SLVELHHRIEPYVGQIVSKWRKGLSEGRKNLAGISMWNIDELEDTRRLLISWSKRSRTPGEANR


 


IETDEPFGSSLLQHIQNVKDDRLKQMANLIIMTALGFKYDKEEKDRYKRWKETYPACQIILFEN


 


LNRYLFNLDRSRRENSRLMKWAHRSIPRTVSMQGEMFGLQVGDVRSEYSSRFHAKTGAPGIRCH


 


ALTEEDLKAGSNTLKRLIEDGFINESELAYLKKGDIIPSQGGELFVTLSKRYKKDSDNNELTVI


 


HADINAAQNLQKRFWQQNSEVYRVPCQLARMGEDKLYIPKSQTETIKKYFGKGSFVKNNTEQEV


 


YKWEKSEKMKIKTDTTFDLQDLDGFEDISKTIELAQEQQKKYLTMFRDPSGYFFNNETWRPQKE


 


YWSIVNNIIKSCLKKKILSNKVEL


 


In some embodiments, the Cas12b is BTCas12b.BTCas12b (Bacillus


thermoamylovorans) NCB1 Reference Sequence: WP_041902512


MATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKV


 


SKAEIQAELWDFVLKMQKCNSFTHEVDKDVVFNILRELYEELVPSSVEKKGEANQLSNKE


 


LYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAE


 


YGLIPLFIPFTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEE


 


YEKVEKEHKTLEERIKEDIQAFKSLEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREII


 


QKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYAT


 


FCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTV


 


QLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGT


 


LGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKFVNE


 


KPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLE


 


FPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFE


 


DITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGK


 


EVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQ


 


LNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERS


 


RFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKL


 


QDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKLVTTHADINAAQNLQ


 


KRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWGNAGK


 


LKLKKGSSKQSSSELVDSDLLKDSEDLASELKGEKLMLYRDPSGNVEPSDKWMAAGVHHG


 


KLERILISKLTNQYSISTIEDDSSKQSM






In some embodiments, a napDNAbp refers to Cas12c. In some embodiments, the Cas12c protein is a Cas12c1 or a variant of Cas12c1. In some embodiments, the Cas12 protein is a Cas12c2 or a variant of Cas12c2. In some embodiments, the Cas12 protein is a Cas12c protein from Oleiphilus sp. HI0009 (i.e., OspCas12c) or a variant of OspCas12c. These Cas12c molecules have been described in Yan et al., “Functionally Diverse Type V CRISPR-Cas Systems,” Science, 2019 Jan. 4; 363: 88-91; the entire contents of which is hereby incorporated by reference. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring Cas12c1, Cas12c2, or OspCas12c protein. In some embodiments, the napDNAbp is a naturally-occurring Cas12c1, Cas12c2, or OspCas12c protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to any Cas12c1, Cas12c2, or OspCas12c protein described herein. It should be appreciated that Cas12c1, Cas12c2, or OspCas12c from other bacterial species may also be used in accordance with the present disclosure.








Cas12c1



MQTKKTHLHLISAKASRKYRRTIACLSDTAKKDLERRKQSGAADPAQELSCLKTIKFKLEVPEG


 


SKLPSFDRISQIYNALETIEKGSLSYLLFALILSGFRIFPNSSAAKTFASSSCYKNDQFASQIK


 


EIFGEMVKNFIPSELESILKKGRRKNNKDWTEENIKRVLNSEFGRKNSEGSSALFDSFLSKFSQ


 


ELFRKFDSWNEVNKKYLEAAELLDSMLASYGPFDSVCKMIGDSDSRNSLPDKSTIAFTNNAEIT


 


VDIESSVMPYMAIAALLREYRQSKSKAAPVAYVQSHLTTTNGNGLSWFFKFGLDLIRKAPVSSK


 


QSTSDGSKSLQELFSVPDDKLDGLKFIKEACEALPEASLLCGEKGELLGYQDFRTSFAGHIDSW


 


VANYVNRLFELIELVNQLPESIKLPSILTQKNHNLVASLGLQEAEVSHSLELFEGLVKNVRQTL


 


KKLAGIDISSSPNEQDIKEFYAFSDVLNRLGSIRNQIENAVQTAKKDKIDLESAIEWKEWKKLK


 


KLPKLNGLGGGVPKQQELLDKALESVKQIRHYQRIDFERVIQWAVNEHCLETVPKFLVDAEKKK


 


INKESSTDFAAKENAVRFLLEGIGAAARGKTDSVSKAAYNWFVVNNFLAKKDLNRYFINCQGCI


 


YKPPYSKRRSLAFALRSDNKDTIEVVWEKFETFYKEISKEIEKFNIFSQEFQTFLHLENLRMKL


 


LLRRIQKPIPAEIAFFSLPQEYYDSLPPNVAFLALNQEITPSEYITQFNLYSSFLNGNLILLRR


 


SRSYLRAKFSWVGNSKLIYAAKEARLWKIPNAYWKSDEWKMILDSNVLVFDKAGNVLPAPTLKK


 


VCEREGDLRLFYPLLRQLPHDWCYRNPFVKSVGREKNVIEVNKEGEPKVASALPGSLFRLIGPA


 


PFKSLLDDCFFNPLDKDLRECMLIVDQEISQKVEAQKVEASLESCTYSIAVPIRYHLEEPKVSN


 


QFENVLAIDQGEAGLAYAVFSLKSIGEAETKPIAVGTIRIPSIRRLIHSVSTYRKKKQRLQNFK


 


QNYDSTAFIMRENVTGDVCAKIVGLMKEFNAFPVLEYDVKNLESGSRQLSAVYKAVNSHFLYFK


 


EPGRDALRKQLWYGGDSWTIDGIEIVTRERKEDGKEGVEKIVPLKVFPGRSVSARFTSKTCSCC


 


GRNVFDWLFTEKKAKTNKKFNVNSKGELTTADGVIQLFEADRSKGPKFYARRKERTPLTKPIAK


 


GSYSLEEIERRVRTNLRRAPKSKQSRDTSQSQYFCVYKDCALHFSGMQADENAAINIGRRFLTA


 


LRKNRRSDFPSNVKISDRLLDN


 


Cas12c2


MTKHSIPLHAFRNSGADARKWKGRIALLAKRGKETMRTLQFPLEMSEPEAAAINTTPFAVAYNA


 


IEGTGKGTLFDYWAKLHLAGFRFFPSGGAATIFRQQAVFEDASWNAAFCQQSGKDWPWLVPSKL


 


YERFTKAPREVAKKDGSKKSIEFTQENVANESHVSLVGASITDKTPEDQKEFFLKMAGALAEKE


 


DSWKSANEDRIVAMKVIDEFLKSEGLHLPSLENIAVKCSVETKPDNATVAWHDAPMSGVQNLAI


 


GVFATCASRIDNIYDLNGGKLSKLIQESATTPNVTALSWLFGKGLEYFRTTDIDTIMQDFNIPA


 


SAKESIKPLVESAQAIPTMTVLGKKNYAPFRPNFGGKIDSWIANYASRLMLLNDILEQIEPGFE


 


LPQALLDNETLMSGIDMTGDELKELIEAVYAWVDAAKQGLATLLGRGGNVDDAVQTFEQFSAMM


 


DTLNGTLNTISARYVRAVEMAGKDEARLEKLIECKFDIPKWCKSVPKLVGISGGLPKVEEEIKV


 


MNAAFKDVRARMFVRFEEIAAYVASKGAGMDVYDALEKRELEQIKKLKSAVPERAHIQAYRAVL


 


HRIGRAVQNCSEKTKQLFSSKVIEMGVFKNPSHLNNFIFNQKGAIYRSPFDRSRHAPYQLHADK


 


LLKNDWLELLAEISATLMASESTEQMEDALRLERTRLQLQLSGLPDWEYPASLAKPDIEVEIQT


 


ALKMQLAKDTVTSDVLQRAFNLYSSVLSGLTFKLLRRSFSLKMRFSVADTTQLIYVPKVCDWAI


 


PKQYLQAEGEIGIAARVVTESSPAKMVTEVEMKEPKALGHFMQQAPHDWYFDASLGGTQVAGRI


 


VEKGKEVGKERKLVGYRMRGNSAYKTVLDKSLVGNTELSQCSMIIEIPYTQTVDADFRAQVQAG


 


LPKVSINLPVKETITASNKDEQMLFDRFVAIDLGERGLGYAVFDAKTLELQESGHRPIKAITNL


 


LNRTHHYEQRPNQRQKFQAKFNVNLSELRENTVGDVCHQINRICAYYNAFPVLEYMVPDRLDKQ


 


LKSVYESVTNRYIWSSTDAHKSARVQFWLGGETWEHPYLKSAKDKKPLVLSPGRGASGKGTSQT


 


CSCCGRNPFDLIKDMKPRAKIAVVDGKAKLENSELKLFERNLESKDDMLARRHRNERAGMEQPL


 


TPGNYTVDEIKALLRANLRRAPKNRRTKDTTVSEYHCVFSDCGKTMHADENAAVNIGGKFIADI


 


EK


 


OspCas12c


MTKLRHRQKKLTHDWAGSKKREVLGSNGKLQNPLLMPVKKGQVTEFRKAFSAYARATKGEMTDG


 


RKNMFTHSFEPFKTKPSLHQCELADKAYQSLHSYLPGSLAHFLLSAHALGFRIFSKSGEATAFQ


 


ASSKIEAYESKLASELACVDLSIQNLTISTLFNALTTSVRGKGEETSADPLIARFYTLLTGKPL


 


SRDTQGPERDLAEVISRKIASSFGTWKEMTANPLQSLQFFEEELHALDANVSLSPAFDVLIKMN


 


DLQGDLKNRTIVFDPDAPVFEYNAEDPADIIIKLTARYAKEAVIKNQNVGNYVKNAITTTNANG


 


LGWLLNKGLSLLPVSTDDELLEFIGVERSHPSCHALIELIAQLEAPELFEKNVFSDTRSEVQGM


 


IDSAVSNHIARLSSSRNSLSMDSEELERLIKSFQIHTPHCSLFIGAQSLSQQLESLPEALQSGV


 


NSADILLGSTQYMLTNSLVEESIATYQRTLNR1NYLSGVAGQINGAIKRKA1DGEKIHLPAAWS


 


ELISLPFIGQPVIDVESDLAHLKNQYQTLSNEFDTLISALQKNFDLNFNKALLNRTQHFEAMCR


 


STKKNALSKPEIVSYRDLLARLTSCLYRGSLVLRRAGIEVLKKHKIFESNSELREHVHERKHFV


 


FVSPLDRKAKKLLRLTDSRPDLLHVIDEILQHDNLENKDRESLWLVRSGYLLAGLPDQLSSSFI


 


NLPIITQKGDRRLIDLIQYDQINRDAFVMLVTSAFKSNLSGLQYRANKQSFVVTRTLSPYLGSK


 


LVYVPKDKDWLVPSQMFEGRFADILQSDYMVWKDAGRLCVIDTAKHLSNIKKSVFSSEEVLAFL


 


RELPHRTFIQTEVRGLGVNVDGIAFNNGDIPSLKTFSNCVQVKVSRTNTSLVQTLNRWFEGGKV


 


SPPSIQFERAYYKKDDQIHEDAAKRKIRFQMPATELVHASDDAGWTPSYLLGIDPGEYGMGLSL


 


VSINNGEVLDSGFIHINSLINFASKKSNHQTKVVPRQQYKSPYANYLEQSKDSAAGDIAHILDR


 


LIYKLNALPVFEALSGNSQSAADQVWTKVLSFYTWGDNDAQNSIRKQHWFGASHWDIKGMLRQP


 


PTEKKPKPYLAFPGSQVSSYGNSQRCSCCGKNPLEQLREMAKDISIKELKIRNSEIQLFDGTIK


 


LFNPDPSTVIERRRHNLGPSRIPVADRTFKNISPSSLEFKELITTVSRSIRHSPEFIAKKRGIG


 


SEYFCAYSDCNSSLNSEANAAANVAQKFQKQLFFEL






In some embodiments, a napDNAbp refers to Cas12g, Cas12h, or Cas12i, which have been described in, for example, Yan et al., “Functionally Diverse Type V CRISPR-Cas Systems,” Science, 2019 Jan. 4; 363: 88-91; the entire contents of each is hereby incorporated by reference. By aggregating more than 10 terabytes of sequence data, new classifications of Type V Cas proteins were identified that showed weak similarity to previously characterized Class V protein, including Cas12g, Cas12h, and Cas12i. In some embodiments, the Cas12 protein is a Cas12g or a variant of Cas12g. In some embodiments, the Cas12 protein is a Cas12h or a variant of Cas12h. In some embodiments, the Cas12 protein is a Cas12i or a variant of Cas12i. It should be appreciated that other RNA-guided DNA binding proteins may be used as a napDNAbp, and are within the scope of this disclosure. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring Cas12g, Cas12h, or Cas12i protein. In some embodiments, the napDNAbp is a naturally-occurring Cas12g, Cas12h, or Cas12i protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to any Cas12g, Cas12h, or Cas12i protein described herein. It should be appreciated that Cas12g, Cas12h, or Cas12i from other bacterial species may also be used in accordance with the present disclosure. In some embodiments, the Cas12i is a Cas12i1l or a Cas12i2.








Cas12g1



MAQASSTPAVSPRPRPRYREERTLVRKLLPRPGQSKQEFRENVKKLRKAFLQFNADVSGVCQWA


 


IQFRPRYGKPAEPTETFWKFFLEPETSLPPNDSRSPEFRRLQAFEAAAGINGAAALDDPAFTNE


 


LRDSILAVASRPKTKEAQRLFSRLKDYQPAHRMILAKVAAEWIESRYRRAHQNWERNYEEWKKE


 


KQEWEQNHPELTPEIREAFNQIFQQLEVKEKRVRICPAARLLQNKDNCQYAGKNKHSVLCNQFN


 


EFKKNHLQGKAIKFFYKDAEKYLRCGLQSLKPNVQGPFREDWNKYLRYMNLKEETLRGKNGGRL


 


PHCKNLGQECEFNPHTALCKQYQQQLSSRPDLVQHDELYRKWRREYWREPRKPVFRYPSVKRHS


 


IAKIFGENYFQADFKNSVVGLRLDSMPAGQYLEFAFAPWPRNYRPQPGETEISSVHLHFVGTRP


 


RIGFRFRVPHKRSRFDCTQEELDELRSRTFPRKAQDQKFLEAARKRLLETFPGNAEQELRLLAV


 


DLGTDSARAAFFIGKTFQQAFPLKIVKIEKLYEQWPNQKQAGDRRDASSKQPRPGLSRDHVGRH


 


LQKMRAQASEIAQKRQELTGTPAPETTTDQAAKKATLQPFDLRGLTVHTARMIRDWARLNARQI


 


IQLAEENQVDLIVLESLRGFRPPGYENLDQEKKRRVAFFAHGRIRRKVTEKAVERGMRVVTVPY


 


LASSKVCAECRKKQKDNKQWEKNKKRGLFKCEGCGSQAQVDENAARVLGRVFWGEIELPTAIP


 


Cas12h1


MKVHRTPRSQLLKTKQYEGSFVEWYRDLQEDRKKFASLLFRWAAFGYAAREDDGATYTSPSQAT


 


LERRLLLGDAEDVAIKFLDVLFKGGAPSSSCYSLFYEDFALRDKAKYSGAKREFIEGLATMPLD


 


KIIERIRQDEQLSKIPAEEWLILGAEYSPEEIWEQVAPRIVNVDRSLGKQLRERLGIKCRRPHD


 


AGYCKILMEVVARQLRSHNETYHEYLNQTHEMKTKVANNLTNEFDLVCEFAEVLEEKNYGLGWY


 


VLWQGVKQALKEQKKPTKIQIAVDQLRQPKFAGLLTAKWRALKGAYDTWKLKKRLEKRKAFPYM


 


PNWDNDYQIPVGLTGLGVFTLEVKRTEVVVDLKEHGKLFCSHSHYFGDLTAEKHPSRYHLKFRH


 


KLKLRKRDSRVEPTIGPWIEAALREITIQKKPNGVFYLGLPYALSHGIDNFQIAKRFFSAAKPD


 


KEVINGLPSEMVVGAADLNLSNIVAPVKARIGKGLEGPLHALDYGYGELIDGPKILTPDGPRCG


 


ELISLKRDIVEIKSAIKEFKACQREGLTMSEETTTWLSEVESPSDSPRCMIQSRIADTSRRLNS


 


FKYQMNKEGYQDLAEALRLLDAMDSYNSLLESYQKMHLSPGEQSPKEAKHDTKKASFKDLLRRR


 


VAHTIVEYFDDCDIVFFEDLDGPSDSDSRNNALVKLLSPRTLLLYIRQALEKRGIGMVEVAKDG


 


TSQNNPISGHVGWRNKQNKSEIYFYEDKELLVMDADEVGAMNILCRGLNHSVCPYSFVTKAPEK


 


KNDEKKEGDYGKRVKRFLKDRYGSSNVRFLVASMGFVTVTTKRPKDALVGKRLYYHGGELVTHD


 


LHNRMKDEIKYLVEKEVLARRVSLSDSTIKSYKSFAHV


 


Cas12i1


MSNKEKNASETRKAYTTKMIPRSHDRMKLLGNFMDYLMDGTPIFFELWNQFGGGIDRDIISGTA


 


NKDKISDDLLLAVNWFKVMPINSKPQGVSPSNLANLFQQYSGSEPDIQAQEYFASNFDTEKHQW


 


KDMRVEYERLLAELQLSRSDMHHDLKLMYKEKCIGLSLSTAHYITSVMFGTGAKNNRQTKHQFY


 


SKVIQLLEESTQINSVEQLASIILKAGDCDSYRKLRIRCSRKGATPSILKIVQDYELGTNHDDE


 


VNVPSLIANLKEKLGRFEYECEWKCMEKIKAFLASKVGPYYLGSYSAMLENALSPIKGMTTKNC


 


KFVLKQIDAKNDIKYENEPFGKIVEGFFDSPYFESDTNVKWVLHPHHIGESNIKTLWEDLNAIH


 


SKYEEDIASLSEDKKEKRIKVYQGDVCQTINTYCEEVGKEAKTPLVQLLRYLYSRKDDIAVDKI


 


IDGITFLSKKHKVEKQKINPVIQKYPSFNFGNNSKLLGKIISPKDKLKHNLKCNRNQVDNYIWI


 


EIKVLNTKTMRWEKHHYALSSTRFLEEVYYPATSENPPDALAARFRTKTNGYEGKPALSAEQIE


 


QIRSAPVGLRKVKKRQMRLEAARQQNLLPRYTWGKDFNINICKRGNNFEVTLATKVKKKKEKNY


 


KVVLGYDANIVRKNTYAAIEAHANGDGVIDYNDIPVKPIESGFVTVESQVRDKSYDQLSYNGVK


 


LLYCKPHVESRRSFLEKYRNGTMKDNRGNNIQIDFMKDFEAIADDETSLYYFNMKYCKLLQSSI


 


RNHSSQAKEYREEIFELLRDGKLSVLKLSSLSNLSFVMFKVAKSLIGTYFGHLLKKPKNSKSDV


 


KAPPITDEDKQKADPEMFALRLALEEKRLNKVKSKKEVIANKIVAKALELRDKYGPVLIKGENI


 


SDTTKKGKKSSTNSFLMDWLARGVANKVKEMVMMHQGLEFVEVNPNFTSHQDPFVHKNPENTFR


 


ARYSRCTPSELTEKNRKEILSFLSDKPSKRPTNAYYNEGAMAFLATYGLKKNDVLGVSLEKFKQ


 


IMANILHQRSEDQLLFPSRGGMFYLATYKLDADATSVNWNGKQFWVCNADLVAAYNVGLVDIQK


 


DFKKK


 


Cas12i2


MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFGGITPEIVRFSTEQEKQQQD


 


IALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGESYYWNDCRQQY


 


YDLCRELGVEVSDLTHDLEILCREKCLAVATESNQNNSIISVLFGTGEKEDRSVKLRITKKILE


 


AISNLKEIPKNVAPIQEIILNVAKATKETFRQVYAGNLGAPSTLEKFIAKDGQKEFDLKKLQTD


 


LKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMFNKAHTALKIKSTRNYNFAKQRLEQ


 


FKEIQSLNNLLVVKKLNDFFDSEFFSGEETYTICVHHLGGKDLSKLYKAWEDDPADPENAIVVI


 


CDDLKNNFKKEPIRNILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKANPSVLGNQGFTWTN


 


AVILPEKAQRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAAGNSPVDTCQFR


 


TPRFGYHLPKLTDQTAIRVNKKHVKAAKTEARIRLAIQQGTLPVSNLKITEISATINSKGQVRI


 


PVKFDVGRQKGTLQIGDRFCGYDQNQTASHAYSLWEVVKEGQYHKELGCFVRFISSGDIVSITE


 


NRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKKEIVTVEAKEKFDAICKYQPRLY


 


KFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVTRLGSLSLSTLETVKAVKGIIYSYE


 


STALNASKNNPISDEQRKEFDPELFALLEKLELIRTRKKKQKVERIANSLIQTCLENNIKFIRG


 


EGDLSTTNNATKKKANSRSMDWLARGVFNKIRQLAPMHNITLFGCGSLYTSHQDPLVHRNPDKA


 


MKCRWAAIPVKDIGDWVLRKLSQNLRAKNIGTGEYYHQGVKEFLSHYELQDLEEELLKWRSDRK


 


SNIPCWVLQNRLAEKLGNKEAVVYIPVRGGRIYFATHKVATGAVSIVFDQKQVWVCNADHVAAA


 


NIALTVKGIGEQSSDEENPDGSRIKLQLTS






Representative nucleic acid and protein sequences of the base editors follow:








BhCas12b GGSGG-ABE8-Xten20 at P153 (“GGSGGS” is disclosed as SEQ ID NO: 273)



(SEQ ID NO: 274)










 


GATCCTTCATCCTGAAGATCGAGCCCAACGAGGAAGTGAAGAAAGGCCTCTGGAAAACCCACGA


 


GGTGCTGAACCACGGAATCGCCTACTACATGAATATCCTGAAGCTGATCCGGCAAGAGGCCATC


 


TACGAGCACCACGAGCAGGACCCCAAGAATCCCAAGAAGGTGTCCAAGGCCGAGATCCAGGCCG


 


AGCTGTGGGATTTCGTGCTGAAGATGCAGAAGTGCAACAGCTTCACACACGAGGTGGACAAGGA


 


CGAGGTGTTCAACATCCTGAGAGAGCTGTACGAGGAACTGGTGCCCAGCAGCGTGGAAAAGAAG


 


GGCGAAGCCAACCAGCTGAGCAACAAGTTTCTGTACCCTCTGGTGGACCCCAACAGCCAGTCTG


 


GAAAGGGAACAGCCAGCAGCGGCAGAAAGCCCAGATGGTACAACCTGAAGATTGCCGGCGATCC


 


Cggaggctctggaggaagc





 








 








 








 








 








 








 








 








 


CTCCTGGGAAGAAGAGAAGAAGAAGTGGGAAGAAGATAAGAAAAAGGACCCGCTGGCCAAGATC


 


CTGGGCAAGCTGGCTGAGTACGGACTGATCCCTCTGTTCATCCCCTACACCGACAGCAACGAGC


 


CCATCGTGAAAGAAATCAAGTGGATGGAAAAGTCCCGGAACCAGAGCGTGCGGCGGCTGGATAA


 


GGACATGTTCATTCAGGCCCTGGAACGGTTCCTGAGCTGGGAGAGCTGGAACCTGAAAGTGAAA


 


GAGGAATACGAGAAGGTCGAGAAAGAGTACAAGACCCTGGAAGAGAGGATCAAAGAGGACATCC


 


AGGCTCTGAAGGCTCTGGAACAGTATGAGAAAGAGCGGCAAGAACAGCTGCTGCGGGACACCCT


 


GAACACCAACGAGTACCGGCTGAGCAAGAGAGGCCTTAGAGGCTGGCGGGAAATCATCCAGAAA


 


TGGCTGAAAATGGACGAGAACGAGCCCTCCGAGAAGTACCTGGAAGTGTTCAAGGACTACCAGC


 


GGAAGCACCCTAGAGAGGCCGGCGATTACAGCGTGTACGAGTTCCTGTCCAAGAAAGAGAACCA


 


CTTCATCTGGCGGAATCACCCTGAGTACCCCTACCTGTACGCCACCTTCTGCGAGATCGACAAG


 


AAAAAGAAGGACGCCAAGCAGCAGGCCACCTTCACACTGGCCGATCCTATCAATCACCCTCTGT


 


GGGTCCGATTCGAGGAAAGAAGCGGCAGCAACCTGAACAAGTACAGAATCCTGACCGAGCAGCT


 


GCACACCGAGAAGCTGAAGAAAAAGCTGACAGTGCAGCTGGACCGGCTGATCTACCCTACAGAA


 


TCTGGCGGCTGGGAAGAGAAGGGCAAAGTGGACATTGTGCTGCTGCCCAGCCGGCAGTTCTACA


 


ACCAGATCTTCCTGGACATCGAGGAAAAGGGCAAGCACGCCTTCACCTACAAGGATGAGAGCAT


 


CAAGTTCCCTCTGAAGGGCACACTCGGCGGAGCCAGAGTGCAGTTCGACAGAGATCACCTGAGA


 


AGATACCCTCACAAGGTGGAAAGCGGCAACGTGGGCAGAATCTACTTCAACATGACCGTGAACA


 


TCGAGCCTACAGAGTCCCCAGTGTCCAAGTCTCTGAAGATCCACCGGGACGACTTCCCCAAGGT


 


GGTCAACTTCAAGCCCAAAGAACTGACCGAGTGGATCAAGGACAGCAAGGGCAAGAAACTGAAG


 


TCCGGCATCGAGTCCCTGGAAATCGGCCTGAGAGTGATGAGCATCGACCTGGGACAGAGACAGG


 


CCGCTGCCGCCTCTATTTTCGAGGTGGTGGATCAGAAGCCCGACATCGAAGGCAAGCTGTTTTT


 


CCCAATCAAGGGCACCGAGCTGTATGCCGTGCACAGAGCCAGCTTCAACATCAAGCTGCCCGGC


 


GAGACACTGGTCAAGAGCAGAGAAGTGCTGCGGAAGGCCAGAGAGGACAATCTGAAACTGATGA


 


ACCAGAAGCTCAACTTCCTGCGGAACGTGCTGCACTTCCAGCAGTTCGAGGACATCACCGAGAG


 


AGAGAAGCGGGTCACCAAGTGGATCAGCAGACAAGAGAACAGCGACGTGCCCCTGGTGTACCAG


 


GATGAGCTGATCCAGATCCGCGAGCTGATGTACAAGCCTTACAAGGACTGGGTCGCCTTCCTGA


 


AGCAGCTCCACAAGAGACTGGAAGTCGAGATCGGCAAAGAAGTGAAGCACTGGCGGAAGTCCCT


 


GAGCGACGGAAGAAAGGGCCTGTACGGCATCTCCCTGAAGAACATCGACGAGATCGATCGGACC


 


CGGAAGTTCCTGCTGAGATGGTCCCTGAGGCCTACCGAACCTGGCGAAGTGCGTAGACTGGAAC


 


CCGGCCAGAGATTCGCCATCGACCAGCTGAATCACCTGAACGCCCTGAAAGAAGATCGGCTGAA


 


GAAGATGGCCAACACCATCATCATGCACGCCCTGGGCTACTGCTACGACGTGCGGAAGAAGAAA


 


TGGCAGGCTAAGAACCCCGCCTGCCAGATCATCCTGTTCGAGGATCTGAGCAACTACAACCCCT


 


ACGAGGAAAGGTCCCGCTTCGAGAACAGCAAGCTCATGAAGTGGTCCAGACGCGAGATCCCCAG


 


ACAGGTTGCACTGCAGGGCGAGATCTATGGCCTGCAAGTGGGAGAAGTGGGCGCTCAGTTCAGC


 


AGCAGATTCCACGCCAAGACAGGCAGCCCTGGCATCAGATGTAGCGTCGTGACCAAAGAGAAGC


 


TGCAGGACAATCGGTTCTTCAAGAATCTGCAGAGAGAGGGCAGACTGACCCTGGACAAAATCGC


 


CGTGCTGAAAGAGGGCGATCTGTACCCAGACAAAGGCGGCGAGAAGTTCATCAGCCTGAGCAAG


 


GATCGGAAGTGCGTGACCACACACGCCGACATCAACGCCGCTCAGAACCTGCAGAAGCGGTTCT


 


GGACAAGAACCCACGGCTTCTACAAGGTGTACTGCAAGGCCTACCAGGTGGACGGCCAGACCG


 


TGTACATCCCTGAGAGCAAGGACCAGAAGCAGAAGATCATCGAAGAGTTCGGCGAGGGCTACT


 


TCATTCTGAAGGACGGGGTGTACGAATGGGTCAACGCCGGCAAGCTGAAAATCAAGAAGGGCAG


 


CTCCAAGCAGAGCAGCAGCGAGCTGGTGGATAGCGACATCCTGAAAGACAGCTTCGACCTGGCC


 


TCCGAGCTGAAAGGCGAAAAGCTGATGCTGTACAGGGACCCCAGCGGCAATGTGTTCCCCAGCG


 


ACAAATGGATGGCCGCTGGCGTGTTCTTCGGAAAGCTGGAACGCATCCTGATCAGCAAGCTGAC


 


CAACCAGTACTCCATCAGCACCATCGAGGACGACAGCAGCAAGCAGTCTATGAAAAGGCCGGCG


 








 


ACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAA


 


(SEQ ID NO: 275)



MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYE



 


HHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVEKKGE


 


ANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPGGSGGSSEVEFSHEYWMRHALTL


 


AKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATLYV


 


TFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLC


 


RFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSGSWEEEKKKWEEDKKKDPLAKILG


 


KLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEE


 


YEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWL


 


KMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKK


 


KDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESG


 


GWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRY


 


PHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKELTEWIKDSKGKKLKSG


 


IESLEIGLRVMSIDLGORQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGET


 


LVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDE


 


LIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRK


 


FLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQ


 


AKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPROVALQGEIYGLQVGEVGAQFSSR


 


FHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDR


 


KCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFIL


 


KDGVYEWVNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKW


 


MAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSMKRPAATKKAGQAKKKKGSYPYDVPDYAY


 


PYDVPDYAYPYDVPDYA


 


BhCas12b GGSGGS-ABE8-Xten20 at K255 (“GGSGGS” is disclosed as SEQ ID NO: 273)


(SEQ ID NO: 276)










 


GATCCTTCATCCTGAAGATCGAGCCCAACGAGGAAGTGAAGAAAGGCCTCTGGAAAACCCACGA


 


GGTGCTGAACCACGGAATCGCCTACTACATGAATATCCTGAAGCTGATCCGGCAAGAGGCCATC


 


TACGAGCACCACGAGCAGGACCCCAAGAATCCCAAGAAGGTGTCCAAGGCCGAGATCCAGGCCG


 


AGCTGTGGGATTTCGTGCTGAAGATGCAGAAGTGCAACAGCTTCACACACGAGGTGGACAAGGA


 


CGAGGTGTTCAACATCCTGAGAGAGCTGTACGAGGAACTGGTGCCCAGCAGCGTGGAAAAGAAG


 


GGCGAAGCCAACCAGCTGAGCAACAAGTTTCTGTACCCTCTGGTGGACCCCAACAGCCAGTCTG


 


GAAAGGGAACAGCCAGCAGCGGCAGAAAGCCCAGATGGTACAACCTGAAGATTGCCGGCGATCC


 


CTCCTGGGAAGAAGAGAAGAAGAAGTGGGAAGAAGATAAGAAAAAGGACCCGCTGGCCAAGATC


 


CTGGGCAAGCTGGCTGAGTACGGACTGATCCCTCTGTTCATCCCCTACACCGACAGCAACGAGC


 


CCATCGTGAAAGAAATCAAGTGGATGGAAAAGTCCCGGAACCAGAGCGTGCGGCGGCTGGATAA


 


GGACATGTTCATTCAGGCCCTGGAACGGTTCCTGAGCTGGGAGAGCTGGAACCTGAAAGTGAAA


 


GAGGAATACGAGAAGGTCGAGAAAGAGTACAAGACCCTGGAAGAGAGGATCAAAggaggctctg


 


gaggaagc





 








 








 








 








 








 








 








 


TCTGGATCTGAAACACCTGGCACAAGCGAGAGCGCCACCCCTGAGAGCTCTGGCGAGGACATCC


 


AGGCTCTGAAGGCTCTGGAACAGTATGAGAAAGAGCGGCAAGAACAGCTGCTGCGGGACACCCT


 


GAACACCAACGAGTACCGGCTGAGCAAGAGAGGCCTTAGAGGCTGGCGGGAAATCATCCAGAAA


 


TGGCTGAAAATGGACGAGAACGAGCCCTCCGAGAAGTACCTGGAAGTGTTCAAGGACTACCAGC


 


GGAAGCACCCTAGAGAGGCCGGCGATTACAGCGTGTACGAGTTCCTGTCCAAGAAAGAGAACCA


 


CTTCATCTGGCGGAATCACCCTGAGTACCCCTACCTGTACGCCACCTTCTGCGAGATCGACAAG


 


AAAAAGAAGGACGCCAAGCAGCAGGCCACCTTCACACTGGCCGATCCTATCAATCACCCTCTGT


 


GGGTCCGATTCGAGGAAAGAAGCGGCAGCAACCTGAACAAGTACAGAATCCTGACCGAGCAGCT


 


GCACACCGAGAAGCTGAAGAAAAAGCTGACAGTGCAGCTGGACCGGCTGATCTACCCTACAGAA


 


TCTGGCGGCTGGGAAGAGAAGGGCAAAGTGGACATTGTGCTGCTGCCCAGCCGGCAGTTCTACA


 


ACCAGATCTTCCTGGACATCGAGGAAAAGGGCAAGCACGCCTTCACCTACAAGGATGAGAGCAT


 


CAAGTTCCCTCTGAAGGGCACACTCGGCGGAGCCAGAGTGCAGTTCGACAGAGATCACCTGAGA


 


AGATACCCTCACAAGGTGGAAAGCGGCAACGTGGGCAGAATCTACTTCAACATGACCGTGAACA


 


TCGAGCCTACAGAGTCCCCAGTGTCCAAGTCTCTGAAGATCCACCGGGACGACTTCCCCAAGGT


 


GGTCAACTTCAAGCCCAAAGAACTGACCGAGTGGATCAAGGACAGCAAGGGCAAGAAACTGAAG


 


TCCGGCATCGAGTCCCTGGAAATCGGCCTGAGAGTGATGAGCATCGACCTGGGACAGAGACAGG


 


CCGCTGCCGCCTCTATTTTCGAGGTGGTGGATCAGAAGCCCGACATCGAAGGCAAGCTGTTTTT


 


CCCAATCAAGGGCACCGAGCTGTATGCCGTGCACAGAGCCAGCTTCAACATCAAGCTGCCCGGC


 


GAGACACTGGTCAAGAGCAGAGAAGTGCTGCGGAAGGCCAGAGAGGACAATCTGAAACTGATGA


 


ACCAGAAGCTCAACTTCCTGCGGAACGTGCTGCACTTCCAGCAGTTCGAGGACATCACCGAGAG


 


AGAGAAGCGGGTCACCAAGTGGATCAGCAGACAAGAGAACAGCGACGTGCCCCTGGTGTACCAG


 


GATGAGCTGATCCAGATCCGCGAGCTGATGTACAAGCCTTACAAGGACTGGGTCGCCTTCCTGA


 


AGCAGCTCCACAAGAGACTGGAAGTCGAGATCGGCAAAGAAGTGAAGCACTGGCGGAAGTCCCT


 


GAGCGACGGAAGAAAGGGCCTGTACGGCATCTCCCTGAAGAACATCGACGAGATCGATCGGACC


 


CGGAAGTTCCTGCTGAGATGGTCCCTGAGGCCTACCGAACCTGGCGAAGTGCGTAGACTGGAAC


 


CCGGCCAGAGATTCGCCATCGACCAGCTGAATCACCTGAACGCCCTGAAAGAAGATCGGCTGAA


 


GAAGATGGCCAACACCATCATCATGCACGCCCTGGGCTACTGCTACGACGTGCGGAAGAAGAAA


 


TGGCAGGCTAAGAACCCCGCCTGCCAGATCATCCTGTTCGAGGATCTGAGCAACTACAACCCCT


 


ACGAGGAAAGGTCCCGCTTCGAGAACAGCAAGCTCATGAAGTGGTCCAGACGCGAGATCCCCAG


 


ACAGGTTGCACTGCAGGGCGAGATCTATGGCCTGCAAGTGGGAGAAGTGGGCGCTCAGTTCAGC


 


AGCAGATTCCACGCCAAGACAGGCAGCCCTGGCATCAGATGTAGCGTCGTGACCAAAGAGAAGC


 


TGCAGGACAATCGGTTCTTCAAGAATCTGCAGAGAGAGGGCAGACTGACCCTGGACAAAATCGC


 


CGTGCTGAAAGAGGGCGATCTGTACCCAGACAAAGGCGGCGAGAAGTTCATCAGCCTGAGCAAG


 


GATCGGAAGTGCGTGACCACACACGCCGACATCAACGCCGCTCAGAACCTGCAGAAGCGGTTCT


 


GGACAAGAACCCACGGCTTCTACAAGGTGTACTGCAAGGCCTACCAGGTGGACGGCCAGACCGT


 


GTACATCCCTGAGAGCAAGGACCAGAAGCAGAAGATCATCGAAGAGTTCGGCGAGGGCTACTTC


 


ATTCTGAAGGACGGGGTGTACGAATGGGTCAACGCCGGCAAGCTGAAAATCAAGAAGGGCAGCT


 


CCAAGCAGAGCAGCAGCGAGCTGGTGGATAGCGACATCCTGAAAGACAGCTTCGACCTGGCCTC


 


CGAGCTGAAAGGCGAAAAGCTGATGCTGTACAGGGACCCCAGCGGCAATGTGTTCCCCAGCGAC


 


AAATGGATGGCCGCTGGCGTGTTCTTCGGAAAGCTGGAACGCATCCTGATCAGCAAGCTGACCA


 


ACCAGTACTCCATCAGCACCATCGAGGACGACAGCAGCAAGCAGTCTATGAAAAGGCCGGCGGC


 








 


GCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAA


 


(SEQ ID NO: 277)



MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYE



 


HHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVEKKGE


 


ANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILG


 


KLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEE


 


YEKVEKEYKTLEERIKGGSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGE


 


GWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVR


 


NAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDGSSG


 


SETPGTSESATPESSGEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWL


 


KMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKK


 


KDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESG


 


GWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRY


 


PHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKELTEWIKDSKGKKLKSG


 


IESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGET


 


LVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDE


 


LIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRK


 


FLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQ


 


AKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSR


 


FHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDR


 


KCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFIL


 


KDGVYEWVNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKW


 


MAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSMKRPAATKKAGQAKKKKGSYPYDVPDYAY


 


PYDVPDYAYPYDVPDYA


 


BhCas 12b GGSGGS-ABE8-Xten20 at D306 (“GGSGGS” is disclosed as SEQ ID NO: 273)


(SEQ ID NO: 278)










 


GATCCTTCATCCTGAAGATCGAGCCCAACGAGGAAGTGAAGAAAGGCCTCTGGAAAACCCACGA


 


GGTGCTGAACCACGGAATCGCCTACTACATGAATATCCTGAAGCTGATCCGGCAAGAGGCCATC


 


TACGAGCACCACGAGCAGGACCCCAAGAATCCCAAGAAGGTGTCCAAGGCCGAGATCCAGGCCG


 


AGCTGTGGGATTTCGTGCTGAAGATGCAGAAGTGCAACAGCTTCACACACGAGGTGGACAAGGA


 


CGAGGTGTTCAACATCCTGAGAGAGCTGTACGAGGAACTGGTGCCCAGCAGCGTGGAAAAGAAG


 


GGCGAAGCCAACCAGCTGAGCAACAAGTTTCTGTACCCTCTGGTGGACCCCAACAGCCAGTCTG


 


GAAAGGGAACAGCCAGCAGCGGCAGAAAGCCCAGATGGTACAACCTGAAGATTGCCGGCGATCC


 


CTCCTGGGAAGAAGAGAAGAAGAAGTGGGAAGAAGATAAGAAAAAGGACCCGCTGGCCAAGATC


 


CTGGGCAAGCTGGCTGAGTACGGACTGATCCCTCTGTTCATCCCCTACACCGACAGCAACGAGC


 


CCATCGTGAAAGAAATCAAGTGGATGGAAAAGTCCCGGAACCAGAGCGTGCGGCGGCTGGATAA


 


GGACATGTTCATTCAGGCCCTGGAACGGTTCCTGAGCTGGGAGAGCTGGAACCTGAAAGTGAAA


 


GAGGAATACGAGAAGGTCGAGAAAGAGTACAAGACCCTGGAAGAGAGGATCAAAGAGGACATCC


 


AGGCTCTGAAGGCTCTGGAACAGTATGAGAAAGAGCGGCAAGAACAGCTGCTGCGGGACACCCT


 


GAACACCAACGAGTACCGGCTGAGCAAGAGAGGCCTTAGAGGCTGGCGGGAAATCATCCAGAAA


 








 








 








 








 








 








 








 








 








 


CCTGAGAGCTCTGGCGAGAACGAGCCCTCCGAGAAGTACCTGGAAGTGTTCAAGGACTACCAGC


 


GGAAGCACCCTAGAGAGGCCGGCGATTACAGCGTGTACGAGTTCCTGTCCAAGAAAGAGAACCA


 


CTTCATCTGGCGGAATCACCCTGAGTACCCCTACCTGTACGCCACCTTCTGCGAGATCGACAAG


 


AAAAAGAAGGACGCCAAGCAGCAGGCCACCTTCACACTGGCCGATCCTATCAATCACCCTCTGT


 


GGGTCCGATTCGAGGAAAGAAGCGGCAGCAACCTGAACAAGTACAGAATCCTGACCGAGCAGCT


 


GCACACCGAGAAGCTGAAGAAAAAGCTGACAGTGCAGCTGGACCGGCTGATCTACCCTACAGAA


 


TCTGGCGGCTGGGAAGAGAAGGGCAAAGTGGACATTGTGCTGCTGCCCAGCCGGCAGTTCTACA


 


ACCAGATCTTCCTGGACATCGAGGAAAAGGGCAAGCACGCCTTCACCTACAAGGATGAGAGCAT


 


CAAGTTCCCTCTGAAGGGCACACTCGGCGGAGCCAGAGTGCAGTTCGACAGAGATCACCTGAGA


 


AGATACCCTCACAAGGTGGAAAGCGGCAACGTGGGCAGAATCTACTTCAACATGACCGTGAACA


 


TCGAGCCTACAGAGTCCCCAGTGTCCAAGTCTCTGAAGATCCACCGGGACGACTTCCCCAAGGT


 


GGTCAACTTCAAGCCCAAAGAACTGACCGAGTGGATCAAGGACAGCAAGGGCAAGAAACTGAAG


 


TCCGGCATCGAGTCCCTGGAAATCGGCCTGAGAGTGATGAGCATCGACCTGGGACAGAGACAGG


 


CCGCTGCCGCCTCTATTTTCGAGGTGGTGGATCAGAAGCCCGACATCGAAGGCAAGCTGTTTTT


 


CCCAATCAAGGGCACCGAGCTGTATGCCGTGCACAGAGCCAGCTTCAACATCAAGCTGCCCGGC


 


GAGACACTGGTCAAGAGCAGAGAAGTGCTGCGGAAGGCCAGAGAGGACAATCTGAAACTGATGA


 


ACCAGAAGCTCAACTTCCTGCGGAACGTGCTGCACTTCCAGCAGTTCGAGGACATCACCGAGAG


 


AGAGAAGCGGGTCACCAAGTGGATCAGCAGACAAGAGAACAGCGACGTGCCCCTGGTGTACCAG


 


GATGAGCTGATCCAGATCCGCGAGCTGATGTACAAGCCTTACAAGGACTGGGTCGCCTTCCTGA


 


AGCAGCTCCACAAGAGACTGGAAGTCGAGATCGGCAAAGAAGTGAAGCACTGGCGGAAGTCCCT


 


GAGCGACGGAAGAAAGGGCCTGTACGGCATCTCCCTGAAGAACATCGACGAGATCGATCGGACC


 


CGGAAGTTCCTGCTGAGATGGTCCCTGAGGCCTACCGAACCTGGCGAAGTGCGTAGACTGGAAC


 


CCGGCCAGAGATTCGCCATCGACCAGCTGAATCACCTGAACGCCCTGAAAGAAGATCGGCTGAA


 


GAAGATGGCCAACACCATCATCATGCACGCCCTGGGCTACTGCTACGACGTGCGGAAGAAGAAA


 


TGGCAGGCTAAGAACCCCGCCTGCCAGATCATCCTGTTCGAGGATCTGAGCAACTACAACCCCT


 


ACGAGGAAAGGTCCCGCTTCGAGAACAGCAAGCTCATGAAGTGGTCCAGACGCGAGATCCCCAG


 


ACAGGTTGCACTGCAGGGCGAGATCTATGGCCTGCAAGTGGGAGAAGTGGGCGCTCAGTTCAGC


 


AGCAGATTCCACGCCAAGACAGGCAGCCCTGGCATCAGATGTAGCGTCGTGACCAAAGAGAAGC


 


TGCAGGACAATCGGTTCTTCAAGAATCTGCAGAGAGAGGGCAGACTGACCCTGGACAAAATCGC


 


CGTGCTGAAAGAGGGCGATCTGTACCCAGACAAAGGCGGCGAGAAGTTCATCAGCCTGAGCAAG


 


GATCGGAAGTGCGTGACCACACACGCCGACATCAACGCCGCTCAGAACCTGCAGAAGCGGTTCT


 


GGACAAGAACCCACGGCTTCTACAAGGTGTACTGCAAGGCCTACCAGGTGGACGGCCAGACCGT


 


GTACATCCCTGAGAGCAAGGACCAGAAGCAGAAGATCATCGAAGAGTTCGGCGAGGGCTACTTC


 


ATTCTGAAGGACGGGGTGTACGAATGGGTCAACGCCGGCAAGCTGAAAATCAAGAAGGGCAGCT


 


CCAAGCAGAGCAGCAGCGAGCTGGTGGATAGCGACATCCTGAAAGACAGCTTCGACCTGGCCTC


 


CGAGCTGAAAGGCGAAAAGCTGATGCTGTACAGGGACCCCAGCGGCAATGTGTTCCCCAGCGAC


 


AAATGGATGGCCGCTGGCGTGTTCTTCGGAAAGCTGGAACGCATCCTGATCAGCAAGCTGACCA


 


ACCAGTACTCCATCAGCACCATCGAGGACGACAGCAGCAAGCAGTCTATGAAAAGGCCGGCGGC


 








 


GCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAA


 


(SEQ ID NO: 279)



MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYE



 


HHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVEKKGE


 


ANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILG


 


KLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEE


 


YEKVEKEYKTLEERI KEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWL


 


KMDGGSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTA


 


HAEIMALRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV


 


LHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPE


 


SSGENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKK


 


KDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESG


 


GWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRY


 


PHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKELTEWIKDSKGKKLKSG


 


IESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGET


 


LVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDE


 


LIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRK


 


FLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQ


 


AKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPROVALQGEIYGLQVGEVGAQFSSR


 


FHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDR


 


KCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFIL


 


KDGVYEWVNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKW


 


MAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSMKRPAATKKAGQAKKKKGSYPYDVPDYAY


 


PYDVPDYAYPYDVPDYA


 


BhCas12b GGSGGS-ABE8-Xten20 at D980 (“GGSGGS” is disclosed as SEQ ID NO: 273)


(SEQ ID NO: 280)










 


GATCCTTCATCCTGAAGATCGAGCCCAACGAGGAAGTGAAGAAAGGCCTCTGGAAAACCCACGA


 


GGTGCTGAACCACGGAATCGCCTACTACATGAATATCCTGAAGCTGATCCGGCAAGAGGCCATC


 


TACGAGCACCACGAGCAGGACCCCAAGAATCCCAAGAAGGTGTCCAAGGCCGAGATCCAGGCCG


 


AGCTGTGGGATTTCGTGCTGAAGATGCAGAAGTGCAACAGCTTCACACACGAGGTGGACAAGGA


 


CGAGGTGTTCAACATCCTGAGAGAGCTGTACGAGGAACTGGTGCCCAGCAGCGTGGAAAAGAAG


 


GGCGAAGCCAACCAGCTGAGCAACAAGTTTCTGTACCCTCTGGTGGACCCCAACAGCCAGTCTG


 


GAAAGGGAACAGCCAGCAGCGGCAGAAAGCCCAGATGGTACAACCTGAAGATTGCCGGCGATCC


 


CTCCTGGGAAGAAGAGAAGAAGAAGTGGGAAGAAGATAAGAAAAAGGACCCGCTGGCCAAGATC


 


CTGGGCAAGCTGGCTGAGTACGGACTGATCCCTCTGTTCATCCCCTACACCGACAGCAACGAGC


 


CCATCGTGAAAGAAATCAAGTGGATGGAAAAGTCCCGGAACCAGAGCGTGCGGCGGCTGGATAA


 


GGACATGTTCATTCAGGCCCTGGAACGGTTCCTGAGCTGGGAGAGCTGGAACCTGAAAGTGAAA


 


GAGGAATACGAGAAGGTCGAGAAAGAGTACAAGACCCTGGAAGAGAGGATCAAAGAGGACATCC


 


AGGCTCTGAAGGCTCTGGAACAGTATGAGAAAGAGCGGCAAGAACAGCTGCTGCGGGACACCCT


 


GAACACCAACGAGTACCGGCTGAGCAAGAGAGGCCTTAGAGGCTGGCGGGAAATCATCCAGAAA


 


TGGCTGAAAATGGACGAGAACGAGCCCTCCGAGAAGTACCTGGAAGTGTTCAAGGACTACCAGC


 


GGAAGCACCCTAGAGAGGCCGGCGATTACAGCGTGTACGAGTTCCTGTCCAAGAAAGAGAACCA


 


CTTCATCTGGCGGAATCACCCTGAGTACCCCTACCTGTACGCCACCTTCTGCGAGATCGACAAG


 


AAAAAGAAGGACGCCAAGCAGCAGGCCACCTTCACACTGGCCGATCCTATCAATCACCCTCTGT


 


GGGTCCGATTCGAGGAAAGAAGCGGCAGCAACCTGAACAAGTACAGAATCCTGACCGAGCAGCT


 


GCACACCGAGAAGCTGAAGAAAAAGCTGACAGTGCAGCTGGACCGGCTGATCTACCCTACAGAA


 


TCTGGCGGCTGGGAAGAGAAGGGCAAAGTGGACATTGTGCTGCTGCCCAGCCGGCAGTTCTACA


 


ACCAGATCTTCCTGGACATCGAGGAAAAGGGCAAGCACGCCTTCACCTACAAGGATGAGAGCAT


 


CAAGTTCCCTCTGAAGGGCACACTCGGCGGAGCCAGAGTGCAGTTCGACAGAGATCACCTGAGA


 


AGATACCCTCACAAGGTGGAAAGCGGCAACGTGGGCAGAATCTACTTCAACATGACCGTGAACA


 


TCGAGCCTACAGAGTCCCCAGTGTCCAAGTCTCTGAAGATCCACCGGGACGACTTCCCCAAGGT


 


GGTCAACTTCAAGCCCAAAGAACTGACCGAGTGGATCAAGGACAGCAAGGGCAAGAAACTGAAG


 


TCCGGCATCGAGTCCCTGGAAATCGGCCTGAGAGTGATGAGCATCGACCTGGGACAGAGACAGG


 


CCGCTGCCGCCTCTATTTTCGAGGTGGTGGATCAGAAGCCCGACATCGAAGGCAAGCTGTTTTT


 


CCCAATCAAGGGCACCGAGCTGTATGCCGTGCACAGAGCCAGCTTCAACATCAAGCTGCCCGGC


 


GAGACACTGGTCAAGAGCAGAGAAGTGCTGCGGAAGGCCAGAGAGGACAATCTGAAACTGATGA


 


ACCAGAAGCTCAACTTCCTGCGGAACGTGCTGCACTTCCAGCAGTTCGAGGACATCACCGAGAG


 


AGAGAAGCGGGTCACCAAGTGGATCAGCAGACAAGAGAACAGCGACGTGCCCCTGGTGTACCAG


 


GATGAGCTGATCCAGATCCGCGAGCTGATGTACAAGCCTTACAAGGACTGGGTCGCCTTCCTGA


 


AGCAGCTCCACAAGAGACTGGAAGTCGAGATCGGCAAAGAAGTGAAGCACTGGCGGAAGTCCCT


 


GAGCGACGGAAGAAAGGGCCTGTACGGCATCTCCCTGAAGAACATCGACGAGATCGATCGGACC


 


CGGAAGTTCCTGCTGAGATGGTCCCTGAGGCCTACCGAACCTGGCGAAGTGCGTAGACTGGAAC


 


CCGGCCAGAGATTCGCCATCGACCAGCTGAATCACCTGAACGCCCTGAAAGAAGATCGGCTGAA


 


GAAGATGGCCAACACCATCATCATGCACGCCCTGGGCTACTGCTACGACGTGCGGAAGAAGAAA


 


TGGCAGGCTAAGAACCCCGCCTGCCAGATCATCCTGTTCGAGGATCTGAGCAACTACAACCCCT


 


ACGAGGAAAGGTCCCGCTTCGAGAACAGCAAGCTCATGAAGTGGTCCAGACGCGAGATCCCCAG


 


ACAGGTTGCACTGCAGGGCGAGATCTATGGCCTGCAAGTGGGAGAAGTGGGCGCTCAGTTCAGC


 


AGCAGATTCCACGCCAAGACAGGCAGCCCTGGCATCAGATGTAGCGTCGTGACCAAAGAGAAGC


 


TGCAGGACAATCGGTTCTTCAAGAATCTGCAGAGAGAGGGCAGACTGACCCTGGACAAAATCGC


 


CGTGCTGAAAGAGGGCGATCTGTACCCAGACAAAGGCGGCGAGAAGTTCATCAGCCTGAGCAAG


 


GATCGGAAGTGCGTGACCACACACGCCGACATCAACGCCGCTCAGAACCTGCAGAAGCGGTTCT


 


GGACAAGAACCCACGGCTTCTACAAGGTGTACTGCAAGGCCTACCAGGTGGACggaggctctgg


 








 








 








 








 








 








 








 








 


CTGGATCTGAAACACCTGGCACAAGCGAGAGCGCCACCCCTGAGAGCTCTGGCGGCCAGACCGT


 


GTACATCCCTGAGAGCAAGGACCAGAAGCAGAAGATCATCGAAGAGTTCGGCGAGGGCTACTTC


 


ATTCTGAAGGACGGGGTGTACGAATGGGTCAACGCCGGCAAGCTGAAAATCAAGAAGGGCAGCT


 


CCAAGCAGAGCAGCAGCGAGCTGGTGGATAGCGACATCCTGAAAGACAGCTTCGACCTGGCCTC


 


CGAGCTGAAAGGCGAAAAGCTGATGCTGTACAGGGACCCCAGCGGCAATGTGTTCCCCAGCGAC


 


AAATGGATGGCCGCTGGCGTGTTCTTCGGAAAGCTGGAACGCATCCTGATCAGCAAGCTGACCA


 


ACCAGTACTCCATCAGCACCATCGAGGACGACAGCAGCAAGCAGTCTATGAAAAGGCCGGCGGC


 








 


GCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAA


 


(SEQ ID NO: 281)



MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYE



 


HHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVEKKGE


 


ANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILG


 


KLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEE


 


YEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWL


 


KMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKK


 


KDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESG


 


GWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRY


 


PHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKELTEWIKDSKGKKLKSG


 


IESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGET


 


LVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDE


 


LIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRK


 


FLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQ


 


AKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSR


 


FHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDR


 


KCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGGSGGSSEVEFSHEYWMRHALTLAKRA


 


RDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATLYVTFEP


 


CVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFR


 


MPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSGGQTVYIPESKDQKQKIIEEFGEGYFIL


 


KDGVYEWVNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKW


 


MAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSMKRPAATKKAGQAKKKKGSYPYDVPDYAY


 


PYDVPDYAYPYDVPDYA


 


BhCas12b GGSGGS-ABE8-Xten20 at K1019 (“GGSGGS” is disclosed as SEQ ID NO: 273)


(SEQ ID NO: 282)










 


GATCCTTCATCCTGAAGATCGAGCCCAACGAGGAAGTGAAGAAAGGCCTCTGGAAAACCCACGA


 


GGTGCTGAACCACGGAATCGCCTACTACATGAATATCCTGAAGCTGATCCGGCAAGAGGCCATC


 


TACGAGCACCACGAGCAGGACCCCAAGAATCCCAAGAAGGTGTCCAAGGCCGAGATCCAGGCCG


 


AGCTGTGGGATTTCGTGCTGAAGATGCAGAAGTGCAACAGCTTCACACACGAGGTGGACAAGGA


 


CGAGGTGTTCAACATCCTGAGAGAGCTGTACGAGGAACTGGTGCCCAGCAGCGTGGAAAAGAAG


 


GGCGAAGCCAACCAGCTGAGCAACAAGTTTCTGTACCCTCTGGTGGACCCCAACAGCCAGTCTG


 


GAAAGGGAACAGCCAGCAGCGGCAGAAAGCCCAGATGGTACAACCTGAAGATTGCCGGCGATCC


 


CTCCTGGGAAGAAGAGAAGAAGAAGTGGGAAGAAGATAAGAAAAAGGACCCGCTGGCCAAGATC


 


CTGGGCAAGCTGGCTGAGTACGGACTGATCCCTCTGTTCATCCCCTACACCGACAGCAACGAGC


 


CCATCGTGAAAGAAATCAAGTGGATGGAAAAGTCCCGGAACCAGAGCGTGCGGCGGCTGGATAA


 


GGACATGTTCATTCAGGCCCTGGAACGGTTCCTGAGCTGGGAGAGCTGGAACCTGAAAGTGAAA


 


GAGGAATACGAGAAGGTCGAGAAAGAGTACAAGACCCTGGAAGAGAGGATCAAAGAGGACATCC


 


AGGCTCTGAAGGCTCTGGAACAGTATGAGAAAGAGCGGCAAGAACAGCTGCTGCGGGACACCCT


 


GAACACCAACGAGTACCGGCTGAGCAAGAGAGGCCTTAGAGGCTGGCGGGAAATCATCCAGAAA


 


TGGCTGAAAATGGACGAGAACGAGCCCTCCGAGAAGTACCTGGAAGTGTTCAAGGACTACCAGC


 


GGAAGCACCCTAGAGAGGCCGGCGATTACAGCGTGTACGAGTTCCTGTCCAAGAAAGAGAACCA


 


CTTCATCTGGCGGAATCACCCTGAGTACCCCTACCTGTACGCCACCTTCTGCGAGATCGACAAG


 


AAAAAGAAGGACGCCAAGCAGCAGGCCACCTTCACACTGGCCGATCCTATCAATCACCCTCTGT


 


GGGTCCGATTCGAGGAAAGAAGCGGCAGCAACCTGAACAAGTACAGAATCCTGACCGAGCAGCT


 


GCACACCGAGAAGCTGAAGAAAAAGCTGACAGTGCAGCTGGACCGGCTGATCTACCCTACAGAA


 


TCTGGCGGCTGGGAAGAGAAGGGCAAAGTGGACATTGTGCTGCTGCCCAGCCGGCAGTTCTACA


 


ACCAGATCTTCCTGGACATCGAGGAAAAGGGCAAGCACGCCTTCACCTACAAGGATGAGAGCAT


 


CAAGTTCCCTCTGAAGGGCACACTCGGCGGAGCCAGAGTGCAGTTCGACAGAGATCACCTGAGA


 


AGATACCCTCACAAGGTGGAAAGCGGCAACGTGGGCAGAATCTACTTCAACATGACCGTGAACA


 


TCGAGCCTACAGAGTCCCCAGTGTCCAAGTCTCTGAAGATCCACCGGGACGACTTCCCCAAGGT


 


GGTCAACTTCAAGCCCAAAGAACTGACCGAGTGGATCAAGGACAGCAAGGGCAAGAAACTGAAG


 


TCCGGCATCGAGTCCCTGGAAATCGGCCTGAGAGTGATGAGCATCGACCTGGGACAGAGACAGG


 


CCGCTGCCGCCTCTATTTTCGAGGTGGTGGATCAGAAGCCCGACATCGAAGGCAAGCTGTTTTT


 


CCCAATCAAGGGCACCGAGCTGTATGCCGTGCACAGAGCCAGCTTCAACATCAAGCTGCCCGGC


 


GAGACACTGGTCAAGAGCAGAGAAGTGCTGCGGAAGGCCAGAGAGGACAATCTGAAACTGATGA


 


ACCAGAAGCTCAACTTCCTGCGGAACGTGCTGCACTTCCAGCAGTTCGAGGACATCACCGAGAG


 


AGAGAAGCGGGTCACCAAGTGGATCAGCAGACAAGAGAACAGCGACGTGCCCCTGGTGTACCAG


 


GATGAGCTGATCCAGATCCGCGAGCTGATGTACAAGCCTTACAAGGACTGGGTCGCCTTCCTGA


 


AGCAGCTCCACAAGAGACTGGAAGTCGAGATCGGCAAAGAAGTGAAGCACTGGCGGAAGTCCCT


 


GAGCGACGGAAGAAAGGGCCTGTACGGCATCTCCCTGAAGAACATCGACGAGATCGATCGGACC


 


CGGAAGTTCCTGCTGAGATGGTCCCTGAGGCCTACCGAACCTGGCGAAGTGCGTAGACTGGAAC


 


CCGGCCAGAGATTCGCCATCGACCAGCTGAATCACCTGAACGCCCTGAAAGAAGATCGGCTGAA


 


GAAGATGGCCAACACCATCATCATGCACGCCCTGGGCTACTGCTACGACGTGCGGAAGAAGAAA


 


TGGCAGGCTAAGAACCCCGCCTGCCAGATCATCCTGTTCGAGGATCTGAGCAACTACAACCCCT


 


ACGAGGAAAGGTCCCGCTTCGAGAACAGCAAGCTCATGAAGTGGTCCAGACGCGAGATCCCCAG


 


ACAGGTTGCACTGCAGGGCGAGATCTATGGCCTGCAAGTGGGAGAAGTGGGCGCTCAGTTCAGC


 


AGCAGATTCCACGCCAAGACAGGCAGCCCTGGCATCAGATGTAGCGTCGTGACCAAAGAGAAGC


 


TGCAGGACAATCGGTTCTTCAAGAATCTGCAGAGAGAGGGCAGACTGACCCTGGACAAAATCGC


 


CGTGCTGAAAGAGGGCGATCTGTACCCAGACAAAGGCGGCGAGAAGTTCATCAGCCTGAGCAAG


 


GATCGGAAGTGCGTGACCACACACGCCGACATCAACGCCGCTCAGAACCTGCAGAAGCGGTTCT


 


GGACAAGAACCCACGGCTTCTACAAGGTGTACTGCAAGGCCTACCAGGTGGACGGCCAGACCGT


 


GTACATCCCTGAGAGCAAGGACCAGAAGCAGAAGATCATCGAAGAGTTCGGCGAGGGCTACTTC


 


ATTCTGAAGGACGGGGTGTACGAATGGGTCAACGCCGGCAAGggaggctctggaggaagc





 








 








 








 








 








 








 








 








 


ACACCTGGCACAAGCGAGAGCGCCACCCCTGAGAGCTCTGGCCTGAAAATCAAGAAGGGCAGCT


 


CCAAGCAGAGCAGCAGCGAGCTGGTGGATAGCGACATCCTGAAAGACAGCTTCGACCTGGCCTC


 


CGAGCTGAAAGGCGAAAAGCTGATGCTGTACAGGGACCCCAGCGGCAATGTGTTCCCCAGCGAC


 


AAATGGATGGCCGCTGGCGTGTTCTTCGGAAAGCTGGAACGCATCCTGATCAGCAAGCTGACCA


 


ACCAGTACTCCATCAGCACCATCGAGGACGACAGCAGCAAGCAGTCTATGAAAAGGCCGGCGGC


 








 


GCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAA


 


(SEQ ID NO: 283)



MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYE



 


HHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVEKKGE


 


ANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILG


 


KLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEE


 


YEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWL


 


KMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKK


 


KDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESG


 


GWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVOFDRDHLRRY


 


PHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKELTEWIKDSKGKKLKSG


 


IESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGET


 


LVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDE


 


LIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRK


 


FLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQ


 


AKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSR


 


FHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDR


 


KCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFIL


 


KDGVYEWVNAGKGGSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNR


 


AIGLHDPTAHAEIMALRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKT


 


GAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETP


 


GTSESATPESSGLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKW


 


MAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSMKRPAATKKAGQAKKKKGSYPYDVPDYAY


 


PYDVPDYAYPYDVPDYA






For the sequences above, the Kozak sequence is bolded and underlined; marks the N-terminal nuclear localization signal (NLS); lower case characters denote the GGGSGGS linker (SEQ ID NO: 284); _ _ _ marks the sequence encoding ABE8, unmodified sequence encodes BhCas12b; double underling denotes the Xten20 linker; single underlining denotes the C-terminal NLS; GGATCC denotes the GS linker; and italicized characters represent the coding sequence of the 3× hemagglutinin (HA) tag.
In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein may be a Cas12j/CasΦ protein. Cas12j/CasΦ is described in Pausch et al., “CRISPR-CasΦ from huge phages is a hypercompact genome editor,” Science, 17 Jul. 2020, Vol. 369, Issue 6501, pp. 333-337, which is incorporated herein by reference in its entirety. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to a naturally-occurring Cas12j/CasΦ protein. In some embodiments, the napDNAbp is a naturally-occurring Cas12j/CasΦ protein. In some embodiments, the napDNAbp is a nuclease inactive (“dead”) Cas12j/CasΦ protein. It should be appreciated that Cas12j/CasΦ from other species may also be used in accordance with the present disclosure.
Exemplary Cas12j/CasΦ amino acid sequences follow:








>CasΦ-1



MADTPTLFTQFLRHHLPGQRFRKDILKQAGRILANKGEDATIAFLRGKSEESPPDFQPP


 


VKCPIIACSRPLTEWPIYQASVAIQGYVYGQSLAEFEASDPGCSKDGLLGWFDKTGVCTDYFSV


 


QGLNLIFQNARKRYIGVQTKVTNRNEKRHKKLKRINAKRIAEGLPELTSDEPESALDETGHLID


 


PPGLNTNIYCYQQVSPKPLALSEVNQLPTAYAGYSTSGDDPIQPMVTKDRLSISKGQPGYIPEH


 


QRALLSQKKHRRMRGYGLKARALLVIVRIQDDWAVIDLRSLLRNAYWRRIVQTKEPSTITKLLK


 


LVTGDPVLDATRMVATFTYKPGIVQVRSAKCLKNKQGSKLFSERYLNETVSVTSIDLGSNNLVA


 


VATYRLVNGNTPELLQRFTLPSHLVKDFERYKQAHDTLEDSIQKTAVASLPQGQQTEIRMWSMY


 


GFREAQERVCQELGLADGSIPWNVMTATSTILTDLFLARGGDPKKCMFTSEPKKKKNSKQVLYK


 


IRDRAWAKMYRTLLSKETREAWNKALWGLKRGSPDYARLSKRKEELARRCVNYTISTAEKRAQC


 


GRTIVALEDLNIGFFHGRGKQEPGWVGLFTRKKENRWLMQALHKAFLELAHHRGYHVIEVNPAY


 


TSQTCPVCRHCDPDNRDQHNREAFHCIGCGFRGNADLDVATHNIAMVAITGESLKRARGSVASK


 


TPQPLAAE*


 


>CasΦ-2


MPKPAVESEFSKVLKKHFPGERFRSSYMKRGGKILAAQGEEAVVAYLQGKSEEEPPNFQPPAKC


 


HVVTKSRDFAEWPIMKASEAIQRYTYALSTTERAACKPGKSSESHAAWFAATGVSNHGYSHVOG


 


LNLIFDHTLGRYDGVLKKVQLRNEKARARLESINASRADEGLPEIKAEEEEVATNETGHLLQPP


 


GINPSFYVYQTISPQAYRPRDEIVLPPEYAGYVRDPNAPIPLGVVRNRCDIQKGCPGYIPEWQR


 


EAGTAISPKTGKAVTVPGLSPKKNKRMRRYWRSEKEKAQDALLVTVRIGTDWVVIDVRGLLRNA


 


RWRTIAPKDISLNALLDLFTGDPVIDVRRNIVTFTYTLDACGTYARKWTLKGKQTKATLDKLTA


 


TQTVALVAIDLGQTNPISAGISRVTQENGALQCEPLDRFTLPDDLLKDISAYRIAWDRNEEELR


 


ARSVEALPEAQQAEVRALDGVSKETARTQLCADFGLDPKRLPWDKMSSNTTFISEALLSNSVSR


 


DQVFETPAPKKGAKKKAPVEVMRKDRTWARAYKPRLSVEAQKLKNEALWALKRTSPEYLKLSRR


 


KEELCRRSINYVIEKTRRRTQCQIVIPVIEDLNVRFFHGSGKRLPGWDNFFTAKKENRWFTOGL


 


HKAFSDLRTHRSFYVFEVRPERTSITCPKCGHCEVGNRDGEAFQCLSCGKTCNADLDVATHNLT


 


QVALTGKTMPKREEPRDAQGTAPARKTKKASKSKAPPAEREDQTPAQEPSQTS


 


>CasΦ-3


MEKEITELTKIRREFPNKKFSSTDMKKAGKLLKAEGPDAVRDFLNSCQEIIGDFKPPVKTNIVS


 


ISRPFEEWPVSMVGRAIQEYYFSLTKEELESVHPGTSSEDHKSFFNITGLSNYNYTSVQGLNLI


 


FKNAKAIYDGTLVKANNKNKKLEKKFNEINHKRSLEGLPIITPDFEEPFDENGHLNNPPGINRN


 


IYGYQGCAAKVFVPSKHKMVSLPKEYEGYNRDPNLSLAGFRNRLEIPEGEPGHVPWFQRMDIPE


 


GQIGHVNKIQRFNFVHGKNSGKVKFSDKTGRVKRYHHSKYKDATKPYKFLEESKKVSALDSILA


 


IITIGDDWVVFDIRGLYRNVFYRELAQKGLTAVQLLDLFTGDPVIDPKKGVVTFSYKEGVVPVF


 


SQKIVPRFKSRDTLEKLTSQGPVALLSVDLGQNEPVAARVCSLKNINDKITLDNSCRISFLDDY


 


KKQIKDYRDSLDELEIKIRLEAINSLETNQQVEIRDLDVFSADRAKANTVDMFDIDPNLISWDS


 


MSDARVSTQISDLYLKNGGDESRVYFEINNKRIKRSDYNISQLVRPKLSDSTRKNLNDSIWKLK


 


RTSEEYLKLSKRKLELSRAVVNYTIRQSKLLSGINDIVIILEDLDVKKKFNGRGIRDIGWDNFE


 


SSRKENRWFIPAFHKAFSELSSNRGLCVIEVNPAWTSATCPDCGFCSKENRDGINFTCRKCGVS


 


YHADIDVATLNIARVAVLGKPMSGPADRERLGDTKKPRVARSRKTMKRKDISNSTVEAMVTA*


 


>CasΦ-4


MYSLEMADLKSEPSLLAKLLRDRFPGKYWLPKYWKLAEKKRLTGGEEAACEYMADKQLDSPPPN


 


FRPPARCVILAKSRPFEDWPVHRVASKAQSPPIGLSEQGFAALRAAPPSTADARRDWLRSEGAS


 


EDDLMALEAQLLETIMGNAISLHGGVLKKIDNANVKAAKRLSGRNEARLNKGLQELPPEQEGSA


 


YGADGLLVNPPGLNLNIYCRKSCCPKPVKNTARFVGHYPGYLRDSDSILISGTMDRLTIIEGMP


 


GHIPAWQREQGLVKPGGRRRRLSGSESNMRQKVDPSTGPRRSTRSGTVNRSNQRTGRNGDPLLV


 


EIRMKEDWVLLDARGLLRNLRWRESKRGLSCDHEDLSLSGLLALFSGDPVIDPVRNEVVFLYGE


 


GIIPVRSTKPVGTRQSKKLLERQASMGPLTLISCDLGQTNLIAGRASAISLTHGSLGVRSSVRI


 


ELDPEIIKSFERLRKDADRLETEILTAAKETLSDEQRGEVNSHEKDSPQTAKASLCRELGLHPP


 


SLPWGQMGPSTTFIADMLISHGRDDDAFLSHGEFPTLEKRKKFDKRFCLESRPLLSSETRKALN


 


ESLWEVKRTSSEYARLSQRKKEMARRAVNFVVEISRRKTGLSNVIVNIEDLNVRIFHGGGKQAP


 


GWDGFFRPKSENRWFIQAIHKAFSDLAAHHGIPVIESDPQRTSMTCPECGHCDSKNRNGVRFLC


 


KGCGASMDADFDAACRNLERVALTGKPMPKPSTSCERLLSATTGKVCSDHSLSHDAIEKAS*


 


>CasΦ-5


MSSLPTPLELLKQKHADLFKGLQFSSKDNKMAGKVLKKDGEEAALAFLSERGVSRGELPNFRPP


 


AKTLVVAQSRPFEEFPIYRVSEAIQLYVYSLSVKELETVPSGSSTKKEHQRFFQDSSVPDFGYT


 


SVQGLNKIFGLARGIYLGVITRGENQLQKAKSKHEALNKKRRASGEAETEFDPTPYEYMTPERK


 


LAKPPGVNHSIMCYVDISVDEFDFRNPDGIVLPSEYAGYCREINTAIEKGTVDRLGHLKGGPGY


 


IPGHQRKESTTEGPKINFRKGRIRRSYTALYAKRDSRRVRQGKLALPSYRHHMMRLNSNAESAI


 


LAVIFFGKDWVVFDLRGLLRNVRWRNLFVDGSTPSTLLGMFGDPVIDPKRGVVAFCYKEQIVPV


 


VSKSITKMVKAPELLNKLYLKSEDPLVLVAIDLGQTNPVGVGVYRVMNASLDYEVVTRFALESE


 


LLREIESYRQRTNAFEAQIRAETFDAMTSEEQEEITRVRAFSASKAKENVCHRFGMPVDAVDWA


 


TMGSNTIHIAKWVMRHGDPSLVEVLEYRKDNEIKLDKNGVPKKVKLTDKRIANLTSIRLRFSQE


 


TSKHYNDTMWELRRKHPVYQKLSKSKADFSRRVVNSIIRRVNHLVPRARIVFIIEDLKNLGKVE


 


HGSGKRELGWDSYFEPKSENRWFIQVLHKAFSETGKHKGYYIIECWPNWTSCTCPKCSCCDSEN


 


RHGEVFRCLACGYCNTDFGAPDNLVKLAGKGLPGPKKRCKGSSKGKNPKLARSSETGVSV


 


TESGAPKVKKSSPTQTSQSSSQSAP*


 


>CasΦ-6


MNKIEKEKTPLAKLMNENFAGLRFPFAIIKQAGKKLLKEGELKTIEYMTGKGSIEPLPNFKPPV


 


KCLIVAKRRDLKYFPICKASCEIQSYVYSLNYKDEMDYFSTPMTSQKQHEEFFKKSGLNIEYQN


 


VAGLNLIFNNVKNTYNGVILKVKNRNEKLKKKAIKNNYEFEEIKTFNDDGCLINKPGINNVIYC


 


FQSISPKILKNITHLPKEYNDYDCSVDRNIIQKYVSRLDIPESQPGHVPEWQRKLPEFNNTNNP


 


RRRRKWYSNGRNISKGYSVDQVNQAKIEDSLLAQIKIGEDWIILDIRGLLRDLNRRELISYKNK


 


LTIKDVLGFFSDYPIIDIKKNLVTFCYKEGVIQVVSQKSIGNKKSKQLLEKLIENKPIALVSID


 


LGQTNPVSVKISKLNKINNKISIESFTYRFLNEEILKEIEKYRKDYDKLELKLINEA


 


>CasΦ-7


MSNTAVSTREHMSNKTTPPSPLSLLLRAHFPGLKFESQDYKIAGKKLRDGGPEAVISYLTGKGQ


 


AKLKDVKPPAKAFVIAQSRPFIEWDLVRVSRQIQEKIFGIPATKGRPKQDGLSETAFNEAVASL


 


EVDGKSKLNEETRAAFYEVLGLDAPSLHAQAQNALIKSAISIREGVLKKVENRNEKNLSKTKRR


 


KEAGEEATFVEEKAHDERGYLIHPPGVNQTIPGYQAVVIKSCPSDFIGLPSGCLAKESAEALTD


 


YLPHDRMTIPKGQPGYVPEWQHPLLNRRKNRRRRDWYSASLNKPKATCSKRSGTPNRKNSRTDQ


 


IQSGRFKGAIPVLMRFQDEWVIIDIRGLLRNARYRKLLKEKSTIPDLLSLFTGDPSIDMRQGVC


 


TFIYKAGQACSAKMVKTKNAPEILSELTKSGPVVLVSIDLGQTNPIAAKVSRVTQLSDGQLSHE


 


TLLRELLSNDSSDGKEIARYRVASDRLRDKLANLAVERLSPEHKSEILRAKNDTPALCKARVCA


 


ALGLNPEMIAWDKMTPYTEFLATAYLEKGGDRKVATLKPKNRPEMLRRDIKFKGTEGVRIEVSP


 


EAAEAYREAQWDLQRTSPEYLRLSTWKQELTKRILNQLRHKAAKSSQCEVVVMAFEDLNIKMMH


 


GNGKWADGGWDAFFIKKRENRWFMQAFHKSLTELGAHKGVPTIEVTPHRTSITCTKCGHCDKAN


 


RDGERFACQKCGFVAHADLEIATDNIERVALTGKPMPKPESERSGDAKKSVGARKAAFKPEEDA


 


EAAE*


 


>CasΦ-8


MTKPTVSQFLTPGFKLIRNHSRTAGLKLKNEGEEACKKFVRENEIPKDECPNFQGGPAIANIIA


 


KSREFTEWEIYQSSLAIQEVIFTLPKDKLPEPILKEEWRAQWLSEHGLDTVPYKEAAGLNLIIK


 


NAVNTYKGVQVKVDNKNKNNLAKINRKNEIAKLNGEQEISFEEIKAFDDKGYLLQKPSPNKSIY


 


CYQSVSPKPFITSKYHNVNLPEEYIGYYRKSNEPIVSPYQFDRLRIPIGEPGYVPKWQYTFLSK


 


KENKRRKLSKRIKNVSPILGIICIKKDWCVFDMRGLLRTNHWKKYHKPTDSINDLFDYFTGDPV


 


IDTKANVVRFRYKMENGIVNYKPVREKKGKELLENICDQNGSCKLATVDVGQNNPVAIGLFELK


 


KVNGELTKTLISRHPTPIDFCNKITAYRERYDKLESSIKLDAIKQLTSEQKIEVDNYNNNFTPQ


 


NTKQIVCSKLNINPNDLPWDKMISGTHFISEKAQVSNKSEIYETSTDKGKTKDVMKSDYKWFQD


 


YKPKLSKEVRDALSDIEWRLRRESLEFNKLSKSREQDARQLANWISSMCDVIGIENLVKKNNFE


 


GGSGKREPGWDNFYKPKKENRWWINAIHKALTELSQNKGKRVILLPAMRTSITCPKCKYCDSKN


 


RNGEKFNCLKCGIELNADIDVATENLATVAITAQSMPKPTCERSGDAKKPVRARKAKAPEFHDK


 


LAPSYTVVLREAV*


 


>CasΦ-9


MRSSREIGDKILMRQPAEKTAFQVFRQEVIGTQKLSGGDAKTAGRLYKQGKMEAAREWLLKGAR


 


DDVPPNFQPPAKCLVVAVSHPFEEWDISKTNHDVQAYIYAQPLQAEGHLNGLSEKWEDTSADQH


 


KLWFEKTGVPDRGLPVQAINKIAKAAVNRAFGVVRKVENRNEKRRSRDNRIAEHNRENGLTEVV


 


REAPEVATNADGFLLHPPGIDPSILSYASVSPVPYNSSKHSFVRLPEEYQAYNVEPDAPIPQEV


 


VEDRFAIPPGQPGYVPEWQRLKCSTNKHRRMRQWSNQDYKPKAGRRAKPLEFQAHLTRERAKGA


 


LLVVMRIKEDWVVFDVRGLLRNVEWRKVLSEEAREKLTLKGLLDLETGDPVIDTKRGIVTFLYK


 


AEITKILSKRTVKTKNARDLLLRLTEPGEDGLRREVGLVAVDLGQTHPIAAAIYRIGRTSAGAL


 


ESTVLHRQGLREDQKEKLKEYRKRHTALDSRLRKEAFETLSVEQQKEIVTVSGSGAQITKDKVC


 


NYLGVDPSTLPWEKMGSYTHFISDDFLRRGGDPNIVHFDRQPKKGKVSKKSQRIKRSDSQWVGR


 


MRPRLSQETAKARMEADWAAQNENEEYKRLARSKQELARWCVNTLLQNTRCITQCDEIVVVIED


 


LNVKSLHGKGAREPGWDNFFTPKTENRWFIQILHKTFSELPKHRGEHVIEGCPLRTSITCPACS


 


YCDKNSRNGEKFVCVACGATFHADFEVATYNLVRLATTGMPMPKSLERQGGGEKAGGARKARKK


 


AKQVEKIVVQANANVTMNGASLHSP*


 


>CasΦ-10


MDMLDTETNYATETPAQQQDYSPKPPKKAQRAPKGFSKKARPEKKPPKPITLFTQKHFSGVRFL


 


KRVIRDASKILKLSESRTITFLEQAIERDGSAPPDVTPPVHNTIMAVTRPFEEWPEVILSKALQ


 


KHCYALTKKIKIKTWPKKGPGKKCLAAWSARTKIPLIPGQVQATNGLFDRIGSIYDGVEKKVTN


 


RNANKKLEYDEATKEGRNPAVPEYETAYNIDGTLINKPGYNPNLYITQSRTPRLITEADRPLVE


 


KILWQMVEKKTQSRNQARRARLEKAAHLQGLPVPKFVPEKVDRSQKIEIRTIDPLDKIEPYMPQ


 


DRMAIKASQDGHVPYWQRPFLSKRRNRRVRAGWGKQVSSIQAWLTGALLVIVRLGNEAFLADIR


 


GALRNAQWRKLLKPDATYQSLFNLFTGDPVVNTRTNHLTMAYREGVVNIVKSRSFKGRQTREHL


 


LTLLGQGKTVAGVSFDLGQKHAAGLLAAHFGLGEDGNPVFTPIQACELPQRYLDSLTNYRNRYD


 


ALTLDMRRQSLLALTPAQQQEFADAQRDPGGQAKRACCLKLNLNPDEIRWDLVSGISTMISDLY


 


IERGGDPRDVHQQVETKPKGKRKSEIRILKIRDGKWAYDFRPKIADETRKAQREQLWKLQKASS


 


EFERLSRYKINIARAIANWALQWGRELSGCDIVIPVLEDLNVGSKFFDGKGKWLLGWDNRFTPK


 


KENRWFIKVLHKAVAELAPHRGVPVYEVMPHRTSMTCPACHYCHPTNREGDRFECQSCHVVKNT


 


DRDVAPYNILRVAVEGKTLDRWQAEKKPQAEPDRPMILIDNQES*


The asterisk (*) in the sequences above denotes a STOP codon.


Alternatively, CasΦ-1 is also termed Casl2j ortholog 1. Tims,


CasΦ-1- CasΦ-10 may also be referred to as Casl2j


orthologs 1-10, respectively.






Guide Polynucleotides

In an embodiment, the guide polynucleotide is a guide RNA. As used herein, the term “guide RNA (gRNA)” and its grammatical equivalents can refer to an RNA which can be specific for a target DNA and can form a complex with Cas protein. An RNA/Cas complex can assist in “guiding” Cas protein to a target DNA. Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA,” or simply “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M. et al., Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti, J. J. et al., Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E. et al., Nature 471:602-607(2011); and “Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M. et al, Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences can be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase.
In some embodiments, the guide polynucleotide is at least one single guide RNA (“sgRNA” or “gRNA”). In some embodiments, the guide polynucleotide is at least one tracrRNA. In some embodiments, the guide polynucleotide does not require PAM sequence to guide the polynucleotide-programmable DNA-binding domain (e.g., Cas9 or Cpf1) to the target nucleotide sequence.
The polynucleotide programmable nucleotide binding domain (e.g., a CRISPR-derived domain) of the base editors disclosed herein can recognize a target polynucleotide sequence by associating with a guide polynucleotide. A guide polynucleotide (e.g., gRNA) is typically single-stranded and can be programmed to site-specifically bind (i.e., via complementary base pairing) to a target sequence of a polynucleotide, thereby directing a base editor that is in conjunction with the guide nucleic acid to the target sequence. A guide polynucleotide can be DNA. A guide polynucleotide can be RNA. As will be appreciated by one having skill in the art, in a guide polynucleotide sequence uracil (U) replaces thymine (T) in the sequence. In some embodiments, the guide polynucleotide comprises natural nucleotides (e.g., adenosine). In some embodiments, the guide polynucleotide comprises non-natural (or unnatural) nucleotides (e.g., peptide nucleic acid or nucleotide analogs). In some embodiments, the targeting region of a guide nucleic acid sequence can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. A targeting region of a guide nucleic acid can be between 10-30 nucleotides in length, or between 15-25 nucleotides in length, or between 15-20 nucleotides in length. In some embodiments, a guide polynucleotide may be truncated by 1, 2, 3, 4, etc. nucleotides, particularly at the 5′ end. By way of nonlimiting example, a guide polynucleotide of 20 nucleotides in length may be truncated by 1, 2, 3, 4, etc. nucleotides, particularly at the 5′ end.
In some embodiments, a guide polynucleotide comprises two or more individual polynucleotides, which can interact with one another via for example complementary base pairing (e.g., a dual guide polynucleotide). For example, a guide polynucleotide can comprise a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). For example, a guide polynucleotide can comprise one or more trans-activating CRISPR RNA (tracrRNA).
In type II CRISPR systems, targeting of a nucleic acid by a CRISPR protein (e.g., Cas9) typically requires complementary base pairing between a first RNA molecule (crRNA) comprising a sequence that recognizes the target sequence and a second RNA molecule (trRNA) comprising repeat sequences which forms a scaffold region that stabilizes the guide RNA-CRISPR protein complex. Such dual guide RNA systems can be employed as a guide polynucleotide to direct the base editors disclosed herein to a target polynucleotide sequence.
In some embodiments, the base editor provided herein utilizes a single guide polynucleotide (e.g., sgRNA). In some embodiments, the base editor provided herein utilizes a dual guide polynucleotide (e.g., dual gRNAs). In some embodiments, the base editor provided herein utilizes one or more guide polynucleotide (e.g., multiple gRNA). In some embodiments, a single guide polynucleotide is utilized for different base editors described herein. For example, a single guide polynucleotide can be utilized for a cytidine base editor and an adenosine base editor.
In other embodiments, a guide polynucleotide can comprise both the polynucleotide targeting portion of the nucleic acid and the scaffold portion of the nucleic acid in a single molecule (i.e., a single-molecule guide nucleic acid). For example, a single-molecule guide polynucleotide can be a single guide RNA (sgRNA or gRNA). Herein the term guide polynucleotide sequence contemplates any single, dual or multi-molecule nucleic acid capable of interacting with and directing a base editor to a target polynucleotide sequence.
Typically, a guide polynucleotide (e.g., crRNA/trRNA complex or a gRNA) comprises a “polynucleotide-targeting segment” that includes a sequence capable of recognizing and binding to a target polynucleotide sequence, and a “protein-binding segment” that stabilizes the guide polynucleotide within a polynucleotide programmable nucleotide binding domain component of a base editor. In some embodiments, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to a DNA polynucleotide, thereby facilitating the editing of a base in DNA. In other embodiments, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to an RNA polynucleotide, thereby facilitating the editing of a base in RNA. Herein a “segment” refers to a section or region of a molecule, e.g., a contiguous stretch of nucleotides in the guide polynucleotide. A segment can also refer to a region/section of a complex such that a segment can comprise regions of more than one molecule. For example, where a guide polynucleotide comprises multiple nucleic acid molecules, the protein-binding segment of can include all or a portion of multiple separate molecules that are for instance hybridized along a region of complementarity. In some embodiments, a protein-binding segment of a DNA-targeting RNA that comprises two separate molecules can comprise (i) base pairs 40-75 of a first RNA molecule that is 100 base pairs in length; and (ii) base pairs 10-25 of a second RNA molecule that is 50 base pairs in length. The definition of “segment,” unless otherwise specifically defined in a particular context, is not limited to a specific number of total base pairs, is not limited to any particular number of base pairs from a given RNA molecule, is not limited to a particular number of separate molecules within a complex, and can include regions of RNA molecules that are of any total length and can include regions with complementarity to other molecules.
A guide RNA or a guide polynucleotide can comprise two or more RNAs, e.g., CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA). A guide RNA or a guide polynucleotide can sometimes comprise a single-chain RNA, or single guide RNA (sgRNA) formed by fusion of a portion (e.g., a functional portion) of crRNA and tracrRNA. A guide RNA or a guide polynucleotide can also be a dual RNA comprising a crRNA and a tracrRNA. Furthermore, a crRNA can hybridize with a target DNA.
As discussed above, a guide RNA or a guide polynucleotide can be an expression product. For example, a DNA that encodes a guide RNA can be a vector comprising a sequence coding for the guide RNA. A guide RNA or a guide polynucleotide can be transferred into a cell by transfecting the cell with an isolated guide RNA or plasmid DNA comprising a sequence coding for the guide RNA and a promoter. A guide RNA or a guide polynucleotide can also be transferred into a cell in other way, such as using virus-mediated gene delivery.
A guide RNA or a guide polynucleotide can be isolated. For example, a guide RNA can be transfected in the form of an isolated RNA into a cell or organism. A guide RNA can be prepared by in vitro transcription using any in vitro transcription system known in the art. A guide RNA can be transferred to a cell in the form of isolated RNA rather than in the form of plasmid comprising encoding sequence for a guide RNA.
A guide RNA or a guide polynucleotide can comprise three regions: a first region at the 5′ end that can be complementary to a target site in a chromosomal sequence, a second internal region that can form a stem loop structure, and a third 3′ region that can be single-stranded. A first region of each guide RNA can also be different such that each guide RNA guides a fusion protein to a specific target site. Further, second and third regions of each guide RNA can be identical in all guide RNAs.
A first region of a guide RNA or a guide polynucleotide can be complementary to sequence at a target site in a chromosomal sequence such that the first region of the guide RNA can base pair with the target site. In some embodiments, a first region of a guide RNA can comprise from or from about 10 nucleotides to 25 nucleotides (i.e., from 10 nucleotides to nucleotides; or from about 10 nucleotides to about 25 nucleotides; or from 10 nucleotides to about 25 nucleotides; or from about 10 nucleotides to 25 nucleotides) or more. For example, a region of base pairing between a first region of a guide RNA and a target site in a chromosomal sequence can be or can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more nucleotides in length. In some embodiments, a first region of a guide RNA can be or can be about 19, 20, or 21 nucleotides in length.
A guide RNA or a guide polynucleotide can also comprise a second region that forms a secondary structure. For example, a secondary structure formed by a guide RNA can comprise a stem (or hairpin) and a loop. A length of a loop and a stem can vary. For example, a loop can range from or from about 3 to 10 nucleotides in length, and a stem can range from or from about 6 to 20 base pairs in length. A stem can comprise one or more bulges of 1 to 10 or about 10 nucleotides. The overall length of a second region can range from or from about 16 to 60 nucleotides in length. For example, a loop can be or can be about 4 nucleotides in length and a stem can be or can be about 12 base pairs.
A guide RNA or a guide polynucleotide can also comprise a third region at the 3′ end that can be essentially single-stranded. For example, a third region is sometimes not complementarity to any chromosomal sequence in a cell of interest and is sometimes not complementarity to the rest of a guide RNA. Further, the length of a third region can vary. A third region can be more than or more than about 4 nucleotides in length. For example, the length of a third region can range from or from about 5 to 60 nucleotides in length.
A guide RNA or a guide polynucleotide can target any exon or intron of a gene target. In some embodiments, a guide can target exon 1 or 2 of a gene; in other embodiments, a guide can target exon 3 or 4 of a gene. A composition can comprise multiple guide RNAs that all target the same exon or in some embodiments, multiple guide RNAs that can target different exons. An exon and an intron of a gene can be targeted.
A guide RNA or a guide polynucleotide can target a nucleic acid sequence of or of about 20 nucleotides. A target nucleic acid can be less than or less than about 20 nucleotides. A target nucleic acid can be at least or at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or anywhere between 1-100 nucleotides in length. A target nucleic acid can be at most or at most about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or anywhere between 1-100 nucleotides in length. A target nucleic acid sequence can be or can be about 20 bases immediately 5′ of the first nucleotide of the PAM. A guide RNA can target a nucleic acid sequence. A target nucleic acid can be at least or at least about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, or 1-100 nucleotides.
A guide polynucleotide, for example, a guide RNA, can refer to a nucleic acid that can hybridize to another nucleic acid, for example, the target nucleic acid or protospacer in a genome of a cell. A guide polynucleotide can be RNA. A guide polynucleotide can be DNA. The guide polynucleotide can be programmed or designed to bind to a sequence of nucleic acid site-specifically. A guide polynucleotide can comprise a polynucleotide chain and can be called a single guide polynucleotide. A guide polynucleotide can comprise two polynucleotide chains and can be called a double guide polynucleotide. A guide RNA can be introduced into a cell or embryo as an RNA molecule. For example, an RNA molecule can be transcribed in vitro and/or can be chemically synthesized. An RNA can be transcribed from a synthetic DNA molecule, e.g., a gBlocks® gene fragment. A guide RNA can then be introduced into a cell or embryo as an RNA molecule. A guide RNA can also be introduced into a cell or embryo in the form of a non-RNA nucleic acid molecule, e.g., a DNA molecule. For example, a DNA encoding a guide RNA can be operably linked to promoter control sequence for expression of the guide RNA in a cell or embryo of interest. An RNA coding sequence can be operably linked to a promoter sequence that is recognized by RNA polymerase III (Pol III). Plasmid vectors that can be used to express guide RNA include, but are not limited to, px330 vectors and px333 vectors. In some embodiments, a plasmid vector (e.g., px333 vector) can comprise at least two guide RNA-encoding DNA sequences.
Methods for selecting, designing, and validating guide polynucleotides, e.g., guide RNAs and targeting sequences are described herein and known to those skilled in the art. For example, to minimize the impact of potential substrate promiscuity of a deaminase domain in the nucleobase editor system (e.g., an AID domain), the number of residues that could unintentionally be targeted for deamination (e.g., off-target C residues that could potentially reside on ssDNA within the target nucleic acid locus) may be minimized. In addition, software tools can be used to optimize the gRNAs corresponding to a target nucleic acid sequence, e.g., to minimize total off-target activity across the genome. For example, for each possible targeting domain choice using S. pyogenes Cas9, all off-target sequences (preceding selected PAMs, e.g., NAG or NGG) may be identified across the genome that contain up to certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base-pairs. First regions of gRNAs complementary to a target site can be identified, and all first regions (e.g., crRNAs) can be ranked according to its total predicted off-target score; the top-ranked targeting domains represent those that are likely to have the greatest on-target and the least off-target activity. Candidate targeting gRNAs can be functionally evaluated by using methods known in the art and/or as set forth herein.
As a non-limiting example, target DNA hybridizing sequences in crRNAs of a guide RNA for use with Cas9s may be identified using a DNA sequence searching algorithm. gRNA design may be carried out using custom gRNA design software based on the public tool cas-offinder as described in Bae S., Park J., & Kim J.-S. Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473-1475 (2014). This software scores guides after calculating their genome-wide off-target propensity. Typically matches ranging from perfect matches to 7 mismatches are considered for guides ranging in length from 17 to 24. Once the off-target sites are computationally-determined, an aggregate score is calculated for each guide and summarized in a tabular output using a web-interface. In addition to identifying potential target sites adjacent to PAM sequences, the software also identifies all PAM adjacent sequences that differ by 1, 2, 3 or more than 3 nucleotides from the selected target sites. Genomic DNA sequences for a target nucleic acid sequence, e.g., a target gene may be obtained and repeat elements may be screened using publicly available tools, for example, the RepeatMasker program. RepeatMasker searches input DNA sequences for repeated elements and regions of low complexity. The output is a detailed annotation of the repeats present in a given query sequence.
Following identification, first regions of guide RNAs, e.g., crRNAs, may be ranked into tiers based on their distance to the target site, their orthogonality and presence of 5′ nucleotides for close matches with relevant PAM sequences (for example, a 5′ G based on identification of close matches in the human genome containing a relevant PAM e.g., NGG PAM for S. pyogenes, NNGRRT or NNGRRV PAM for S. aureus). As used herein, orthogonality refers to the number of sequences in the human genome that contain a minimum number of mismatches to the target sequence. A “high level of orthogonality” or “good orthogonality” may, for example, refer to 20-mer targeting domains that have no identical sequences in the human genome besides the intended target, nor any sequences that contain one or two mismatches in the target sequence. Targeting domains with good orthogonality may be selected to minimize off-target DNA cleavage.
In some embodiments, a reporter system may be used for detecting base-editing activity and testing candidate guide polynucleotides. In some embodiments, a reporter system may comprise a reporter gene based assay where base editing activity leads to expression of the reporter gene. For example, a reporter system may include a reporter gene comprising a deactivated start codon, e.g., a mutation on the template strand from 3′-TAC-5′ to 3′-CAC-5′. Upon successful deamination of the target C, the corresponding mRNA will be transcribed as 5′-AUG-3′ instead of 5′-GUG-3′, enabling the translation of the reporter gene. Suitable reporter genes will be apparent to those of skill in the art. Non-limiting examples of reporter genes include gene encoding green fluorescence protein (GFP), red fluorescence protein (RFP), luciferase, secreted alkaline phosphatase (SEAP), or any other gene whose expression are detectable and apparent to those skilled in the art. The reporter system can be used to test many different gRNAs, e.g., in order to determine which nucleotide residue(s) with respect to the target DNA sequence the respective deaminase will target. sgRNAs that target non-template strand nucleotide residues can also be tested in order to assess off-target effects of a specific base editing protein, e.g., a Cas9 deaminase fusion protein. In some embodiments, such gRNAs can be designed such that the mutated start codon will not be base-paired with the gRNA. The guide polynucleotides can comprise standard nucleotides, modified nucleotides (e.g., pseudouridine), nucleotide isomers, and/or nucleotide analogs. In some embodiments, the guide polynucleotide can comprise at least one detectable label. The detectable label can be a fluorophore (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluors, Halo tags, or any other suitable fluorescent dye), a detection tag (e.g., biotin, digoxigenin, and the like), quantum dots, or gold particles.
The guide polynucleotides can be synthesized chemically and/or enzymatically. For example, the guide RNA can be synthesized using standard phosphoramidite-based solid-phase synthesis methods. Alternatively, the guide RNA can be synthesized in vitro by operably linking DNA encoding the guide RNA to a promoter control sequence that is recognized by a phage RNA polymerase. Examples of suitable phage promoter sequences include T7, T3, SP6 promoter sequences, or variations thereof. In embodiments in which the guide RNA comprises two separate molecules (e.g., crRNA and tracrRNA), the crRNA can be chemically synthesized and the tracrRNA can be enzymatically synthesized.
In some embodiments, a base editor system may comprise multiple guide polynucleotides, e.g., gRNAs. For example, the gRNAs may target the base editor to one or more target loci (e.g., at least one (1) gRNA, at least 2 gRNA, at least 5 gRNA, at least 10 gRNA, at least 20 gRNA, at least 30 g RNA, or at least 50 gRNA). In some embodiments, multiple gRNA sequences can be tandemly arranged and are preferably separated by a direct repeat.
A DNA sequence encoding a guide RNA or a guide polynucleotide can also be part of a vector. In some embodiments, a vector comprises additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences, etc.), selectable marker sequences (e.g., GFP or antibiotic resistance genes such as puromycin), origins of replication, and the like. A DNA molecule encoding a guide RNA or a guide polynucleotide can be circular or linear.
In some embodiments, one or more components of a base editor system may be encoded by DNA sequences. Such DNA sequences may be introduced into an expression system, e.g., a cell, together or separately. For example, DNA sequences encoding a polynucleotide programmable nucleotide binding domain and a guide RNA may be introduced into a cell, each DNA sequence can be part of a separate molecule (e.g., one vector containing the polynucleotide programmable nucleotide binding domain coding sequence and a second vector containing the guide RNA coding sequence) or both can be part of a same molecule (e.g., one vector containing coding (and regulatory) sequence for both the polynucleotide programmable nucleotide binding domain and the guide RNA).
A guide polynucleotide can comprise one or more modifications to provide a nucleic acid with a new or enhanced feature. A guide polynucleotide can comprise a nucleic acid affinity tag. A guide polynucleotide can comprise synthetic nucleotide, synthetic nucleotide analog, nucleotide derivatives, and/or modified nucleotides.
In some embodiments, a gRNA or a guide polynucleotide can comprise modifications. A modification can be made at any location of a gRNA or a guide polynucleotide. More than one modification can be made to a single gRNA or a guide polynucleotide. A gRNA or a guide polynucleotide can undergo quality control after a modification. In some embodiments, quality control can include PAGE, HPLC, MS, or any combination thereof.
A modification of a gRNA or a guide polynucleotide can be a substitution, insertion, deletion, chemical modification, physical modification, stabilization, purification, or any combination thereof.
A gRNA or a guide polynucleotide can also be modified by 5′adenylate, 5′ guanosine-triphosphate cap, 5′N7-Methylguanosine-triphosphate cap, 5′triphosphate cap, 3′phosphate, 3′thiophosphate, 5′phosphate, 5′thiophosphate, Cis-Syn thymidine dimer, trimers, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9,3′-3′ modifications, 5′-5′ modifications, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, dual biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3′DABCYL, black hole quencher 1, black hole quencer 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linkers, 2′-deoxyribonucleoside analog purine, 2′-deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2′-O-methyl ribonucleoside analog, sugar modified analogs, wobble/universal bases, fluorescent dye label, 2′-fluoro RNA, 2′-O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5′-triphosphate, 5′-methylcytidine-5′-triphosphate, or any combination thereof.
In some embodiments, a modification is permanent. In other embodiments, a modification is transient. In some embodiments, multiple modifications are made to a gRNA or a guide polynucleotide. A gRNA or a guide polynucleotide modification can alter physiochemical properties of a nucleotide, such as their conformation, polarity, hydrophobicity, chemical reactivity, base-pairing interactions, or any combination thereof.
A modification can also be a phosphorothioate substitute. In some embodiments, a natural phosphodiester bond can be susceptible to rapid degradation by cellular nucleases and; a modification of internucleotide linkage using phosphorothioate (PS) bond substitutes can be more stable towards hydrolysis by cellular degradation. A modification can increase stability in a gRNA or a guide polynucleotide. A modification can also enhance biological activity. In some embodiments, a phosphorothioate enhanced RNA gRNA can inhibit RNase A, RNase T1, calf serum nucleases, or any combinations thereof. These properties can allow the use of PS-RNA gRNAs to be used in applications where exposure to nucleases is of high probability in vivo or in vitro. For example, phosphorothioate (PS) bonds can be introduced between the last 3-5 nucleotides at the 5′- or ″-end of a gRNA which can inhibit exonuclease degradation. In some embodiments, phosphorothioate bonds can be added throughout an entire gRNA to reduce attack by endonucleases.
In some embodiments, the guide RNA is designed to disrupt a splice site (i.e., a splice acceptor (SA) or a splice donor (SD). In some embodiments, the guide RNA is designed such that the base editing results in a premature STOP codon. Tables 4A-4B provide a non-exhaustive list of gRNA target sequences designed to disrupt a splice site or to result in a premature STOP codon.







TABLE 4A







gRNAs: Splice Site and STOP Codons












Gene
Description
Targeting sequence
SEQ ID NO:
gRNA Spacer Sequence
SEQ ID NO:















VISTA
Exon 1 SD (pos6)
CCTTACCTAGGGACGCAGCC
295
CCUUACCUAGGGACGCAGCC
803



Exon 1 STOP (pos7)
GGATCCCCAGCGCCAGCTGC
296
GGAUCCCCAGCGCCAGCUGC
804



Exon 1 STOP (pos5)
AGCGCCAGCTGCCGGCCTCC
297
AGCGCCAGCUGCCGGCCUCC
805



Exon 1 STOP (pos4)
GCGCCAGCTGCCGGCCTCCA
298
GCGCCAGCUGCCGGCCUCCA
806



Exon 2 STOP (pos8)
CCTGGCTCAGCGCCACGGGC
299
CCUGGCUCAGCGCCACGGGC
807



Exon 2 STOP (pos5)
GCTGCAGGTGCAGACAGGTG
300
GCUGCAGGUGCAGACAGGUG
808



Exon 2 STOP (pos7)
GCGGTACCACGTCTTGTAGA
301
GCGGUACCACGUCUUGUAGA
809



Exon 3 SA (pos4)
TGCCTGTGGGAACAAACAGA
302
UGCCUGUGGGAACAAACAGA
810



Exon 3 SD (pos5)
CTTACTTTCACTATCCTGGG
303
CUUACUUUCACUAUCCUGGG
811



Exon 3 SD (pos8)
TCCCTTACTTTCACTATCCT
304
UCCCUUACUUUCACUAUCCU
812



Exon 3 STOP (pos5)
CTCCCAGGATAGTGAAAGTA
305
CUCCCAGGAUAGUGAAAGUA
813



Exon 4 SA (pos7)
TGATGTCTGAAAGGGCAGAG
306
UGAUGUCUGAAAGGGCAGAG
814



Exon 5 STOP (pos5)
TGCCCAGGAGCTGGTGCGGA
307
UGCCCAGGAGCUGGUGCGGA
815



Exon 6 SA (pos4)
TTGCTGCCACAGAACCAGAA
308
UUGCUGCCACAGAACCAGAA
816



Exon 6 STOP (pos4)
ATTCAAGGGATTGAAAACCC
309
AUUCAAGGGAUUGAAAACCC
817



Exon 6 STOP (pos8)
ACCTGCCCAGGGGATACCCG
310
ACCUGCCCAGGGGAUACCCG
818



Exon 6 STOP (pos7)
CAGCGGCAGCCTTCTGAGTC
311
CAGCGGCAGCCUUCUGAGUC
819


 


TRAC
Exon 1 STOP 1 (pos5)
GCTACAAACAAGCTCATCTT
312
GCUACAAACAAGCUCAUCUU
820



Exon 1 STOP 2 (pos6)
CCAGCCAAGTACGTAAGTAG
313
CCAGCCAAGUACGUAAGUAG
821



Exon 2 SA (pos9)
CTGGATATCTGTGGGACAAG
314
CUGGAUAUCUGUGGGACAAG
822



Exon 2 SD
CTTACCTGGGCTGGGGAAGA
315
CUUACCUGGGCUGGGGAAGA
823



Exon 4 SA
TTCGTATCTGTAAAACCAAG
316
UUCGUAUCUGUAAAACCAAG
824



Exon 4 STOP
TTTCAAAACCTGTCAGTGAT
317
UUUCAAAACCUGUCAGUGAU
825



Exon 4 STOP
TTCAAAACCTGTCAGTGATT
318
UUCAAAACCUGUCAGUGAUU
826


 


Tim-3
Exon 2 SA (pos6)
GGACCCTGCATAGAGAGAGA
319
GGACCCUGCAUAGAGAGAGA
827



Exon 2 STOP (pos5)
TGCCCCAGCAGACGGGCACG
320
UGCCCCAGCAGACGGGCACG
828



Exon 3 SD (pos5)
GTTACCTGGGCCATGTCCCC
321
GUUACCUGGGCCAUGUCCCC
829



Exon 4 SD (pos5)
CTTACTGTTAGATTTATATC
322
CUUACUGUUAGAUUUAUAUC
830



Exon 4 SD (pos4)
TTACTGTTAGATTTATATCA
323
UUACUGUUAGAUUUAUAUCA
831



Exon 5 SA (pos5)
TTTGCTATGGAAACACAAAC
324
UUUGCUAUGGAAACACAAAC
832



Exon 5 STOP (pos8)
TCCATAGCAAATATCCACAT
325
UCCAUAGCAAAUAUCCACAU
833



Exon 7 STOP (pos5)
GCAGCAACCCTCACAACCTT
326
GCAGCAACCCUCACAACCUU
834



Exon 7 STOP (pos 4)
CAGCAACCCTCACAACCTTT
327
CAGCAACCCUCACAACCUUU
835


 


TIGIT
Exon 1 STOP (pos4)
AGGCAGGCTCCCCTCGCCTC
328
AGGCAGGCUCCCCUCGCCUC
836



Exon 2 STOP (5&8)
GGAGCAGCAGGACCAGCTTC
329
GGAGCAGCAGGACCAGCUUC
837



Exon 2 SD (pos9)
CAGGAATACCTGAGCTTTCT
330
CAGGAAUACCUGAGCUUUCU
838



Exon 3 STOP (pos7)
AGGTTCCAGATTCCATTGCT
331
AGGUUCCAGAUUCCAUUGCU
839



Exon 1 STOP
CTGGGCCCAGGGGCTGAGGC
332
CUGGGCCCAGGGGCUGAGGC
840



Exon 2 STOP
GATCGAGTGGCCCCAGGTCC
333
GAUCGAGUGGCCCCAGGUCC
841


 


TGFbRII
Exon 1 SD (JMG79)
TCACCCGACTTCTGAACGTG
334
UCACCCGACUUCUGAACGUG
842



Exon 3 SD (JMG83)
TTACCTGCCCACTGTTAGCC
335
UUACCUGCCCACUGUUAGCC
843



Exon 2 STOP (JMG80)
GAAGCCACAGGAAGTCTGTG
336
GAAGCCACAGGAAGUCUGUG
844



Exon 3 STOP (JMG81)
ACTCCAGTTCCTGACGGCTG
337
ACUCCAGUUCCUGACGGCUG
845



Exon 3 STOP (JMG82)
ACCTACAGGAGTACCTGACG
338
ACCUACAGGAGUACCUGACG
846



Exon 4 STOP (JMG84)
TTCCCAGAGCACCAGAGCCA
339
UUCCCAGAGCACCAGAGCCA
847



Exon 1 STOP (JMG85)
ACGTTCAGAAGTCGGGTGAG
340
ACGUUCAGAAGUCGGGUGAG
848



Exon 3 STOP (pos8)
TTCAGAGCAGTTTGAGACAG
341
UUCAGAGCAGUUUGAGACAG
849


RFXANK
Exon 2 SA (JMG8)
CCTGCTGGGAAACAGACAAC
342
CCUGCUGGGAAACAGACAAC
850


 



Exon 2 SD (JMG9)
CACTCACAGTCTAGGGTGGC
343
CACUCACAGUCUAGGGUGGC
851



Exon 2 STOP (pos8)
CAACCGGCAGCGAGGGAACG
344
CAACCGGCAGCGAGGGAACG
1504



Exon 3 SA (pos7)
ACAGGGCTGGGGCAGGACAG
345
ACAGGGCUGGGGCAGGACAG
852



Exon 3 STOP (pos8)
CATCCACCAGCTCGCAGCAC
346
CAUCCACCAGCUCGCAGCAC
853



Exon 3 STOP (pos7)
ATCCACCAGCTCGCAGCACA
347
AUCCACCAGCUCGCAGCACA
854



Exon 3 STOP (pos6)
TCCACCAGCTCGCAGCACAG
348
UCCACCAGCUCGCAGCACAG
855



Exon 3 STOP (pos5)
CCACCAGCTCGCAGCACAGG
349
CCACCAGCUCGCAGCACAGG
856



Exon 4 SA (JMG10)
TGTCACCTGGCAGGAGGAGG
350
UGUCACCUGGCAGGAGGAGG
857



Exon 4 SA (pos6)
GTCACCTGGCAGGAGGAGGC
351
GUCACCUGGCAGGAGGAGGC
858



Exon 5 SA (pos7)
GGCACCCTGCAGGGAGAAGA
352
GGCACCCUGCAGGGAGAAGA
859



Exon 5 SA (JMG11)
GCACCCTGCAGGGAGAAGAA
353
GCACCCUGCAGGGAGAAGAA
860



Exon 6 SA (pos4)
ATTCTGTCGTGGGTAGGGGC
354
AUUCUGUCGUGGGUAGGGGC
861



Exon 6 SA (JMG12)
CTCCATTCTGTCGTGGGTAG
355
CUCCAUUCUGUCGUGGGUAG
862



Exon 7 SA (pos8)
CCTCGGGCTGCAAAGGAGAG
356
CCUCGGGCUGCAAAGGAGAG
863



Exon 7 SA (pos5)
CGGGCTGCAAAGGAGAGGGG
357
CGGGCUGCAAAGGAGAGGGG
864



Exon 7 SD (pos6)
GCTGACCTTTCCGGTATCCC
358
GCUGACCUUUCCGGUAUCCC
865



Exon 7 SD (pos5)
CTGACCTTTCCGGTATCCCA
359
CUGACCUUUCCGGUAUCCCA
866



Exon 8 SA (pos8)
TGTTGCACTGAGATGGGGCA
360
UGUUGCACUGAGAUGGGGCA
867



Exon 8 SA (pos9)
CTGTTGCACTGAGATGGGGC
361
CUGUUGCACUGAGAUGGGGC
868


 


PVRIG
Exon 1 STOP (pos7)
GCCCTGCAGCCCCCAGAACC
362
GCCCUGCAGCCCCCAGAACC
869


(CD112R)
Exon 1 SD (pos5)
CTCACCCGCAGTGACACACA
363
CUCACCCGCAGUGACACACA
870



Exon 1 STOP (pos8)
GCAGCACCCAGGGCAGGACC
364
GCAGCACCCAGGGCAGGACC
1505



Exon 1 STOP (pos7)
CAGCACCCAGGGCAGGACCA
365
CAGCACCCAGGGCAGGACCA
1506



Exon 2 SA (pos5)
GTCCCTGTGGAACAGCAGCA
366
GUCCCUGUGGAACAGCAGCA
871



Exon 2 STOP (pos8)
GTGGGTTCAAGTTCGGATGG
367
GUGGGUUCAAGUUCGGAUGG
872



Exon 2 SD (pos 7)
GCCCCACCTGGGTCTGAGCT
368
GCCCCACCUGGGUCUGAGCU
873



Exon 2 SD (pos8)
GGCCCCACCTGGGTCTGAGC
369
GGCCCCACCUGGGUCUGAGC
874



Exon 2 SD (pos4)
CCACCTGGGTCTGAGCTGGG
370
CCACCUGGGUCUGAGCUGGG
875



Exon 2 STOP (pos8)
AGGCCTCCCAGGAGCCCTCA
371
AGGCCUCCCAGGAGCCCUCA
876



Exon 2 STOP (pos4)
CTCCCAGGAGCCCTCAGGGA
372
CUCCCAGGAGCCCUCAGGGA
877



Exon 2 STOP (pos4)
CCCCCAGCTCACAGTCACCA
373
CCCCCAGCUCACAGUCACCA
878



Exon 3 SD (pos8)
GGTCTCACCGGTGCTTATGT
374
GGUCUCACCGGUGCUUAUGU
879



Exon 3 STOP (pos9)
TGCTGCGCCGACATAAGCAC
375
UGCUGCGCCGACAUAAGCAC
880



Exon 4 SA (pos8)
GGCAGGGCTGGGAGAGAGCA
376
GGCAGGGCUGGGAGAGAGCA
881



Exon 4 STOP (pos9)
CGAGAGCACGAGCATGGGTG
377
CGAGAGCACGAGCAUGGGUG
882



Exon 4 STOP (pos6)
GAGCACGAGCATGGGTGAGG
378
GAGCACGAGCAUGGGUGAGG
883



Exon 4 STOP (pos5)
AGCACGAGCATGGGTGAGGA
379
AGCACGAGCAUGGGUGAGGA
884



Exon 4 STOP (pos4)
GCACGAGCATGGGTGAGGAG
380
GCACGAGCAUGGGUGAGGAG
885



Exon 4 SD (pos5)
CTCACCCATGCTCGTGCTCT
381
CUCACCCAUGCUCGUGCUCU
886



Exon 5 SA (pos6)
GGTGCCTGCGCGGGGGAAGG
382
GGUGCCUGCGCGGGGGAAGG
887



Exon 5 SA (pos5)
GTGCCTGCGCGGGGGAAGGA
383
GUGCCUGCGCGGGGGAAGGA
888



Exon 5 SA (pos9)
CTTGGTGCCTGCGCGGGGGA
384
CUUGGUGCCUGCGCGGGGGA
889



Exon 5 STOP (pos6)
GGCCCCAGGGCCCTGCCGCC
385
GGCCCCAGGGCCCUGCCGCC
890



Exon 5 STOP (pos9)
TCTACGCTCAGGCAGGGGAG
386
UCUACGCUCAGGCAGGGGAG
891



Exon 5 STOP (pos4)
CCACCAGGACGGCCCCCCAT
387
CCACCAGGACGGCCCCCCAU
892



Exon 5 STOP (pos5)
AGGCCCAGGCGGCAGGGCCC
388
AGGCCCAGGCGGCAGGGCCC
1507



Exon 5 STOP (pos4)
GGCCCAGGCGGCAGGGCCCT
389
GGCCCAGGCGGCAGGGCCCU
893


 


PDCD1
Exon 1 STOP 2 (pos9)
ACGACTGGCCAGGGCGCCTG
390
ACGACUGGCCAGGGCGCCUG
894



Exon 1 STOP 4 (pos7)
CACCGCCCAGACGACTGGCC
391
CACCGCCCAGACGACUGGCC
895



Exon 1 STOP (pos4)
CTACAACTGGGCTGGCGGCC
392
CUACAACUGGGCUGGCGGCC
896



Exon 1 SD
CACCTACCTAAGAACCATCC
202
CACCUACCUAAGAACCAUCC
897



Exon 2 SA
GGAGTCTGAGAGATGGAGAG
393
GGAGUCUGAGAGAUGGAGAG
898



Exon 2 STOP 1 (pos8)
CAGCAACCAGACGGACAAGC
394
CAGCAACCAGACGGACAAGC
1508



Exon 2 STOP 2 (pos9)
GTGTCACACAACTGCCCAAC
395
GUGUCACACAACUGCCCAAC
899



Exon 3 STOP 1 (pos8)
AGCCGGCCAGTTCCAAACCC
396
AGCCGGCCAGUUCCAAACCC
900



Exon 3 STOP (pos7)
CAGTTCCAAACCCTGGTGGT
397
CAGUUCCAAACCCUGGUGGU
901



Exon 3 STOP 2 (pos5)
CGGCCAGTTCCAAACCCTGG
398
CGGCCAGUUCCAAACCCUGG
902



Exon 3 STOP (pos5)
GGACCCAGACTAGCAGCACC
399
GGACCCAGACUAGCAGCACC
903



Exon 3 SD
GACGTTACCTCGTGCGGCCC
400
GACGUUACCUCGUGCGGCCC
904



Exon 4 SA
TCCCTGCAGAGAAACACACT
401
UCCCUGCAGAGAAACACACU
905



Exon 4 SD
GAGACTCACCAGGGGCTGGC
402
GAGACUCACCAGGGGCUGGC
906



Exon 5 SA
CCTCCTTCTTTGAGGAGAAA
403
CCUCCUUCUUUGAGGAGAAA
907



Exon 2 STOP (pos 7)
GGGGTTCCAGGGCCTGTCTG
203
GGGGUUCCAGGGCCUGUCUG
908



Exon 3 SA
TTCTCTCTGGAAGGGCACAA
404
UUCUCUCUGGAAGGGCACAA
909



Exon 5 STOP 1 (pos 8)
CCAGTGGCGAGAGAAGACCC
405
CCAGUGGCGAGAGAAGACCC
910



Exon 5 STOP 2 (pos 5)
TGCCCAGCCACTGAGGCCTG
406
UGCCCAGCCACUGAGGCCUG
911



Exon 1 STOP 1 (pos8)
CGACTGGCCAGGGCGCCTGT
407
CGACUGGCCAGGGCGCCUGU
912



Exon 1 STOP 3 (pos6)
ACCGCCCAGACGACTGGCCA
408
ACCGCCCAGACGACUGGCCA
913


 


Lag3
Exon 1 STOP (pos8)
GTTTCTGCAGCCGCTTTGGG
409
GUUUCUGCAGCCGCUUUGGG
914



Exon 1 SD (pos4)
TTACCTGGAGCCACCCAAAG
410
UUACCUGGAGCCACCCAAAG
915



Exon 2 SA (pos4)
TCACTAGGTGAGCAAAAGAG
411
UCACUAGGUGAGCAAAAGAG
916



Exon 2 STOP (pos8)
GCCTCTCCAGCCAGGGGCTG
412
GCCUCUCCAGCCAGGGGCUG
917



Exon 2 STOP (pos 6)
CTTGGCAGCATCAGCCAGAC
413
CUUGGCAGCAUCAGCCAGAC
918



Exon 3 SA (pos4)
CCACTGGGCGGGAAAGAGAA
414
CCACUGGGCGGGAAAGAGAA
919



Exon 3 SD (pos6)
ACATACTCGAGGCCTGGCCC
415
ACAUACUCGAGGCCUGGCCC
920



Exon 3 STOP (pos5)
CCTGCAGCCCCGCGTCCAGC
416
CCUGCAGCCCCGCGUCCAGC
92



Exon 3 STOP (pos7)
CGCGTCCAGCTGGATGAGCG
417
CGCGUCCAGCUGGAUGAGCG
922



Exon 3 STOP (pos6)
TGGGCCAGGCCTCGAGTATG
418
UGGGCCAGGCCUCGAGUAUG
923



Exon 4 SD (pos4)
GGGAGTTACCCAGAACAGTG
419
GGGAGUUACCCAGAACAGUG
924



Exon 4 STOP (pos8)
CCTGCCCCAAGTCAGCCCCA
420
CCUGCCCCAAGUCAGCCCCA
925



Exon 4 STOP (pos9)
GCCAGGGCCGAGTCCCTGTC
421
GCCAGGGCCGAGUCCCUGUC
926



Exon 4 STOP (pos8)
CCAGGGCCGAGTCCCTGTCO
422
CCAGGGCCGAGUCCCUGUCC
927



Exon 4 STOP (pos4)
GCCCCAGGGCCCAGAGTCCA
423
GCCCCAGGGCCCAGAGUCCA
928



Exon 5 STOP (pos9)
ATGTGAGCCAGGCCCAGGCT
424
AUGUGAGCCAGGCCCAGGCU
929



Exon 5 STOP (pos 8)
GAGGAGTCCACTTGGCAGTG
425
GAGGAGUCCACUUGGCAGUG
930



Exon 6 SA (pos7)
GAGTCACTGAAAAGAGTAGA
426
GAGUCACUGAAAAGAGUAGA
931



Exon 6 STOP (pos6)
CTGGACAAGAACGCTTTGTG
427
CUGGACAAGAACGCUUUGUG
932



Exon 6 STOP (pos7)
CCATCCCAGAGGAGTTTCTC
428
CCAUCCCAGAGGAGUUUCUC
933



Exon 6 STOP (pos4)
TGGCAATGCCAGCTGTACCA
429
UGGCAAUGCCAGCUGUACCA
934



Exon 6 STOP (pos4)
TACCAGGGGGAGAGGCTTCT
430
UACCAGGGGGAGAGGCUUCU
935



Exon 6 STOP (pos8)
GGCATTGCCAAGGCTGGGAA
431
GGCAUUGCCAAGGCUGGGAA
936



Exon 7 SA (pos6)
GGCACCTATGGAGAAAGTAC
432
GGCACCUAUGGAGAAAGUAC
937



Exon 7 STOP (pos4)
AGACAGGTGAGCCAGGGACA
433
AGACAGGUGAGCCAGGGACA
938



Exon 7 SD (pos7)
GGCTCACCTGTCTTCTCCAA
434
GGCUCACCUGUCUUCUCCAA
939



Exon 8 SA (pos8)
GTCGCCACTGTGAGAAGAGA
435
GUCGCCACUGUGAGAAGAGA
940



Exon 8 STOP (pos8)
GCAGGCTCAGAGCAAGATAG
436
GCAGGCUCAGAGCAAGAUAG
941



Exon 8 STOP (pos8)
GCTGGAGCAAGAACCGGAGC
437
GCUGGAGCAAGAACCGGAGC
942


 


CTLA-4
Exon 1 SD (pos 6)
ACTCACCTTTGCAGAAGACA
438
ACUCACCUUUGCAGAAGACA
943



Exon 1 SD
CACTCACCTTTGCAGAAGAC
439
CACUCACCUUUGCAGAAGAC
944



Exon 1 STOP (pos5)
AGGGCCAGGTCCTGGTAGCC
440
AGGGCCAGGUCCUGGUAGCC
945



Exon 2 STOP
GGCCCAGCCTGCTGTGGTAC
441
GGCCCAGCCUGCUGUGGUAC
946



Exon 2 STOP (pos 8)
GCTTCGGCAGGCTGACAGCC
442
GCUUCGGCAGGCUGACAGCC
947



Exon 2 STOP
TATCCAAGGACTGAGGGCCA
443
UAUCCAAGGACUGAGGGCCA
948



Exon 2 STOP
GGAACCCAGATTTATGTAAT
444
GGAACCCAGAUUUAUGUAAU
949



Exon 2 SD
GCTCACCAATTACATAAATC
445
GCUCACCAAUUACAUAAAUC
950



Exon 2 SD
CTCACCAATTACATAAATCT
446
CUCACCAAUUACAUAAAUCU
951



Exon 1 STOP
CTCAGCTGAACCTGGCTACC
447
CUCAGCUGAACCUGGCUACC
952


 


Chi3l1
Exon 1 STOP (pos8)
GGCGTCTCAAACAGGTATCT
448
GGCGUCUCAAACAGGUAUCU
953



Exon 1 SA (pos7)
CAAAGCCTGAAGAGAAATCC
449
CAAAGCCUGAAGAGAAAUCC
954



Exon 3 SA (pos6)
AGAGCCTGAAGGAGAAGTCT
450
AGAGCCUGAAGGAGAAGUCU
955



Exon 3 STOP (pos4)
TCCCAGTACCGGGAAGGCGA
451
UCCCAGUACCGGGAAGGCGA
956



Exon 4 SA (pos6)
GGTTCCTGTGGAGCACAGGG
452
GGUUCCUGUGGAGCACAGGG
957



Exon 4 SA (pos9)
TGGGGTTCCTGTGGAGCACA
453
UGGGGUUCCUGUGGAGCACA
958



Exon 6 SA (pos8)
TCATTTCCTAGATGGGAGAC
454
UCAUUUCCUAGAUGGGAGAC
959



Exon 6 SA (pos4)
TTCCTAGATGGGAGACAGGC
455
UUCCUAGAUGGGAGACAGGC
960



Exon 8 SA (pos9)
CCAGGTGTCTGAGGAGGAAG
456
CCAGGUGUCUGAGGAGGAAG
961



Exon 8 SA (pos5)
GTGTCTGAGGAGGAAGGGGA
457
GUGUCUGAGGAGGAAGGGGA
962



Exon 9 SA (pos6)
TAGTCCTGGGTGGGGTAGGG
458
UAGUCCUGGGUGGGGUAGGG
963



Exon 9 SA (pos5)
AGTCCTGGGTGGGGTAGGGT
459
AGUCCUGGGUGGGGUAGGGU
964



Exon 9 SD (pos6)
CATTACCTCATAGTAGGCAA
460
CAUUACCUCAUAGUAGGCAA
965



Exon 9 SD (pos7)
CCATTACCTCATAGTAGGCA
461
CCAUUACCUCAUAGUAGGCA
966



Exon 10 SA (pos7)
ACAGATCTGAGCAGATAACA
462
ACAGAUCUGAGCAGAUAACA
967



Exon 10 STOP (pos 7)
TCCTACCCACTGGTTGCCCT
463
UCCUACCCACUGGUUGCCCU
968



Exon 11 STOP (pos7)
AGGTGCAGTACCTGAAGGAC
464
AGGUGCAGUACCUGAAGGAC
969



Exon 11 STOP (pos5)
CAGGCAGCTGGCGGGCGCCA
465
CAGGCAGCUGGCGGGCGCCA
970



Exon 11 STOP (pos7)
GACTTCCAGGGCTCCTTCTG
466
GACUUCCAGGGCUCCUUCUG
971


 


CD96
Exon 1 STOP (pos5)
CATCCAGATACATTTTGTCA
467
CAUCCAGAUACAUUUUGUCA
972



Exon 2 STOP (pos5)
ACCTGCCAAACACAGACAGT
468
ACCUGCCAAACACAGACAGU
973



Exon 2 STOP (pos7)
CGTGCAGATGCAATGGTCCA
469
CGUGCAGAUGCAAUGGUCCA
974



Exon 3 SA (pos6)
TGTAACTGTAACAAAACATA
470
UGUAACUGUAACAAAACAUA
975



Exon 3 SD (pos6)
ACTTACCACCGACCATGCAT
471
ACUUACCACCGACCAUGCAU
976



Exon 5 SD (pos5)
CTTACCAAAAACCTTGACTG
472
CUUACCAAAAACCUUGACUG
977



Exon 5 STOP (pos6)
CCAGTCCAAATCTTCGATGA
473
CCAGUCCAAAUCUUCGAUGA
978



Exon 5 STOP (pos7)
CAGTCCAAATCTTCGATGAT
474
CAGUCCAAAUCUUCGAUGAU
979



Exon 7 STOP (pos4)
AAACCATGTGATATTTGCTT
475
AAACCAUGUGAUAUUUGCUU
980



Exon 8 STOP (pos6)
ATGTTCCACACTTTATTTCC
476
AUGUUCCACACUUUAUUUCC
981



Exon 10 SD (pos4)
TCACGTTGAGGAGTGGTGTT
477
UCACGUUGAGGAGUGGUGUU
982



Exon 13 SA (pos7)
CATTGTCTAGGGATATAAAG
478
CAUUGUCUAGGGAUAUAAAG
983



Exon 13 SA (pos8)
ACATTGTCTAGGGATATAAA
479
ACAUUGUCUAGGGAUAUAAA
984



Exon 13 SA (pos9)
GACATTGTCTAGGGATATAA
480
GACAUUGUCUAGGGAUAUAA
985



Exon 14 STOP (pos4)
TGGCCAGGACATTCCATCTT
481
UGGCCAGGACAUUCCAUCUU
986



Exon 15 SA (pos6)
CCATTCTAGGAACAAAATAT
482
CCAUUCUAGGAACAAAAUAU
987


 


Cblb
Exon 1 STOP
GAGCTTCCAAGTCTTCTCCA
483
GAGCUUCCAAGUCUUCUCCA
988



Exon 1 STOP (JMG44)
TCCCCGAAAAGGTCGAATTT
484
UCCCCGAAAAGGUCGAAUUU
989



Exon 2 STOP
ATGAAGAACAGTCACAGGAC
485
AUGAAGAACAGUCACAGGAC
990



Exon 3 SA
GATTTCGTCTGTAGGCACAA
486
GAUUUCGUCUGUAGGCACAA
991



Exon 4 SD
TAAACTTACCTGAAACAGCC
487
UAAACUUACCUGAAACAGCC
992



Exon 4 STOP
ATTCAGACAGTGCCTTCATG
488
AUUCAGACAGUGCCUUCAUG
993



Exon 6 STOP
GTTGCACTCGATTGGGACAG
489
GUUGCACUCGAUUGGGACAG
994



Exon 6 STOP
TTATTTCAAGCCCTGATTGA
490
UUAUUUCAAGCCCUGAUUGA
995



Exon 7 SD
TTACCTGTGTAACTTTTATA
491
UUACCUGUGUAACUUUUAUA
996



Exon 8 SA (pos8)
ATTGTTCCTGGAATTTGGGG
492
AUUGUUCCUGGAAUUUGGGG
997



Exon 8 SD (JMG48)
ATTATACCTGCCATGCCGTA
493
AUUAUACCUGCCAUGCCGUA
998



Exon 8 SA (pos 5) (JMG46)
GTTCCTGGAATTTGGGGAGG
494
GUUCCUGGAAUUUGGGGAGG
999



Exon 8 STOP (JMG47)
CTGCCATGCCGTAAGGCAAG
495
CUGCCAUGCCGUAAGGCAAG
1000



Exon 10 SD (JMG49)
TCTACCTTTGGTGAACCCGT
496
UCUACCUUUGGUGAACCCGU
1001



Exon 11 SD (JMG50)
CTTACCTTAGCTCCTTCTAA
497
CUUACCUUAGCUCCUUCUAA
1002



Exon 11 STOP
GGGATGTCGACTCCTAGGGG
498
GGGAUGUCGACUCCUAGGGG
1003



Exon 11 STOP
CGAGGGCACCATGCTTCAAG
499
CGAGGGCACCAUGCUUCAAG
1004



Exon 12 SD
AAACTCACTTTATGCTAGGG
500
AAACUCACUUUAUGCUAGGG
1005



Exon 12 SD (JMG51)
CTCACTTTATGCTAGGGAGG
501
CUCACUUUAUGCUAGGGAGG
1006



Exon 16 SA (JMG52)
CTTCACCTGCATTTAAAGAA
502
CUUCACCUGCAUUUAAAGAA
1007



Exon 4 STOP (JMG45)
CCACCAGATTAGCTCTGGCC
503
CCACCAGAUUAGCUCUGGCC
1008



Exon 10 SD (pos4)
CTACCTTTGGTGAACCCGTT
504
CUACCUUUGGUGAACCCGUU
1009


 


BTLA
Exon 1 STOP (pos6)
ATGTTCCAGATGTCCAGATA
505
AUGUUCCAGAUGUCCAGAUA
1010



Exon 1 STOP (pos5)
TGTTCCAGATGTCCAGATAT
506
UGUUCCAGAUGUCCAGAUAU
1011



Exon 2 STOP (pos8)
AGATAGACAAACAAGTTGGA
507
AGAUAGACAAACAAGUUGGA
1012



Exon 2 STOP (pos9)
AGCTTGCACCAAGTCACATG
508
AGCUUGCACCAAGUCACAUG
1013



Exon 3 SD (pos6)
ACCCACCTTGGTGCCTTCTC
509
ACCCACCUUGGUGCCUUCUC
1014


 


B2M (BE)
Exon 1 SD
ACTCACGCTGGATAGCCTCC
510
ACUCACGCUGGAUAGCCUCC
1015



Exon 2 SA (pos9)
TGGAGTACCTGAGGAATATC
511
UGGAGUACCUGAGGAAUAUC
1016



Exon 2 STOP (pos6)
TTACCCCACTTAACTATCTT
512
UUACCCCACUUAACUAUCUU
1017



Exon 3 SA
TCGATCTATGAAAAAGACAG
513
UCGAUCUAUGAAAAAGACAG
1018



Exon 2 STOP
TACCCCACTTAACTATCT
514
UACCCCACUUAACUAUCU
1019


 


B2M (ABE)
Exon 1 SD 1 (pos 5)
ACTCACGCTGGATAGCCTCC
510
ACUCACGCUGGAUAGCCUCC
1015



Exon 2 SA (pos 4)
CTCAGGTACTCCAAAGATTC
515
CUCAGGUACUCCAAAGAUUC
1020



Exon 2 SD (pos 4)
CTTACCCCACTTAACTATCT
516
CUUACCCCACUUAACUAUCU
1021


 


TET2
Exon 1 STOP 1 (pos 8)
CATTTGCCAGACAGAACCTC
517
CAUUUGCCAGACAGAACCUC
1022



Exon 1 STOP 2 (pos 4)
AAACAAGACCAAAAGGCTAA
518
AAACAAGACCAAAAGGCUAA
1023



Exon 1 STOP 3 (pos 7)
GTAAGCCAAGAAAGAAATCC
519
GUAAGCCAAGAAAGAAAUCC
1024



Exon 1 STOP 4 (pos 5)
GCTTCAGATTCTGAATGAGC
520
GCUUCAGAUUCUGAAUGAGC
1025



Exon 1 STOP 5 (pos 7)
TTAAAACAAAATGAAATGAA
521
UUAAAACAAAAUGAAAUGAA
1026



Exon 1 STOP 6 (pos 7)
GTTCCTCAGCTTCCTTCAGA
522
GUUCCUCAGCUUCCUUCAGA
1027



Exon 1 STOP 7 (pos 8)
CAAAGAGCAAGAGATTCTGA
523
CAAAGAGCAAGAGAUUCUGA
1028



Exon 1 STOP 8 (pos 7)
AAAGAGCAAGAGATTCTGAA
524
AAAGAGCAAGAGAUUCUGAA
1029



Exon 1 STOP 9 (pos 4)
ACACAGCACTATCTGAAACC
525
ACACAGCACUAUCUGAAACC
1030



Exon 1 STOP 10 (pos 5)
CACCCAGAAAACAACACAGC
526
CACCCAGAAAACAACACAGC
1509



Exon 1 STOP 11 (pos 4)
TACCAAGTTGAAATGAATCA
527
UACCAAGUUGAAAUGAAUCA
1031



Exon 1 STOP 12 (pos 7)
ATGAATCAAGGGCAGTCCCA
528
AUGAAUCAAGGGCAGUCCCA
1032



Exon 1 STOP 13 (pos 5)
AGGGCAGTCCCAAGGTACAG
529
AGGGCAGUCCCAAGGUACAG
1033



Exon 1 STOP 14 (pos 5)
GTTCCAAAAACCCTCACACC
530
GUUCCAAAAACCCUCACACC
1034



Exon 1 STOP 15 (pos 5)
GAAACAGCACTTGAATCAAC
531
GAAACAGCACUUGAAUCAAC
1035



Exon 1 STOP 16 (pos 5)
ATTACAAATAAAGAATAAAG
532
AUUACAAAUAAAGAAUAAAG
1036



Exon 1 STOP 17 (pos 8)
TAATGTCCAAATGGGACTGG
533
UAAUGUCCAAAUGGGACUGG
1037



Exon 1 STOP 18 (pos 6)
CAAAGCAAGATCTTCTTCAC
534
CAAAGCAAGAUCUUCUUCAC
1038



Exon 1 STOP 19 (pos 5)
ACAACAAGCTTCAGTTCTAC
535
ACAACAAGCUUCAGUUCUAC
1039



Exon 1 STOP 20 (pos 6)
CTGCGCAACTTGCTCAGCAA
536
CUGCGCAACUUGCUCAGCAA
1040



Exon 1 STOP 21 (pos 5)
CACTCAGACCCCTCCCCAGA
537
CACUCAGACCCCUCCCCAGA
1041



Exon 1 STOP 22 (pos 6)
TTTTTCCATGTTTTGTTTTC
538
UUUUUCCAUGUUUUGUUUUC
1042



Exon 1 SD (pos 4)
TTACCTACACATCTGCAAGA
539
UUACCUACACAUCUGCAAGA
1043



Exon 3 SD (pos 8)
ACACTTACCCACTTAGCAAT
540
ACACUUACCCACUUAGCAAU
1044



Exon 7 STOP (pos 5)
CATGCAGAATGGCAGCACAT
541
CAUGCAGAAUGGCAGCACAU
1045



Exon 8 STOP 1 (pos 6)
AAGCTCAGGAGGAGAAAAAA
542
AAGCUCAGGAGGAGAAAAAA
1046



Exon 8 STOP 2 (pos 8)
CGCAAGCCAGGCTAAACAGT
543
CGCAAGCCAGGCUAAACAGU
1047



Exon 9 STOP 1 (pos 8)
TTCTCCCCAGTCTCAGCCGA
544
UUCUCCCCAGUCUCAGCCGA
1048



Exon 9 STOP 2 (pos 5)
TGGTCAGGAAAAGCAGCCAT
545
UGGUCAGGAAAAGCAGCCAU
1049



Exon 9 STOP 3 (pos 7)
CTAGTCCAGGGTGTGGCTTC
546
CUAGUCCAGGGUGUGGCUUC
1050


 


Spry 1
Exon 1 STOP 1
CCCCAAAATCAACATGGCAG
547
CCCCAAAAUCAACAUGGCAG
1051



Exon 1 STOP 2
TGTGATCCAGCAGCCTTCTT
548
UGUGAUCCAGCAGCCUUCUU
1052



Exon 1 STOP 3
GACCAGATCAAGGCCATAAG
549
GACCAGAUCAAGGCCAUAAG
1053



Exon 1 STOP 4
CAAGACAAGAAAAGCATGAA
550
CAAGACAAGAAAAGCAUGAA
1054



Exon 1 STOP 5
CTGAACAGGGACTGTTAGGA
551
CUGAACAGGGACUGUUAGGA
1055


 


Spry 2
Exon 1 STOP 1
CCAGAGCTCAGAGTGGCAAC
552
CCAGAGCUCAGAGUGGCAAC
1056



Exon 1 STOP 2
TTGCTGCAGACGCCCCGTGA
553
UUGCUGCAGACGCCCCGUGA
1057



Exon 1 STOP 3
CTGCAGACGCCCCGTGACGG
554
CUGCAGACGCCCCGUGACGG
1058



Exon 1 STOP 4
CGACAAGCAGTGCCTTTGCT
555
CGACAAGCAGUGCCUUUGCU
1059



Exon 1 STOP 5
GCCCAGAACGTGATTGACTA
556
GCCCAGAACGUGAUUGACUA
1060



Exon 1 STOP 6
TGTGCCAGGGGTGTTATGAC
557
UGUGCCAGGGGUGUUAUGAC
1061



Exon 1 STOP 7
CAGATCCAGTCTGATGGCAG
558
CAGAUCCAGUCUGAUGGCAG
1062



Exon 1 STOP 8
TGTACACGATGGTCAGCCAT
559
UGUACACGAUGGUCAGCCAU
1063


 


CIITA
Exon 1 SD (pos 6)
TTTTACCTTGGGGCTCTGAC
560
UUUUACCUUGGGGCUCUGAC
1064



Exon 1 STOP 1 (pos 6)
AGCCCCAAGGTAAAAAGGCC
561
AGCCCCAAGGUAAAAAGGCC
1065



Exon 1 STOP 2 (pos 7)
GAGCCCCAAGGTAAAAAGGC
562
GAGCCCCAAGGUAAAAAGGC
1066



Exon 2 STOP 1 (pos 8)
CAGCTCACAGTGTGCCACCA
563
CAGCUCACAGUGUGCCACCA
1067



Exon 2 STOP 2 (pos 7)
TATGACCAGATGGACCTGGC
564
UAUGACCAGAUGGACCUGGC
1068



Exon 4 STOP 1 (pos 8)
ACTGGACCAGTATGTCTTCC
565
ACUGGACCAGUAUGUCUUCC
1069



Exon 4 STOP 2 (pos 8)
TGTCTTCCAGGACTCCCAGC
566
UGUCUUCCAGGACUCCCAGC
1070



Exon 7 STOP 1 (pos 7)
TTCAACCAGGAGCCAGCCTC
567
UUCAACCAGGAGCCAGCCUC
1071



Exon 7 STOP 2 (pos 4)
GACCAGATTCCCAGTATGTT
568
GACCAGAUUCCCAGUAUGUU
1072



Exon 7 SD (pos 8)
TAACATACTGGGAATCTGGT
569
UAACAUACUGGGAAUCUGGU
1073



Exon 8 SA (pos 8)
AAAGGCACTGCAAGAGACAA
570
AAAGGCACUGCAAGAGACAA
1074



Exon 8 STOP (pos 8)
CTCTGGCAAATCTCTGAGGC
571
CUCUGGCAAAUCUCUGAGGC
1075



Exon 9 STOP 1 (pos 4)
AGCCAAGTACCCCCTCCCAG
572
AGCCAAGUACCCCCUCCCAG
1076



Exon 9 STOP 2 (pos 7)
ACCTCCCGAGCAAACATGAC
573
ACCUCCCGAGCAAACAUGAC
1077



Exon 9 SD (pos 6)
CCTTACCTGTCATGTTTGCT
574
CCUUACCUGUCAUGUUUGCU
1078



Exon 10 SA (pos 5)
TGCTCTGGAGATGGAGAAGC
575
UGCUCUGGAGAUGGAGAAGC
1079



Exon 10 STOP 1 (pos 7)
CCCACCCAATGCCCGGCAGC
576
CCCACCCAAUGCCCGGCAGC
1080



Exon 10 STOP 2 (pos 4)
AGGCCATTTTGGAAGCTTGT
577
AGGCCAUUUUGGAAGCUUGU
1081



Exon 11 SA (pos 8)
ACCGGCTCTGCAAAGGCCAG
578
ACCGGCUCUGCAAAGGCCAG
1082



Exon 11 STOP 1 (pos 6)
TGGTGCAGGCCAGGCTGGAG
579
UGGUGCAGGCCAGGCUGGAG
1083



Exon 11 STOP 3 (pos 7)
GAACGGCAGCTGGCCCAAGG
580
GAACGGCAGCUGGCCCAAGG
1084



Exon 11 STOP 4 (pos 5)
GGCCCAAGGAGGCCTGGCTG
581
GGCCCAAGGAGGCCUGGCUG
1085



Exon 11 STOP 5 (pos 5)
GACACGAGTGATTGCTGTGC
582
GACACGAGUGAUUGCUGUGC
1086



Exon 11 STOP 5 (pos 6)
CTGGTCAGGGCAAGAGCTAT
583
CUGGUCAGGGCAAGAGCUAU
1087



Exon 11 STOP 5 (pos 8)
GGGCCCACAGCCACTCGTGG
584
GGGCCCACAGCCACUCGUGG
1088



Exon 11 STOP 6 (pos 4)
TTCCAGAAGAAGCTGCTCCG
585
UUCCAGAAGAAGCUGCUCCG
1089



Exon 11 STOP 7 (pos 8)
CCTGGTCCAGAGCCTGAGCA
586
CCUGGUCCAGAGCCUGAGCA
1090



Exon 11 STOP 8 (pos 8)
CAGACATCAAAGTACCCTAC
587
CAGACAUCAAAGUACCCUAC
1091



Exon 11 STOP 9 (pos 5)
ACATCAAAGTACCCTACAGG
588
ACAUCAAAGUACCCUACAGG
1092



Exon 11 STOP 10 (pos 4)
CGCCCAGGTCCTCACGTCTG
589
CGCCCAGGUCCUCACGUCUG
1093



Exon 11 STOP 11 (pos 8)
CTTAGTCCAACACCCACCGC
590
CUUAGUCCAACACCCACCGC
1094



Exon 11 STOP 12 (pos 8)
CCTCCTGCAATGCTTCCTGG
591
CCUCCUGCAAUGCUUCCUGG
1095



Exon 11 STOP 13 (pos 8)
GAGCCAGCCACAGGGCCCCC
592
GAGCCAGCCACAGGGCCCCC
1510



Exon 11 STOP 14 (pos 6)
GGAAGCAGAAGGTGCTTGCG
593
GGAAGCAGAAGGUGCUUGCG
1096



Exon 11 STOP 15 (pos 6)
GGCTGCAGCCGGGGACACTG
594
GGCUGCAGCCGGGGACACUG
1097



Exon 11 STOP 16 (pos 4)
CTGCCAAATTCCAGCCTCCT
595
CUGCCAAAUUCCAGCCUCCU
1098



Exon 11 STOP 17 (pos 8)
GGCGGGCCAAGACTTCTCCC
596
GGCGGGCCAAGACUUCUCCC
1099



Exon 12 STOP 1 (pos 6)
AGACTCAGAGGTGAGAGGAG
597
AGACUCAGAGGUGAGAGGAG
1100



Exon 14 SA (pos 4)
AGCCTAGGAGGCAAAGAGCA
598
AGCCUAGGAGGCAAAGAGCA
1101



Exon 14 STOP 1 (pos 5)
CCCCCAGGCTTTCCCCAAAC
599
CCCCCAGGCUUUCCCCAAAC
1102



Exon 14 SD (pos 4)
TCACTCCAGATGCTGCAGGG
600
UCACUCCAGAUGCUGCAGGG
1103



Exon 15 SA (pos 4)
AGGCTGCAGGTGGAATCAGA
601
AGGCUGCAGGUGGAAUCAGA
1104



Exon 15 STOP 1 (pos 8)
CTTCCCCCAGCTGAAGTCCT
602
CUUCCCCCAGCUGAAGUCCU
1105



Exon 15 SD (pos 7)
CACTCACTTGAGGGTTTCCA
603
CACUCACUUGAGGGUUUCCA
1106



Exon 16 SA (pos 5)
CAGACTGCGGGGACACAGTG
604
CAGACUGCGGGGACACAGUG
1107



Exon 16 SD 1 (pos 8)
CCACTCACCTTAGCCTGAGC
605
CCACUCACCUUAGCCUGAGC
1108



Exon 16 SD 2 (pos 7)
CACTCACCTTAGCCTGAGCA
606
CACUCACCUUAGCCUGAGCA
1109



Exon 17 SA (pos 8)
GTACAAGCTGTCGGAAACAG
607
GUACAAGCUGUCGGAAACAG
1110



Exon 17 SD 1 (pos 8)
ACACTCACTCCATCACCCGG
608
ACACUCACUCCAUCACCCGG
1111



Exon 17 SD 2 (pos 7)
CACTCACTCCATCACCCGGA
609
CACUCACUCCAUCACCCGGA
1112



Exon 18 STOP (pos 5)
CGTCCAGTACAACAAGTTCA
610
CGUCCAGUACAACAAGUUCA
1113



Exon 19 SA 1 (pos 8)
CCACATCCTGCAAGGGGGGA
611
CCACAUCCUGCAAGGGGGGA
1114



Exon 19 SA 2 (pos 7)
CACATCCTGCAAGGGGGGAT
612
CACAUCCUGCAAGGGGGGAU
1115



Exon 19 STOP 1 (pos 8)
TGGGCGTCCACATCCTGCAA
613
UGGGCGUCCACAUCCUGCAA
1116



Exon 19 STOP 2 (pos 7)
GGGCGTCCACATCCTGCAAG
614
GGGCGUCCACAUCCUGCAAG
1117



Exon 19 STOP 3 (pos 6)
GGCGTCCACATCCTGCAAGG
615
GGCGUCCACAUCCUGCAAGG
1118



Exon 19 STOP 4 (pos 5)
GCGTCCACATCCTGCAAGGG
616
GCGUCCACAUCCUGCAAGGG
1119


 


CD7
Exon 1 STOP (pos 4)
GCCCAAGGTAAGAGCTTCCC
617
GCCCAAGGUAAGAGCUUCCC
1120



Exon 1 SD 1 (pos 8)
GCTCTTACCTTGGGCAGCCA
618
GCUCUUACCUUGGGCAGCCA
1121



Exon 1 SD 2 (pos 9)
AGCTCTTACCTTGGGCAGCC
619
AGCUCUUACCUUGGGCAGCC
1122



Exon 2 SA 1 (pos 8)
TGCACCTCTGGGGAGGACCT
620
UGCACCUCUGGGGAGGACCU
1123



Exon 2 SA 2 (pos 9)
CTGCACCTCTGGGGAGGACC
621
CUGCACCUCUGGGGAGGACC
1124



Exon 2 STOP 1 (pos 7)
CGCCTGCAGCTGTCGGACAC
622
CGCCUGCAGCUGUCGGACAC
1125



Exon 2 STOP 2 (pos 8)
CACCTGCCAGGCCATCACGG
623
CACCUGCCAGGCCAUCACGG
1126



Exon 2 SD 1 (pos 6)
CCCTACCTGTCACCAGGACC
624
CCCUACCUGUCACCAGGACC
1127



Exon 2 SD 2 (pos 5)
CCTACCTGTCACCAGGACCA
625
CCUACCUGUCACCAGGACCA
1128



Exon 3 SA (pos 4)
CCTCTGAGAAGGAAAAAAGA
626
CCUCUGAGAAGGAAAAAAGA
1129



Exon 3 STOP 1 (pos9)
CAGAGGAACAGTCCCAAGGA
627
CAGAGGAACAGUCCCAAGGA
1130


 


CD33
Exon 1 SD 1 (pos 7)
CACTCACCTGCCCACAGCAG
628
CACUCACCUGCCCACAGCAG
1131



Exon 1 SD 2 (pos 8)
CCACTCACCTGCCCACAGCA
629
CCACUCACCUGCCCACAGCA
1132



Exon 1 SD (pos 9)
GCCACTCACCTGCCCACAGC
630
GCCACUCACCUGCCCACAGC
1133



Exon 2 SA 1 (pos 8)
AGGGCCCCTGTGGGGAAACG
631
AGGGCCCCUGUGGGGAAACG
1134



Exon 2 SA 2 (pos 7)
GGGCCCCTGTGGGGAAACGA
632
GGGCCCCUGUGGGGAAACGA
1135



Exon 2 STOP 1 (pos 8)
GCAAGTGCAGGAGTCAGTGA
633
GCAAGUGCAGGAGUCAGUGA
1136



Exon 2 STOP 2 (pos 6)
CGGAACCAGTAACCATGAAC
634
CGGAACCAGUAACCAUGAAC
1137



Exon 2 STOP 3 (pos 5)
GGAACCAGTAACCATGAACT
635
GGAACCAGUAACCAUGAACU
1138



Exon 2 STOP 4 (pos 4)
GAACCAGTAACCATGAACTG
636
GAACCAGUAACCAUGAACUG
1139



Exon 2 STOP 5 (pos 8)
GCTAGATCAAGAAGTACAGG
637
GCUAGAUCAAGAAGUACAGG
1140



Exon 2 STOP 6 (pos 8)
AGAAGTACAGGAGGAGACTC
638
AGAAGUACAGGAGGAGACUC
1141



Exon 3 SA 1 (pos 6)
CAAGTCTAGTGAGGAGAAAG
639
CAAGUCUAGUGAGGAGAAAG
1142



Exon 3 SA 2 (pos 5)
AAGTCTAGTGAGGAGAAAGA
640
AAGUCUAGUGAGGAGAAAGA
1143



Exon 3 SA 3 (pos 4)
AGTCTAGTGAGGAGAAAGAG
641
AGUCUAGUGAGGAGAAAGAG
1144



Exon 3 STOP 1 (pos 7)
ACAGGCCCAGGACACAGAGC
642
ACAGGCCCAGGACACAGAGC
1511



Exon 3 STOP 2 (pos 7)
ACCTGTCAGGTGAAGTTCGC
643
ACCUGUCAGGUGAAGUUCGC
1145



Exon 3 SD 1 (pos 6)
ACTTACAGGTGACGTTGAGC
644
ACUUACAGGUGACGUUGAGC
1146



Exon 4 SA 1 (pos 6)
AACATCTAGGAGAGGAAGAG
645
AACAUCUAGGAGAGGAAGAG
1147



Exon 4 STOP 1 (pos 7)
GTTCCACAGAACCCAACAAC
646
GUUCCACAGAACCCAACAAC
1148



Exon 4 SD 1 (pos 7)
TTCCTACCTGAGCCATCTCC
647
UUCCUACCUGAGCCAUCUCC
1149



Exon 5 SD (pos 8)
ATGCTCACATGAAGAAGATG
648
AUGCUCACAUGAAGAAGAUG
1150



Exon 5 STOP 1 (pos 7)
GGGAAACAAGAGACCAGAGC
649
GGGAAACAAGAGACCAGAGC
1512



Exon 6 SA 1 (pos 6)
TCACTCTGATGGGAGACACC
650
UCACUCUGAUGGGAGACACC
1151



Exon 6 SA 2 (pos 5)
CACTCTGATGGGAGACACCA
651
CACUCUGAUGGGAGACACCA
1152



Exon 6 SA 1 (pos 4)
TTTCTTATGGAGAGGAAAGA
652
UUUCUUAUGGAGAGGAAAGA
1153


 


CD52
Exon 1 STOP (pos 4)
GTACAGGTAAGAGCAACGCC
653
GUACAGGUAAGAGCAACGCC
1154



Exon 1 SD (pos7)
CTCTTACCTGTACCATAACC
654
CUCUUACCUGUACCAUAACC
1155



Exon 1 SD (pos 4)
TTACCTGTACCATAACCAGG
655
UUACCUGUACCAUAACCAGG
1156



Exon 2 SA (pos 6)
TGTATCTGTAGGAGGAGAAG
656
UGUAUCUGUAGGAGGAGAAG
1157



Exon 2 SA (pos 5)
GTATCTGTAGGAGGAGAAGT
657
GUAUCUGUAGGAGGAGAAGU
1158



Exon 2 STOP (pos 7)
CAGATACAAACTGGACTCTC
658
CAGAUACAAACUGGACUCUC
1159


 


CD123
Exon 1 SD (pos 6)
TCTTACCTTCCTTCGTTTGC
659
UCUUACCUUCCUUCGUUUGC
1160



Exon 2 SA 1 (pos 8)
TTTGGATCTAAAACGGTGAC
660
UUUGGAUCUAAAACGGUGAC
1161



Exon 2 SA 2 (pos 4)
GATCTAAAACGGTGACAGGT
661
GAUCUAAAACGGUGACAGGU
1162



Exon 2 STOP 1 (pos 8)
AAAGGCTCAGCAGTTGACCT
662
AAAGGCUCAGCAGUUGACCU
1163



Exon 2 SD (pos 6)
ATTTACCGGCATAGAATAGT
663
AUUUACCGGCAUAGAAUAGU
1164



Exon 3 SA (pos 8)
TCACTGCCTAAGAGAGACAT
664
UCACUGCCUAAGAGAGACAU
1165



Exon 3 STOP 1 (pos 6)
AGGATCCACGTGGAGAATGG
665
AGGAUCCACGUGGAGAAUGG
1166



Exon 3 STOP 2 (pos 5)
GGATCCACGTGGAGAATGGT
666
GGAUCCACGUGGAGAAUGGU
1167



Exon 3 SD (pos 6)
TCTCACTGTTCTCAGGGAAG
667
UCUCACUGUUCUCAGGGAAG
1168



Exon 4 STOP 1 (pos 6)
CCTGCCCAAGGCTTCCCACC
668
CCUGCCCAAGGCUUCCCACC
1169



Exon 4 STOP 2 (pos 5)
CTGCCCAAGGCTTCCCACCT
669
CUGCCCAAGGCUUCCCACCU
1170



Exon 5 SA 1 (pos 6)
GCCTGCTGCGGTAAGCGGTA
670
GCCUGCUGCGGUAAGCGGUA
1171



Exon 5 STOP 1 (pos 7)
GATGCTCAGGGAACACGTAT
671
GAUGCUCAGGGAACACGUAU
1172



Exon 5 STOP 2 (pos 5)
TTCTCAAAGTTCCCACATCC
672
UUCUCAAAGUUCCCACAUCC
1173



Exon 5 STOP 3 (pos 4)
TCACAGATTGGTGAGTAGCC
673
UCACAGAUUGGUGAGUAGCC
1174



Exon 7 SD (pos 5)
CTCACCTGTTCTGTGATTAC
674
CUCACCUGUUCUGUGAUUAC
1175



Exon 8 STOP 1 (pos 7)
TCCTTCCAGCTACTCAATCC
675
UCCUUCCAGCUACUCAAUCC
1176



Exon 8 STOP 2 (pos 8)
CACAGTACAAATAAGAGCCC
676
CACAGUACAAAUAAGAGCCC
1177



Exon 8 STOP 3 (pos 6)
CCCCCCAGCGCTTCGGTGAG
677
CCCCCCAGCGCUUCGGUGAG
1178



Exon 8 STOP 4 (pos 5)
CCCCCAGCGCTTCGGTGAGT
678
CCCCCAGCGCUUCGGUGAGU
1179



Exon 8 SD (pos 8)
CCACTCACCGAAGCGCTGGG
679
CCACUCACCGAAGCGCUGGG
1180



Exon 10 SA (pos 4)
TACCTCGGAGGAAAGAGAAA
680
UACCUCGGAGGAAAGAGAAA
1181



Exon 10 STOP (pos 8)
CAGCTTCCAAAACGACAAGC
681
CAGCUUCCAAAACGACAAGC
1182



Exon 10 SD (pos 7)
AACATACCAGCTTGTCGTTT
682
AACAUACCAGCUUGUCGUUU
1183



Exon 11 SA 1 (pos 8)
AGACCACCTGCAGAGACGAG
683
AGACCACCUGCAGAGACGAG
1184



Exon 11 SA 2 (pos 5)
CCACCTGCAGAGACGAGAGG
684
CCACCUGCAGAGACGAGAGG
1185


 


TRBC1
Exon 1 STOP 1 (pos 8)
CCACACCCAAAAGGCCACAC
685
CCACACCCAAAAGGCCACAC
1500



Exon 1 STOP 2 (pos 5)
CCCACCAGCTCAGCTCCACG
686
CCCACCAGCUCAGCUCCACG
1186



Exon 1 STOP 3 (pos 7)
CGCTGTCAAGTCCAGTTCTA
687
CGCUGUCAAGUCCAGUUCUA
1187



Exon 1 STOP 4 (pos 6)
GCTGTCAAGTCCAGTTCTAC
688
GCUGUCAAGUCCAGUUCUAC
1188



Exon 1 STOP 5 (pos 5)
CACCCAGATCGTCAGCGCCG
689
CACCCAGAUCGUCAGCGCCG
1189



Exon 1 SD (pos 8)
CCACTCACCTGCTCTACCCC
690
CCACUCACCUGCUCUACCCC
1190



Exon 2 SA (pos 8)
CCACAGTCTGAAAGAAAGCA
691
CCACAGUCUGAAAGAAAGCA
1191



Exon 3 SA (pos 5)
GACACTGTTGGCACGGAGGA
692
GACACUGUUGGCACGGAGGA
1192



Exon 3 SD (pos 4)
TTACCATGGCCATCAACACA
693
UUACCAUGGCCAUCAACACA
1193


 


TRBC2
Exon 1 STOP 1 (pos 8)
CCACACCCAAAAGGCCACAC
685
CCACACCCAAAAGGCCACAC
1500



Exon 1 STOP 2 (pos 5)
CCCACCAGCTCAGCTCCACG
686
CCCACCAGCUCAGCUCCACG
1186



Exon 1 STOP 3 (pos 7)
CGCTGTCAAGTCCAGTTCTA
687
CGCUGUCAAGUCCAGUUCUA
1187



Exon 1 STOP 4 (pos 6)
GCTGTCAAGTCCAGTTCTAC
688
GCUGUCAAGUCCAGUUCUAC
1188



Exon 1 STOP 5 (pos 5)
CACCCAGATCGTCAGCGCCG
689
CACCCAGAUCGUCAGCGCCG
1189



Exon 2 SA (pos 8)
CCACAGTCTGAAAGAAAACA
694
CCACAGUCUGAAAGAAAACA
1194



Exon 2 SA (pos 7)
CACAGTCTGAAAGAAAACAG
695
CACAGUCUGAAAGAAAACAG
1195



Exon 3 SD (pos 4)
TTACCATGGCCATCAGCACG
696
UUACCAUGGCCAUCAGCACG
1196



Exon 1 SD (pos 8)
CCACTCACCTGCTCTACCCC
690
CCACUCACCUGCUCUACCCC
1190


 


CISH
Exon 1 STOP
TCTGCGTTCAGGGGTAAGCG
697
UCUGCGUUCAGGGGUAAGCG
1197



Exon 1 SD
GCGCTTACCCCTGAACGCAG
698
GCGCUUACCCCUGAACGCAG
1198



Exon 2 STOP 2
GACTGGGCAGCGGCCCCTGT
699
GACUGGGCAGCGGCCCCUGU
1199



Exon 2 STOP 1
GGACTGGGCAGCGGCCCCTG
700
GGACUGGGCAGCGGCCCCUG
1200



Exon 2 STOP 3
GTCATGCAGCCCTTGCCTGC
701
GUCAUGCAGCCCUUGCCUGC
1201



Exon 2 STOP 4
TCATGCAGCCCTTGCCTGCT
702
UCAUGCAGCCCUUGCCUGCU
1202



Exon 2 STOP 5
CATGCAGCCCTTGCCTGCTG
703
CAUGCAGCCCUUGCCUGCUG
1203



Exon 2 SD 1
CTCACCAGATTCCCGAAGGT
704
CUCACCAGAUUCCCGAAGGU
1204



Exon 2 SD 2
CAGACTCACCAGATTCCCGA
705
CAGACUCACCAGAUUCCCGA
1205



Exon 3 SA 1 (pos 4)
AGCCTAGGCAAGTGCAGAGG
706
AGCCUAGGCAAGUGCAGAGG
1206



Exon 3 SA 2 (pos 5)
CAGCCTAGGCAAGTGCAGAG
707
CAGCCUAGGCAAGUGCAGAG
1207



Exon 3 SA 3 (pos 7)
ACCAGCCTAGGCAAGTGCAG
708
ACCAGCCUAGGCAAGUGCAG
1208



Exon 3 STOP 1 (pos 8)
TGGAACCCCAATACCAGCCT
709
UGGAACCCCAAUACCAGCCU
1209



Exon 3 STOP 2 (pos 7)
CACCTGCAGAAGATGCCAGA
710
CACCUGCAGAAGAUGCCAGA
1210


 


ACAT1
Exon 1 SD 1 (pos 7)
CGCTCACCTGCACCAGCCTC
711
CGCUCACCUGCACCAGCCUC
1211



Exon 3 SA (pos 5)
CTTCCTGGCAAGACACAAGA
712
CUUCCUGGCAAGACACAAGA
1212



Exon 3 STOP (pos 5)
AATTCAGGGAGCCATTGAAA
713
AAUUCAGGGAGCCAUUGAAA
1213



Exon 3 SD (pos 8)
CTACTGACCTGCCTTTTCAA
714
CUACUGACCUGCCUUUUCAA
1214



Exon 5 STOP (pos 7)
GCCTCTCAAAGTCTTATGTG
715
GCCUCUCAAAGUCUUAUGUG
1215



Exon 7 STOP (pos 4)
TTCCCATGCTGCTTTACTTC
716
UUCCCAUGCUGCUUUACUUC
1216



Exon 8 STOP (pos 8)
TTTAGGTCAACCAGATGTAG
717
UUUAGGUCAACCAGAUGUAG
1217



Exon 9 SA (pos 9)
TGTGCCTGAAAGCAAAAATG
718
UGUGCCUGAAAGCAAAAAUG
1218



Exon 9 SD (pos 4)
TTACCTACTATTCTTGCCAG
719
UUACCUACUAUUCUUGCCAG
1219



Exon 10 SA (pos 6)
AAATGCTGTTTAAAAAAAGG
720
AAAUGCUGUUUAAAAAAAGG
1220



Exon 11 STOP (pos 4)
CCCCAAAAAGTGAATATCAA
721
CCCCAAAAAGUGAAUAUCAA
1221


 


Cyp11a1
Exon 1 STOP 1 (pos 4)
GTCCAGAATTTCCAGAAGTA
722
GUCCAGAAUUUCCAGAAGUA
1222



Exon 2 SA 1 (pos 4)
TCCCTGGAGGGGTGGGGGAG
723
UCCCUGGAGGGGUGGGGGAG
1223



Exon 2 SD 1 (pos 4)
TCACTTCAACAGGACTCCTA
724
UCACUUCAACAGGACUCCUA
1224



Exon 3 SD 1 (pos 6)
CCTTACACTCAAAGGCAAAG
725
CCUUACACUCAAAGGCAAAG
1225



Exon 4 SA (pos 5)
ATGGCTGCAGGGAGAGGAAG
726
AUGGCUGCAGGGAGAGGAAG
1226



Exon 4 STOP 1 (pos 8)
GGAGCGCCAGGGGATGCTGG
727
GGAGCGCCAGGGGAUGCUGG
1227



Exon 4 STOP 2 (pos 8)
TCACGTCCCATGCAGCCACA
728
UCACGUCCCAUGCAGCCACA
1228



Exon 6 SA (pos 8)
TGGACGTCTGGTGGGGAGTA
729
UGGACGUCUGGUGGGGAGUA
1229



Exon 8 STOP 1 (pos 6)
ACTCACATTGATGAGGAAGA
730
ACUCACAUUGAUGAGGAAGA
1230



Exon 9 SA (pos 7)
CAGCATCTGAGAAAGGCAGA
731
CAGCAUCUGAGAAAGGCAGA
1231



Exon 9 STOP 1 (pos 5)
AATCCAACACCTCAGCGATG
732
AAUCCAACACCUCAGCGAUG
1232



Exon 9 STOP 2 (pos 4)
ATCCAACACCTCAGCGATGT
733
AUCCAACACCUCAGCGAUGU
1233


 


GATA3
Exon 1 STOP 1 (pos 8)
CGCGGCGCAGTACCCGCTGC
734
CGCGGCGCAGUACCCGCUGC
1234



Exon 1 SD 1 (pos 7)
CACTCACCGTGGTGGGTCGG
735
CACUCACCGUGGUGGGUCGG
1235



Exon 1 SD 2 (pos 6)
ACTCACCGTGGTGGGTCGGA
736
ACUCACCGUGGUGGGUCGGA
1236



Exon 2 SA 1 (pos 8)
TGGCTCCCTGTGGGGCAACG
737
UGGCUCCCUGUGGGGCAACG
1237



Exon 2 STOP 2 (pos 5)
GATTCCAGGGGGAGGCGGTG
738
GAUUCCAGGGGGAGGCGGUG
1238



Exon 2 SD 1 (pos 8)
GCTCCTACCTGTGCTGGACC
739
GCUCCUACCUGUGCUGGACC
1239



Exon 3 STOP 1 (pos 7)
TCGCCGCCACAGTGGGGTCG
740
UCGCCGCCACAGUGGGGUCG
1240



Exon 4 SA (pos 5)
CAGACTGAGAGTGGGGAGAG
741
CAGACUGAGAGUGGGGAGAG
1241



Exon 4 STOP 1 (pos 7)
CCTCCTCCAGAGTGTGGTTG
742
CCUCCUCCAGAGUGUGGUUG
1242


 


NR4A1
Exon 1 STOP 1 (pos 8)
AGCCATCCCAGGGAGAGAGC
743
AGCCAUCCCAGGGAGAGAGC
1243



Exon 1 STOP 2 (pos 7)
GCCATCCCAGGGAGAGAGCT
744
GCCAUCCCAGGGAGAGAGCU
1244



Exon 1 STOP 3 (pos 6)
CCATCCCAGGGAGAGAGCTG
745
CCAUCCCAGGGAGAGAGCUG
1245



Exon 1 STOP 4 (pos 5)
CTCACAGGCCACCCACCAGC
746
CUCACAGGCCACCCACCAGC
1246



Exon 2 STOP 1 (pos 8)
CCGCTTCCAGAAGTGCCTGG
747
CCGCUUCCAGAAGUGCCUGG
1247



Exon 2 STOP 2 (pos 5)
CTTCCAGAAGTGCCTGGCGG
748
CUUCCAGAAGUGCCUGGCGG
1248



Exon 3 SA 1 (pos 5)
ACAACTGCAAAGGAATGGGT
749
ACAACUGCAAAGGAAUGGGU
1249



Exon 3 SA 2 (pos 4)
CAACTGCAAAGGAATGGGTA
750
CAACUGCAAAGGAAUGGGUA
1250



Exon 4 SA (pos 4)
GAACTAGGAAGACGGTCCAG
751
GAACUAGGAAGACGGUCCAG
1251



Exon 4 STOP 1 (pos 8)
GGCTGACCAGGACCTGTTGC
752
GGCUGACCAGGACCUGUUGC
1252



Exon 4 SD 1 (pos 5)
CTCACCTGTACGCCAGGCGG
753
CUCACCUGUACGCCAGGCGG
1253



Exon 4 SD 2 (pos 8)
GCTCTCACCTGTACGCCAGG
754
GCUCUCACCUGUACGCCAGG
1254



Exon 5 SA (pos 8)
CTTAGACCTGGCAGGCAGAT
755
CUUAGACCUGGCAGGCAGAU
1255



Exon 5 STOP 1 (pos 5)
CAATCCAGTCCCCGAAGCCA
756
CAAUCCAGUCCCCGAAGCCA
1256



Exon 5 STOP 2 (pos 4)
AATCCAGTCCCCGAAGCCAC
757
AAUCCAGUCCCCGAAGCCAC
1257



Exon 5 SD 1 (pos 6)
ACTCACCGGTGATGAGGACA
758
ACUCACCGGUGAUGAGGACA
1258



Exon 5 SD 2 (pos 5)
CTCACCGGTGATGAGGACAA
759
CUCACCGGUGAUGAGGACAA
1259



Exon 6 SA (pos 6)
CCGGTCTGCGGGAAGGGTAC
760
CCGGUCUGCGGGAAGGGUAC
1260



Exon 6 STOP 1 (pos 8)
TGGGCTGCAGGAGCCGCGGC
761
UGGGCUGCAGGAGCCGCGGC
1261


 


NR4A2
Exon 1 STOP 1 (pos 7)
TTGTACCAAATGCCCCTGTC
762
UUGUACCAAAUGCCCCUGUC
1262



Exon 1 STOP 2 (pos 8)
CGGACAGCAGTCCTCCATTA
763
CGGACAGCAGUCCUCCAUUA
1263



Exon 1 STOP 3 (pos 6)
AGGTGCAGCACAGCCCCATG
764
AGGUGCAGCACAGCCCCAUG
1264



Exon 1 STOP 4 (pos 5)
GGTGCAGCACAGCCCCATGT
765
GGUGCAGCACAGCCCCAUGU
1265



Exon 1 STOP 5 (pos 7)
AGTTGCCAGATGCGCTTCGA
766
AGUUGCCAGAUGCGCUUCGA
1266



Exon 1 STOP 6 (pos 6)
GTTGCCAGATGCGCTTCGAC
767
GUUGCCAGAUGCGCUUCGAC
1267



Exon 1 STOP 7 (pos 5)
GTCTCAGCTGCTCGACACGC
768
GUCUCAGCUGCUCGACACGC
1268



Exon 3 SD (pos 7)
TTCTTACCCTGGAATAGTCC
769
UUCUUACCCUGGAAUAGUCC
1269



Exon 4 SD (pos 5)
ATTACCTGTATGCTAATCGA
770
AUUACCUGUAUGCUAAUCGA
1270



Exon 5 STOP 1 (pos 4)
TTGCAATGCGTTCGTGGCTT
771
UUGCAAUGCGUUCGUGGCUU
1271



Exon 5 SD (pos 6)
ACTGACCTGTGACCATAGCC
772
ACUGACCUGUGACCAUAGCC
1272


 


NR4A3
Exon 2 SA (pos 4)
TATCTGCAGGGACAGAGAAA
773
UAUCUGCAGGGACAGAGAAA
1273



Exon 2 STOP 1 (pos 8)
TGCGGCGCAGACATACAGCT
774
UGCGGCGCAGACAUACAGCU
1274



Exon 2 STOP 2 (pos 6)
CCCCGCAGGCGGGGGCGTTA
775
CCCCGCAGGCGGGGGCGUUA
1275



Exon 3 STOP 1 (pos 4)
TTTCAGAAGTGTCTCAGTGT
776
UUUCAGAAGUGUCUCAGUGU
1276



Exon 5 SD (pos 5)
ATTACCTGATGGAAAGTCTG
777
AUUACCUGAUGGAAAGUCUG
1277



Exon 6 STOP 1 (pos 4)
CTTCAGTGCCTTCGTGGATT
778
CUUCAGUGCCUUCGUGGAUU
1278



Exon 7 SA (pos 4)
TTTCTGCAGAGGGATAGAGA
779
UUUCUGCAGAGGGAUAGAGA
1279



Exon 7 STOP 1 (pos 8)
AGACCACCAGAGTAAGGGAC
780
AGACCACCAGAGUAAGGGAC
1280


 


MCJ
Exon 1 STOP (pos 6)
ACTTGCAGCCCTCGGCCAAA
781
ACUUGCAGCCCUCGGCCAAA
1281


 


FAS
Exon 1 SD (pos 9)
AGGGCTCACCAGAGGTAGGA
782
AGGGCUCACCAGAGGUAGGA
1282



Exon 3 SA (pos 6)
TTCACCTGCCCAAGGAAAAA
783
UUCACCUGCCCAAGGAAAAA
1283



Exon 4 SA (pos 7)
CTAAGCCTAGAAAATCAGTT
784
CUAAGCCUAGAAAAUCAGUU
1284



Exon 5 SA (pos 5)
ACATCTAGAAAAAAAAATAC
785
ACAUCUAGAAAAAAAAAUAC
1285



Exon 5 SD (pos 5)
ATTACCTTCCTCTTTGCACT
786
AUUACCUUCCUCUUUGCACU
1286



Exon 6 SA (pos 5)
GATCCTGTAGGTTGGAACAT
787
GAUCCUGUAGGUUGGAACAU
1287



Exon 6 STOP 1 (pos 4)
AAGCCACCCCAAGTTAGATC
788
AAGCCACCCCAAGUUAGAUC
1288



Exon 6 SD (pos 7)
AACTTACCCCAAACAATTAG
789
AACUUACCCCAAACAAUUAG
1289



Exon 7 SD (pos 8)
ATACCTACAGGATTTAAAGT
790
AUACCUACAGGAUUUAAAGU
1290



Exon 8 SA (pos 8)
GTTTCCTAGAAAGCAAAAAA
791
GUUUCCUAGAAAGCAAAAAA
1291



Exon 9 STOP 1 (pos 6)
AAGTTCAACTGCTTCGTAAT
792
AAGUUCAACUGCUUCGUAAU
1292



Exon 9 STOP (pos 5)
AATTCAGACTATCATCCTCA
793
AAUUCAGACUAUCAUCCUCA
1293


 


SELPG/
Exon1 STOP 1 (pos 6)
GCTTGCAGCTGTGGGACACC
794
GCUUGCAGCUGUGGGACACC
1294


PSGL1
Exon1 STOP 2 (pos 8)
GACCACTCAACCAGTGCCCA
795
GACCACUCAACCAGUGCCCA
1295



Exon1 STOP 3 (pos 8)
GGAGGCACAGACCACTCCAC
796
GGAGGCACAGACCACUCCAC
1296



Exon1 STOP 4 (pos 5)
GGCACAGACAACTCGACTGA
797
GGCACAGACAACUCGACUGA
1297



Exon1 STOP 5 (pos 8)
GGAGGCACAGACCACTCCAC
796
GGAGGCACAGACCACUCCAC
1296



Exon1 STOP 6 (pos 4)
GCACAGACCACTCAACCCAC
798
GCACAGACCACUCAACCCAC
1298



Exon1 STOP 7 (pos 8)
GACCACTCAACCCACAGGCC
799
GACCACUCAACCCACAGGCC
1299



Exon1 STOP 8 (pos 8)
GACCACTCAAACCACAGCCA
800
GACCACUCAAACCACAGCCA
1300



Exon1 STOP 9 (pos 8)
GACCACTCAACCCACAGCCA
801
GACCACUCAACCCACAGCCA
1301



Exon1 STOP 10 (pos 8)
GGAGGCACAGACCACTCCAC
796
GGAGGCACAGACCACUCCAC
1296



Exon1 STOP 11 (pos 8)
GACCACTCAACCAGCAGCCA
802
GACCACUCAACCAGCAGCCA
1302
















TABLE 4B







gRNAs: Splice Site and STOP Codons
















SEQ 
Orien-
Target



Gene
gRNA Name
gRNA target
ID NO:
tatation
Bases(s)
Predicted Outcome
















PDCD1
Ex. 1 SD
CACCTACCTAAGAACCATCC
202
Antisense
C7
Splice donor disruption. GT→AT


 


PDCD1
Ex. 2 SA
GGAGTCTGAGAGATGGAGAG
393
Antisense
C6
Splice acceptor disruption.








AG→AA


 


PDCD1
Ex. 3 SA
TTCTCTCTGGAAGGGCACAA
404
Antisense
C7
Splice acceptor disruption.








AG→AA


 


PDCD1
Ex. 3 SD
GACGTTACCTCGTGCGGCCC
400
Antisense
C8
Splice donor disruption. GT→AT


 


PDCD1
Ex. 4 SA
CCTGCAGAGAAACACACTTG
1303
Antisense
C2
Splice acceptor disruption.








AG→AA


 


PDCD1
Ex. 2 pmSTOP
GGGGTTCCAGGGCCCTGTCTG
203
Antisense
C7, C8
pmSTOP induction: TGG (Trp)→








TAG, TGA, TAA


 


PDCD1
Ex. 3 pmSTOP_1
CAGTTCCAAACCCTGGTGGT
397
Sense
C7
pmSTOP induction: CAA (Gln)→








TAA


 


PDCD1
Ex. 3 pmSTOP_2 
GGACCCAGACTAGCAGCACC
399
Antisense
C5, C5
pmSTOP induction: TGG (Trp)→








TAG, TGA, TAA


 


TRAC
Ex. 1 SD
CTTACCTGGGCTGGGGAAGA
315
Antisense
C5
Splice donor disruption. GT→AT


 


TRAC
Ex. 3 SA
TTCGTATCTGTAAAACCAAG
316
Antisense
C8
Splice acceptor disruption.








AG→AA


 


TRAC
Ex. 3 pmSTOP_1
TTTCCAAAACCTGTCAGTGAT
317
Sense
C4
pmSTOP induction: CAA (Gln)→








TAA


 


TRAC
Ex. 3 pmSTOP_2 
TTCAAAACCTGTCAGTGATT
318
Sense
C3
pmSTOP induction: CAA (Gln)→








TAA









Protospacer Adjacent Motif

The term “protospacer adjacent motif (PAM)” or PAM-like motif refers to a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. In some embodiments, the PAM can be a 5′ PAM (i.e., located upstream of the 5′ end of the protospacer). In other embodiments, the PAM can be a 3′ PAM (i.e., located downstream of the 5′ end of the protospacer). The PAM sequence is essential for target binding, but the exact sequence depends on a type of Cas protein. The PAM sequence can be any PAM sequence known in the art. Suitable PAM sequences include, but are not limited to, NGG, NGA, NGC, NGN, NGT, NGTT, NGCG, NGAG, NGAN, NGNG, NGCN, NGCG, NGTN, NNGRRT, NNNRRT, NNGRR(N), TTTV, TYCV, TYCV, TATV, NNNNGATT, NNAGAAW, or NAAAAC. Y is a pyrimidine; N is any nucleotide base; W is A or T.
A base editor provided herein can comprise a CRISPR protein-derived domain that is capable of binding a nucleotide sequence that contains a canonical or non-canonical protospacer adjacent motif (PAM) sequence. A PAM site is a nucleotide sequence in proximity to a target polynucleotide sequence. Some aspects of the disclosure provide for base editors comprising all or a portion of CRISPR proteins that have different PAM specificities.
For example, typically Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region, where the “N” in “NGG” is adenine (A), thymine (T), guanine (G), or cytosine (C), and the G is guanine. A PAM can be CRISPR protein-specific and can be different between different base editors comprising different CRISPR protein-derived domains. A PAM can be 5′ or 3′ of a target sequence. A PAM can be upstream or downstream of a target sequence. A PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. Often, a PAM is between 2-6 nucleotides in length.
In some embodiments, the PAM is an “NRN” PAM where the “N” in “NRN” is adenine (A), thymine (T), guanine (G), or cytosine (C), and the R is adenine (A) or guanine (G); or the PAM is an “NYN” PAM, wherein the “N” in NYN is adenine (A), thymine (T), guanine (G), or cytosine (C), and the Y is cytidine (C) or thymine (T), for example, as described in R. T. Walton et al., 2020, Science, 10.1126/science.aba8853 (2020), the entire contents of which are incorporated herein by reference.
Several PAM variants are described in Table 5 below.







TABLE 5







Cas9 proteins and corresponding PAM sequences










Variant
PAM







spCas9
NGG



spCas9-VRQR
NGA



spCas9-VRER
NGCG



xCas9 (sp)
NGN



saCas9
NNGRRT



saCas9-KKH
NNNRRT



spCas9-MQKSER
NGCG



spCas9-MQKSER
NGCN



spCas9-LRKIQK
NGTN



spCas9-LRVSQK
NGTN



spCas9-LRVSQL
NGTN



spCas9-MQKFRAER
NGC



Cpf1
5′ (TTTV)



SpyMac
5′-NAA-3′










In some embodiments, the PAM is NGC. In some embodiments, the NGC PAM is recognized by a Cas9 variant. In some embodiments, the NGC PAM variant includes one or more amino acid substitutions selected from D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (collectively termed “MQKFRAER”).
In some embodiments, the PAM is NGT. In some embodiments, the NGT PAM is recognized by a Cas9 variant. In some embodiments, the NGT PAM variant is generated through targeted mutations at one or more residues 1335, 1337, 1135, 1136, 1218, and/or 1219. In some embodiments, the NGT PAM variant is created through targeted mutations at one or more residues 1219, 1335, 1337, 1218. In some embodiments, the NGT PAM variant is created through targeted mutations at one or more residues 1135, 1136, 1218, 1219, and 1335. In some embodiments, the NGT PAM variant is selected from the set of targeted mutations provided in Tables 6A and 6B below.







TABLE 6A







NGT PAM Variant Mutations at residues 1219, 1335, 1337, 1218











Variant
E1219V
R1335Q
T1337
G1218














1
F
V
T



2
F
V
R


3
F
V
Q


4
F
V
L


5
F
V
T
R


6
F
V
R
R


7
F
V
Q
R


8
F
V
L
R


9
L
L
T


10
L
L
R


11
L
L
Q


12
L
L
L


13
F
I
T


14
F
I
R


15
F
I
Q


16
F
I
L


17
F
G
C


18
H
L
N


19
F
G
C
A


20
H
L
N
V


21
L
A
W


22
L
A
F


23
L
A
Y


24
I
A
W


25
I
A
F


26
I
A
Y
















TABLE 6B







NGT PAM Variant Mutations at residues


1135, 1136, 1218, 1219, and 1335












Variant
D1135L
S1136R
G1218S
E1219V
R1335Q















27
G






28
V


29
I


30

A


31

W


32

H


33

K


34


K


35


R


36


Q


37


T


38


N


39



I


40



A


41



N


42



Q


43



G


44



L


45



S


46



T


47




L


48




I


49




V


50




N


51




S


52




T


53




F


54




Y


55
N1286Q
I1331F









In some embodiments, the NGT PAM variant is selected from variant 5, 7, 28, 31, or 36 in Table 6A and Table 6B. In some embodiments, the variants have improved NGT PAM recognition.
In some embodiments, the NGT PAM variants have mutations at residues 1219, 1335, 1337, and/or 1218. In some embodiments, the NGT PAM variant is selected with mutations for improved recognition from the variants provided in Table 7 below.







TABLE 7







NGT PAM Variant Mutations at residues


1219, 1335, 1337, and 1218











Variant
E1219V
R1335Q
T1337
G1218





1
F
V
T



2
F
V
R


3
F
V
Q


4
F
V
L


5
F
V
T
R


6
F
V
R
R


7
F
V
Q
R


8
F
V
L
R









In some embodiments, the NGT PAM is selected from the variants provided in Table 8 below.







TABLE 8







NGT PAM variants
















NGTN variant
D1135
S1136
G1218
E1219
A1322R
R1335
T1337



















Variant 1
LRKIQK
L
R
K
I
—
Q
K


Variant 2
LRSVQK
L
R
S
V
—
Q
K


Variant 3
LRSVQL
L
R
S
V
—
Q
L


Variant 4
LRKIRQK
L
R
K
I
R
Q
K


Variant 5
LRSVRQK
L
R
S
V
R
Q
K


Variant 6
LRSVRQL
L
R
S
V
R
Q
L









In some embodiments the NGTN variant is variant 1. In some embodiments, the NGTN variant is variant 2. In some embodiments, the NGTN variant is variant 3. In some embodiments, the NGTN variant is variant 4. In some embodiments, the NGTN variant is variant 5. In some embodiments, the NGTN variant is variant 6.
In some embodiments, the Cas9 domain is a Cas9 domain from Streptococcus pyogenes (SpCas9). In some embodiments, the SpCas9 domain is a nuclease active SpCas9, a nuclease inactive SpCas9 (SpCas9d), or a SpCas9 nickase (SpCas9n). In some embodiments, the SpCas9 comprises a D9X mutation, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid except for D. In some embodiments, the SpCas9 comprises a D9A mutation, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence having an NGG, a NGA, or a NGCG PAM sequence.
In some embodiments, the SpCas9 domain comprises one or more of a D1135X, a R1335X, and a T1337X mutation, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1135E, R1335Q, and T1337R mutation, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises a D1135E, a R1335Q, and a T1337R mutation, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises one or more of a D1135X, a R1335X, and a T1337X mutation, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1135V, a R1335Q, and a T1337R mutation, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises a D1135V, a R1335Q, and a T1337R mutation, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises one or more of a D1135X, a G1218X, a R1335X, and a T1337X mutation, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1135V, a G1218R, a R1335Q, and a T1337R mutation, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises a D1135V, a G1218R, a R1335Q, and a T1337R mutation, or corresponding mutations in any of the amino acid sequences provided herein.
In some embodiments, the Cas9 domains of any of the fusion proteins provided herein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a Cas9 polypeptide described herein. In some embodiments, the Cas9 domains of any of the fusion proteins provided herein comprises the amino acid sequence of any Cas9 polypeptide described herein. In some embodiments, the Cas9 domains of any of the fusion proteins provided herein consists of the amino acid sequence of any Cas9 polypeptide described herein.
In some examples, a PAM recognized by a CRISPR protein-derived domain of a base editor disclosed herein can be provided to a cell on a separate oligonucleotide to an insert (e.g., an AAV insert) encoding the base editor. In such embodiments, providing PAM on a separate oligonucleotide can allow cleavage of a target sequence that otherwise would not be able to be cleaved, because no adjacent PAM is present on the same polynucleotide as the target sequence.
In an embodiment, S. pyogenes Cas9 (SpCas9) can be used as a CRISPR endonuclease for genome engineering. However, others can be used. In some embodiments, a different endonuclease can be used to target certain genomic targets. In some embodiments, synthetic SpCas9-derived variants with non-NGG PAM sequences can be used. Additionally, other Cas9 orthologues from various species have been identified and these “non-SpCas9s” can bind a variety of PAM sequences that can also be useful for the present disclosure. For example, the relatively large size of SpCas9 (approximately 4kb coding sequence) can lead to plasmids carrying the SpCas9 cDNA that cannot be efficiently expressed in a cell. Conversely, the coding sequence for Staphylococcus aureus Cas9 (SaCas9) is approximately 1 kilobase shorter than SpCas9, possibly allowing it to be efficiently expressed in a cell. Similar to SpCas9, the SaCas9 endonuclease is capable of modifying target genes in mammalian cells in vitro and in mice in vivo. In some embodiments, a Cas protein can target a different PAM sequence. In some embodiments, a target gene can be adjacent to a Cas9 PAM, 5′-NGG, for example. In other embodiments, other Cas9 orthologs can have different PAM requirements. For example, other PAMs such as those of S. thermophilus (5′-NNAGAA for CRISPR1 and 5′-NGGNG for CRISPR3) and Neisseria meningitidis (5′-NNNNGATT) can also be found adjacent to a target gene.
In some embodiments, for a S. pyogenes system, a target gene sequence can precede (i.e., be 5′ to) a 5′-NGG PAM, and a 20-nt guide RNA sequence can base pair with an opposite strand to mediate a Cas9 cleavage adjacent to a PAM. In some embodiments, an adjacent cut can be or can be about 3 base pairs upstream of a PAM. In some embodiments, an adjacent cut can be or can be about 10 base pairs upstream of a PAM. In some embodiments, an adjacent cut can be or can be about 0-20 base pairs upstream of a PAM. For example, an adjacent cut can be next to, 1, 2,3,4,5,6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs upstream of a PAM. An adjacent cut can also be downstream of a PAM by 1 to 30 base pairs. The sequences of exemplary SpCas9 proteins capable of binding a PAM sequence follow:
The amino acid sequence of an exemplary PAM-binding SpCas9 is as follows:








MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL



 


KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY


 


HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY


 


NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNE


 


DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS


 


MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD


 


GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI


 


PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS


 


LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD


 


SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA


 


HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTE


 


KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ


 


TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR


 


LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK


 


FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS


 


KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK


 


SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS


 


MPQVNTVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG


 


KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS


 


AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV


 


ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD


 


ATLIHQSITGLYETRIDLSQLGGD






The amino acid sequence of an exemplary PAM-binding SpCas9n is as follows:








MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL



 


KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY


 


HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY


 


NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF


 


DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS


 


MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD


 


GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI


 


PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS


 


LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD


 


SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA


 


HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF


 


KEDIQKAQVSGQGDSLHEHIANLAGSPALKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ


 


TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR


 


LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK


 


FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS


 


KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK


 


SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS


 


MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG


 


KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS


 


AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLEVEQHKHYLDEIIEQISEFSKRV


 


ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD


 


ATLIHQSITGLYETRIDLSQLGGD






The amino acid sequence of an exemplary PAM-binding SpEQR Cas9 is as follows:








MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL



 


KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESVLVEEDKKHERHPIFGNIVDEVAY


 


HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY


 


NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNE


 


DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS


 


MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD


 


GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI


 


PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS


 


LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD


 


SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA


 


HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF


 


KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ


 


TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR


 


LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK


 


FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS


 


KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK


 


SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS


 


MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFESPTVAYSVLVVAKVEKG


 


KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS


 


AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLHVEQHKHYLDEIIEQISEFSKRV


 


ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLD


 


ATLIHQSITGLYETRIDLSQLGGD






In the above sequence, residues E1135, Q1335, and R1337, which can be mutated from D1135, R1335, and T1337 to yield a SpEQR Cas9, are underlined and in bold.
The amino acid sequence of an exemplary PAM-binding SpVQR Cas9 is as follows:








MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSTKKNLIGALLFDSGETAEATRL



 


KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY


 


HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY


 


NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNE


 


DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS


 


MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD


 


GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI


 


PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS


 


LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD


 


SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA


 


HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF


 


KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ


 


TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR


 


LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK


 


FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS


 


KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK


 


SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS


 


MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGEVSPTVAYSVLVVAKVEKG


 


KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS


 


AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV


 


ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLD


 


ATLIHQSITGLYETRIDLSQLGGD






In the above sequence, residues V1135, Q1335, and R1337, which can be mutated from D1135, R1335, and T1337 to yield a SpVQR Cas9, are underlined and in bold.
The amino acid sequence of an exemplary PAM-binding SpVRER Cas9 is as follows:








MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL



 


KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY


 


HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY


 


NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNE


 


DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS


 


MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD


 


GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI


 


PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS


 


LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD


 


SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA


 


HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF


 


KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ


 


TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR


 


LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK


 


FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS


 


KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK


 


SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS


 


MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKG


 


KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS


 


ARELQKGNELALPSKYVNFLILASHYEKLKGSPEDNEQKQLHVEQHKHYLDEIIEQISEFSKRV


 


ILADANLDKVLSAYNKHRDKPIREQAENIIHLETLTNLGAPAAFKYFDTTIDRKEYRSTKEVLD


 


ATLIHQSITGLYETRIDLSQLGGD.






In the above sequence, residues V1135, R1218, Q1335, and R1337, which can be mutated from D1135, G1218, R1335, and T1337 to yield a SpVRER Cas9, are underlined and in bold.
In some embodiments, engineered SpCas9 variants are capable of recognizing protospacer adjacent motif (PAM) sequences flanked by a 3′ H (non-G PAM) (see Tables 3A-3D). In some embodiments, the SpCas9 variants recognize NRNH PAMs (where R is A or G and H is A, C or T). In some embodiments, the non-G PAM is NRRH, NRTH, or NRCH (see e.g., Miller, S. M., et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs, Nat. Biotechnol. (2020), the contents of which is incorporated herein by reference in its entirety).
In some embodiments, the Cas9 domain is a recombinant Cas9 domain. In some embodiments, the recombinant Cas9 domain is a SpyMacCas9 domain. In some embodiments, the SpyMacCas9 domain is a nuclease active SpyMacCas9, a nuclease inactive SpyMacCas9 (SpyMacCas9d), or a SpyMacCas9 nickase (SpyMacCas9n). In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SpyMacCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence having a NAA PAM sequence.
The sequence of an exemplary Cas9 A homolog of Spy Cas9 in Streptococcus macacae with native 5′-NAAN-3′ PAM specificity is known in the art and described, for example, by Jakimo et al., (www.biorxiv.org/content/biorxiv/early/2018/09/27/429654.full.pdf), and is provided below.








SpyMacCas9



MDKKYSLGLDLGTNSVGWAVLTDDYKVPSKKHKVLGNTDRHSIKKNLLGALLEGSGETAE


 


ATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG


 


NIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSD


 


VDKLFTQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGN


 


LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA


 


GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH


 


AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE


 


VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL


 


SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYAHLFDDKVMKQLKRRRYTGWG


 


RLSRKLINGIRDKQSGKTILDELKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGHSL


 


HEQIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTQKGQKNSRERM


 


KRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHI


 


VPQSFTKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT


 


KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK


 


LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM


 


IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIKKRPLIETNGETGEIVWDKGRDHA


 


TVRKVLSMPQVNIVKKTEIQTVGQNGGLFDDNPKSPLEVTPSKLVPLKKELNPKKYGGYQ


 


KPTTAYPVLLITDTKQLIPISVMNKKQFEQNPVKFLRDRGYQQVGKNDFIKLPKYTLVDI


 


GDGIKRLWASSKEIHKGNQLVVSKKSQILLYHAHHLDSDLSNDYLQNHNQQFDVLFNEII


 


SFSKKCKLGKEHIQKIENVYSNKKNSASIEELAESFIKLLGFTQLGATSPFNFLGVKLNQ


 


KQYKGKKDYILPCTEGTLIRQSITGLYETRVDLSKIGED.






In some embodiments, a variant Cas9 protein harbors, H840A, P475A, W476A, N477A, D1125A, W1126A, and D1218A mutations such that the polypeptide has a reduced ability to cleave a target DNA or RNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA). As another non-limiting example, in some embodiments, the variant Cas9 protein harbors D10A, H840A, P475A, W476A, N477A, D1125A, W1126A, and D1218A mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA). In some embodiments, when a variant Cas9 protein harbors W476A and WI 126A mutations or when the variant Cas9 protein harbors P475A, W476A, N477A, D1125A, W1126A, and D1218A mutations, the variant Cas9 protein does not bind efficiently to a PAM sequence. Thus, in some such cases, when such a variant Cas9 protein is used in a method of binding, the method does not require a PAM sequence. In other words, in some embodiments, when such a variant Cas9 protein is used in a method of binding, the method can include a guide RNA, but the method can be performed in the absence of a PAM sequence (and the specificity of binding is therefore provided by the targeting segment of the guide RNA). Other residues can be mutated to achieve the above effects (i.e., inactivate one or the other nuclease portions). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987 can be altered (i.e., substituted). Also, mutations other than alanine substitutions are suitable.
In some embodiments, a CRISPR protein-derived domain of a base editor can comprise all or a portion of a Cas9 protein with a canonical PAM sequence (NGG). In other embodiments, a Cas9-derived domain of a base editor can employ a non-canonical PAM sequence. Such sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); R. T. Walton et al. “Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants” Science 10.1126/science.aba8853 (2020); Hu et al. “Evolved Cas9 variants with broad PAM compatibility and high DNA specificity,” Nature, 2018 Apr. 5, 556(7699), 57-63; Miller et al., “Continuous evolution of SpCas9 variants compatible with non-G PAMs” Nat. Biotechnol., 2020 April; 38(4):471-481; the entire contents of each are hereby incorporated by reference.
Cas9 Domains with Reduced Exclusivity
Typically, Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region, where the “N” in “NGG” is adenosine (A), thymidine (T), or cytosine (C), and the G is guanosine. This may limit the ability to edit desired bases within a genome. In some embodiments, the base editing fusion proteins provided herein may need to be placed at a precise location, for example a region comprising a target base that is upstream of the PAM. See e.g., Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016), the entire contents of which are hereby incorporated by reference. Accordingly, in some embodiments, any of the fusion proteins provided herein may contain a Cas9 domain that is capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference.
Fusion Proteins with Internal Insertions
Provided herein are fusion proteins comprising a heterologous polypeptide fused to a nucleic acid programmable nucleic acid binding protein, for example, a napDNAbp. A heterologous polypeptide can be a polypeptide that is not found in the native or wild-type napDNAbp polypeptide sequence. The heterologous polypeptide can be fused to the napDNAbp at a C-terminal end of the napDNAbp, an N-terminal end of the napDNAbp, or inserted at an internal location of the napDNAbp. In some embodiments, the heterologous polypeptide is inserted at an internal location of the napDNAbp. In some embodiments, the heterologous polypeptide is a deaminase or a functional fragment thereof. For example, a fusion protein can comprise a deaminase flanked by an N-terminal fragment and a C-terminal fragment of a Cas9 or Cas12 (e.g., Cas12b/C2c1), polypeptide. The deaminase in a fusion protein can be a cytidine deaminase. In some embodiments, the cytidine deaminase is an APOBEC deaminase (e.g., APOBEC1). The deaminase in a fusion protein can be an adenosine deaminase. In some embodiments, the adenosine deaminase is a TadA (e.g., TadA*7.10 or TadA*8). In some embodiments, the TadA is a TadA*8 or a TadA*9. TadA sequences (e.g., TadA7.10 or TadA*8) as described herein are suitable deaminases for the above-described fusion proteins.
In some embodiments, the fusion protein comprises the structure:

    
    
        NH2-[N-terminal fragment of a napDNAbp]-[deaminase]-[C-terminal fragment of a napDNAbp]-COOH;
        NH2-[N-terminal fragment of a Cas9]-[adenosine deaminase]-[C-terminal fragment of a Cas9]-COOH;
        NH2-[N-terminal fragment of a Cas12]-[adenosine deaminase]-[C-terminal fragment of a Cas12]-COOH;
        NH2-[N-terminal fragment of a Cas9]-[cytidine deaminase]-[C-terminal fragment of a Cas9]-COOH;
        NH2-[N-terminal fragment of a Cas12]-[cytidine deaminase]-[C-terminal fragment of a Cas12]-COOH;
        wherein each instance of “]-[” is an optional linker.
    
    


The deaminase can be a circular permutant deaminase. For example, the deaminase can be a circular permutant adenosine deaminase. In some embodiments, the deaminase is a circular permutant TadA, circularly permutated at amino acid residue 116 as numbered in the TadA reference sequence. In some embodiments, the deaminase is a circular permutant TadA, circularly permutated at amino acid residue 136 as numbered in the TadA reference sequence. In some embodiments, the deaminase is a circular permutant TadA, circularly permutated at amino acid residue 65 as numbered in the TadA reference sequence.
The fusion protein can comprise more than one deaminase. The fusion protein can comprise, for example, 1, 2, 3, 4, 5 or more deaminases. In some embodiments, the fusion protein comprises one deaminase. In some embodiments, the fusion protein comprises two deaminases. The two or more deaminases in a fusion protein can be an adenosine deaminase. cytidine deaminase, or a combination thereof. The two or more deaminases can be homodimers. The two or more deaminases can be heterodimers. The two or more deaminases can be inserted in tandem in the napDNAbp. In some embodiments, the two or more deaminases may not be in tandem in the napDNAbp.
In some embodiments, the napDNAbp in the fusion protein is a Cas9 polypeptide or a fragment thereof. The Cas9 polypeptide can be a variant Cas9 polypeptide. In some embodiments, the Cas9 polypeptide is a Cas9 nickase (nCas9) polypeptide or a fragment thereof. In some embodiments, the Cas9 polypeptide is a nuclease dead Cas9 (dCas9) polypeptide or a fragment thereof. The Cas9 polypeptide in a fusion protein can be a full-length Cas9 polypeptide. In some cases, the Cas9 polypeptide in a fusion protein may not be a full length Cas9 polypeptide. The Cas9 polypeptide can be truncated, for example, at a N-terminal or C-terminal end relative to a naturally-occurring Cas9 protein. The Cas9 polypeptide can be a circularly permuted Cas9 protein. The Cas9 polypeptide can be a fragment, a portion, or a domain of a Cas9 polypeptide, that is still capable of binding the target polynucleotide and a guide nucleic acid sequence.
In some embodiments, the Cas9 polypeptide is a Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), or fragments or variants thereof.
The Cas9 polypeptide of a fusion protein can comprise an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring Cas9 polypeptide.
The Cas9 polypeptide of a fusion protein can comprise an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the Cas9 amino acid sequence set forth below (called the “Cas9 reference sequence” below): MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQTLDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 223) (single underline: HNH domain; double underline: RuvC domain).
Fusion proteins comprising a heterologous catalytic domain flanked by N- and C-terminal fragments of a Cas9 polypeptide are also useful for base editing in the methods as described herein. Fusion proteins comprising Cas9 and one or more deaminase domains, e.g., adenosine deaminase, or comprising an adenosine deaminase domain flanked by Cas9 sequences are also useful for highly specific and efficient base editing of target sequences. In an embodiment, a chimeric Cas9 fusion protein contains a heterologous catalytic domain (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) inserted within a Cas9 polypeptide. In some embodiments, the fusion protein comprises an adenosine deaminase domain and a cytidine deaminase domain inserted within a Cas9. In some embodiments, an adenosine deaminase is fused within a Cas9 and a cytidine deaminase is fused to the C-terminus. In some embodiments, an adenosine deaminase is fused within Cas9 and a cytidine deaminase fused to the N-terminus. In some embodiments, a cytidine deaminase is fused within Cas9 and an adenosine deaminase is fused to the C-terminus. In some embodiments, a cytidine deaminase is fused within Cas9 and an adenosine deaminase fused to the N-terminus.
Exemplary structures of a fusion protein with an adenosine deaminase and a cytidine deaminase and a Cas9 are provided as follows:

    
    
        NH2-[Cas9(adenosine deaminase)]-[cytidine deaminase]-COOH;
        NH2-[cytidine deaminase]-[Cas9(adenosine deaminase)]-COOH;
        NH2-[Cas9(cytidine deaminase)]-[adenosine deaminase]-COOH; or
        NH2-[adenosine deaminase]-[Cas9(cytidine deaminase)]-COOH.
    
    


In some embodiments, the “-” used in the general architecture above indicates the presence of an optional linker.
In various embodiments, the catalytic domain has DNA modifying activity (e.g., deaminase activity), such as adenosine deaminase activity. In some embodiments, the adenosine deaminase is a TadA (e.g., TadA*7.10). In some embodiments, the TadA is a TadA*8. In some embodiments, a TadA*8 is fused within Cas9 and a cytidine deaminase is fused to the C-terminus. In some embodiments, a TadA*8 is fused within Cas9 and a cytidine deaminase fused to the N-terminus. In some embodiments, a cytidine deaminase is fused within Cas9 and a TadA*8 is fused to the C-terminus. In some embodiments, a cytidine deaminase is fused within Cas9 and a TadA*8 fused to the N-terminus. Exemplary structures of a fusion protein with a TadA*8 and a cytidine deaminase and a Cas9 are provided as follows:

    
    
        NH2-[Cas9(TadA*8)]-[cytidine deaminase]-COOH;
        NH2-[cytidine deaminase]-[Cas9(TadA*8)]-COOH;
        NH2-[Cas9(cytidine deaminase)]-[TadA*8]-COOH; or
        NH2-[TadA*8]-[Cas9(cytidine deaminase)]-COOH.
    
    


In some embodiments, the “-” used in the general architecture above indicates the presence of an optional linker.
The heterologous polypeptide (e.g., deaminase) can be inserted in the napDNAbp (e.g., Cas9 or Cas12 (e.g., Cas12b/C2c1)) at a suitable location, for example, such that the napDNAbp retains its ability to bind the target polynucleotide and a guide nucleic acid. A deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) can be inserted into a napDNAbp without compromising function of the deaminase (e.g., base editing activity) or the napDNAbp (e.g., ability to bind to target nucleic acid and guide nucleic acid). A deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) can be inserted in the napDNAbp at, for example, a disordered region or a region comprising a high temperature factor or B-factor as shown by crystallographic studies. Regions of a protein that are less ordered, disordered, or unstructured, for example solvent exposed regions and loops, can be used for insertion without compromising structure or function. A deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase)can be inserted in the napDNAbp in a flexible loop region or a solvent-exposed region. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted in a flexible loop of the Cas9 or the Cas12b/C2c1 polypeptide.
In some embodiments, the insertion location of a deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is determined by B-factor analysis of the crystal structure of Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted in regions of the Cas9 polypeptide comprising higher than average B-factors (e.g., higher B factors compared to the total protein or the protein domain comprising the disordered region). B-factor or temperature factor can indicate the fluctuation of atoms from their average position (for example, as a result of temperature-dependent atomic vibrations or static disorder in a crystal lattice). A high B-factor (e.g., higher than average B-factor) for backbone atoms can be indicative of a region with relatively high local mobility. Such a region can be used for inserting a deaminase without compromising structure or function. A deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) can be inserted at a location with a residue having a Ca atom with a B-factor that is 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or greater than 200% more than the average B-factor for the total protein. A deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) can be inserted at a location with a residue having a Ca atom with a B-factor that is 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%,200% or greater than 200% more than the average B-factor for a Cas9 protein domain comprising the residue. Cas9 polypeptide positions comprising a higher than average B-factor can include, for example, residues 768, 792, 1052, 1015, 1022, 1026, 1029, 1067, 1040, 1054, 1068, 1246, 1247, and 1248 as numbered in the above Cas9 reference sequence. Cas9 polypeptide regions comprising a higher than average B-factor can include, for example, residues 792-872, 792-906, and 2-791 as numbered in the above Cas9 reference sequence.
A heterologous polypeptide (e.g., deaminase) can be inserted in the napDNAbp at an amino acid residue selected from the group consisting of 768, 791, 792, 1015, 1016, 1022, 1023, 1026, 1029, 1040, 1052, 1054, 1067, 1068, 1069, 1246, 1247, and 1248 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the heterologous polypeptide is inserted between amino acid positions 768-769, 791-792, 792-793, 1015-1016, 1022-1023, 1026-1027, 1029-1030, 1040-1041, 1052-1053, 1054-1055, 1067-1068, 1068-1069, 1247-1248, or 1248-1249 as numbered in the above Cas9 reference sequence or corresponding amino acid positions thereof. In some embodiments, the heterologous polypeptide is inserted between amino acid positions 769-770, 792-793, 793-794, 1016-1017, 1023-1024, 1027-1028, 1030-1031, 1041-1042, 1053-1054, 1055-1056, 1068-1069, 1069-1070, 1248-1249, or 1249-1250 as numbered in the above Cas9 reference sequence or corresponding amino acid positions thereof. In some embodiments, the heterologous polypeptide replaces an amino acid residue selected from the group consisting of 768, 791, 792, 1015, 1016, 1022, 1023, 1026, 1029, 1040, 1052, 1054, 1067, 1068, 1069, 1246, 1247, and 1248 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. It should be understood that the reference to the above Cas9 reference sequence with respect to insertion positions is for illustrative purposes. The insertions as discussed herein are not limited to the Cas9 polypeptide sequence of the above Cas9 reference sequence, but include insertion at corresponding locations in variant Cas9 polypeptides, for example a Cas9 nickase (nCas9), nuclease dead Cas9 (dCas9), a Cas9 variant lacking a nuclease domain, a truncated Cas9, or a Cas9 domain lacking partial or complete HNH domain.
A heterologous polypeptide (e.g., deaminase) can be inserted in the napDNAbp at an amino acid residue selected from the group consisting of: 768, 792, 1022, 1026, 1040, 1068, and 1247 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the heterologous polypeptide is inserted between amino acid positions 768-769, 792-793, 1022-1023, 1026-1027, 1029-1030, 1040-1041, 1068-1069, or 1247-1248 as numbered in the above Cas9 reference sequence or corresponding amino acid positions thereof. In some embodiments, the heterologous polypeptide is inserted between amino acid positions 769-770, 793-794, 1023-1024, 1027-1028, 1030-1031, 1041-1042, 1069-1070, or 1248-1249 as numbered in the above Cas9 reference sequence or corresponding amino acid positions thereof. In some embodiments, the heterologous polypeptide replaces an amino acid residue selected from the group consisting of: 768, 792, 1022, 1026, 1040, 1068, and 1247 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.
A heterologous polypeptide (e.g., deaminase) can be inserted in the napDNAbp at an amino acid residue as described herein, or a corresponding amino acid residue in another Cas9 polypeptide. In an embodiment, a heterologous polypeptide (e.g., deaminase) can be inserted in the napDNAbp at an amino acid residue selected from the group consisting of: 1002, 1003, 1025, 1052-1056, 1242-1247, 1061-1077, 943-947, 686-691, 569-578, 530-539, and 1060-1077 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. The deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) can be inserted at the N-terminus or the C-terminus of the residue or replace the residue. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the C-terminus of the residue.
In some embodiments, an adenosine deaminase (e.g., TadA) is inserted at an amino acid residue selected from the group consisting of: 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, an adenosine deaminase (e.g., TadA) is inserted in place of residues 792-872, 792-906, or 2-791 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the adenosine deaminase is inserted at the N-terminus of an amino acid selected from the group consisting of: 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the adenosine deaminase is inserted at the C-terminus of an amino acid selected from the group consisting of: 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the adenosine deaminase is inserted to replace an amino acid selected from the group consisting of: 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.
In some embodiments, a cytidine deaminase (e.g., APOBEC1) is inserted at an amino acid residue selected from the group consisting of: 1016, 1023, 1029, 1040, 1069, and 1247 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the cytidine deaminase is inserted at the N-terminus of an amino acid selected from the group consisting of: 1016, 1023, 1029, 1040, 1069, and 1247 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the cytidine deaminase is inserted at the C-terminus of an amino acid selected from the group consisting of: 1016, 1023, 1029, 1040, 1069, and 1247 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the cytidine deaminase is inserted to replace an amino acid selected from the group consisting of: 1016, 1023, 1029, 1040, 1069, and 1247 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.
In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 768 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the N-terminus of amino acid residue 768 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the C-terminus of amino acid residue 768 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid residue 768 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.
In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 791 or is inserted at amino acid residue 792, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the N-terminus of amino acid residue 791 or is inserted at the N-terminus of amino acid 792, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the C-terminus of amino acid 791 or is inserted at the N-terminus of amino acid 792, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid 791, or is inserted to replace amino acid 792, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.
In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 1016 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the N-terminus of amino acid residue 1016 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the C-terminus of amino acid residue 1016 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid residue 1016 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.
In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 1022, or is inserted at amino acid residue 1023, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the N-terminus of amino acid residue 1022 or is inserted at the N-terminus of amino acid residue 1023, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the C-terminus of amino acid residue 1022 or is inserted at the C-terminus of amino acid residue 1023, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid residue 1022, or is inserted to replace amino acid residue 1023, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.
In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 1026, or is inserted at amino acid residue 1029, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the N-terminus of amino acid residue 1026 or is inserted at the N-terminus of amino acid residue 1029, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the C-terminus of amino acid residue 1026 or is inserted at the C-terminus of amino acid residue 1029, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid residue 1026, or is inserted to replace amino acid residue 1029, as numbered in the above Cas9 reference sequence, or corresponding amino acid residue in another Cas9 polypeptide.
In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 1040 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the N-terminus of amino acid residue 1040 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the C-terminus of amino acid residue 1040 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid residue 1040 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.
In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 1052, or is inserted at amino acid residue 1054, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the N-terminus of amino acid residue 1052 or is inserted at the N-terminus of amino acid residue 1054, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the C-terminus of amino acid residue 1052 or is inserted at the C-terminus of amino acid residue 1054, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid residue 1052, or is inserted to replace amino acid residue 1054, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.
In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 1067, or is inserted at amino acid residue 1068, or is inserted at amino acid residue 1069, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the N-terminus of amino acid residue 1067 or is inserted at the N-terminus of amino acid residue 1068 or is inserted at the N-terminus of amino acid residue 1069, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the C-terminus of amino acid residue 1067 or is inserted at the C-terminus of amino acid residue 1068 or is inserted at the C-terminus of amino acid residue 1069, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid residue 1067, or is inserted to replace amino acid residue 1068, or is inserted to replace amino acid residue 1069, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.
In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at amino acid residue 1246, or is inserted at amino acid residue 1247, or is inserted at amino acid residue 1248, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the N-terminus of amino acid residue 1246 or is inserted at the N-terminus of amino acid residue 1247 or is inserted at the N-terminus of amino acid residue 1248, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted at the C-terminus of amino acid residue 1246 or is inserted at the C-terminus of amino acid residue 1247 or is inserted at the C-terminus of amino acid residue 1248, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted to replace amino acid residue 1246, or is inserted to replace amino acid residue 1247, or is inserted to replace amino acid residue 1248, as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.
In some embodiments, a heterologous polypeptide (e.g., deaminase) is inserted in a flexible loop of a Cas9 polypeptide. The flexible loop portions can be selected from the group consisting of 530-537, 569-570, 686-691, 943-947, 1002-1025, 1052-1077, 1232-1247, or 1298-1300 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. The flexible loop portions can be selected from the group consisting of: 1-529, 538-568, 580-685, 692-942, 948-1001, 1026-1051, 1078-1231, or 1248-1297 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.
A heterologous polypeptide (e.g., adenine deaminase) can be inserted into a Cas9 polypeptide region corresponding to amino acid residues: 1017-1069, 1242-1247, 1052-1056, 1060-1077, 1002-1003, 943-947, 530-537, 568-579, 686-691, 1242-1247, 1298-1300, 1066-1077, 1052-1056, or 1060-1077 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.
A heterologous polypeptide (e.g., adenine deaminase) can be inserted in place of a deleted region of a Cas9 polypeptide. The deleted region can correspond to an N-terminal or C-terminal portion of the Cas9 polypeptide. In some embodiments, the deleted region corresponds to residues 792-872 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deleted region corresponds to residues 792-906 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deleted region corresponds to residues 2-791 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deleted region corresponds to residues 1017-1069 as numbered in the above Cas9 reference sequence, or corresponding amino acid residues thereof.
Exemplary internal fusions base editors are provided in Table 9 below:







TABLE 9







Insertion loci in Cas9 proteins









BE ID
Modification
Other ID





IBE001
Cas9 TadA ins 1015
ISLAY01


IBE002
Cas9 TadA ins 1022
ISLAY02


IBE003
Cas9 TadA ins 1029
ISLAY03


IBE004
Cas9 TadA ins 1040
ISLAY04


IBE005
Cas9 TadA ins 1068
ISLAY05


IBE006
Cas9 TadA ins 1247
ISLAY06


IBE007
Cas9 TadA ins 1054
ISLAY07


IBE008
Cas9 TadA ins 1026
ISLAY08


IBE009
Cas9 TadA ins 768
ISLAY09


IBE020
delta HNH TadA 792
ISLAY20


IBE021
N-term fusion single TadA helix truncated 165-end
ISLAY21


IBE029
TadA-Circular Permutant116 ins1067
ISLAY29


IBE031
TadA- Circular Permutant 136 ins1248
ISLAY31


IBE032
TadA- Circular Permutant 136ins 1052
ISLAY32


IBE035
delta 792-872 TadA ins
ISLAY35


IBE036
delta 792-906 TadA ins
ISLAY36


IBE043
TadA-Circular Permutant 65 ins1246
ISLAY43


IBE044
TadA ins C-term truncate 2791
ISLAY44









A heterologous polypeptide (e.g., deaminase) can be inserted within a structural or functional domain of a Cas9 polypeptide. A heterologous polypeptide (e.g., deaminase) can be inserted between two structural or functional domains of a Cas9 polypeptide. A heterologous polypeptide (e.g., deaminase) can be inserted in place of a structural or functional domain of a Cas9 polypeptide, for example, after deleting the domain from the Cas9 polypeptide. The structural or functional domains of a Cas9 polypeptide can include, for example, RuvC I, RuvC II, RuvC III, Rec1, Rec2, PI, or HNH.
In some embodiments, the Cas9 polypeptide lacks one or more domains selected from the group consisting of: RuvC I, RuvC II, RuvC III, Rec1, Rec2, PI, or HNH domain. In some embodiments, the Cas9 polypeptide lacks a nuclease domain. In some embodiments, the Cas9 polypeptide lacks an HNH domain. In some embodiments, the Cas9 polypeptide lacks a portion of the HNH domain such that the Cas9 polypeptide has reduced or abolished HNH activity. In some embodiments, the Cas9 polypeptide comprises a deletion of the nuclease domain, and the deaminase is inserted to replace the nuclease domain. In some embodiments, the HNH domain is deleted and the deaminase is inserted in its place. In some embodiments, one or more of the RuvC domains is deleted and the deaminase is inserted in its place.
A fusion protein comprising a heterologous polypeptide can be flanked by a N-terminal and a C-terminal fragment of a napDNAbp. In some embodiments, the fusion protein comprises a deaminase flanked by a N-terminal fragment and a C-terminal fragment of a Cas9 polypeptide. The N terminal fragment or the C terminal fragment can bind the target polynucleotide sequence. The C-terminus of the N terminal fragment or the N-terminus of the C terminal fragment can comprise a part of a flexible loop of a Cas9 polypeptide. The C-terminus of the N terminal fragment or the N-terminus of the C terminal fragment can comprise a part of an alpha-helix structure of the Cas9 polypeptide. The N-terminal fragment or the C-terminal fragment can comprise a DNA binding domain. The N-terminal fragment or the C-terminal fragment can comprise a RuvC domain. The N-terminal fragment or the C-terminal fragment can comprise an HNH domain. In some embodiments, neither of the N-terminal fragment and the C-terminal fragment comprises an HNH domain.
In some embodiments, the C-terminus of the N terminal Cas9 fragment comprises an amino acid that is in proximity to a target nucleobase when the fusion protein deaminates the target nucleobase. In some embodiments, the N-terminus of the C terminal Cas9 fragment comprises an amino acid that is in proximity to a target nucleobase when the fusion protein deaminates the target nucleobase. The insertion location of different deaminases can be different in order to have proximity between the target nucleobase and an amino acid in the C-terminus of the N terminal Cas9 fragment or the N-terminus of the C terminal Cas9 fragment. For example, the insertion position of an deaminase can be at an amino acid residue selected from the group consisting of: 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.
The N-terminal Cas9 fragment of a fusion protein (i.e. the N-terminal Cas9 fragment flanking the deaminase in a fusion protein) can comprise the N-terminus of a Cas9 polypeptide. The N-terminal Cas9 fragment of a fusion protein can comprise a length of at least about: 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids. The N-terminal Cas9 fragment of a fusion protein can comprise a sequence corresponding to amino acid residues: 1-56, 1-95, 1-200, 1-300, 1-400, 1-500, 1-600, 1-700, 1-718, 1-765, 1-780, 1-906, 1-918, or 1-1100 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. The N-terminal Cas9 fragment can comprise a sequence comprising at least: 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to amino acid residues: 1-56, 1-95, 1-200, 1-300, 1-400, 1-500, 1-600, 1-700, 1-718, 1-765, 1-780, 1-906, 1-918, or 1-1100 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.
The C-terminal Cas9 fragment of a fusion protein (i.e. the C-terminal Cas9 fragment flanking the deaminase in a fusion protein) can comprise the C-terminus of a Cas9 polypeptide. The C-terminal Cas9 fragment of a fusion protein can comprise a length of at least about: 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids. The C-terminal Cas9 fragment of a fusion protein can comprise a sequence corresponding to amino acid residues: 1099-1368, 918-1368, 906-1368, 780-1368, 765-1368, 718-1368, 94-1368, or 56-1368 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide. The N-terminal Cas9 fragment can comprise a sequence comprising at least: 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to amino acid residues: 1099-1368, 918-1368, 906-1368, 780-1368, 765-1368, 718-1368, 94-1368, or 56-1368 as numbered in the above Cas9 reference sequence, or a corresponding amino acid residue in another Cas9 polypeptide.
The N-terminal Cas9 fragment and C-terminal Cas9 fragment of a fusion protein taken together may not correspond to a full-length naturally occurring Cas9 polypeptide sequence, for example, as set forth in the above Cas9 reference sequence.
The fusion protein described herein can effect targeted deamination with reduced deamination at non-target sites (e.g., off-target sites), such as reduced genome wide spurious deamination. The fusion protein described herein can effect targeted deamination with reduced bystander deamination at non-target sites. The undesired deamination or off-target deamination can be reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% compared with, for example, an end terminus fusion protein comprising the deaminase fused to a N terminus or a C terminus of a Cas9 polypeptide. The undesired deamination or off-target deamination can be reduced by at least one-fold, at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least tenfold, at least fifteen fold, at least twenty fold, at least thirty fold, at least forty fold, at least fifty fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, or at least hundred fold, compared with, for example, an end terminus fusion protein comprising the deaminase fused to a N terminus or a C terminus of a Cas9 polypeptide.
In some embodiments, the deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) of the fusion protein deaminates no more than two nucleobases within the range of an R-loop. In some embodiments, the deaminase of the fusion protein deaminates no more than three nucleobases within the range of the R-loop. In some embodiments, the deaminase of the fusion protein deaminates no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobases within the range of the R-loop. An R-loop is a three-stranded nucleic acid structure including a DNA:RNA hybrid, a DNA:DNA or an RNA: RNA complementary structure and the associated with single-stranded DNA. As used herein, an R-loop may be formed when a target polynucleotide is contacted with a CRISPR complex or a base editing complex, wherein a portion of a guide polynucleotide, e.g. a guide RNA, hybridizes with and displaces with a portion of a target polynucleotide, e.g. a target DNA. In some embodiments, an R-loop comprises a hybridized region of a spacer sequence and a target DNA complementary sequence. An R-loop region may be of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobase pairs in length. In some embodiments, the R-loop region is about 20 nucleobase pairs in length. It should be understood that, as used herein, an R-loop region is not limited to the target DNA strand that hybridizes with the guide polynucleotide. For example, editing of a target nucleobase within an R-loop region may be to a DNA strand that comprises the complementary strand to a guide RNA, or may be to a DNA strand that is the opposing strand of the strand complementary to the guide RNA. In some embodiments, editing in the region of the R-loop comprises editing a nucleobase on non-complementary strand (protospacer strand) to a guide RNA in a target DNA sequence.
The fusion protein described herein can effect target deamination in an editing window different from canonical base editing. In some embodiments, a target nucleobase is from about 1 to about 20 bases upstream of a PAM sequence in the target polynucleotide sequence. In some embodiments, a target nucleobase is from about 2 to about 12 bases upstream of a PAM sequence in the target polynucleotide sequence. In some embodiments, a target nucleobase is from about 1 to 9 base pairs, about 2 to 10 base pairs, about 3 to 11 base pairs, about 4 to 12 base pairs, about 5 to 13 base pairs, about 6 to 14 base pairs, about 7 to 15 base pairs, about 8 to 16 base pairs, about 9 to 17 base pairs, about 10 to 18 base pairs, about 11 to 19 base pairs, about 12 to 20 base pairs, about 1 to 7 base pairs, about 2 to 8 base pairs, about 3 to 9 base pairs, about 4 to 10 base pairs, about 5 to 11 base pairs, about 6 to 12 base pairs, about 7 to 13 base pairs, about 8 to 14 base pairs, about 9 to 15 base pairs, about 10 to 16 base pairs, about 11 to 17 base pairs, about 12 to 18 base pairs, about 13 to 19 base pairs, about 14 to 20 base pairs, about 1 to 5 base pairs, about 2 to 6 base pairs, about 3 to 7 base pairs, about 4 to 8 base pairs, about 5 to 9 base pairs, about 6 to 10 base pairs, about 7 to 11 base pairs, about 8 to 12 base pairs, about 9 to 13 base pairs, about 10 to 14 base pairs, about 11 to 15 base pairs, about 12 to 16 base pairs, about 13 to 17 base pairs, about 14 to 18 base pairs, about 15 to 19 base pairs, about 16 to 20 base pairs, about 1 to 3 base pairs, about 2 to 4 base pairs, about 3 to 5 base pairs, about 4 to 6 base pairs, about 5 to 7 base pairs, about 6 to 8 base pairs, about 7 to 9 base pairs, about 8 to 10 base pairs, about 9 to 11 base pairs, about 10 to 12 base pairs, about 11 to 13 base pairs, about 12 to 14 base pairs, about 13 to 15 base pairs, about 14 to 16 base pairs, about 15 to 17 base pairs, about 16 to 18 base pairs, about 17 to 19 base pairs, about 18 to 20 base pairs away or upstream of the PAM sequence. In some embodiments, a target nucleobase is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more base pairs away from or upstream of the PAM sequence. In some embodiments, a target nucleobase is about 1, 2, 3, 4, 5, 6, 7, 8, or 9 base pairs upstream of the PAM sequence. In some embodiments, a target nucleobase is about 2, 3, 4, or 6 base pairs upstream of the PAM sequence.
The fusion protein can comprise more than one heterologous polypeptide. For example, the fusion protein can additionally comprise one or more UGI domains and/or one or more nuclear localization signals. The two or more heterologous domains can be inserted in tandem. The two or more heterologous domains can be inserted at locations such that they are not in tandem in the NapDNAbp.
A fusion protein can comprise a linker between the deaminase and the napDNAbp polypeptide. The linker can be a peptide or a non-peptide linker. For example, the linker can be an XTEN, (GGGS)n (SEQ ID NO: 1308), (GGGGS)n (SEQ ID NO: 109), (G)n, (EAAAK)n (SEQ ID NO: 1309), (GGS)n, SGSETPGTSESATPES (SEQ ID NO: 56). In some embodiments, the fusion protein comprises a linker between the N-terminal Cas9 fragment and the deaminase. In some embodiments, the fusion protein comprises a linker between the C-terminal Cas9 fragment and the deaminase. In some embodiments, the N-terminal and C-terminal fragments of napDNAbp are connected to the deaminase with a linker. In some embodiments, the N-terminal and C-terminal fragments are joined to the deaminase domain without a linker. In some embodiments, the fusion protein comprises a linker between the N-terminal Cas9 fragment and the deaminase, but does not comprise a linker between the C-terminal Cas9 fragment and the deaminase. In some embodiments, the fusion protein comprises a linker between the C-terminal Cas9 fragment and the deaminase, but does not comprise a linker between the N-terminal Cas9 fragment and the deaminase.
In some embodiments, the napDNAbp in the fusion protein is a Cas12 polypeptide, e.g., Cas12b/C2c1, or a fragment thereof. The Cas12 polypeptide can be a variant Cas12 polypeptide. In other embodiments, the N- or C-terminal fragments of the Cas12 polypeptide comprise a nucleic acid programmable DNA binding domain or a RuvC domain. In other embodiments, the fusion protein contains a linker between the Cas12 polypeptide and the catalytic domain. In other embodiments, the amino acid sequence of the linker is GGSGGS (SEQ ID NO: 273) or GSSGSETPGTSESATPESSG (SEQ ID NO: 1310). In other embodiments, the linker is a rigid linker. In other embodiments of the above aspects, the linker is encoded by GGAGGCTCTGGAGGAAGC (SEQ ID NO: 1311) or GGCTCTTCTGGATCTGAAACACCTGGCACAAGCGAGAGCGCCACCCCTGAGAGCTCTGGC (SEQ ID NO: 1312).
Fusion proteins comprising a heterologous catalytic domain flanked by N- and C-terminal fragments of a Cas12 polypeptide are also useful for base editing in the methods as described herein. Fusion proteins comprising Cas12 and one or more deaminase domains, e.g. adenosine deaminase, or comprising an adenosine deaminase domain flanked by Cas12 sequences are also useful for highly specific and efficient base editing of target sequences. In an embodiment, a chimeric Cas12 fusion protein contains a heterologous catalytic domain (e.g. adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) inserted within a Cas12 polypeptide. In some embodiments, the fusion protein comprises an adenosine deaminase domain and a cytidine deaminase domain inserted within a Cas12. In some embodiments, an adenosine deaminase is fused within Cas12 and a cytidine deaminase is fused to the C-terminus. In some embodiments, an adenosine deaminase is fused within Cas12 and a cytidine deaminase fused to the N-terminus. In some embodiments, a cytidine deaminase is fused within Cas12 and an adenosine deaminase is fused to the C-terminus. In some embodiments, a cytidine deaminase is fused within Cas12 and an adenosine deaminase fused to the N-terminus. Exemplary structures of a fusion protein with an adenosine deaminase and a cytidine deaminase and a Cas12 are provided as follows:

    
    
        NH2-[Cas12(adenosine deaminase)]-[cytidine deaminase]-COOH;
        NH2-[cytidine deaminase]-[Cas12(adenosine deaminase)]-COOH;
        NH2-[Cas12(cytidine deaminase)]-[adenosine deaminase]-COOH; or
        NH2-[adenosine deaminase]-[Cas12(cytidine deaminase)]-COOH;
    
    


In some embodiments, the “-” used in the general architecture above indicates the presence of an optional linker.
In various embodiments, the catalytic domain has DNA modifying activity (e.g. deaminase activity), such as adenosine deaminase activity. In some embodiments, the adenosine deaminase is a TadA (e.g., TadA*7.10). In some embodiments, the TadA is a TadA*8. In some embodiments, a TadA*8 is fused within Cas12 and a cytidine deaminase is fused to the C-terminus. In some embodiments, a TadA*8 is fused within Cas12 and a cytidine deaminase fused to the N-terminus. In some embodiments, a cytidine deaminase is fused within Cas12 and a TadA*8 is fused to the C-terminus. In some embodiments, a cytidine deaminase is fused within Cas12 and a TadA*8 fused to the N-terminus. Exemplary structures of a fusion protein with a TadA*8 and a cytidine deaminase and a Cas12 are provided as follows: N-[Cas12(TadA*8)]-[cytidine deaminase]-C; N-[cytidine deaminase]-[Cas12(TadA*8)]-C; N-[Cas12(cytidine deaminase)]-[TadA*8]-C; or N-[TadA*8]-[Cas12(cytidine deaminase)]-C.
In some embodiments, the “-” used in the general architecture above indicates the presence of an optional linker.
In other embodiments, the fusion protein contains one or more catalytic domains. In other embodiments, at least one of the one or more catalytic domains is inserted within the Cas12 polypeptide or is fused at the Cas12 N-terminus or C-terminus. In other embodiments, at least one of the one or more catalytic domains is inserted within a loop, an alpha helix region, an unstructured portion, or a solvent accessible portion of the Cas12 polypeptide. In other embodiments, the Cas12 polypeptide is Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, or Cas12j/CasΦ. In other embodiments, the Cas12 polypeptide has at least about 85% amino acid sequence identity to Bacillus hisashii Cas12b, Bacillus thermoamylovorans Cas12b, Bacillus sp. V3-13 Cas12b, or Alicyclobacillus acidiphilus Cas12b. In other embodiments, the Cas12 polypeptide has at least about 90% amino acid sequence identity to Bacillus hisashii Cas12b, Bacillus thermoamylovorans Cas12b, Bacillus sp. V3-13 Cas12b, or Alicyclobacillus acidiphilus Cas12b. In other embodiments, the Cas12 polypeptide has at least about 95% amino acid sequence identity to Bacillus hisashii Cas12b, Bacillus thermoamylovorans Cas12b, Bacillus sp. V3-13 Cas12b, or Alicyclobacillus acidiphilus Cas12b. In other embodiments, the Cas12 polypeptide contains or consists essentially of a fragment of Bacillus hisashii Cas12b, Bacillus thermoamylovorans Cas12b, Bacillus sp. V3-13 Cas12b, or Alicyclobacillus acidiphilus Cas12b.
In other embodiments, the catalytic domain is inserted between amino acid positions 153-154, 255-256, 306-307, 980-981, 1019-1020, 534-535, 604-605, or 344-345 of BhCas12b or a corresponding amino acid residue of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, or Cas12j/CasΦ. In other embodiments, the catalytic domain is inserted between amino acids P153 and S154 of BhCas12b. In other embodiments, the catalytic domain is inserted between amino acids K255 and E256 of BhCas12b. In other embodiments, the catalytic domain is inserted between amino acids D980 and G981 of BhCas12b. In other embodiments, the catalytic domain is inserted between amino acids K1019 and L1020 of BhCas12b. In other embodiments, the catalytic domain is inserted between amino acids F534 and P535 of BhCas12b. In other embodiments, the catalytic domain is inserted between amino acids K604 and G605 of BhCas12b. In other embodiments, the catalytic domain is inserted between amino acids H344 and F345 of BhCas12b. In other embodiments, catalytic domain is inserted between amino acid positions 147 and 148, 248 and 249, 299 and 300, 991 and 992, or 1031 and 1032 of BvCas12b or a corresponding amino acid residue of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, or Cas12j/CasΦ. In other embodiments, the catalytic domain is inserted between amino acids P147 and D148 of BvCas12b. In other embodiments, the catalytic domain is inserted between amino acids G248 and G249 of BvCas12b. In other embodiments, the catalytic domain is inserted between amino acids P299 and E300 of BvCas12b. In other embodiments, the catalytic domain is inserted between amino acids G991 and E992 of BvCas12b. In other embodiments, the catalytic domain is inserted between amino acids K1031 and M1032 of BvCas12b. In other embodiments, the catalytic domain is inserted between amino acid positions 157 and 158, 258 and 259, 310 and 311, 1008 and 1009, or 1044 and 1045 of AaCas12b or a corresponding amino acid residue of Cas12a, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, or Cas12j/CasΦ. In other embodiments, the catalytic domain is inserted between amino acids P157 and G158 of AaCas12b. In other embodiments, the catalytic domain is inserted between amino acids V258 and G259 of AaCas12b. In other embodiments, the catalytic domain is inserted between amino acids D310 and P311 of AaCas12b. In other embodiments, the catalytic domain is inserted between amino acids G1008 and E1009 of AaCas12b. In other embodiments, the catalytic domain is inserted between amino acids G1044 and K1045 at of AaCas12b.
In other embodiments, the fusion protein contains a nuclear localization signal (e.g., a bipartite nuclear localization signal). In other embodiments, the amino acid sequence of the nuclear localization signal is MAPKKKRKVGIHGVPAA (SEQ ID NO: 1313). In other embodiments of the above aspects, the nuclear localization signal is encoded by the following sequence:
ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCC (SEQ ID NO: 1314). In other embodiments, the Cas12b polypeptide contains a mutation that silences the catalytic activity of a RuvC domain. In other embodiments, the Cas12b polypeptide contains D574A, D829A and/or D952A mutations. In other embodiments, the fusion protein further contains a tag (e.g., an influenza hemagglutinin tag).
In some embodiments, the fusion protein comprises a napDNAbp domain (e.g., Cas12-derived domain) with an internally fused nucleobase editing domain (e.g., all or a portion of a deaminase domain, e.g., an adenosine deaminase domain). In some embodiments, the napDNAbp is a Cas12b. In some embodiments, the base editor comprises a BhCas12b domain with an internally fused TadA*8 domain inserted at the loci provided in Table 10 below.







TABLE 10







Insertion loci in Cas12b proteins










Insertion site
Inserted between aa















BhCas12b





position 1
153
PS



position 2
255
KE



position 3
306
DE



position 4
980
DG



position 5
1019
KL



position 6
534
FP



position 7
604
KG



position 8
344
HF



BvCas12b



position 1
147
PD



position 2
248
GG



position 3
299
PE



position 4
991
GE



position 5
1031
KM



AaCas12b



position 1
157
PG



position 2
258
VG



position 3
310
DP



position 4
1008
GE



position 5
1044
GK










By way of nonlimiting example, an adenosine deaminase (e.g., TadA*8.13) may be inserted into a BhCas12b to produce a fusion protein (e.g., TadA*8.13-BhCas12b) that effectively edits a nucleic acid sequence.
In some embodiments, the base editing system described herein is an ABE with TadA inserted into a Cas9. Sequences of relevant ABEs with TadA inserted into a Cas9 are provided.







101 Cas9 TadAins 1015


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFREDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIRK


 


YPKLESEFVYGDYKVGSSGSETPGTSESATPESSGSEVEFSHEYWMRHAL


 


TLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQG


 


GLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGS


 


LMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSST


 


DYDVRKMTAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE


 


TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLTIKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


102 Cas9 TadAins 1022


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNTVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDELLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAAKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREPNNYHRAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIGSSGSETPGTSESATPESSGSEVEFSHE


 


YWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGDHDPTAHAE


 


IMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNA


 


KTGAAGSDMDVLHYPGMNHRVEITEGILADECAALLCYFERMPRQVFNAQ


 


KKAQSSTDAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE


 


TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


103 Cas9 TadAins 1029


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERRPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDEAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDELLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIRDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHARDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGSSGSETPGTSESATPESSGS


 


EVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH


 


DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV


 


VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADEGAALLCYFFRMP


 


RQVFNAQKKAQSSTDGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE


 


TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


103 Cas9 TadAins 1040


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSGSSGSETPGT


 


SESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVI


 


GEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCA


 


GAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADEC


 


AALLCYFFRMPRQVFNAQKKAQSSTDNIMNFFKTEITLANGEIRKRPLIE


 


TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


105 Cas9 TadAins 1068


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLANGEIRKRPLIETNGEGSSGSETPGTSESATPESSGSEVEFSHEYWMR


 


HALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMAL


 


RQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGA


 


AGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQ


 


SSTDTGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


106 Cas9 TadAins 1247


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDEAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDELLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHRAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV


 


QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE


 


KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK


 


YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGGSS


 


GSFTPGTSESATPESSGSEVEFSHEVWMRHATTTAKRARDRREVPVGAVL


 


VLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTE


 


EPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITE


 


GILADECAALLCYFFRMPRQVFNAQKKAQSSTDSPEDNEQKQLHVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLET


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


107 Cas9 TadAins 1054


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDEAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDELLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLIEGIRDKQSGKTILDFLKSDGFAEREFMQLIRDD


 


SLTFKEDIQKAQVSGQGDSLHEHIAMLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLONGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREIENYHRAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLANGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDERE


 


VPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLID


 


ATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMN


 


HRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDGEIRKRPLIE


 


TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


108 Cas9 TadAins 1026


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIRDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEGSSGSETPGTSESATPESSGSEVE


 


FSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPT


 


AHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFG


 


VRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQV


 


FNAQKKAQSSTDQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE


 


TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


109 Cas9 TadAins 768


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIRDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQGSSGSETPGTSESATPESSGSEVEFSHEYWMR


 


HALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMAL


 


RQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGA


 


AGSLMDVLHYPGMNHRVEITEGILADEGAALLCYFFRMPRTTQKGQKNSR


 


ERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL


 


DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKK


 


MKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQIT


 


KHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREI


 


NNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQE


 


IGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGR


 


DFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP


 


KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP


 


IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL


 


PSKYVNFLYLASHYEKLKGSPEDNEQKQLEVEQHKHYLDEIIEQISEFSK


 


RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFD


 


TTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


110.1 Cas9 TadAins 1250


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDELLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV


 


QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE


 


KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK


 


YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPG


 


SSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGA


 


VLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYV


 


TFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEI


 


TEGILADECAALLCYFFRMPREDNEQKQLFVEQHKHYLDEIIEQISEFSK


 


RVILADANIDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFD


 


TTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


110.2 Cas9 TadAins 1250


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALAHMIKFRGHFLIEGDLEPDNSDVDKLFTQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NEKSNFDLAEDAKLQLSKDTYDDDLDELLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLEREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWEFEEVVDKGASAQSFTERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTERKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAEREFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPEEIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQEEKLYLYYLQNGRDMYVDQELDIERLSDYDVDHIVPQSFLKDD


 


SIDEKVLTRSDKNRGKSDNVPSEEVVKKMKEYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMETKYDEEDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV


 


QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE


 


KGKSKKLKSVKELLGITIMERSSFEKEPIDFLEAKGYKEVKKDLIIKLPK


 


YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPG


 


SSGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVP


 


VGAVLVLENRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDAT


 


LYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMEHR


 


VEITEGILADECAALLCYFFRMPREDEEQKQLFVEQHKHYLDEIIEQISE


 


FSKRVILADANLDKVLSAYNKHRDKPIREQAEEIIHLFTLTNLGAPAAFK


 


YFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


110.3 Cas9TadAins 1250


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV


 


QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE


 


KGKSKKLKSVKELLGITLMERSSFEKNPLDFLEAKGYKEVKKDLILKEPK


 


YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPG


 


SSGSSGSETPGTSESATPESGSSSGSEVEFSHEYWMRHALTLAKRARDER


 


EVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLI


 


DATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVIHYPGM


 


NHRVEITEGILADECAALLCYFFRMPREDNEQKQLFVEQHKHYLDEIIEQ


 


ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA


 


AFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


110.4 Cas9 Tad Ains 1250


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSEFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDEAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFALKRTRKSEETITPWNFEEWDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV


 


QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE


 


KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK


 


YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPG


 


SSGSSGSETPGTSESATPESGSSSGSEVEFSHEYWMRHALTLAKRARDER


 


EVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLI


 


DATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGM


 


NHRVEITEGILADECAALLCYFFRMRREDNEQKQLFVEQHKHYLDEIIEQ


 


ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA


 


AFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


110.5 Cas9 TadAins 1249


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDELLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLEREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFALKRTRKSEETITPWEFEEWDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV


 


QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE


 


KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK


 


YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSGS


 


SGSSGSETPGTSESATPESGSSSGSEVEFSHEYWMRHALTLAKRARDERE


 


VPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLID


 


ATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMN


 


HRVEITEGILADECAALLCYFFRMRRPEDNEQKQLFVEQHKHYLDEIIEQ


 


ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA


 


AFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


110.5 Cas9 TadAins delta 59-66 1250


MDKKYSIGIALGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDEAD


 


LRDIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDELLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV


 


QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE


 


KGKSKKLKSVKELLGITIMERSSEEKNPIDFLEAKGIKEVKKDLIIKLPK


 


YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPG


 


SSGSSGSETPGTSESATPESGSSGSEVEFSHEYWMRHALTLAKRARDERE


 


VPVGAVLVLNNRVIGEGWNRAHAEIMALRQGGLVMQNYRLIDATLYVTFE


 


PCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEG


 


ILADECAALLCYFFRMPRQVFNAQKKAQSSTDEDNEQKQLFVEQHKHYLD


 


EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTN


 


LGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGG


 


110.6 Cas9 TadAins 1251


MDKKYSIGLALGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRDIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRISRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDLNRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV


 


QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE


 


KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK


 


YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE


 


GSSGSSGSETPGTSESATPESGSSSGSEVEFSHEYWMRHALTLAKRARDE


 


REVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRL


 


IDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPG


 


MNHRVEITEGILADECAALLCYFFRMRRDNEQKQLFVEQHKHYLDEIIEQ


 


ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA


 


AFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


110.7 Cas9 TadAins 1252


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRISRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDLNRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV


 


QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE


 


KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK


 


YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE


 


DGSSGSSGSETPGTSESATPESGSSSGSEVEFSHEYWMRHALTLAKRARD


 


EREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYR


 


LIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP


 


GMNHRVEITEGILADECAALLCYFFRMRRNEQKQLFVEQHKHYLDEIIEQ


 


ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA


 


AFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


110.8 Cas9 TadAins delta 59-66 C-truncate 1250


MDKKYSIGLATGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRISRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV


 


QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE


 


KGKSKKLKSVKELLGITIMERSSEEKNPIDFLEAKGIKEVKKDLIIKLPK


 


YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPG


 


SSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGA


 


VLVLNNRVIGEGWNRAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMC


 


AGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADE


 


CAALLCYFFRMPRQEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA


 


NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKR


 


YTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


111.1 Cas9 TadAins 997


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRDIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALSHE


 


YWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAE


 


IMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNA


 


KTGAAGSLMDVIHYPGMNHRVEITEGILADEGAALLCYFFRMPRQVFNAQ


 


KKAQSSTDGSSGSETPGTSESATPESSGIKKYPKLESEFVYGDYKVYDVR


 


KMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET


 


GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKL


 


IARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIM


 


ERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGE


 


LQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI


 


IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLG


 


APAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


111.2 Cas9 TadAins 997


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRDIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALSHE


 


YWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAE


 


IMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNA


 


KTGAAGSLMDVIHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQ


 


KKAQSSTDGSSGSSGSETPGTSESATPESSGGSSIKKYPKLESEFVYGDY


 


KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLI


 


ETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPK


 


RNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKEL


 


LGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRM


 


LASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHK


 


HYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLF


 


TLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLS


 


QLGGD


 


112 delta HNH Tad A


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNTVDEVAYHEKYPTIYHLRKKLVDSTDEAD


 


LRDIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSEVEFSHE


 


YWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAE


 


IMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNA


 


KTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQ


 


KKAQSSTDGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEND


 


KLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTAL


 


IKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFK


 


TEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKK


 


TEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVA


 


KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK


 


LPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKG


 


SPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH


 


RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL


 


IHQSITGLYETRIDLSQLGGD


 


113 N-term single TadA helix trunc 165-end


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG


 


LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG


 


RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR


 


MPRSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSV


 


GWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR


 


TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH


 


PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRG


 


HFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARL


 


SKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQL


 


SKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAP


 


LSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG


 


ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHL


 


GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMT


 


RKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYE


 


YFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE


 


DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDIL


 


EDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKL


 


INGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQ


 


GDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE


 


NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL


 


QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGK


 


SDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGF


 


IKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF


 


RKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY


 


DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN


 


GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNS


 


DKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI


 


TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS


 


AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYL


 


DEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT


 


NLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG


 


GD


 


114 N-term single TadA helix trunc 165-end delta


59-65


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRTAH


 


AEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVR


 


NAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRSGGS


 


SGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITD


 


EYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYT


 


RRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV


 


DEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGD


 


LNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE


 


NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDD


 


DLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIK


 


RYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFY


 


KFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAIL


 


RRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI


 


TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNE


 


LTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE


 


CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTL


 


TLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK


 


QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEH


 


IANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKG


 


QKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMY


 


VDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSE


 


EVVKKMKNYWKQLLNAKLLTQRKEDNLTKAEKGGLSELDKAGELKKQLVE


 


TRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFY


 


KVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIA


 


KSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIV


 


WDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK


 


KDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS


 


FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKG


 


NELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQI


 


SEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAA


 


FKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


115.1 Cas9 TadAins 1004


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREV


 


PVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDA


 


TLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH


 


RVEITEGILADEGAALLCYFFRMPRQLESEFVYGDYKVYDVRKMIAKSEQ


 


EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG


 


RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD


 


PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN


 


PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA


 


LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS


 


KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF


 


DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


115.2 Cas9 TadAins 1005


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDEAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NEKSNFDLAEDAKLQLSKDTYDDDLDELLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAAKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDERE


 


VPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLID


 


ATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMN


 


HRVEITEGILADECAALLCYFFRMPRQESEFVYGDYKVYDVRKMIAKSEQ


 


EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG


 


RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD


 


PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN


 


PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA


 


LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS


 


KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF


 


DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


115.3 Cas9 TadAins 1006


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NEKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLEGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDER


 


EVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLI


 


DATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVIHYPGM


 


NHRVEITEGILADECAALLCYFFRMPRQSEFVYGDYKVYDVRKMIAKSEQ


 


EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG


 


RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD


 


PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN


 


PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA


 


LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS


 


KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF


 


DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


115.4 Cas9 TadAins 1007


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDEAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDELLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLEREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWEFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTERKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAEREFMQLIRDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPEEIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQEEKLYLYYLQNGRDMYVDQELDIERLSDYDVDHIVPQSFLKDD


 


SIDEKVLTRSDKNRGKSDNVPSEEVVKKMKEYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDE


 


REVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRL


 


IDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPG


 


MEHRVEITEGILADECAALLCYFFRMPRQEFVYGDYKVYDVRKMIAKSEQ


 


EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG


 


RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD


 


PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN


 


PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA


 


LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS


 


KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF


 


DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


116.1 Cas9 TadAins C-term truncate2 792


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDELLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLEREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWEFEEVVDKGASAQSFIERMTNFDK


 


NLPEEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTERKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLIEGIRDKQSGKTILDFLKSDGFAEREFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPEEIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGGSSGSETP


 


GTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLENR


 


VIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQEYRLIDATLYVTFEPCVM


 


CAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILAD


 


ECAALLCYFFRMPRQSQILKEHPVENTQLQEEKLYLYYLQNGRDMYVDQE


 


LDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVK


 


KMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI


 


TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVRE


 


LENYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ


 


EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG


 


RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD


 


PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN


 


PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA


 


LPSKYVNFLYLASHYEKLKGSPEDEEQKQLFVEQHKHYLDEIIEQISEFS


 


KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF


 


DTTTDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


116.2 Cas9 TadAins C-term truncate2 791


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDEAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDELLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAEREFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSSGSETPG


 


TSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV


 


IGEGWERAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMC


 


AGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMEHRVEITEGILADE


 


GAALLCYFFRMPRQGSQILKEHPVENTQLQEEKLYLYYLQNGRDMYVDQE


 


LDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVK


 


KMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI


 


TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVRE


 


INNYHHAHDAYLNAVVGTALLKRYPKLESEEVYGDYKVYDVRKMIAKSEQ


 


EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG


 


RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD


 


PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN


 


PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA


 


LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS


 


KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF


 


DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


116.3 Cas9 TadAins C-term truncate2 790


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKEGSSGSETPGT


 


SESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVI


 


GEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCA


 


GAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADEC


 


AALLCYFFRMPRQLGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE


 


LDINRLSDYDVDHLVFQSHLKDDSIDNKVLIRSDKNKGKSDNVPSEEVVK


 


KMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI


 


TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVRE


 


INNYHHAHDAYLNAVVGTALLKKYPKLESEEVYGDYKVYDVRKMIAKSEQ


 


EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG


 


RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD


 


PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN


 


PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA


 


LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS


 


KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF


 


DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


117 Cas9 delta 1017-1069


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSEFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDEAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFALKRTRKSEETITPWNFEEWDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYSSGSEVEFSHEYWMRHALTLAKRARDEREVPVGA


 


VLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYV


 


TFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEI


 


TEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDGEIVWDKGRDFATVR


 


KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF


 


DSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEA


 


KGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVN


 


FLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD


 


ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK


 


RYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD


 


118 Cas9 TadA-CP116ins 1067


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLANGEIRKRPLIETNMNHRVEITEGILADECAALLCYFFRMPRQVFNAQ


 


KKAQSSTDGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRAR


 


DEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNY


 


RLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHY


 


PGGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


119 Cas9 TadAins 701


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDELLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPV


 


GAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATL


 


YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRV


 


EITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDLTFKEDIQKAQVS


 


GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA


 


RENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLY


 


YLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNR


 


GKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA


 


GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVS


 


DFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYK


 


VYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE


 


TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


120 Cas9 TadACP136ins 1248


MDKKYSIGLALGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRDIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRISRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDLNRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV


 


QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE


 


KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK


 


YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSMN


 


HRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDGSSGSETPGT


 


SESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVI


 


GEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCA


 


GAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


121 Cas9TadACP136ins 1052


MDKKYSTGLALGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSTKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRISRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDLNRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLAMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDGSSGS


 


ETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVI


 


NNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEP


 


CVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGNGEIRKRPLIE


 


TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLTIKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


122 Cas9 TadACP136ins 1041


MDKKYSTGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSTKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDEAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRISRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDLNRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSMNHRVEITEG


 


ILADECAALLCYFERMPRQVFNAQKKAQSSTDGSSGSETPGTSESATPES


 


SGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAI


 


GLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRI


 


GRVVFGVRNAKTGAAGSLMDVLHYPGNIMNFFKTEITLANGEIRKRPLIE


 


TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLTTKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


123 Cas9 TadACP139ins 1299


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDEAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRISRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDLNRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV


 


QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE


 


KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK


 


YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE


 


DNEQKQLFVEQHKHYLDEIIEQISEFSKRVTLADANLDKVLSAYNKHRMN


 


HRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDGSSGSETPGT


 


SESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVI


 


GEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCA


 


GAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


124 Cas9 delta 792-872 TadAins


MDKKYSTGLALGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSTKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYLDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRISRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVLEMARENQTTQKGQKNSRERMKRIEEGIKELGSEVEFSHE


 


YWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAE


 


IMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNA


 


KTGAAGSLMDVLHYPGMNHRVEITEGILADEGAALLCYFFRMPRQVFNAQ


 


KKAQSSTDEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA


 


GFIKRQLVETRQTTKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVS


 


DFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYK


 


VYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLTE


 


TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


125 Cas9 delta 792-906 TadAins


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSEVEFSHE


 


YWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAE


 


IMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNA


 


KTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQ


 


KKAQSSTDGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK


 


LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALI


 


KKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT


 


EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVISMPQVNIVKKT


 


EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAK


 


VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKL


 


PKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGS


 


PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR


 


DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI


 


HQSITGLYETRIDLSQLGGD


 


126 TadA CP65ins 1003


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDEAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR


 


VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRM


 


PRQVFNAQKKAQSSTDGSSGSETPGTSESATPESSGSEVEFSHEYWMRHA


 


LTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPLESEFVYGDYK


 


VYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE


 


TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


127 TadA CP65ins 1016


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDELLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLEREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFTKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVM


 


CAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILAD


 


ECAALLCYFFRMPRQVFNAQKKAQSSTDGSSGSETPGTSESATPESSGSE


 


VEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHD


 


PYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE


 


TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


128 TadA CP65ins 1022


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDELLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMITAHAEIMALRQGGLVMQNYRLIDATLYV


 


TFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEI


 


TEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDGSSGSETPGTSESAT


 


PESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWN


 


RAIGLHDPAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE


 


TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


129 TadA CP65ins 1029


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEEYKELKPILEKMDGTEELLVKLNKEDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEITAHAEIMALRQGGLVMQNYRL


 


IDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPG


 


MNHRVEITEGILADEGAALLCYFERMPRQVFNAQKKAQSSTDGSSGSETP


 


GTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNR


 


VIGEGWNRAIGLHDPGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE


 


TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLTTKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


130 TadA CP65ins 1041


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESELVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDEAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDELFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEEKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDELLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLEREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGETILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANDAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSTAHAEIMALR


 


QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAA


 


GSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQS


 


STDGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREV


 


PVGAVLVLNNRVIGEGWNRAIGLHDPNIMNFFKTEITLANGEIRKRPLIE


 


TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLTIKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


131 TadA CP65ins 1054


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERRPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALARMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDELLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERI4TNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLANTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG


 


RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR


 


MPRQVFNAQKKAQSSTDGSSGSETPGTSESATPESSGSEVEFSHEYWMRH


 


ALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPGEIRKRPLIE


 


TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR


 


NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL


 


GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML


 


ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD


 


132 TadA CP65ins 1246


MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA


 


LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR


 


LEESFLVEEDKKHERHPIFGNTVDEVAYHEKYPTIYHLRKKLVDSTDKAD


 


LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP


 


INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NEKSNFDLAEDAKLQLSKDTYDDDLDHLLAQIGDQYADLFLAAKNLSDAI


 


LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI


 


FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR


 


KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY


 


YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK


 


NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD


 


LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI


 


IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ


 


LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD


 


SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV


 


MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP


 


VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD


 


SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL


 


TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK


 


YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI


 


TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV


 


QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE


 


KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK


 


YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGTAH


 


AEIMALRQGGLVMQNYRLIDATLYVIFEPCVMCAGAMIHSRIGRVVFGVR


 


NAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFN


 


AQKKAQSSTDGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKR


 


ARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPSPEDNEQKQLFVEQHKH


 


YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT


 


LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ


 


LGGD






In some embodiments, adenosine deaminase base editors were generated to insert TadA or variants thereof into the Cas9 polypeptide at the identified positions.
Exemplary, yet nonlimiting, fusion proteins are described in International PCT Application Nos. PCT/US2020/016285 and U.S. Provisional Application Nos. 62/852,228 and 62/852,224, the contents of which are incorporated by reference herein in their entireties.
A to G Editing
In some embodiments, a base editor described herein comprises a fusion protein comprising an adenosine deaminase domain. Such an adenosine deaminase domain of a base editor can facilitate the editing of an adenine (A) nucleobase to a guanine (G) nucleobase by deaminating the A to form inosine (I), which exhibits base pairing properties of G. Adenosine deaminase is capable of deaminating (i.e., removing an amine group) adenine of a deoxyadenosine residue in deoxyribonucleic acid (DNA).
In some embodiments, the nucleobase editors provided herein can be made by fusing together one or more protein domains, thereby generating a fusion protein. In certain embodiments, the fusion proteins provided herein comprise one or more features that improve the base editing activity (e.g., efficiency, selectivity, and specificity) of the fusion proteins. For example, the fusion proteins provided herein can comprise a Cas9 domain that has reduced nuclease activity. In some embodiments, the fusion proteins provided herein can have a Cas9 domain that does not have nuclease activity (dCas9), or a Cas9 domain that cuts one strand of a duplexed DNA molecule, referred to as a Cas9 nickase (nCas9). Without wishing to be bound by any particular theory, the presence of the catalytic residue (e.g., H840) maintains the activity of the Cas9 to cleave the non-edited (e.g., non-deaminated) strand containing a T opposite the targeted A. Mutation of the catalytic residue (e.g., D10 to A10) of Cas9 prevents cleavage of the edited strand containing the targeted A residue. Such Cas9 variants are able to generate a single-strand DNA break (nick) at a specific location based on the gRNA-defined target sequence, leading to repair of the non-edited strand, ultimately resulting in a T to C change on the non-edited strand. In some embodiments, an A-to-G base editor further comprises an inhibitor of inosine base excision repair, for example, a uracil glycosylase inhibitor (UGI) domain or a catalytically inactive inosine specific nuclease. Without wishing to be bound by any particular theory, the UGI domain or catalytically inactive inosine specific nuclease can inhibit or prevent base excision repair of a deaminated adenosine residue (e.g., inosine), which can improve the activity or efficiency of the base editor.
A base editor comprising an adenosine deaminase can act on any polynucleotide, including DNA, RNA and DNA-RNA hybrids. In certain embodiments, a base editor comprising an adenosine deaminase can deaminate a target A of a polynucleotide comprising RNA. For example, the base editor can comprise an adenosine deaminase domain capable of deaminating a target A of an RNA polynucleotide and/or a DNA-RNA hybrid polynucleotide. In an embodiment, an adenosine deaminase incorporated into a base editor comprises all or a portion of adenosine deaminase acting on RNA (ADAR, e.g., ADAR1 or ADAR2). In another embodiment, an adenosine deaminase incorporated into a base editor comprises all or a portion of adenosine deaminase acting on tRNA (ADAT). A base editor comprising an adenosine deaminase domain can also be capable of deaminating an A nucleobase of a DNA polynucleotide. In an embodiment an adenosine deaminase domain of a base editor comprises all or a portion of an ADAT comprising one or more mutations which permit the ADAT to deaminate a target A in DNA. For example, the base editor can comprise all or a portion of an ADAT from Escherichia coli (EcTadA) comprising one or more of the following mutations: D108N, A106V, D147Y, E155V, L84F, H123Y, I156F, or a corresponding mutation in another adenosine deaminase.
The adenosine deaminase can be derived from any suitable organism (e.g., E. coli). In some embodiments, the adenosine deaminase is from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is from E. coli. In some embodiments, the adenine deaminase is a naturally-occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). The corresponding residue in any homologous protein can be identified by e.g., sequence alignment and determination of homologous residues. The mutations in any naturally-occurring adenosine deaminase (e.g., having homology to ecTadA) that correspond to any of the mutations described herein (e.g., any of the mutations identified in ecTadA) can be generated accordingly.
Adenosine Deaminases
In some embodiments, the fusion proteins of the invention comprise one or more adenosine deaminase domains. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine in a deoxyadenosine residue of DNA. The adenosine deaminase may be derived from any suitable organism (e.g., E. coli). In some embodiments, the adenine deaminase is a naturally-occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). One of skill in the art will be able to identify the corresponding residue in any homologous protein, e.g., by sequence alignment and determination of homologous residues. Accordingly, one of skill in the art would be able to generate mutations in any naturally-occurring adenosine deaminase (e.g., having homology to ecTadA) that corresponds to any of the mutations described herein, e.g., any of the mutations identified in ecTadA. In some embodiments, the adenosine deaminase is from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is from E. coli. 
The invention provides adenosine deaminase variants that have increased efficiency (>50-60%) and specificity. In particular, the adenosine deaminase variants described herein are more likely to edit a desired base within a polynucleotide, and are less likely to edit bases that are not intended to be altered (i.e., “bystanders”).
In some embodiments, the adenosine deaminase is a TadA deaminase. In particular embodiments, the TadA is any one of the TadA described in PCT/US2017/045381 (WO 2018/027078), which is incorporated herein by reference in its entirety.
A wild type TadA(wt) adenosine deaminase has the following sequence (also termed TadA reference sequence):







MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPI


 


GRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSR


 


IGRVVFGARDARTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSD


 


FFRMRRQEIKAQKKAQSSTD






In some embodiments the adenosine deaminase is a full-length E. coli TadA deaminase. For example, in certain embodiments, the adenosine deaminase comprises the amino acid sequence:







MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNN


 


RVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPC


 


VMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGI


 


LADECAALLSDFFRMRRQEIKAQKKAQSSTD.






In some embodiments, the adenosine deaminase is from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is from Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Salmonella typhimurium (S. typhimurium), Shewanella putrefaciens (S. putrefaciens), Haemophilus influenzae (H. influenzae), Caulobacter crescentus (C. crescentus), Geobacter sulfurreducens (G. sulfurreducens), or Bacillus subtilis. In some embodiments, the adenosine deaminase is from E. coli. 
It should be appreciated, however, that additional adenosine deaminases useful in the present application would be apparent to the skilled artisan and are within the scope of this disclosure. For example, the adenosine deaminase may be a homolog of adenosine deaminase acting on tRNA (ADAT). Without limitation, the amino acid sequences of exemplary AD AT homologs include the following:








Staphylococcus aureus (S. aureus) TadA:



MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIE


RAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNH


RAIVDKGVLKEACSTLLTTFFKNLRANKKSTN


 


Bacillus subtilis (B. subtilis) TadA:


MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEMLVIDEACK


ALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEV


VSGVLEEECGGMLSAFFRELRKKKKAARKNLSE


 


Salmonella typhimurium (S. typhimurium) TadA:


MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHD


PTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSL


IDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV


 


Shewanella putrefaciens (S. putrefaciens) TadA:


MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKL


ENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTS


GVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE


 


Haemophilus influenzae F3031 (H. influenzae) TadA:


MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTAHAEI


IALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDY


KMNHTLEITSGVIAEECSQKLSTFFQKRREEKKIEKALLKSLSDK


 


Caulobacter crescentus (C. crescentus) TadA:


MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEI


AAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQP


TCHWRPEVTGGVLADESADLLRGFFRARRKAKI


 


Geobacter sulfurreducens (G. sulfurreducens) TadA:


MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEM


IAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADP


RLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP


 


An embodiment of E. Coli TadA (ecTadA) includes the following:


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR


QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH


RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD






In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in any of the adenosine deaminases provided herein. It should be appreciated that adenosine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). The disclosure provides any deaminase domains with a certain percent identify plus any of the mutations or combinations thereof described herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to a reference sequence, or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues as compared to any one of the amino acid sequences known in the art or described herein.
It should be appreciated that any of the mutations provided herein (e.g., based on the TadA reference sequence) can be introduced into other adenosine deaminases, such as E. coli TadA (ecTadA), S. aureus TadA (saTadA), or other adenosine deaminases (e.g., bacterial adenosine deaminases). It would be apparent to the skilled artisan that additional deaminases may similarly be aligned to identify homologous amino acid residues that can be mutated as provided herein. Thus, any of the mutations identified in the TadA reference sequence can be made in other adenosine deaminases (e.g., ecTada) that have homologous amino acid residues. It should also be appreciated that any of the mutations provided herein can be made individually or in any combination in the TadA reference sequence or another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises a D108X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D108G, D108N, D108V, D108A, or D108Y mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase. It should be appreciated, however, that additional deaminases may similarly be aligned to identify homologous amino acid residues that can be mutated as provided herein.
In some embodiments, the adenosine deaminase comprises an A106X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A106V mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g. ecTadA).
In some embodiments, the adenosine deaminase comprises a E155X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a E155D, E155G, or E155V mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises a D147X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D147Y, mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises an A106X, E155X, or D147X, mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an E155D, E155G, or E155V mutation. In some embodiments, the adenosine deaminase comprises a D147Y.
It should be appreciated that any of the mutations provided herein (e.g., based on the ecTadA amino acid sequence of TadA reference sequence) may be introduced into other adenosine deaminases, such as S. aureus TadA (saTadA), or other adenosine deaminases (e.g., bacterial adenosine deaminases). It would be apparent to the skilled artisan how to are homologous to the mutated residues in ecTadA. Thus, any of the mutations identified in ecTadA may be made in other adenosine deaminases that have homologous amino acid residues. It should also be appreciated that any of the mutations provided herein may be made individually or in any combination in ecTadA or another adenosine deaminase.
For example, an adenosine deaminase may contain a D108N, a A106V, a E155V, and/or a D147Y mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). In some embodiments, an adenosine deaminase comprises the following group of mutations (groups of mutations are separated by a “;”) in TadA reference sequence, or corresponding mutations in another adenosine deaminase: D108N and A106V; D108N and E155V; D108N and D147Y; A106V and E155V; A106V and D147Y; E155V and D147Y; D108N, A106V, and E155V; D108N, A106V, and D147Y; D108N, E155V, and D147Y; A106V, E155V, and D147Y; and D108N, A106V, E155V, and D147Y. It should be appreciated, however, that any combination of corresponding mutations provided herein may be made in an adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises one or more of a H8X, T17X, L18X, W23X, L34X, W45X, R51X, A56X, E59X, E85X, M94X, I95X, V102X, F104X, A106X, R107X, D108X, K110X, M118X, N127X, A138X, F149X, M151X, R153X, Q154X, 1156X, and/or K157X mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H8Y, T17S, L18E, W23L, L34S, W45L, R51H, A56E, or A56S, E59G, E85K, or E85G, M94L, I95L, V102A, F104L, A106V, R107C, or R107H, or R107P, D108G, or D108N, or D108V, or D108A, or D108Y, K1101, M118K, N127S, A138V, F149Y, M151V, R153C, Q154L, 1156D, and/or K157R mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises one or more of a H8X, D108X, and/or N127X mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, where X indicates the presence of any amino acid. In some embodiments, the adenosine deaminase comprises one or more of a H8Y, D108N, and/or N127S mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises one or more of H8X, R26X, M61X, L68X, M70X, A106X, D108X, A109X, N127X, D147X, R152X, Q154X, E155X, K161X, Q163X, and/or T166X mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H8Y, R26W, M61I, L68Q, M70V, A106T, D108N, A109T, N127S, D147Y, R152C, Q154H or Q154R, E155G or E155V or E155D, K161Q, Q163H, and/or T166P mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8X, D108X, N127X, D147X, R152X, and Q154X in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8X, M61X, M70X, D108X, N127X, Q154X, E155X, and Q163X in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five, mutations selected from the group consisting of H8X, D108X, N127X, E155X, and T166X in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.
In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8X, A106X, and D108X, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8X, R26X, L68X, D108X, N127X, D147X, and E155X, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.
In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations selected from the group consisting of H8X, R126X, L68X, D108X, N127X, D147X, and E155X in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8X, D108X, A109X, N127X, and E155X in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.
In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8Y, D108N, N127S, D147Y, R152C, and Q154H in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8Y, M61I, M70V, D108N, N127S, Q154R, E155G and Q163H in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, or five, mutations selected from the group consisting of H8Y, D108N, N127S, E155V, and T166P in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8Y, A106T, D108N, N127S, E155D, and K161Q in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8Y, R26W, L68Q, D108N, N127S, D147Y, and E155V in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, or five, mutations selected from the group consisting of H8Y, D108N, A109T, N127S, and E155G in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises one or more of the or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D108N, D108G, or D108V mutation in TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a A106V and D108N mutation in TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises R107C and D108N mutations in TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a H8Y, D108N, N127S, D147Y, and Q154H mutation in TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a H8Y, D108N, N127S, D147Y, and E155V mutation in TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D108N, D147Y, and E155V mutation in TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a H8Y, D108N, and N127S mutation in TadA reference sequence, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a A106V, D108N, D147Y, and E155V mutation in TadA reference sequence, or corresponding mutations in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises one or more of S2X, H8X, I49X, L84X, H123X, N127X, I156X, and/or K160X mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of S2A, H8Y, I49F, L84F, H123Y, N127S, I156F, and/or K160S mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises an L84X mutation adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an L84F mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises an H123X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an H123Y mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises an I156X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an I156F mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations selected from the group consisting of L84X, A106X, D108X, H123X, D147X, E155X, and I156X in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of S2X, I49X, A106X, D108X, D147X, and E155X in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8X, A106X, D108X, N127X, and K160X in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.
In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations selected from the group consisting of L84F, A106V, D108N, H123Y, D147Y, E155V, and 1156F in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of S2A, I49F, A106V, D108N, D147Y, and E155V in TadA reference sequence.
In some embodiments, the adenosine deaminase comprises one, two, three, four, or five mutations selected from the group consisting of H8Y, A106T, D108N, N127S, and K160S in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises one or more of a E25X, R26X, R107X, A142X, and/or A143X mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of E25M, E25D, E25A, E25R, E25V, E25S, E25Y, R26G, R26N, R26Q, R26C, R26L, R26K, R107P, R107K, R107A, R107N, R107W, R107H, R107S, A142N, A142D, A142G, A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q, and/or A143R mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of the mutations described herein corresponding to TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises an E25X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an E25M, E25D, E25A, E25R, E25V, E25S, or E25Y mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises an R26X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises R26G, R26N, R26Q, R26C, R26L, or R26K mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises an R107X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R107P, R107K, R107A, R107N, R107W, R107H, or R107S mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises an A142X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A142N, A142D, A142G, mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises an A143X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q, and/or A143R mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises one or more of a H36X, N37X, P48X, I49X, R51X, M70X, N72X, D77X, E134X, S146X, Q154X, K157X, and/or K161X mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H36L, N37T, N37S, P48T, P48L, I49V, R51H, R51L, M70L, N72S, D77G, E134G, S146R, S146C, Q154H, K157N, and/or K161T mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA).
In some embodiments, the adenosine deaminase comprises an H36X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an H36L mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises an N37X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an N37T or N37S mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises an P48X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an P48T or P48L mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises an R51X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R51H or R51L mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises an S146X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an S146R or S146C mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises an K157X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a K157N mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises an P48X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a P48S, P48T, or P48A mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises an A142X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a A142N mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises an W23X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a W23R or W23L mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase.
In some embodiments, the adenosine deaminase comprises an R152X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a R152P or R52H mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase.
In one embodiment, the adenosine deaminase may comprise the mutations H36L, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F, and K157N. In some embodiments, the adenosine deaminase comprises the following combination of mutations relative to TadA reference sequence, where each mutation of a combination is separated by a “_” and each combination of mutations is between parentheses:

    
    
        (A106V_D108N),
        (R107C_D108N),
        (H8Y_D108N_N127S_D147Y_Q154H),
        (H8Y_D108N_N127S_D147Y_E155V),
        (D108N_D147Y_E155V),
        (H8Y_D108N_N127S),
        (H8Y_D108N_N127S_D147Y_Q154H),
        (A106V_D108N_D147Y_E155V),
        (D108Q_D147Y_E155V),
        (D108M_D147Y_E155V),
        (D108L_D147Y_E155V),
        (D108K_D147Y_E155V),
        (D108I_D147Y_E155V),
        (D108F_D147Y_E155V),
        (A106V_D108N_D147Y),
        (A106V_D108M_D147Y_E155V),
        (E59A_A106V_D108N_D147Y_E155V),
        (E59A cat dead_A106V_D108N_D147Y_E155V),
        (L84F_A106V_D108N_H123Y_D147Y_E155V_I156Y),
        (L84F_A106V_D108N_H123Y_D147Y_E155V_I156F),
        (D103A_D104N),
        (G22P_D103A_D104N),
        (D103A_D104N_S138A),
        (R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F),
        (E25G_R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F),
        (E25D_R26G_L84F_A106V_R107K_D108N_H123Y_A142N_A143G_D147Y_E155V_I156F),
        (R26Q_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F),
        (E25M_R26G_L84F_A106V_R107P_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F),
        (R26C_L84F_A106V_R107H_D108N_H123Y_A142N_D147Y_E155V_I156F),
        (L84F_A106V_D108N_H123Y_A142N_A143L_D147Y_E155V_I156F),
        (R26G_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F),
        (E25A_R26G_L84F_A106V_R107N_D108N_H123Y_A142N_A143E_D147Y_E155V_I156F),
        (R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F),
        (A106V_D108N_A142N_D147Y_E155V),
        (R26G_A106V_D108N_A142N_D147Y_E155V),
        (E25D_R26G_A106V_R107K_D108N_A142N_A143G_D147Y_E155V),
        (R26G_A106V_D108N_R107H_A142N_A143D_D147Y_E155V),
        (E25D_R26G_A106V_D108N_A142N_D147Y_E155V),
        (A106V_R107K_D108N_A142N_D147Y_E155V),
        (A106V_D108N_A142N_A143G_D147Y_E155V),
        (A106V_D108N_A142N_A143L_D147Y_E155V),
        (H36L_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N),
        (N37T_P48T_M70L_L84F_A106V_D108N_H123Y_D147Y_149V_E155V_I156F),
        (N37S_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K161T),
        (H36L_L84F_A106V_D108N_H123Y_D147Y_Q154H_E155V_I156F),
        (N72S_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F),
        (H36L_P48L_L84F_A106V_D108N_H123Y_E134G_D147Y_E155V_I156F),
        (H36L_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K157N)
        (H36L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F),
        (L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T),
        (N37S_R51H_D77G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F),
        (R51L_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K157N),
        (D24G_Q71R_L84F_H96L_A106V_D108N_H123Y_D147Y_E155V_I156F_K160E),
        (H36L_G67V_L84F_A106V_D108N_H123Y_S146T_D147Y_E155V_I156F),
        (Q71L_L84F_A106V_D108N_H123Y_L137M_A143E_D147Y_E155V_I156F),
        (E25G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_Q159L),
        (L84F_A91T_F104I_A106V_D108N_H123Y_D147Y_E155V_I156F),
        (N72D_L84F_A106V_D108N_H123Y_G125A_D147Y_E155V_I156F),
        (P48S_L84F_S97C_A106V_D108N_H123Y_D147Y_E155V_I156F),
        (W23G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F),
        (D24G_P48L_Q71R_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_Q159L),
        (L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F),
        (H36L_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N),
        (N37S_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F_K161T),
        (L84F_A106V_D108N_D147Y_E155V_I156F),
        (R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K161T),
        (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K161T),
        (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K160E_K161T),
        (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K160E),
        (R74Q_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F),
        (R74A_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F),
        (L84F_A106V_D108N_H123Y_D147Y_E155V_I156F),
        (R74Q_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F),
        (L84F_R98Q_A106V_D108N_H123Y_D147Y_E155V_I156F),
        (L84F_A106V_D108N_H123Y_R129Q_D147Y_E155V_I156F),
        (P48S_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F),
        (P48S_A142N),
        (P48T_I49V_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F_L157N),
        (P48T_I49V_A142N),
        (H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N),
        (H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F
        (H36L_P48T_I49V_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N),
        (H36L_P48T_I49V_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N),
        (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N),
        (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N),
        (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F_K157N),
        (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N),
        (W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N),
        (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T),
        (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152H_E155V_I156F_K157N),
        (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N),
        (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N),
        (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_E155V_I156F_K157N),
        (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_R152P_E155V_I156F_K157N),
        (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T),
        (W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F
        _K157N), (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_R152P_E155 V_I156F_K157N).
    
    


In some embodiments, the TadA deaminase is TadA variant. In some embodiments, the TadA variant is TadA*7.10. In particular embodiments, the fusion proteins comprise a single TadA*7.10 domain (e.g., provided as a monomer). In other embodiments, the fusion protein comprises TadA*7.10 and TadA(wt), which are capable of forming heterodimers. In one embodiment, a fusion protein of the invention comprises a wild-type TadA linked to TadA*7.10, which is linked to Cas9 nickase.
In some embodiments, TadA*7.10 comprises at least one alteration. In some embodiments, the adenosine deaminase comprises an alteration in the following sequence:







TadA*7.10


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAI


 


GLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSR


 


IGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCY


 


FFRMPRQVFNAQKKAQSSTD






In some embodiments, TadA*7.10 comprises an alteration at amino acid 82 and/or 166. In particular embodiments, TadA*7.10 comprises one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and/or Q154R. In other embodiments, a variant of TadA*7.10 comprises a combination of alterations selected from the group of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+176Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.
In some embodiments, an adenosine deaminase variant (e.g., TadA*8) comprises a deletion. In some embodiments, an adenosine deaminase variant comprises a deletion of the C terminus. In particular embodiments, an adenosine deaminase variant comprises a deletion of the C terminus beginning at residue 149, 150, 151, 152, 153, 154, 155, 156, and 157, relative to TadA*7.10, the TadA reference sequence, or a corresponding mutation in another TadA.
In other embodiments, an adenosine deaminase variant (e.g., TadA*8) is a monomer comprising one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and/or Q154R, relative to TadA*7.10, the TadA reference sequence, or a corresponding mutation in another TadA. In other embodiments, the adenosine deaminase variant (TadA*8) is a monomer comprising a combination of alterations selected from the group of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+176Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R, relative to TadA*7.10, the TadA reference sequence, or a corresponding mutation in another TadA.
In other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8) each having one or more of the following alterations Y147T, Y147R, Q154S, Y123H, V82S, T166R, and/or Q154R, relative to TadA*7.10, the TadA reference sequence, or a corresponding mutation in another TadA. In other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8) each having a combination of alterations selected from the group of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+176Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R, relative to TadA*7.10, the TadA reference sequence, or a corresponding mutation in another TadA.
In other embodiments, the adenosine deaminase variant is a heterodimer of a wild-type adenosine deaminase domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising one or more of the following alterations Y147T, Y147R, Q154S, Y123H, V82S, T166R, and/or Q154R, relative to TadA*7.10, the TadA reference sequence, or a corresponding mutation in another TadA. In other embodiments, the adenosine deaminase variant is a heterodimer of a wild-type adenosine deaminase domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising a combination of alterations selected from the group of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R, relative to TadA*7.10, the TadA reference sequence, or a corresponding mutation in another TadA.
In other embodiments, the adenosine deaminase variant is a heterodimer of a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising one or more of the following alterations Y147T, Y147R, Q154S, Y123H, V82S, T166R, and/or Q154R, relative to TadA*7.10, the TadA reference sequence, or a corresponding mutation in another TadA. In other embodiments, the adenosine deaminase variant is a heterodimer of a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising a combination of alterations selected from the group of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R, relative to TadA*7.10, the TadA reference sequence, or a corresponding mutation in another TadA.
In particular embodiments, an adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from Staphylococcus aureus (S. aureus) TadA, Bacillus subtilis (B. subtilis) TadA, Salmonella typhimurium (S. typhimurium) TadA, Shewanella putrefaciens (S. putrefaciens) TadA, Haemophilus influenzae F3031 (H. influenzae) TadA, Caulobacter crescentus (C. crescentus) TadA, Geobacter sulfurreducens (G. sulfurreducens) TadA, or TadA*7.10.
In some embodiments, an adenosine deaminase is a TadA*8. In one embodiment, an adenosine deaminase is a TadA*8 that comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:







MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAI


 


GLHDPTAHAEIMALRQGGLVMQNYRLIDATIYVTFEPCVMCAGAMIHSR


 


IGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCT


 


FFRMPRQVFNAQKKAQSSTD






In some embodiments, the TadA*8 is truncated. In some embodiments, the truncated TadA*8 is missing 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to the full length TadA*8. In some embodiments, the truncated TadA*8 is missing 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to the full length TadA*8. In some embodiments the adenosine deaminase variant is a full-length TadA*8.
In some embodiments the TadA*8 is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24.
In other embodiments, a base editor of the disclosure comprising an adenosine deaminase variant (e.g., TadA*8) monomer comprising one or more of the following alterations: R26C, V88A, A109S, T111R, D119N, H122N, Y147D, F149Y, T166I and/or D167N, relative to TadA*7.10, the TadA reference sequence, or a corresponding mutation in another TadA. In other embodiments, the adenosine deaminase variant (TadA*8) monomer comprises a combination of alterations selected from the group of: R26C+A109S+T111R+D119N+H122N+Y147D+F149Y+T166I+D167N; V88A+A109S+T111R+D119N+H122N+F149Y+T166I+D167N; R26C+A109S+T111R+D119N+H122N+F149Y+T166I+D167N; V88A+T111R+D119N+F149Y; and A109S+T111R+D119N+H122N+Y147D+F149Y+T166I+D167N, relative to TadA*7.10, the TadA reference sequence, or a corresponding mutation in another TadA.
In other embodiments, a base editor comprises a heterodimer of a wild-type adenosine deaminase domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising one or more of the following alterations R26C, V88A, A109S, T111R, D119N, H122N, Y147D, F149Y, T166I and/or D167N, relative to TadA*7.10, the TadA reference sequence, or a corresponding mutation in another TadA. In other embodiments, the base editor comprises a heterodimer of a wild-type adenosine deaminase domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising a combination of alterations selected from the group of: R26C+A109S+T111R+D119N+H122N+Y147D+F149Y+T166I+D167N; V88A+A109S+T111R+D119N+H122N+F149Y+T166I+D167N; R26C+A109S+T111R+D119N+H122N+F149Y+T166I+D167N; V88A+T111R+D119N+F149Y; and A109S+T111R+D119N+H122N+Y147D+F149Y+T166I+D167N, relative to TadA*7.10, the TadA reference sequence, or a corresponding mutation in another TadA.
In other embodiments, a base editor comprises a heterodimer of a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising one or more of the following alterations R26C, V88A, A109S, T111R, D119N, H122N, Y147D, F149Y, T166I and/or D167N, relative to TadA*7.10, the TadA reference sequence, or a corresponding mutation in another TadA. In other embodiments, the base editor comprises a heterodimer of a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising a combination of alterations selected from the group of: R26C+A109S+T111R+D119N+H122N+Y147D+F149Y+T166I+D167N; V88A+A109S+T111R+D119N+H122N+F149Y+T166I+D167N; R26C+A109S+T 11R+D119N+H122N+F149Y+T166I+D167N; V88A+T111R+D119N+F149Y; and A109S+T111R+D119N+H122N+Y147D+F149Y+T166I+D167N, relative to TadA*7.10, the TadA reference sequence, or a corresponding mutation in another TadA.
In some embodiments, the TadA*8 is a variant as shown in Table 11. Table 11 shows certain amino acid position numbers in the TadA amino acid sequence and the amino acids present in those positions in the TadA-7.10 adenosine deaminase. Table 11 also shows amino acid changes in TadA variants relative to TadA-7.10 following phage-assisted non-continuous evolution (PANCE) and phage-assisted continuous evolution (PACE), as described in M. Richter et al., 2020, Nature Biotechnology, doi.org/10.1038/s41587-020-0453-z, the entire contents of which are incorporated by reference herein. In some embodiments, the TadA*8 is TadA*8a, TadA*8b, TadA*8c, TadA*8d, or TadA*8e. In some embodiments, the TadA*8 is TadA*8e.







TABLE 11







Additional TadA*8 Variants









TadA



TadA amino acid number




















26
88
109
111
119
122
147
149
166
167



TadA-7.10
R
V
A
T
D
H
Y
F
T
D






















PANCE 1




R








PANCE 2



S/T
R


PACE
TadA-8a
C

S
R
N
N
D
Y
I
N



TadA-8b

A
S/T
R
N
N

Y
I
N



TadA-8c
C

S
R
N
N

Y
I
N



TadA-8d

A

R
N


Y



TadA-8e


S
R
N
N
D
Y
I
N









In one embodiment, a fusion protein of the invention comprises a wild-type TadA is linked to an adenosine deaminase variant described herein (e.g., TadA*8), which is linked to Cas9 nickase. In particular embodiments, the fusion proteins comprise a single TadA*8 domain (e.g., provided as a monomer). In other embodiments, the fusion protein comprises TadA*8 and TadA(wt), which are capable of forming heterodimers.
In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in any of the adenosine deaminases provided herein. It should be appreciated that adenosine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). The disclosure provides any deaminase domains with a certain percent identity plus any of the mutations or combinations thereof described herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to a reference sequence, or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues as compared to any one of the amino acid sequences known in the art or described herein.
In particular embodiments, a TadA*8 comprises one or more mutations at any of the following positions shown in bold. In other embodiments, a TadA*8 comprises one or more mutations at any of the positions shown with underlining:









MSEVEFSHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV IGEGWNRAIG
 50



 


LHDPTAHAEI MALRQGGLVM QNYRLIDATL YVTEEPCVMC AGAMIHSRIG
100


 


RVVFGVRNAK TGAAGSLMDV LHYPGMNHRV EITEGILADE CAALLCYFFR
150


 


MPRQVFNAQK KAQSSTD







For example, the TadA*8 comprises alterations at amino acid position 82 and/or 166 (e.g., V82S, T166R) alone or in combination with any one or more of the following Y147T, Y147R, Q154S, Y123H, and/or Q154R, relative to TadA*7.10, the TadA reference sequence, or a corresponding mutation in another TadA. In particular embodiments, a combination of alterations is selected from the group of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R, relative to TadA*7.10, the TadA reference sequence, or a corresponding mutation in another TadA.
In some embodiments, the adenosine deaminase is TadA*8, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:







MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAI


 


GLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSR


 


IGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCT


 


FFRMPRQVFNAQKKAQSSTD






In some embodiments, the TadA*8 is truncated. In some embodiments, the truncated TadA*8 is missing 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to the full length TadA*8. In some embodiments, the truncated TadA*8 is missing 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to the full length TadA*8. In some embodiments the adenosine deaminase variant is a full-length TadA*8.
In one embodiment, a fusion protein of the invention comprises a wild-type TadA is linked to an adenosine deaminase variant described herein (e.g., TadA*8), which is linked to Cas9 nickase. In particular embodiments, the fusion proteins comprise a single TadA*8 domain (e.g., provided as a monomer). In other embodiments, the base editor comprises TadA*8 and TadA(wt), which are capable of forming heterodimers.
In particular embodiments, the fusion proteins comprise a single (e.g., provided as a monomer) TadA*8. In some embodiments, the TadA*8 is linked to a Cas9 nickase. In some embodiments, the fusion proteins of the invention comprise as a heterodimer of a wild-type TadA (TadA(wt)) linked to a TadA*8. In other embodiments, the fusion proteins of the invention comprise as a heterodimer of a TadA*7.10 linked to a TadA*8. In some embodiments, the base editor is ABE8 comprising a TadA*8 variant monomer. In some embodiments, the base editor is ABE8 comprising a heterodimer of a TadA*8 and a TadA(wt). In some embodiments, the base editor is ABE8 comprising a heterodimer of a TadA*8 and TadA*7.10. In some embodiments, the base editor is ABE8 comprising a heterodimer of a TadA*8. In some embodiments, the TadA*8 is selected from Table 11, 13 or 14. In some embodiments, the ABE8 is selected from Table 13, 14 or 16.
In some embodiments, the adenosine deaminase is a TadA*9 variant. In some embodiments, the adenosine deaminase is a TadA*9 variant selected from the variants described below and with reference to the following sequence (termed TadA*7.10).









(SEQ ID NO: 8)



        10         20         30         40



MSEVEFSHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV


 



        50         60         70         80



IGEGWNRAIG LHDPTAHAEI MALRQGGLVM QNYRLIDATL


 



         90        100       110        120



YVTFEPCVMC AGAMIHSRIG RVVFGVRNAK TGAAGSLMDV


 



       130        140        150        160



LHYPGMNHRV EITEGILADE CAALLCYFFR MPRQVFNAQK


 



KAQSSTD






In some embodiments, an adenosine deaminase comprises one or more of the following alterations: R21N, R23H, E25F, N38G, L51W, P54C, M70V, Q71M, N72K, Y73S, V82T, M94V, P124W, T133K, D139L, D139M, C146R, and A158K. The one or more alternations are shown in the sequence above in underlining and bold font.
In some embodiments, an adenosine deaminase comprises one or more of the following combinations of alterations: V82S+Q154R+Y147R; V82S+Q154R+Y123H; V82S+Q154R+Y147R+Y123H; Q154R+Y147R+Y123H+I76Y+V82S; V82S+I76Y; V82S+Y147R; V82S+Y147R+Y123H; V82S+Q154R+Y123H; Q154R+Y147R+Y123H+I76Y; V82S+Y147R; V82S+Y147R+Y123H; V82S+Q154R+Y123H; V82S+Q154R+Y147R; V82S+Q154R+Y147R; Q154R+Y147R+Y123H+I76Y; Q154R+Y147R+Y123H+I76Y+V82S; I76Y_V82S_Y123H_Y147R Q154R; Y147R+Q154R+H123H; and V82S+Q154R.
In some embodiments, an adenosine deaminase comprises one or more of the following combinations of alterations: E25F+V82S+Y123H, T133K+Y147R+Q154R; E25F+V82S+Y123H+Y147R+Q154R; L51W+V82S+Y123H+C146R+Y147R+Q154R; Y73S+V82S+Y123H+Y147R+Q154R; P54C+V82S+Y123H+Y147R+Q154R; N38G+V82T+Y123H+Y147R+Q154R; N72K+V82S+Y123H+D139L+Y147R+Q154R; E25F+V82S+Y123H+D139M+Y147R+Q154R; Q71M+V82S+Y123H+Y147R+Q154R; E25F+V82S+Y123H+T133K+Y147R+Q154R; E25F+V82S+Y123H+Y147R+Q154R; V82S+Y123H+P124W+Y147R+Q154R; L51W+V82S+Y123H+C146R+Y147R+Q154R; P54C+V82S+Y123H+Y147R+Q154R; Y73S+V82S+Y123H+Y147R+Q154R; N38G+V82T+Y123H+Y147R+Q154R; R23H+V82S+Y123H+Y147R+Q154R; R21N+V82S+Y123H+Y147R+Q154R; V82S+Y123H+Y147R+Q154R+A158K; N72K+V82S+Y123H+D139L+Y147R+Q154R; E25F+V82S+Y123H+D139M+Y147R+Q154R; and M70V+V82S+M94V+Y123H+Y147R+Q154R
In some embodiments, an adenosine deaminase comprises one or more of the following combinations of alterations: Q71M+V82S+Y123H+Y147R+Q154R; E25F+I76Y+V82S+Y123H+Y147R+Q154R; I76Y+V82T+Y123H+Y147R+Q154R; N38G+I76Y+V82S+Y123H+Y147R+Q154R; R23H+I76Y+V82S+Y123H+Y147R+Q154R; P54C+176Y+V82S+Y123H+Y147R+Q154R; R21N+176Y+V82S+Y123H+Y147R+Q154R; I76Y+V82S+Y123H+D139M+Y147R+Q154R; Y73S+I76Y+V82S+Y123H+Y147R+Q154R; E25F+176Y+V82S+Y123H+Y147R+Q154R; 176Y+V82T+Y123H+Y147R+Q154R; N38G+176Y+V82S+Y123H+Y147R+Q154R; R23H+176Y+V82S+Y123H+Y147R+Q154R; P54C+I76Y+V82S+Y123H+Y147R+Q154R; R21N+I76Y+V82S+Y123H+Y147R+Q154R; 176Y+V82S+Y123H+D139M+Y147R+Q154R; Y73S+176Y+V82S+Y123H+Y147R+Q154R; and V82S+Q154R; N72K_V82S+Y123H+Y147R+Q154R; Q71M_V82S+Y123H+Y147R+Q154R; V82S+Y123H+T133K+Y147R+Q154R; V82S+Y123H+T133K+Y147R+Q154R+A158K; M70V+Q71M+N72K+V82S+Y123H+Y147R+Q154R; N72K_V82S+Y123H+Y147R+Q154R; Q71M_V82S+Y123H+Y147R+Q154R; M70V+V82S+M94V+Y123H+Y147R+Q154R; V82S+Y123H+T133K+Y147R+Q154R; V82S+Y123H+T133K+Y147R+Q154R+A158K; and M70V+Q71M+N72K+V82S+Y123H+Y147R+Q154R. In some embodiments, the adenosine deaminase is expressed as a monomer. In other embodiments, the adenosine deaminase is expressed as a heterodimer. In some embodiments, the deaminase or other polypeptide sequence lacks a methionine, for example when included as a component of a fusion protein. This can alter the numbering of positions. However, the skilled person will understand that such corresponding mutations refer to the same mutation, e.g., Y73S and Y72S and D139M and D138M.
In some embodiments, the TadA*9 variant comprises the alterations described in Table 17 as described herein. In some embodiments, the TadA*9 variant is a monomer. In some embodiments, the TadA*9 variant is a heterodimer with a wild-type TadA adenosine deaminase. In some embodiments, the TadA*9 variant is a heterodimer with another TadA variant (e.g., TadA*8, TadA*9). Additional details of TadA*9 adenosine deaminases are described in International PCT Application No. PCT/2020/049975, which is incorporated herein by reference for its entirety.
Any of the mutations provided herein and any additional mutations (e.g., based on the ecTadA amino acid sequence) can be introduced into any other adenosine deaminases. Any of the mutations provided herein can be made individually or in any combination in TadA reference sequence or another adenosine deaminase (e.g., ecTadA).
Details of A to G nucleobase editing proteins are described in International PCT Application No. PCT/2017/045381 (WO2018/027078) and Gaudelli, N. M., et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature, 551, 464-471 (2017), the entire contents of which are hereby incorporated by reference.
C to T Editing
In some embodiments, a base editor disclosed herein comprises a fusion protein comprising cytidine deaminase capable of deaminating a target cytidine (C) base of a polynucleotide to produce uridine (U), which has the base pairing properties of thymine. In some embodiments, for example where the polynucleotide is double-stranded (e.g., DNA), the uridine base can then be substituted with a thymidine base (e.g., by cellular repair machinery) to give rise to a C:G to a T:A transition. In other embodiments, deamination of a C to U in a nucleic acid by a base editor cannot be accompanied by substitution of the U to a T.
The deamination of a target C in a polynucleotide to give rise to a U is a non-limiting example of a type of base editing that can be executed by a base editor described herein. In another example, a base editor comprising a cytidine deaminase domain can mediate conversion of a cytosine (C) base to a guanine (G) base. For example, a U of a polynucleotide produced by deamination of a cytidine by a cytidine deaminase domain of a base editor can be excised from the polynucleotide by a base excision repair mechanism (e.g., by a uracil DNA glycosylase (UDG) domain), producing an abasic site. The nucleobase opposite the abasic site can then be substituted (e.g., by base repair machinery) with another base, such as a C, by for example a translesion polymerase. Although it is typical for a nucleobase opposite an abasic site to be replaced with a C, other substitutions (e.g., A, G or T) can also occur.
Accordingly, in some embodiments a base editor described herein comprises a deamination domain (e.g., cytidine deaminase domain) capable of deaminating a target C to a U in a polynucleotide. Further, as described below, the base editor can comprise additional domains which facilitate conversion of the U resulting from deamination to, in some embodiments, a T or a G. For example, a base editor comprising a cytidine deaminase domain can further comprise a uracil glycosylase inhibitor (UGI) domain to mediate substitution of a U by a T, completing a C-to-T base editing event. In another example, a base editor can incorporate a translesion polymerase to improve the efficiency of C-to-G base editing, since a translesion polymerase can facilitate incorporation of a C opposite an abasic site (i.e., resulting in incorporation of a G at the abasic site, completing the C-to-G base editing event).
A base editor comprising a cytidine deaminase as a domain can deaminate a target C in any polynucleotide, including DNA, RNA and DNA-RNA hybrids. Typically, a cytidine deaminase catalyzes a C nucleobase that is positioned in the context of a single-stranded portion of a polynucleotide. In some embodiments, the entire polynucleotide comprising a target C can be single-stranded. For example, a cytidine deaminase incorporated into the base editor can deaminate a target C in a single-stranded RNA polynucleotide. In other embodiments, a base editor comprising a cytidine deaminase domain can act on a double-stranded polynucleotide, but the target C can be positioned in a portion of the polynucleotide which at the time of the deamination reaction is in a single-stranded state. For example, in embodiments where the NAGPB domain comprises a Cas9 domain, several nucleotides can be left unpaired during formation of the Cas9-gRNA-target DNA complex, resulting in formation of a Cas9 “R-loop complex”. These unpaired nucleotides can form a bubble of single-stranded DNA that can serve as a substrate for a single-strand specific nucleotide deaminase enzyme (e.g., cytidine deaminase).
In some embodiments, a cytidine deaminase of a base editor can comprise all or a portion of an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. APOBEC is a family of evolutionarily conserved cytidine deaminases. Members of this family are C-to-U editing enzymes. The N-terminal domain of APOBEC like proteins is the catalytic domain, while the C-terminal domain is a pseudocatalytic domain. More specifically, the catalytic domain is a zinc dependent cytidine deaminase domain and is important for cytidine deamination. APOBEC family members include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D (“APOBEC3E” now refers to this), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and Activation-induced (cytidine) deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of an APOBEC1 deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC2 deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of is an APOBEC3 deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of an APOBEC3A deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3B deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3C deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3D deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3E deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3F deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3G deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3H deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC4 deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of activation-induced deaminase (AID). In some embodiments a deaminase incorporated into a base editor comprises all or a portion of cytidine deaminase 1 (CDA1). It should be appreciated that a base editor can comprise a deaminase from any suitable organism (e.g., a human or a rat). In some embodiments, a deaminase domain of a base editor is from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase domain of the base editor is derived from rat (e.g., rat APOBEC1). In some embodiments, the deaminase domain of the base editor is human APOBEC1. In some embodiments, the deaminase domain of the base editor is pmCDA1.
The amino acid and nucleic acid sequences of PmCDA1 are shown herein below.








tr|A5H718|A5H718_PETMA Cytosine deaminase OS = Petromyzon marinus



OX = 7757 PE = 2 SV = 1 amino acid sequence:


MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERG


 


IHAEIFSIRKVEEYLRDNPGQETINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYY


 


EKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELS


 


IMIQVKILHTTKSPAV


 


Nucleic acid sequence: >EF094822.1 Petromyzon marinus isolate PmCDA.21


cytosine deaminase mRNA, complete cds:


TGACACGACACAGCCGTGTATATGAGGAAGGGTAGCTGGATGGGGGGGGGGGGAATACGTTCAG


 


AGAGGACATTAGCGAGCGTCTTGTTGGTGGCCTTGAGTCTAGACACCTGCAGACATGACCGACG


 


CTGAGTACGTGAGAATCCATGAGAAGTTGGACATCTACACGTTTAAGAAACAGTTTTTCAACAA


 


CAAAAAATCCGTGTCGCATAGATGCTACGTTCTCTTTGAATTAAAACGACGGGGTGAACGTAGA


 


GCGTGTTTTTGGGGCTATGCTGTGAATAAACCACAGAGCGGGACAGAAAGTGGAATTCACGCCG


 


AAATCTTTAGCATTAGAAAAGTCGAAGAATACCTGCGCGACAACCCCGGACAATTCACGATAAA


 


TTGGTACTCATCCTGGAGTCCTTGTGCAGATTGCGCTGAAAAGATCTTAGAATGGTATAACCAG


 


GAGCTGCGGGGGAACGGCCACACTTTGAAAATCTGGGCTTGCAAACTCTATTACGAGAAAAATG


 


CGAGGAATCAAATTGGGCTGTGGAACCTCAGAGATAACGGGGTTGGGTTGAATGTAATGGTAAG


 


TGAACACTACCAATGTTGCAGGAAAATATTCATCCAATCGTCGCACAATCAATTGAATGAGAAT


 


AGATGGCTTGAGAAGACTTTGAAGCGAGCTGAAAAACGACGGAGCGAGTTGTCCATTATGATTC


 


AGGTAAAAATACTCCACACCACTAAGAGTCCTGCTGTTTAAGAGGCTATGCGGATGGTTTTC






The amino acid and nucleic acid sequences of the coding sequence (CDS) of human activation-induced cytidine deaminase (AID) are shown below.








>tr|Q6QJ80|Q6QJ80_HUMAN Activation-induced cytidine deaminase OS =



Homo sapiens OX = 9606 GN = AICDA PE = 2 SV = 1 amino acid sequence:


MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELL


FLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRK


AEPEGLRRLHRAGVQIAIMTFKAPV






The amino acid and nucleic acid sequences of the coding sequence (CDS) of human activation-induced cytidine deaminase (AID) are shown below.








>tr|Q6QJ80|Q6QJ80_HUMAN Activation-induced cytidine deaminase OS = Homo sapiens



OX = 9606 GN = AICDA PE = 2 SV = 1 amino acid sequence:


MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELL


 


FLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRK


 


AEPEGLRRLHRAGVQIAIMTFKAPV


 


Nucleic acid sequence: >NG_011588.1:5001-15681 Homo sapiens activation induced


cytidine deaminase (AICDA), RefSeqGene (LRG_17) on chromosome 12:


AGAGAACCATCATTAATTGAAGTGAGATTTTTCTGGCCTGAGACTTGCAGGGAGGCAAGAAGAC


 


ACTCTGGACACCACTATGGACAGGTAAAGAGGCAGTCTTCTCGTGGGTGATTGCACTGGCCTTC


 


CTCTCAGAGCAAATCTGAGTAATGAGACTGGTAGCTATCCCTTTCTCTCATGTAACTGTCTGAC


 


TGATAAGATCAGCTTGATCAATATGCATATATATTTTTTGATCTGTCTCCTTTTCTTCTATTCA


 


GATCTTATACGCTGTCAGCCCAATTCTTTCTGTTTCAGACTTCTCTTGATTTCCCTCTTTTTCA


 


TGTGGCAAAAGAAGTAGTGCGTACAATGTACTGATTCGTCCTGAGATTTGTACCATGGTTGAAA


 


CTAATTTATGGTAATAATATTAACATAGCAAATCTTTAGAGACTCAAATCATGAAAAGGTAATA


 


GCAGTACTGTACTAAAAACGGTAGTGCTAATTTTCGTAATAATTTTGTAAATATTCAACAGTAA


 


AACAACTTGAAGACACACTTTCCTAGGGAGGCGTTACTGAAATAATTTAGCTATAGTAAGAAAA


 


TTTGTAATTTTAGAAATGCCAAGCATTCTAAATTAATTGCTTGAAAGTCACTATGATTGTGTCC


 


ATTATAAGGAGACAAATTCATTCAAGCAAGTTATTTAATGTTAAAGGCCCAATTGTTAGGCAGT


 


TAATGGCACTTTTACTATTAACTAATCTTTCCATTTGTTCAGACGTAGCTTAACTTACCTCTTA


 


GGTGTGAATTTGGTTAAGGTCCTCATAATGTCTTTATGTGCAGTTTTTGATAGGTTATTGTCAT


 


AGAACTTATTCTATTCCTACATTTATGATTACTATGGATGTATGAGAATAACACCTAATCCTTA


 


TACTTTACCTCAATTTAACTCCTTTATAAAGAACTTACATTACAGAATAAAGATTTTTTAAAAA


 


TATATTTTTTTGTAGAGACAGGGTCTTAGCCCAGCCGAGGCTGGTCTCTAAGTCCTGGCCCAAG


 


CGATCCTCCTGCCTGGGCCTCCTAAAGTGCTGGAATTATAGACATGAGCCATCACATCCAATAT


 


ACAGAATAAAGATTTTTAATGGAGGATTTAATGTTCTTCAGAAAATTTTCTTGAGGTCAGACAA


 


TGTCAAATGTCTCCTCAGTTTACACTGAGATTTTGAAAACAAGTCTGAGCTATAGGTCCTTGTG


 


AAGGGTCCATTGGAAATACTTGTTCAAAGTAAAATGGAAAGCAAAGGTAAAATCAGCAGTTGAA


 


ATTCAGAGAAAGACAGAAAAGGAGAAAAGATGAAATTCAACAGGACAGAAGGGAAATATATTAT


 


CATTAAGGAGGACAGTATCTGTAGAGCTCATTAGTGATGGCAAAATGACTTGGTCAGGATTATT


 


TTTAACCCGCTTGTTTCTGGTTTGCACGGCTGGGGATGCAGCTAGGGTTCTGCCTCAGGGAGCA


 


CAGCTGTCCAGAGCAGCTGTCAGCCTGCAAGCCTGAAACACTCCCTCGGTAAAGTCCTTCCTAC


 


TCAGGACAGAAATGACGAGAACAGGGAGCTGGAAACAGGCCCCTAACCAGAGAAGGGAAGTAAT


 


GGATCAACAAAGTTAACTAGCAGGTCAGGATCACGCAATTCATTTCACTCTGACTGGTAACATG


 


TGACAGAAACAGTGTAGGCTTATTGTATTTTCATGTAGAGTAGGACCCAAAAATCCACCCAAAG


 


TCCTTTATCTATGCCACATCCTTCTTATCTATACTTCCAGGACACTTTTTCTTCCTTATGATAA


 


GGCTCTCTCTCTCTCCACACACACACACACACACACACACACACACACACACACACACACAAAC


 


ACACACCCCGCCAACCAAGGTGCATGTAAAAAGATGTAGATTCCTCTGCCTTTCTCATCTACAC


 


AGCCCAGGAGGGTAAGTTAATATAAGAGGGATTTATTGGTAAGAGATGATGCTTAATCTGTTTA


 


ACACTGGGCCTCAAAGAGAGAATTTCTTTTCTTCTGTACTTATTAAGCACCTATTATGTGTTGA


 


GCTTATATATACAAAGGGTTATTATATGCTAATATAGTAATAGTAATGGTGGTTGGTACTATGG


 


TAATTACCATAAAAATTATTATCCTTTTAAAATAAAGCTAATTATTATTGGATCTTTTTTAGTA


 


TTCATTTTATGTTTTTTATGTTTTTGATTTTTTAAAAGACAATCTCACCCTGTTACCCAGGCTG


 


GAGTGCAGTGGTGCAATCATAGCTTTCTGCAGTCTTGAACTCCTGGGCTCAAGCAATCCTCCTG


 


CCTTGGCCTCCCAAAGTGTTGGGATACAGTCATGAGCCACTGCATCTGGCCTAGGATCCATTTA


 


GATTAAAATATGCATTTTAAATTTTAAAATAATATGGCTAATTTTTACCTTATGTAATGTGTAT


 


ACTGGCAATAAATCTAGTTTGCTGCCTAAAGTTTAAAGTGCTTTCCAGTAAGCTTCATGTACGT


 


GAGGGGAGACATTTAAAGTGAAACAGACAGCCAGGTGTGGTGGCTCACGCCTGTAATCCCAGCA


 


CTCTGGGAGGCTGAGGTGGGTGGATCGCTTGAGCCCTGGAGTTCAAGACCAGCCTGAGCAACAT


 


GGCAAAACGCTGTTTCTATAACAAAAATTAGCCGGGCATGGTGGCATGTGCCTGTGGTCCCAGC


 


TACTAGGGGGCTGAGGCAGGAGAATCGTTGGAGCCCAGGAGGTCAAGGCTGCACTGAGCAGTGC


 


TTGCGCCACTGCACTCCAGCCTGGGTGACAGGACCAGACCTTGCCTCAAAAAAATAAGAAGAAA


 


AATTAAAAATAAATGGAAACAACTACAAAGAGCTGTTGTCCTAGATGAGCTACTTAGTTAGGCT


 


GATATTTTGGTATTTAACTTTTAAAGTCAGGGTCTGTCACCTGCACTACATTATTAAAATATCA


 


ATTCTCAATGTATATCCACACAAAGACTGGTACGTGAATGTTCATAGTACCTTTATTCACAAAA


 


CCCCAAAGTAGAGACTATCCAAATATCCATCAACAAGTGAACAAATAAACAAAATGTGCTATAT


 


CCATGCAATGGAATACCACCCTGCAGTACAAAGAAGCTACTTGGGGATGAATCCCAAAGTCATG


 


ACGCTAAATGAAAGAGTCAGACATGAAGGAGGAGATAATGTATGCCATACGAAATTCTAGAAAA


 


TGAAAGTAACTTATAGTTACAGAAAGCAAATCAGGGCAGGCATAGAGGCTCACACCTGTAATCC


 


CAGCACTTTGAGAGGCCACGTGGGAAGATTGCTAGAACTCAGGAGTTCAAGACCAGCCTGGGCA


 


ACACAGTGAAACTCCATTCTCCACAAAAATGGGAAAAAAAGAAAGCAAATCAGTGGTTGTCCTG


 


TGGGGAGGGGAAGGACTGCAAAGAGGGAAGAAGCTCTGGTGGGGTGAGGGTGGTGATTCAGGTT


 


CTGTATCCTGACTGTGGTAGCAGTTTGGGGTGTTTACATCCAAAAATATTCGTAGAATTATGCA


 


TCTTAAATGGGTGGAGTTTACTGTATGTAAATTATACCTCAATGTAAGAAAAAATAATGTGTAA


 


GAAAACTTTCAATTCTCTTGCCAGCAAACGTTATTCAAATTCCTGAGCCCTTTACTTCGCAAAT


 


TCTCTGCACTTCTGCCCCGTACCATTAGGTGACAGCACTAGCTCCACAAATTGGATAAATGCAT


 


TTCTGGAAAAGACTAGGGACAAAATCCAGGCATCACTTGTGCTTTCATATCAACCATGCTGTAC


 


AGCTTGTGTTGCTGTCTGCAGCTGCAATGGGGACTCTTGATTTCTTTAAGGAAACTTGGGTTAC


 


CAGAGTATTTCCACAAATGCTATTCAAATTAGTGCTTATGATATGCAAGACACTGTGCTAGGAG


 


CCAGAAAACAAAGAGGAGGAGAAATCAGTCATTATGTGGGAACAACATAGCAAGATATTTAGAT


 


CATTTTGACTAGTTAAAAAAGCAGCAGAGTACAAAATCACACATGCAATCAGTATAATCCAAAT


 


CATGTAAATATGTGCCTGTAGAAAGACTAGAGGAATAAACACAAGAATCTTAACAGTCATTGTC


 


ATTAGACACTAAGTCTAATTATTATTATTAGACACTATGATATTTGAGATTTAAAAAATCTTTA


 


ATATTTTAAAATTTAGAGCTCTTCTATTTTTCCATAGTATTCAAGTTTGACAATGATCAAGTAT


 


TACTCTTTCTTTTTTTTTTTTTTTTTTTTTTTTTGAGATGGAGTTTTGGTCTTGTTGCCCATGC


 


TGGAGTGGAATGGCATGACCATAGCTCACTGCAACCTCCACCTCCTGGGTTCAAGCAAAGCTGT


 


CGCCTCAGCCTCCCGGGTAGATGGGATTACAGGCGCCCACCACCACACTCGGCTAATGTTTGTA


 


TTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTCAGAGG


 


ATCCACCTGCCTCAGCCTCCCAAAGTGCTGGGATTACAGATGTAGGCCACTGCGCCCGGCCAAG


 


TATTGCTCTTATACATTAAAAAACAGGTGTGAGCCACTGCGCCCAGCCAGGTATTGCTCTTATA


 


CATTAAAAAATAGGCCGGTGCAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAAGCCAAGGC


 


GGGCAGAACACCCGAGGTCAGGAGTCCAAGGCCAGCCTGGCCAAGATGGTGAAACCCCGTCTCT


 


ATTAAAAATACAAACATTACCTGGGCATGATGGTGGGCGCCTGTAATCCCAGCTACTCAGGAGG


 


CTGAGGCAGGAGGATCCGCGGAGCCTGGCAGATCTGCCTGAGCCTGGGAGGTTGAGGCTACAGT


 


AAGCCAAGATCATGCCAGTATACTTCAGCCTGGGCGACAAAGTGAGACCGTAACAAAAAAAAAA


 


AAATTTAAAAAAAGAAATTTAGATCAAGATCCAACTGTAAAAAGTGGCCTAAACACCACATTAA


 


AGAGTTTGGAGTTTATTCTGCAGGCAGAAGAGAACCATCAGGGGGTCTTCAGCATGGGAATGGC


 


ATGGTGCACCTGGTTTTTGTGAGATCATGGTGGTGACAGTGTGGGGAATGTTATTTTGGAGGGA


 


CTGGAGGCAGACAGACCGGTTAAAAGGCCAGCACAACAGATAAGGAGGAAGAAGATGAGGGCTT


 


GGACCGAAGCAGAGAAGAGCAAACAGGGAAGGTACAAATTCAAGAAATATTGGGGGGTTTGAAT


 


CAACACATTTAGATGATTAATTAAATATGAGGACTGAGGAATAAGAAATGAGTCAAGGATGGTT


 


CCAGGCTGCTAGGCTGCTTACCTGAGGTGGCAAAGTCGGGAGGAGTGGCAGTTTAGGACAGGGG


 


GCAGTTGAGGAATATTGTTTTGATCATTTTGAGTTTGAGGTACAAGTTGGACACTTAGGTAAAG


 


ACTGGAGGGGAAATCTGAATATACAATTATGGGACTGAGGAACAAGTTTATTTTATTTTTTGTT


 


TCGTTTTCTTGTTGAAGAACAAATTTAATTGTAATCCCAAGTCATCAGCATCTAGAAGACAGTG


 


GCAGGAGGTGACTGTCTTGTGGGTAAGGGTTTGGGGTCCTTGATGAGTATCTCTCAATTGGCCT


 


TAAATATAAGCAGGAAAAGGAGTTTATGATGGATTCCAGGCTCAGCAGGGCTCAGGAGGGCTCA


 


GGCAGCCAGCAGAGGAAGTCAGAGCATCTTCTTTGGTTTAGCCCAAGTAATGACTTCCTTAAAA


 


AGCTGAAGGAAAATCCAGAGTGACCAGATTATAAACTGTACTCTTGCATTTTCTCTCCCTCCTC


 


TCACCCACAGCCTCTTGATGAACCGGAGGAAGTTTCTTTACCAATTCAAAAATGTCCGCTGGGC


 


TAAGGGTCGGCGTGAGACCTACCTGTGCTACGTAGTGAAGAGGCGTGACAGTGCTACATCCTTT


 


TCACTGGACTTTGGTTATCTTCGCAATAAGGTATCAATTAAAGTCGGCTTTGCAAGCAGTTTAA


 


TGGTCAACTGTGAGTGCTTTTAGAGCCACCTGCTGATGGTATTACTTCCATCCTTTTTTGGCAT


 


TTGTGTCTCTATCACATTCCTCAAATCCTTTTTTTTATTTCTTTTTCCATGTCCATGCACCCAT


 


ATTAGACATGGCCCAAAATATGTGATTTAATTCCTCCCCAGTAATGCTGGGCACCCTAATACCA


 


CTCCTTCCTTCAGTGCCAAGAACAACTGCTCCCAAACTGTTTACCAGCTTTCCTCAGCATCTGA


 


ATTGCCTTTGAGATTAATTAAGCTAAAAGCATTTTTATATGGGAGAATATTATCAGCTTGTCCA


 


AGCAAAAATTTTAAATGTGAAAAACAAATTGTGTCTTAAGCATTTTTGAAAATTAAGGAAGAAG


 


AATTTGGGAAAAAATTAACGGTGGCTCAATTCTGTCTTCCAAATGATTTCTTTTCCCTCCTACT


 


CACATGGGTCGTAGGCCAGTGAATACATTCAACATGGTGATCCCCAGAAAACTCAGAGAAGCCT


 


CGGCTGATGATTAATTAAATTGATCTTTCGGCTACCCGAGAGAATTACATTTCCAAGAGACTTC


 


TTCACCAAAATCCAGATGGGTTTACATAAACTTCTGCCCACGGGTATCTCCTCTCTCCTAACAC


 


GCTGTGACGTCTGGGCTTGGTGGAATCTCAGGGAAGCATCCGTGGGGTGGAAGGTCATCGTCTG


 


GCTCGTTGTTTGATGGTTATATTACCATGCAATTTTCTTTGCCTACATTTGTATTGAATACATC


 


CCAATCTCCTTCCTATTCGGTGACATGACACATTCTATTTCAGAAGGCTTTGATTTTATCAAGC


 


ACTTTCATTTACTTCTCATGGCAGTGCCTATTACTTCTCTTACAATACCCATCTGTCTGCTTTA


 


CCAAAATCTATTTCCCCTTTTCAGATCCTCCCAAATGGTCCTCATAAACTGTCCTGCCTCCACC


 


TAGTGGTCCAGGTATATTTCCACAATGTTACATCAACAGGCACTTCTAGCCATTTTCCTTCTCA


 


AAAGGTGCAAAAAGCAACTTCATAAACACAAATTAAATCTTCGGTGAGGTAGTGTGATGCTGCT


 


TCCTCCCAACTCAGCGCACTTCGTCTTCCTCATTCCACAAAAACCCATAGCCTTCCTTCACTCT


 


GCAGGACTAGTGCTGCCAAGGGTTCAGCTCTACCTACTGGTGTGCTCTTTTGAGCAAGTTGCTT


 


AGCCTCTCTGTAACACAAGGACAATAGCTGCAAGCATCCCCAAAGATCATTGCAGGAGACAATG


 


ACTAAGGCTACCAGAGCCGCAATAAAAGTCAGTGAATTTTAGCGTGGTCCTCTCTGTCTCTCCA


 


GAACGGCTGCCACGTGGAATTGCTCTTCCTCCGCTACATCTCGGACTGGGACCTAGACCCTGGC


 


CGCTGCTACCGCGTCACCTGGTTCACCTCCTGGAGCCCCTGCTACGACTGTGCCCGACATGTGG


 


CCGACTTTCTGCGAGGGAACCCCAACCTCAGTCTGAGGATCTTCACCGCGCGCCTCTACTTCTG


 


TGAGGACCGCAAGGCTGAGCCCGAGGGGCTGCGGCGGCTGCACCGCGCCGGGGTGCAAATAGCC


 


ATCATGACCTTCAAAGGTGCGAAAGGGCCTTCCGCGCAGGCGCAGTGCAGCAGCCCGCATTCGG


 


GATTGCGATGCGGAATGAATGAGTTAGTGGGGAAGCTCGAGGGGAAGAAGTGGGCGGGGATTCT


 


GGTTCACCTCTGGAGCCGAAATTAAAGATTAGAAGCAGAGAAAAGAGTGAATGGCTCAGAGACA


 


AGGCCCCGAGGAAATGAGAAAATGGGGCCAGGGTTGCTTCTTTCCCCTCGATTTGGAACCTGAA


 


CTGTCTTCTACCCCCATATCCCCGCCTTTTTTTCCTTTTTTTTTTTTTGAAGATTATTTTTACT


 


GCTGGAATACTTTTGTAGAAAACCACGAAAGAACTTTCAAAGCCTGGGAAGGGCTGCATGAAAA


 


TTCAGTTCGTCTCTCCAGACAGCTTCGGCGCATCCTTTTGGTAAGGGGCTTCCTCGCTTTTTAA


 


ATTTTCTTTCTTTCTCTACAGTCTTTTTTGGAGTTTCGTATATTTCTTATATTTTCTTATTGTT


 


CAATCACTCTCAGTTTTCATCTGATGAAAACTTTATTTCTCCTCCACATCAGCTTTTTCTTCTG


 


CTGTTTCACCATTCAGAGCCCTCTGCTAAGGTTCCTTTTCCCTCCCTTTTCTTTCTTTTGTTGT


 


TTCACATCTTTAAATTTCTGTCTCTCCCCAGGGTTGCGTTTCCTTCCTGGTCAGAATTCTTTTC


 


TCCTTTTTTTTTTTTTTTTTTTTTTTTTTTAAACAAACAAACAAAAAACCCAAAAAAACTCTTT


 


CCCAATTTACTTTCTTCCAACATGTTACAAAGCCATCCACTCAGTTTAGAAGACTCTCCGGCCC


 


CACCGACCCCCAACCTCGTTTTGAAGCCATTCACTCAATTTGCTTCTCTCTTTCTCTACAGCCC


 


CTGTATGAGGTTGATGACTTACGAGACGCATTTCGTACTTTGGGACTTTGATAGCAACTTCCAG


 


GAATGTCACACACGATGAAATATCTCTGCTGAAGACAGTGGATAAAAAACAGTCCTTCAAGTCT


 


TCTCTGTTTTTATTCTTCAACTCTCACTTTCTTAGAGTTTACAGAAAAAATATTTATATACGAC


 


TCTTTAAAAAGATCTATGTCTTGAAAATAGAGAAGGAACACAGGTCTGGCCAGGGACGTGCTGC


 


AATTGGTGCAGTTTTGAATGCAACATTGTCCCCTACTGGGAATAACAGAACTGCAGGACCTGGG


 


AGCATCCTAAAGTGTCAACGTTTTTCTATGACTTTTAGGTAGGATGAGAGCAGAAGGTAGATCC


 


TAAAAAGCATGGTGAGAGGATCAAATGTTTTTATATCAACATCCTTTATTATTTGATTCATTTG


 


AGTTAACAGTGGTGTTAGTGATAGATTTTTCTATTCTTTTCCCTTGACGTTTAGTTTCAAGTAA


 


CACAAACTCTTCCATCAGGCCATGATCTATAGGACCTCCTAATGAGAGTATCTGGGTGATTGTG


 


ACCCCAAACCATCTCTCCAAAGCATTAATATCCAATCATGCGCTGTATGTTTTAATCAGCAGAA


 


GCATGTTTTTATGTTTGTACAAAAGAAGATTGTTATGGGTGGGGATGGAGGTATAGACCATGCA


 


TGGTCACCTTCAAGCTACTTTAATAAAGGATCTTAAAATGGGCAGGAGGACTGTGAACAAGACA


 


CCCTAATAATGGGTTGATGTCTGAAGTAGCAAATCTTCTGGAAACGCAAACTCTTTTAAGGAAG


 


TCCCTAATTTAGAAACACCCACAAACTTCACATATCATAATTAGCAAACAATTGGAAGGAAGTT


 


GCTTGAATGTTGGGGAGAGGAAAATCTATTGGCTCTCGTGGGTCTCTTCATCTCAGAAATGCCA


 


ATCAGGTCAAGGTTTGCTACATTTTGTATGTGTGTGATGCTTCTCCCAAAGGTATATTAACTAT


 


ATAAGAGAGTTGTGACAAAACAGAATGATAAAGCTGCGAACCGTGGCACACGCTCATAGTTCTA


 


GCTGCTTGGGAGGTTGAGGAGGGAGGATGGCTTGAACACAGGTGTTCAAGGCCAGCCTGGGCAA


 


CATAACAAGATCCTGTCTCTCAAAAAAAAAAAAAAAAAAAAGAAAGAGAGAGGGCCGGGCGTGG


 


TGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGCCGGGCGGATCACCTGTGGTCAGGA


 


GTTTGAGACCAGCCTGGCCAACATGGCAAAACCCCGTCTGTACTCAAAATGCAAAAATTAGCCA


 


GGCGTGGTAGCAGGCACCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGCTTGAA


 


CCCAGGAGGTGGAGGTTGCAGTAAGCTGAGATCGTGCCGTTGCACTCCAGCCTGGGCGACAAGA


 


GCAAGACTCTGTCTCAGAAAAAAAAAAAAAAAAGAGAGAGAGAGAGAAAGAGAACAATATTTGG


 


GAGAGAAGGATGGGGAAGCATTGCAAGGAAATTGTGCTTTATCCAACAAAATGTAAGGAGCCAA


 


TAAGGGATCCCTATTTGTCTCTTTTGGTGTCTATTTGTCCCTAACAACTGTCTTTGACAGTGAG


 


AAAAATATTCAGAATAACCATATCCCTGTGCCGTTATTACCTAGCAACCCTTGCAATGAAGATG


 


AGCAGATCCACAGGAAAACTTGAATGCACAACTGTCTTATTTTAATCTTATTGTACATAAGTTT


 


GTAAAAGAGTTAAAAATTGTTACTTCATGTATTCATTTATATTTTATATTATTTTGCGTCTAAT


 


GATTTTTTATTAACATGATTTCCTTTTCTGATATATTGAAATGGAGTCTCAAAGCTTCATAAAT


 


TTATAACTTTAGAAATGATTCTAATAACAACGTATGTAATTGTAACATTGCAGTAATGGTGCTA


 


CGAAGCCATTTCTCTTGATTTTTAGTAAACTTTTATGACAGCAAATTTGCTTCTGGCTCACTTT


 


CAATCAGTTAAATAAATGATAAATAATTTTGGAAGCTGTGAAGATAAAATACCAAATAAAATAA


 


TATAAAAGTGATTTATATGAAGTTAAAATAAAAAATCAGTATGATGGAATAAACTTG






Other exemplary deaminases that can be fused to Cas9 according to aspects of this disclosure are provided below. In embodiments, the deaminases are activation-induced deaminases (AID). It should be understood that, in some embodiments, the active domain of the respective sequence can be used, e.g., the domain without a localizing signal (nuclear localization sequence, without nuclear export signal, cytoplasmic localizing signal).








Human AID:



(SEQ ID NO: 1372)



MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRY



 


ISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRR


 


LHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDA


 


FRTLGL


 


(underline: nuclear localization sequence; double underline: nuclear export signal)


 


Mouse AID:


(SEQ ID NO: 1373)



MDSLLMKQKKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSCSLDFGHLRNKSGCHVELLFLRY



 


ISDWDLDPGRCYRVTWFTSWSPCYDCARHVAEFLRWNPNLSLRIFTARLYFCEDRKAEPEGLRR


 


LHRAGVQIGIMTFKDYFYCWNTFVENRERTFKAWEGLHENSVRLTRQLRRILLPLYEVDDLRDA


 


FRMLGF


 


(underline: nuclear localization sequence; double underline: nuclear export signal)


 


Canine AID:


(SEQ ID NO: 1374)



MDSLLMKQRKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGHLRNKSGCHVELLFLRY



 


ISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFAARLYFCEDRKAEPEGLRR


 


LHRAGVQIAIMTFKDYFYCWNTFVENREKTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDA


 


FRTLGL


 


(underline: nuclear localization sequence; double underline: nuclear export signal)


 


Bovine AID:


(SEQ ID NO: 1375)



MDSLLKKQRQFLYQFKNVRWAKGRHETYLCYVVKRRDSPTSFSLDFGHLRNKAGCHVELLFLRY



 


ISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFTARLYFCDKERKAEPEGLR


 


RLHRAGVQIAIMTEKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRD


 


AFRTLGL


 


(underline: nuclear localization sequence; double underline: nuclear export signal)


 


Rat AID:


(SEQ ID NO: 1376)



MAVGSKPKAALVGPHWERERIWCFLCSTGLGTQQTGQTSRWLRPAATQDPVSPPRSLLMKQRKE



 


LYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGYLRNKSGCHVELLFLRYISDWDLDPGRC


 


YRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLTGWGALPAGLMSPARPSDYFYCWNTF


 


VENHERTFKAWEGLHENSVRLSRRLRRILLPLYEVDDLRDAFRTLGL


 


(underline: nuclear localization sequence; double underline: nuclear export signal)


 


clAID (Canis lupus familiaris):


(SEQ ID NO: 1374)



MDSLLMKQRKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGHLRNKSGCHVELLFLRYISDWDL



 


DPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFAARLYFCEDRKAEPEGLRRLHRAGVQIAIMT


 


FKDYFYCWNTFVENREKTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL


 


btAID (Bos Taurus):


(SEQ ID NO: 1375)



MDSLLKKQRQFLYQFKNVRWAKGRHETYLCYVVKRRDSPTSFSLDFGHLRNKAGCHVELLFLRYISDWDL



 


DPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFTARLYFCDKERKAEPEGLRRLHRAGVQIAIM


 


TFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL


 


mAID (Mus musculus):


(SEQ ID NO: 1377)



MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDL



 


DPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMT


 


FKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL


 


rAPOBEC-1 (Rattus norvegicus):


(SEQ ID NO: 1378)



MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKE



 


TTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVT


 


IQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIAL


 


QSCHYQRLPPHILWATGLK


 


maAPOBEC-1 (Mesocricetus auratus):


(SEQ ID NO: 1379)



MSSETGPVVVDPTLRRRIEPHEFDAFFDQGELRKETCLLYEIRWGGRHNIWRHTGQNTSRHVEINFIEKF



 


TSERYFYPSTRCSIVWFLSWSPCGECSKAITEFLSGHPNVTLFIYAARLYHHTDQRNRQGLRDLISRGVT


 


IRIMTEQEYCYCWRNFVNYPPSNEVYWPRYPNLWMRLYALELYCIHLGLPPCLKIKRRHQYPLTFFRLNL


 


QSCHYQRIPPHILWATGFI


 


ppAPOBEC-1 (Pongo pygmaeus):


(SEQ ID NO: 1380)



MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKF



 


TSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVARLFWHMDQRNRQGLRDLVNSGVT


 


IQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHL


 


QNCHYQTIPPHILLATGLIHPSVTWR


 


ocAPOBEC 1(Oryctolagus cuniculus):


(SEQ ID NO: 1381)



MASEKGPSNKDYTLRRRIEPWEFEVFFDPQELRKEACLLYEIKWGASSKTWRSSGKNTTNHVEVNFLEKL



 


TSEGRLGPSTCCSITWFLSWSPCWECSMAIREFLSQHPGVTLIIFVARLFQHMDRRNRQGLKDLVTSGVT


 


VRVMSVSEYCYCWENFVNYPPGKAAQWPRYPPRWMLMYALELYCIILGLPPCLKISRRHQKQLTFFSLTP


 


QYCHYKMIPPYILLATGLLQPSVPWR


 


mdAPOBEC-1 (Monodelphis domestica):


(SEQ ID NO: 1382)



MNSKTGPSVGDATLRRRIKPWEFVAFFNPQELRKETCLLYEIKWGNQNIWRHSNQNTSQHAEINFMEKFT



 


AERHFNSSVRCSITWFLSWSPCWECSKAIRKFLDHYPNVTLAIFISRLYWHMDQQHRQGLKELVHSGVTI


 


QIMSYSEYHYCWRNFVDYPQGEEDYWPKYPYLWIMLYVLELHCIILGLPPCLKISGSHSNQLALFSLDLQ


 


DCHYQKIPYNVLVATGLVQPFVTWR


 


ppAPOBEC-2 (Pongo pygmaeus):


(SEQ ID NO: 1383)



MAQKEEAAAATEAASQNGEDLENLDDPEKLKELIELPPFEIVTGERLPANFFKFQFRNVEYSSGRNKTFL



 


CYVVEAQGKGGQVQASRGYLEDEHAAAHAEEAFFNTILPAFDPALRYNVTWYVSSSPCAACADRIIKTLS


 


KTKNLRLLILVGRLFMWEELEIQDALKKLKEAGCKLRIMKPQDFEYVWQNFVEQEEGESKAFQPWEDIQE


 


NFLYYEEKLADILK


 


btAPOBEC-2 (Bos Taurus):


(SEQ ID NO: 1384)



MAQKEEAAAAAEPASQNGEEVENLEDPEKLKELIELPPFEIVTGERLPAHYFKFQFRNVEYSSGRNKTFL



 


CYVVEAQSKGGQVQASRGYLEDEHATNHAEEAFFNSIMPTFDPALRYMVTWYVSSSPCAACADRIVKTLN


 


KTKNLRLLILVGRLFMWEEPEIQAALRKLKEAGCRLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQE


 


NFLYYEEKLADILK


 


mAPOBEC-3-(1) (Mus musculus):


(SEQ ID NO: 1385)



MQPQRLGPRAGMGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLGYAKGRKDTFLCYEVTRKDCDSPVSL



 


HHGVFKNKDNIHAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFECAEQIVRFLATHHNLSLDIFSS


 


RLYNVQDPETQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKRLLTNFRYQDSKLQEILRP


 


CYISVPSSSSSTLSNICLTKGLPETRFWVEGRRMDPLSEEEFYSQFYNQRVKHLCYYHRMKPYLCYQLEQ


 


FNGQAPLKGCLLSEKGKQHAEILFLDKIRSMELSQVTITCYLTWSPCPNCAWQLAAFKRDRPDLILHIYT


 


SRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLRRIKES


 


WGLQDLVNDFGNLQLGPPMS


 


Mouse APOBEC-3-(2):


(SEQ ID NO: 1386)



MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLGYAKGRKDTFLCYEVTRKDCDSPVSLHHGVFKNKDNI



 


HAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFECAEQIVRFLATHHNLSLDIFSSRLYNVQDPETQ


 


QNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKRLLTNFRYQDSKLQEILRPCYIPVPSSSSS


 


TLSNICLTKGLPETRFCVEGRRMDPLSEEEFYSQFYNQRVKHLCYYHRMKPYLCYQLEQFNGQAPLKGCL


 


LSEKGKQHAEILFLDKIRSMELSQVTITCYLTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRPF


 


QKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLRRIKESWGLQDLVNDFG


 


NLQLGPPMS


 


(italic: nucleic acid editing domain)


 


Rat APOBEC-3:


(SEQ ID NO: 1387)



MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNRLRYAIDRKDTFLCYEVTRKDCDSPVSLHHGVFKNKDN



 


IHAEICFLYWFHDKVLRVLSPREEFKITWYMSWSPCFECAEQVLRFLATHHNLSLDIFSSRLYNIRDPEN


 


QQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKKLLTNFRYQDSKLQEILRPCYIPVPSSSS


 


STLSNICLTKGLPETRFCVERRRVHLLSEEEFYSQFYNQRVKHLCYYHGVKPYLCYQLEQFNGQAPLKGC


 


LLSEKGKQHAEILFLDKIRSMELSQVIITCYLTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRP


 


FQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLHRIKESWGLQDLVNDF


 


GNLQLGPPMS


 


(italic: nucleic acid editing domain)


 


hAPOBEC-3A (Homo sapiens):


(SEQ ID NO: 1388)



MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRH



 


AELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEAL


 


QMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN (


 


hAPOBEC-3F (Homo sapiens):


(SEQ ID NO: 1389)



MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPRLDAKIFRGQVYSQPEHHAEMCF



 


LSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLAEHPNVTLTISAARLYYYWERDYRRALCRLSQA


 


GARVKIMDDEEFAYCWENFVYSEGQPFMPWYKFDDNYAFLHRTLKEILRNPMEAMYPHIFYFHFKNLRKA


 


YGRNESWLCFTMEVVKHHSPVSWKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNYEVTWYTSWSPC


 


PECAGEVAEFLARHSNVNLTIFTARLYYFWDTDYQEGLRSLSQEGASVEIMGYKDEKYCWENFVYNDDEP


 


FKPWKGLKYNFLFLDSKLQEILE


 


Rhesus macaque APOBEC-3G:


(SEQ ID NO: 1390)



MVEPMDPRTFVSNFNNRPILSGLNTVWLCCEVKTKDPSGPPLDAKIFQGKVYSKAKYHPEMRFLRWFHKW



 


RQLHHDQEYKVTWYVSWSPCTRCANSVATFLAKDPKVTLTIFVARLYYFWKPDYQQALRILCQKRGGPHA


 


TMKIMNYNEFQDCWNKFVDGRGKPFKPRNNLPKHYTLLQATLGELLRHLMDPGTFTSNFNNKPWVSGQHE


 


TYLCYKVERLHNDTWVPLNQHRGFLRNQAPNIHGFPKGRHAELCFLDLIPFWKLDGQQYRVTCFTSWSPC


 


FSCAQEMAKFISNNEHVSLCIFAARIYDDQGRYQEGLRALHRDGAKIAMMNYSEFEYCWDTFVDRQGRPF


 


QPWDGLDEHSQALSGRLRAI


 


(italic: nucleic acid editing domain; underline: cytoplasmic localization signal)


 


Chimpanzee APOBEC-3G:


(SEQ ID NO: 1391)



MKPHFRNPVERMYQDTFSDNFYNRPILSHRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSKLKYHPEMRF



 


FHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDVATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQ


 


KRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTSNFNNEL


 


WVRGRHETYLCYEVERLHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLHQDYRVT


 


CFTSWSPCFSCAQEMAKFISNNKHVSLCIFAARIYDDQGRCQEGLRTLAKAGAKISIMTYSEFKHCWDTF


 


VDHQGCPFQPWDGLEEHSQALSGRLRAILQNQGN


 


(italic: nucleic acid editing domain; underline: cytoplasmic localization signal)


 


Green monkey APOBEC-3G:


(SEQ ID NO: 1392)



MNPQIRNMVEQMEPDIFVYYFNNRPILSGRNTVWLCYEVKTKDPSGPPLDANIFQGKLYPEAKDHPEMKF



 


LHWFRKWRQLHRDQEYEVTWYVSWSPCTRCANSVATFLAEDPKVTLTIFVARLYYFWKPDYQQALRILCQ


 


ERGGPHATMKIMNYNEFQHCWNEFVDGQGKPFKPRKNLPKHYTLLHATLGELLRHVMDPGTFTSNFNNKP


 


WVSGQRETYLCYKVERSHNDTWVLLNQHRGFLRNQAPDRHGFPKGRHAELCFLDLLPFWKLDDQQYRVTC


 


FTSWSPCFSCAQKMAKFISNNKHVSLCIFAARIYDDQGRCQEGLRTLHRDGAKIAVMNYSEFEYCWDTFV


 


DRQGRPFQPWDGLDEHSQALSGRLRAI


 


(italic: nucleic acid editing domain; underline: cytoplasmic localization signal)


 


Human APOBEC-3G:


(SEQ ID NO: 1393)



MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSELKYHPEMRF



 


FHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQ


 


KRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNEP


 


WVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVT


 


CFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTF


 


VDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN


 


(italic: nucleic acid editing domain; underline: cytoplasmic localization signal)


 


Human APOBEC-3F:


(SEQ ID NO: 1389)



MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPRLDAKIFRGQVYSQPEHHAEMCF



 


LSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLAEHPNVTLTISAARLYYYWERDYRRALCRLSQA


 


GARVKIMDDEEFAYCWENFVYSEGQPFMPWYKFDDNYAFLHRTLKEILRNPMEAMYPHIFYFHFKNLRKA


 


YGRNESWLCFTMEVVKHHSPVSWKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNYEVTWYTSWSPC


 


PECAGEVAEFLARHSNVNLTIFTARLYYFWDTDYQEGLRSLSQEGASVEIMGYKDFKYCWENFVYNDDEP


 


FKPWKGLKYNFLFLDSKLQEILE


 


(italic: nucleic acid editing domain)


 


Human APOBEC-3B:


(SEQ ID NO: 1394)



MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGQVYFKPQYHAEMC



 


FLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLSEHPNVTLTISAARLYYYWERDYRRALCRLSQ


 


AGARVTIMDYEEFAYCWENFVYNEGQQFMPWYKFDENYAFLHRTLKEILRYLMDPDTFTFNFNNDPLVLR


 


RRQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFIS


 


WSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVY


 


RQGCPFQPWDGLEEHSQALSGRLRAILQNQGN


 


(italic: nucleic acid editing domain)


 


Rat APOBEC-3B:


(SEQ ID NO: 1395)



MQPQGLGPNAGMGPVCLGCSHRRPYSPIRNPLKKLYQQTFYFHFKNVRYAWGRKNNFLCYEVNGMDCALP



 


VPLRQGVFRKQGHIHAELCFIYWFHDKVLRVLSPMEEFKVTWYMSWSPCSKCAEQVARFLAAHRNLSLAI


 


FSSRLYYYLRNPNYQQKLCRLIQEGVHVAAMDLPEFKKCWNKFVDNDGQPFRPWMRLRINFSFYDCKLQE


 


IFSRMNLLREDVFYLQFNNSHRVKPVQNRYYRRKSYLCYQLERANGQEPLKGYLLYKKGEQHVEILFLEK


 


MRSMELSQVRITCYLTWSPCPNCARQLAAFKKDHPDLILRIYTSRLYFWRKKFQKGLCTLWRSGIHVDVM


 


DLPQFADCWTNFVNPQRPFRPWNELEKNSWRIQRRLRRIKESWGL


 


Bovine APOBEC-3B:


(SEQ ID NO: 1396)



DGWEVAFRSGTVLKAGVLGVSMTEGWAGSGHPGQGACVWTPGTRNTMNLLREVLFKQQFGNQPRVPAPYY



 


RRKTYLCYQLKQRNDLTLDRGCFRNKKQRHAERFIDKINSLDLNPSQSYKIICYITWSPCPNCANELVNE


 


ITRNNHLKLEIFASRLYFHWIKSFKMGLQDLQNAGISVAVMTHTEFEDCWEQFVDNQSRPFQPWDKLEQY


 


SASIRRRLQRILTAPI


 


Chimpanzee APOBEC-3B:


(SEQ ID NO: 1397)



MNPQIRNPMEWMYQRTFYYNFENEPILYGRSYTWLCYEVKIRRGHSNLLWDTGVFRGQMYSQPEHHAEMC



 


FLSWFCGNQLSAYKCFQITWFVSWTPCPDCVAKLAKFLAEHPNVTLTISAARLYYYWERDYRRALCRLSQ


 


AGARVKIMDDEEFAYCWENFVYNEGQPFMPWYKFDDNYAFLHRTLKEIIRHLMDPDTFTFNFNNDPLVLR


 


RHQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFIS


 


WSPCFSWGCAGQVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVY


 


RQGCPFQPWDGLEEHSQALSGRLRAILQVRASSLCMVPHRPPPPPQSPGPCLPLCSEPPLGSLLPTGRPA


 


PSLPFLLTASFSFPPPASLPPLPSLSLSPGHLPVPSFHSLTSCSIQPPCSSRIRETEGWASVSKEGRDLG


 


Human APOBEC-3C:


(SEQ ID NO: 1398)



MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVFRNQVDSETHCHAERC



 


FLSWFCDDILSPNTKYQVTWYTSWSPCPDCAGEVAEFLARHSNVNLTIFTARLYYFQYPCYQEGLRSLSQ


 


EGVAVEIMDYEDFKYCWENFVYNDNEPFKPWKGLKTNFRLLKRRLRESLQ


 


(italic: nucleic acid editing domain)


 


Gorilla APOBEC-3C


(SEQ ID NO: 1399)



MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVFRNQVDSETHCHAERC



 


FLSWECDDILSPNTNYQVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLYYFQDTDYQEGLRSLSQ


 


EGVAVKIMDYKDFKYCWENFVYNDDEPFKPWKGLKYNFRFLKRRLQEILE


 


(italic: nucleic acid editing domain)


 


Human APOBEC-3A:


(SEQ ID NO: 1388)



MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRH



 


AELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEAL


 


QMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN


 


(italic: nucleic acid editing domain)


 


Rhesus macaque APOBEC-3A:


(SEQ ID NO: 1400)



MDGSPASRPRHLMDPNTFTFNFNNDLSVRGRHQTYLCYEVERLDNGTWVPMDERRGFLCNKAKNVPCGDY



 


GCHVELRFLCEVPSWQLDPAQTYRVTWFISWSPCFRRGCAGQVRVFLQENKHVRLRIFAARIYDYDPLYQ


 


EALRTLRDAGAQVSIMTYEEFKHCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAILQNQGN


 


(italic: nucleic acid editing domain)


 


Bovine APOBEC-3A:


(SEQ ID NO: 1401)



MDEYTFTENFNNQGWPSKTYLCYEMERLDGDATIPLDEYKGFVRNKGLDQPEKPCHAELYFLGKIHSWNL



 


DRNQHYRLTCFISWSPCYDCAQKLTTFLKENHHISLHILASRIYTHNRFGCHQSGLCELQAAGARITIMT


 


FEDFKHCWETFVDHKGKPFQPWEGLNVKSQALCTELQAILKTQQN


 


(italic: nucleic acid editing domain)


 


Human APOBEC-3H:


(SEQ ID NO: 1402)



MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENKKKCHAEICFINEIKSMGLDE



 


TQCYQVTCYLTWSPCSSCAWELVDFIKAHDHLNLGIFASRLYYHWCKPQQKGLRLLCGSQVPVEVMGFPK


 


FADCWENFVDHEKPLSFNPYKMLEELDKNSRAIKRRLERIKIPGVRAQGRYMDILCDAEV


 


(italic: nucleic acid editing domain)


 


Rhesus macaque APOBEC-3H:


(SEQ ID NO: 1403)



MALLTAKTFSLQFNNKRRVNKPYYPRKALLCYQLTPQNGSTPTRGHLKNKKKDHAEIRFINKIKSMGLDE



 


TQCYQVTCYLTWSPCPSCAGELVDFIKAHRHLNLRIFASRLYYHWRPNYQEGLLLLCGSQVPVEVMGLPE


 


FTDCWENFVDHKEPPSFNPSEKLEELDKNSQAIKRRLERIKSRSVDVLENGLRSLQLGPVTPSSSIRNSR


 


Human APOBEC-3D:


(SEQ ID NO: 1404)



MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGPVLPKRQSNHRQE



 


VYFRFENHAEMCFLSWFCGNRLPANRRFQITWFVSWNPCLPCVVKVTKFLAEHPNVTLTISAARLYYYRD


 


RDWRWVLLRLHKAGARVKIMDYEDFAYCWENFVCNEGQPFMPWYKFDDNYASLHRTLKEILRNPMEAMYP


 


HIFYFHFKNLLKACGRNESWLCFTMEVTKHHSAVFRKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNT


 


NYEVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLCYFWDTDYQEGLCSLSQEGASVKIMGYKDFV


 


SCWKNFVYSDDEPFKPWKGLQTNFRLLKRRLREILQ


 


(italic: nucleic acid editing domain)


 


Human APOBEC-1:


(SEQ ID NO: 1405)



MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKE



 


TSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVT


 


IQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHL


 


QNCHYQTIPPHILLATGLIHPSVAWR


 


Mouse APOBEC-1:


(SEQ ID NO: 1406)



MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQNTSNHVEVNFLEKF



 


TTERYFRPNTRCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIARLYHHTDQRNRQGLRDLISSGVT


 


IQIMTEQEYCYCWRNFVNYPPSNEAYWPRYPHLWVKLYVLELYCIILGLPPCLKILRRKQPQLTFFTITL


 


QTCHYQRIPPHLLWATGLK


 


Rat APOBEC-1:


(SEQ ID NO: 1378)



MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKF



 


TTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVT


 


IQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIAL


 


QSCHYQRLPPHILWATGLK


 


Human APOBEC-2:


(SEQ ID NO: 1407)



MAQKEEAAVATEAASQNGEDLENLDDPEKLKELIELPPFEIVTGERLPANFFKFQFRNVEYSSGRNKTFL



 


CYVVEAQGKGGQVQASRGYLEDEHAAAHAEEAFFNTILPAFDPALRYNVTWYVSSSPCAACADRIIKTLS


 


KTKNLRLLILVGRLFMWEEPEIQAALKKLKEAGCKLRIMKPQDFEYVWQNFVEQEEGESKAFQPWEDIQE


 


NFLYYEEKLADILK


 


Mouse APOBEC-2:


(SEQ ID NO: 1408)



MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNVEYSSGRNKTFL



 


CYVVEVQSKGGQAQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKYNVTWYVSSSPCAACADRILKTLS


 


KTKNLRLLILVSRLFMWEEPEVQAALKKLKEAGCKLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQE


 


NFLYYEEKLADILK


 


Rat APOBEC-2:


(SEQ ID NO: 1409)



MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNVEYSSGRNKTFL



 


CYvVEAQSKGGQVQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKYNVTWYVSSSPCAACADRILKTLS


 


KTKNLRLLILVSRLFMWEEPEVQAALKKLKEAGCKLRIMKPQDFEYLWQNFVEQEEGESKAFEPWEDIQE


 


NFLYYEEKLADILK


 


Bovine APOBEC-2:


(SEQ ID NO: 1384)



MAQKEEAAAAAEPASQNGEEVENLEDPEKLKELIELPPFEIVTGERLPAHYFKFQFRNVEYSSGRNKTFL



 


CYVVEAQSKGGQVQASRGYLEDEHATNHAEEAFFNSIMPTFDPALRYMVTWYVSSSPCAACADRIVKTLN


 


KTKNLRLLILVGRLFMWEEPEIQAALRKLKEAGCRLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQE


 


NFLYYEEKLADILK


 


Petromyzon marinus CDA1 (pmCDA1):


(SEQ ID NO: 1410)



MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIF



 


SIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWN


 


LRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSFMIQVKILHTTKSPAV


 


Human APOBEC3G D316R D317R:


(SEQ ID NO: 1411)



MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSELKYHPEMRF



 


FHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQ


 


KRDGPRATMKFNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHFMLGEILRHSMDPPTFTFNFNNEPW


 


VRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTC


 


FTSWSPCFSCAQEMAKFISKKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISFTYSEFKHCWDTFVDH


 


QGCPFQPWDGLDEHSQDLSGRLRAILQNQEN


 


Human APOBEC3G chain A:


(SEQ ID NO: 1412)



MDPPTFTFNFNNEPWWGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIP



 


FWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISF


 


TYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQ


 


Human APOBEC3G chain A D120R D121R:


(SEQ ID NO: 1413)



MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVI



 


PFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARTYRRQGRCQEGLRTLAEAGAKIS


 


FMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQ


 


hAPOBEC-4 (Homo sapiens):


(SEQ ID NO: 1414)



MEPIYEEYLANHGTIVKPYYWLSFSLDCSNCPYHIRTGEEARVSLTEFCQIFGFPYGTTFPQTKHLTFYE



 


LKTSSGSLVQKGHASSCTGNYIHPESMLFEMNGYLDSAIYNNDSIRHIILYSNNSPCNEANHCCISKMYN


 


FLITYPGITLSIYFSQLYHTEMDFPASAWNREALRSLASLWPRVVLSPISGGIWHSVLHSFISGVSGSHV


 


FQPILTGRALADRHNAYEINAITGVKPYFTDVLLQTKRNPNTKAQEALESYPLNNAFPGQFFQMPSGQLQ


 


PNLPPDLRAPVVFVLVPLRDLPPMHMGQNPNKPRNIVRHLNMPQMSFQETKDLGRLPTGRSVEIVEITEQ


 


FASSKEADEKKKKKGKK


 


mAPOBEC-4 (Mus musculus):


(SEQ ID NO: 1373)



MDSLLMKQKKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSCSLDFGHLRNKSGCHVELLFLRYISDWDL



 


DPGRCYRVTWFTSWSPCYDCARHVAEFLRWNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIGIMT


 


FKDYFYCWNTFVENRERTFKAWEGLHENSVRLTRQLRRILLPLYEVDDLRDAFRMLGF


 


rAPOBEC-4 (Rattus norvegicus):


(SEQ ID NO: 1415)



MEPLYEEYLTHSGTIVKPYYWLSVSLNCTNCPYHIRTGEEARVPYTEFHQTFGFPWSTYPQTKHLTFYEL



 


RSSSGNLIQKGLASNCTGSHTHPESMLFERDGYLDSLIFHDSNIRHIILYSNNSPCDEANHCCISKMYNF


 


LMNYPEVTLSVFFSQLYHTENQFPTSAWNREALRGLASLWPQVTLSAISGGIWQSILETFVSGISEGLTA


 


VRPFTAGRTLTDRYNAYEINCITEVKPYFTDALHSWQKENQDQKVWAASENQPLHNTTPAQWQPDMSQDC


 


RTPAVFMLVPYRDLPPIHVNPSPQKPRTVVRHLNTLQLSASKVKALRKSPSGRPVKKEEARKGSTRSQEA


 


NETNKSKWKKQTLFIKSNICHLLEREQKKIGILSSWSV


 


mfAPOBEC-4 (Macaca fascicularis):


(SEQ ID NO: 1416)



MEPTYEEYLANHGTIVKPYYWLSFSLDCSNCPYHIRTGEEARVSLTEFCQIFGFPYGTTYPQTKHLTFYE



 


LKTSSGSLVQKGHASSCTGNYIHPESMLFEMNGYLDSAIYNNDSIRHIILYCNNSPCNEANHCCISKVYN


 


FLITYPGITLSIYFSQLYHTEMDFPASAWNREALRSLASLWPRVVLSPISGGIWHSVLHSFVSGVSGSHV


 


FQPILTGRALTDRYNAYEINAITGVKPFFTDVLLHTKRNPNTKAQMALESYPLNNAFPGQSFQMTSGIPP


 


DLRAPVVFVLLPLRDLPPMHMGQDPNKPRNIIRHLNMPQMSFQETKDLERLPTRRSVETVEITERFASSK


 


QAEEKTKKKKGKK


 


pmCDA-1 (Petromyzon marinus):


(SEQ ID NO: 1417)



MAGYECVRVSEKLDFDTFEFQFENLHYATERHRTYVIFDVKPQSAGGRSRRLWGYIINNPNVCHAELILM



 


SMIDRHLESNPGVYAMTWYMSWSPCANCSSKLNPWLKNLLEEQGHTLTMHFSRIYDRDREGDHRGLRGLK


 


HVSNSFRMGVVGRAEVKECLAEYVEASRRTLTWLDTTESMAAKMRRKLFCILVRCAGMRESGIPLHLFTL


 


QTPLLSGRVVWWRV


 


pmCDA-2 (Petromyzon marinus):


(SEQ ID NO: 1418)



MELREVVDCALASCVRHEPLSRVAFLRCFAAPSQKPRGTVILFYVEGAGRGVTGGHAVNYNKQGTSIHAE



 


VLLLSAVRAALLRRRRCEDGEEATRGCTLHCYSTYSPCRDCVEYIQEFGASTGVRVVIHCCRLYELDVNR


 


RRSEAEGVLRSLSRLGRDFRLMGPRDAIALLLGGRLANTADGESGASGNAWVTETNVVEPLVDMTGFGDE


 


DLHAQVQRNKQIREAYANYASAVSLMLGELHVDPDKFPFLAEFLAQTSVEPSGTPRETRGRPRGASSRGP


 


EIGRQRPADFERALGAYGLFLHPRIVSREADREEIKRDLIVVMRKHNYQGP


 


pmCDA-5 (Petromyzon marinus):


(SEQ ID NO: 1419)



MAGDENVRVSEKLDFDTFEFQFENLHYATERHRTYVIFDVKPQSAGGRSRRLWGYIINNPNVCHAELILM



 


SMIDRHLESNPGVYAMTWYMSWSPCANCSSKLNPWLKNLLEEQGHTLMMHFSRIYDRDREGDHRGLRGLK


 


HVSNSFRMGVVGRAEVKECLAEYVEASRRTLTWLDTTESMAAKMRRKLFCILVRCAGMRESGMPLHLFT


 


yCD (Saccharomyces cerevisiae):


(SEQ ID NO: 1420)



MVTGGMASKWDQKGMDIAYEEAALGYKEGGVPIGGCLINNKDGSVLGRGHNMRFQKGSATLHGEISTLEN



 


CGRLEGKVYKDTTLYTTLSPCDMCTGAIIMYGIPRCVVGENVNFKSKGEKYLQTRGHEVVVVDDERCKKI


 


MKQFIDERPQDWFEDIGE


 


rAPOBEC-1 (delta 177-186):


(SEQ ID NO: 1421)



MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKF



 


TTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVT


 


IQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPH


 


ILWATGLK


 


rAPOBEC-1 (delta 202-213):


(SEQ ID NO: 1422)



MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKF



 


TTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVT


 


TQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQHYQRLPPH


 


ILWATGLK


 


Mouse APOBEC-3:


(SEQ ID NO: 1386)



MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLGYAKGRKDTFLCYEVTRKDCDSPVSLHHGVF



 


KNKDNIHAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFECAEQIVRFLATHHNLSLDIFS


 


SRLYNVQDPETQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKRLLTNFRYQDSK


 


LQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVEGRRMDPLSEEEFYSQFYNQRVKHLCYY


 


HWMKPYLCYQLEQFNGQAPLKGCLLSEKGEQHAEILFLDKIRSMELSQVTITCYLTWSPCPNCA


 


WQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKR


 


PFWPWKGLEIISRRTQRRLRRIKESWGLQDLVNDFGNLQLGPPMS


 


(italic: nucleic acid editing domain)






Some aspects of the present disclosure are based on the recognition that modulating the deaminase domain catalytic activity of any of the fusion proteins described herein, for example by making point mutations in the deaminase domain, affect the processivity of the fusion proteins (e.g., base editors). For example, mutations that reduce, but do not eliminate, the catalytic activity of a deaminase domain within a base editing fusion protein can make it less likely that the deaminase domain will catalyze the deamination of a residue adjacent to a target residue, thereby narrowing the deamination window. The ability to narrow the deamination window can prevent unwanted deamination of residues adjacent to specific target residues, which can decrease or prevent off-target effects.
For example, in some embodiments, an APOBEC deaminase incorporated into a base editor can comprise one or more mutations selected from the group consisting of H121X, H122X, R126X, R126X, R118X, W90X, W90X, and R132X of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase, wherein X is any amino acid. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise one or more mutations selected from the group consisting of H121R, H122R, R126A, R126E, R118A, W90A, W90Y, and R132E of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase.
In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise one or more mutations selected from the group consisting of D316X, D317X, R320X, R320X, R313X, W285X, W285X, R326X of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase, wherein X is any amino acid. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising one or more mutations selected from the group consisting of D316R, D317R, R320A, R320E, R313A, W285A, W285Y, R326E of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase.
In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise a H121R and a H122R mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R126A mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R126E mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R118A mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W90A mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W90Y mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R132E mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W90Y and a R126E mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R126E and a R132E mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W90Y and a R132E mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W90Y, R126E, and R132E mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase.
In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a D316R and a D317R mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R320A mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R320E mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R313A mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W285A mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W285Y mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R326E mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W285Y and a R320E mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R320E and a R326E mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W285Y and a R326E mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W285Y, R320E, and R326E mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase.
A number of modified cytidine deaminases are commercially available, including, but not limited to, SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, VRER-BE3, YE1-BE3, EE-BE3, YE2-BE3, and YEE-BE3, which are available from Addgene (plasmids 85169, 85170, 85171, 85172, 85173, 85174, 85175, 85176, 85177). In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of an APOBEC1 deaminase.
Details of C to T nucleobase editing proteins are described in International PCT Application No. PCT/US2016/058344 (WO2017/070632) and Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016), the entire contents of which are hereby incorporated by reference.
Cytidine Deaminases
In some embodiments, the fusion proteins of the invention comprise one or more cytidine deaminase domains. In some embodiments, the cytidine deaminases provided herein are capable of deaminating cytosine or 5-methylcytosine to uracil or thymine. In some embodiments, the cytidine deaminases provided herein are capable of deaminating cytosine in DNA. The cytidine deaminase may be derived from any suitable organism. In some embodiments, the cytidine deaminase is a naturally-occurring cytidine deaminase that includes one or more mutations corresponding to any of the mutations provided herein. One of skill in the art will be able to identify the corresponding residue in any homologous protein, e.g., by sequence alignment and determination of homologous residues. Accordingly, one of skill in the art would be able to generate mutations in any naturally-occurring cytidine deaminase that corresponds to any of the mutations described herein. In some embodiments, the cytidine deaminase is from a prokaryote. In some embodiments, the cytidine deaminase is from a bacterium. In some embodiments, the cytidine deaminase is from a mammal (e.g., human).
In some embodiments, the cytidine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the cytidine deaminase amino acid sequences set forth herein. It should be appreciated that cytidine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). Some embodiments provide a polynucleotide molecule encoding the cytidine deaminase nucleobase editor polypeptide of any previous aspect or as delineated herein. In some embodiments, the polynucleotide is codon optimized.
The disclosure provides any deaminase domains with a certain percent identity plus any of the mutations or combinations thereof described herein. In some embodiments, the cytidine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to a reference sequence, or any of the cytidine deaminases provided herein. In some embodiments, the cytidine deaminase comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues as compared to any one of the amino acid sequences known in the art or described herein.
A fusion protein of the invention second protein comprises two or more nucleic acid editing domains. In some embodiments, the nucleic acid editing domain can catalyze a C to U base change. In some embodiments, the nucleic acid editing domain is a deaminase domain. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 deaminase. In some embodiments, the deaminase is an APOBEC2 deaminase. In some embodiments, the deaminase is an APOBEC3 deaminase. In some embodiments, the deaminase is an APOBEC3A deaminase. In some embodiments, the deaminase is an APOBEC3B deaminase. In some embodiments, the deaminase is an APOBEC3C deaminase. In some embodiments, the deaminase is an APOBEC3D deaminase. In some embodiments, the deaminase is an APOBEC3E deaminase. In some embodiments, the deaminase is an APOBEC3F deaminase. In some embodiments, the deaminase is an APOBEC3G deaminase. In some embodiments, the deaminase is an APOBEC3H deaminase. In some embodiments, the deaminase is an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some embodiments, the deaminase is a vertebrate deaminase. In some embodiments, the deaminase is an invertebrate deaminase. In some embodiments, the deaminase is a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse deaminase. In some embodiments, the deaminase is a human deaminase. In some embodiments, the deaminase is a rat deaminase, e.g., rAPOBEC1. In some embodiments, the deaminase is a Petromyzon marinus cytidine deaminase 1 (pmCDA1). In some embodiments, the deaminase is a human APOBEC3G. In some embodiments, the deaminase is a fragment of the human APOBEC3G. In some embodiments, the deaminase is a human APOBEC3G variant comprising a D316R D317R mutation. In some embodiments, the deaminase is a fragment of the human APOBEC3G and comprising mutations corresponding to the D316R D317R mutations. In some embodiments, the nucleic acid editing domain is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or at least 99.5% identical to the deaminase domain of any deaminase described herein.
In certain embodiments, the fusion proteins provided herein comprise one or more features that improve the base editing activity of the fusion proteins. For example, any of the fusion proteins provided herein may comprise a Cas9 domain that has reduced nuclease activity. In some embodiments, any of the fusion proteins provided herein may have a Cas9 domain that does not have nuclease activity (dCas9), or a Cas9 domain that cuts one strand of a duplexed DNA molecule, referred to as a Cas9 nickase (nCas9).
High Fidelity Cas9 Domains
Some aspects of the disclosure provide high fidelity Cas9 domains. In some embodiments, high fidelity Cas9 domains are engineered Cas9 domains comprising one or more mutations that decrease electrostatic interactions between the Cas9 domain and a sugar-phosphate backbone of a DNA, as compared to a corresponding wild-type Cas9 domain. Without wishing to be bound by any particular theory, high fidelity Cas9 domains that have decreased electrostatic interactions with a sugar-phosphate backbone of DNA may have less off-target effects. In some embodiments, a Cas9 domain (e.g., a wild type Cas9 domain) comprises one or more mutations that decreases the association between the Cas9 domain and a sugar-phosphate backbone of a DNA. In some embodiments, a Cas9 domain comprises one or more mutations that decreases the association between the Cas9 domain and a sugar-phosphate backbone of a DNA by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70%.
In some embodiments, any of the Cas9 fusion proteins provided herein comprise one or more of a N497X, a R661X, a Q695X, and/or a Q926X mutation, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid. In some embodiments, any of the Cas9 fusion proteins provided herein comprise one or more of a N497A, a R661A, a Q695A, and/or a Q926A mutation, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the Cas9 domain comprises a D10A mutation, or a corresponding mutation in any of the amino acid sequences provided herein. Cas9 domains with high fidelity are known in the art and would be apparent to the skilled artisan. For example, Cas9 domains with high fidelity have been described in Kleinstiver, B. P., et al. “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.” Nature 529, 490-495 (2016); and Slaymaker, I. M., et al. “Rationally engineered Cas9 nucleases with improved specificity.” Science 351, 84-88 (2015); the entire contents of each are incorporated herein by reference.
In some embodiments, the modified Cas9 is a high fidelity Cas9 enzyme. In some embodiments, the high fidelity Cas9 enzyme is SpCas9(K855A), eSpCas9(1.1), SpCas9-HF1, or hyper accurate Cas9 variant (HypaCas9). The modified Cas9 eSpCas9(1.1) contains alanine substitutions that weaken the interactions between the HNH/RuvC groove and the non-target DNA strand, preventing strand separation and cutting at off-target sites. Similarly, SpCas9-HF1 lowers off-target editing through alanine substitutions that disrupt Cas9's interactions with the DNA phosphate backbone. HypaCas9 contains mutations (SpCas9 N692A/M694A/Q695A/H698A) in the REC3 domain that increase Cas9 proofreading and target discrimination. All three high fidelity enzymes generate less off-target editing than wildtype Cas9.
An exemplary high fidelity Cas9 is provided below. High Fidelity Cas9 domain mutations relative to Cas9 are shown in bold and underlined.







DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL


 


LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL


 


EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL


 


RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI


 


NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN


 


FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL


 


LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF


 


FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK


 


QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY


 


VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKN


 


LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL


 


LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII


 


KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL


 


KRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMALIHDDS


 


LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM


 


GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV


 


ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPOSFLKDDS


 


IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT


 


KAERGGLSELDKAGFIKRQLVETRAITKHVAQILDSRiYNTKYDENDKLI


 


REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAWGTALIKKY


 


PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT


 


LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ


 


TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK


 


GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY


 


SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED


 


NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP


 


IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS


 


ITGLYETRIDLSQLGGD






Fusion Proteins Comprising a napDNAbp and a Cytidine Deaminase and/or Adenosine Deaminase

Some aspects of the disclosure provide fusion proteins comprising a Cas9 domain or other nucleic acid programmable DNA binding protein (e.g., Cas12) and one or more cytidine deaminase or adenosine deaminase domains, and/or DNA glycosylase domains. It should be appreciated that the Cas9 domain may be any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein. In some embodiments, any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein may be fused with any of the cytidine deaminases and/or adenosine deaminases provided herein. The domains of the base editors disclosed herein can be arranged in any order.
In some embodiments, the fusion protein comprises the following domains A-C, A-D, or A-E:

    
    
        NH2-[A-B-C]-COOH;
        NH2-[A-B-C-D]-COOH; or
        NH2-[A-B-C-D-E]-COOH;

wherein A and C or A, C, and E, each comprises one or more of the following:

        an adenosine deaminase domain or an active fragment thereof,
        a cytidine deaminase domain or an active fragment thereof,
        a DNA glycosylase domain or an active fragment thereof; and
        wherein B or B and D, each comprises one or more domains having nucleic acid sequence specific binding activity.
    
    


In some embodiments, the fusion protein comprises the following structure:

    
    
        NH2-[An-Bo-Cn]-COOH;
        NH2-[An-Bo-Cn-Do]-COOH; or
        NH2-[An-Bo-Cp-Do-Eq]-COOH;

wherein A and C or A, C, and E, each comprises one or more of the following:

        an adenosine deaminase domain or an active fragment thereof,
        a cytidine deaminase domain or an active fragment thereof,
        a DNA glycosylase domain or an active fragment thereof; and

wherein n is an integer: 1, 2, 3, 4, or 5, wherein p is an integer: 0, 1, 2, 3, 4, or 5; wherein q is an integer 0, 1, 2, 3, 4, or 5; and wherein B or B and D each comprises a domain having nucleic acid sequence specific binding activity; and wherein o is an integer: 1, 2, 3, 4, or 5.

    
    


For example, and without limitation, in some embodiments, the fusion protein comprises the structure:

    
    
        NH2-[adenosine deaminase]-[Cas9 domain]-COOH;
        NH2-[Cas9 domain]-[adenosine deaminase]-COOH;
        NH2-[cytidine deaminase]-[Cas9 domain]-COOH;
        NH2-[Cas9 domain]-[cytidine deaminase]-COOH;
        NH2-[cytidine deaminase]-[Cas9 domain]-[adenosine deaminase]-COOH;
        NH2-[adenosine deaminase]-[Cas9 domain]-[cytidine deaminase]-COOH;
        NH2-[adenosine deaminase]-[cytidine deaminase]-[Cas9 domain]-COOH;
        NH2-[cytidine deaminase]-[adenosine deaminase]-[Cas9 domain]-COOH;
        NH2-[Cas9 domain]-[adenosine deaminase]-[cytidine deaminase]-COOH; or
        NH2-[Cas9 domain]-[cytidine deaminase]-[adenosine deaminase]-COOH.
    
    


In some embodiments, any of the Cas12 domains or Cas12 proteins provided herein may be fused with any of the cytidine or adenosine deaminases provided herein. For example, and without limitation, in some embodiments, the fusion protein comprises the structure:

    
    
        NH2-[adenosine deaminase]-[Cas12 domain]-COOH;
        NH2-[Cas12 domain]-[adenosine deaminase]-COOH;
        NH2-[cytidine deaminase]-[Cas12 domain]-COOH;
        NH2-[Cas12 domain]-[cytidine deaminase]-COOH;
        NH2-[cytidine deaminase]-[Cas12 domain]-[adenosine deaminase]-COOH;
        NH2-[adenosine deaminase]-[Cas12 domain]-[cytidine deaminase]-COOH;
        NH2-[adenosine deaminase]-[cytidine deaminase]-[Cas12 domain]-COOH;
        NH2-[cytidine deaminase]-[adenosine deaminase]-[Cas12 domain]-COOH;
        NH2-[Cas12 domain]-[adenosine deaminase]-[cytidine deaminase]-COOH; or
        NH2-[Cas12 domain]-[cytidine deaminase]-[adenosine deaminase]-COOH.
    
    


In some embodiments, the adenosine deaminase is a TadA*8. Exemplary fusion protein structures include the following:

    
    
        NH2-[TadA*8]-[Cas9 domain]-COOH;
        NH2-[Cas9 domain]-[TadA*8]-COOH;
        NH2-[TadA*8]-[Cas12 domain]-COOH; or
        NH2-[Cas12 domain]-[TadA*8]-COOH.
    
    


In some embodiments, the adenosine deaminase of the fusion protein comprises a TadA*8 and a cytidine deaminase and/or an adenosine deaminase. In some embodiments, the TadA*8 is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24.
Exemplary fusion protein structures include the following:

    
    
        NH2-[TadA*8]-[Cas9/Cas12]-[adenosine deaminase]-COOH;
        NH2-[adenosine deaminase]-[Cas9/Cas12]-[TadA*8]-COOH;
        NH2-[TadA*8]-[Cas9/Cas12]-[cytidine deaminase]-COOH; or
        NH2-[cytidine deaminase]-[Cas9/Cas12]-[TadA*8]-COOH.
    
    


In some embodiments, the adenosine deaminase of the fusion protein comprises a TadA*9 and a cytidine deaminase and/or an adenosine deaminase. Exemplary fusion protein structures include the following:

    
    
        NH2-[TadA*9]-[Cas9/Cas12]-[adenosine deaminase]-COOH;
        NH2-[adenosine deaminase]-[Cas9/Cas12]-[TadA*9]-COOH;
        NH2-[TadA*9]-[Cas9/Cas12]-[cytidine deaminase]-COOH; or
        NH2-[cytidine deaminase]-[Cas9/Cas12]-[TadA*9]-COOH.
    
    


In some embodiments, the fusion protein can comprise a deaminase flanked by an N-terminal fragment and a C-terminal fragment of a Cas9 or Cas12 polypeptide. In some embodiments, the fusion protein comprises a cytidine deaminase flanked by an N-terminal fragment and a C-terminal fragment of a Cas9 or Cas12 polypeptide. In some embodiments, the fusion protein comprises an adenosine deaminase flanked by an N-terminal fragment and a C-terminal fragment of a Cas9 or Cas 12 polypeptide.
In some embodiments, the fusion proteins comprising a cytidine deaminase or adenosine deaminase and a napDNAbp (e.g., Cas9 or Cas12 domain) do not include a linker sequence. In some embodiments, a linker is present between the cytidine or adenosine deaminase and the napDNAbp. In some embodiments, the “-” used in the general architecture above indicates the presence of an optional linker. In some embodiments, cytidine or adenosine deaminase and the napDNAbp are fused via any of the linkers provided herein. For example, in some embodiments the cytidine or adenosine deaminase and the napDNAbp are fused via any of the linkers provided herein.
It should be appreciated that the fusion proteins of the present disclosure may comprise one or more additional features. For example, in some embodiments, the fusion protein may comprise inhibitors, cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. In some embodiments, the fusion protein comprises one or more His tags.
Exemplary, yet nonlimiting, fusion proteins are described in International PCT Application Nos. PCT/2017/044935, PCT/US2019/044935, and PCT/US2020/016288, each of which is incorporated herein by reference for its entirety.
Fusion Proteins Comprising a Nuclear Localization Sequence (NLS)
In some embodiments, the fusion proteins provided herein further comprise one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, for example a nuclear localization sequence (NLS). In one embodiment, a bipartite NLS is used. In some embodiments, a NLS comprises an amino acid sequence that facilitates the importation of a protein, that comprises an NLS, into the cell nucleus (e.g., by nuclear transport). In some embodiments, any of the fusion proteins provided herein further comprise a nuclear localization sequence (NLS). In some embodiments, the NLS is fused to the N-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of the Cas9 domain. In some embodiments, the NLS is fused to the C-terminus of the Cas9 domain. In some embodiments, the NLS is fused to the C-terminus of an nCas9 domain or a dCas9 domain. In some embodiments, the NLS is fused to the N-terminus of the Cas12 domain. In some embodiments, the NLS is fused to the C-terminus of the Cas12 domain. In some embodiments, the NLS is fused to the N-terminus of the cytidine or adenosine deaminase. In some embodiments, the NLS is fused to the C-terminus of the cytidine or adenosine deaminase. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS comprises an amino acid sequence of any one of the NLS sequences provided or referenced herein. Additional nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., 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 the amino acid sequence PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 83), KRTADGSEFESPKKKRKV (SEQ ID NO: 84), KRPAATKKAGQAKKKK (SEQ ID NO: 85), KKTELQTTNAENKTKKL (SEQ ID NO: 86), KRGINDRNFWRGENGRKTR (SEQ ID NO: 87), RKSGKIAAIVVKRPRKPKKKRKV (SEQ ID NO: 1424), or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 90).
In some embodiments, the fusion proteins comprising a cytidine or adenosine deaminase, a Cas9 domain, and an NLS do not comprise a linker sequence. In some embodiments, linker sequences between one or more of the domains or proteins (e.g., cytidine or adenosine deaminase, Cas9 domain or NLS) are present. In some embodiments, a linker is present between the cytidine deaminase and adenosine deaminase domains and the napDNAbp. In some embodiments, the “-” used in the general architecture below indicates the presence of an optional linker. In some embodiments, the cytidine deaminase and adenosine deaminase and the napDNAbp are fused via any of the linkers provided herein. For example, in some embodiments the cytidine deaminase and adenosine deaminase and the napDNAbp are fused via any of the linkers provided herein.
In some embodiments, the general architecture of exemplary napDNAbp (e.g., Cas9 or Cas12) fusion proteins with a cytidine or adenosine deaminase and a napDNAbp (e.g., Cas9 or Cas12) domain comprises any one of the following structures, where NLS is a nuclear localization sequence (e.g., any NLS provided herein), NH2 is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein:

    
    
        NH2-NLS-[cytidine deaminase]-[napDNAbp domain]-COOH;
        NH2-NLS [napDNAbp domain]-[cytidine deaminase]-COOH;
        NH2-[cytidine deaminase]-[napDNAbp domain]-NLS-COOH;
        NH2-[napDNAbp domain]-[cytidine deaminase]-NLS-COOH;
        NH2-NLS-[adenosine deaminase]-[napDNAbp domain]-COOH;
        NH2-NLS [napDNAbp domain]-[adenosine deaminase]-COOH;
        NH2-[adenosine deaminase]-[napDNAbp domain]-NLS-COOH;
        NH2-[napDNAbp domain]-[adenosine deaminase]-NLS-COOH;
        NH2-NLS-[cytidine deaminase]-[napDNAbp domain]-[adenosine deaminase]-COOH;
        NH2-NLS-[adenosine deaminase]-[napDNAbp domain]-[cytidine deaminase]-COOH;
        NH2-NLS-[adenosine deaminase][cytidine deaminase]-[napDNAbp domain]-COOH;
        NH2-NLS-[cytidine deaminase]-[adenosine deaminase]-[napDNAbp domain]-COOH;
        NH2-NLS-[napDNAbp domain]-[adenosine deaminase]-[cytidine deaminase]-COOH;
        NH2-NLS-[napDNAbp domain]-[cytidine deaminase]-[adenosine deaminase]-COOH;
        NH2-[cytidine deaminase]-[napDNAbp domain]-[adenosine deaminase]-NLS-COOH;
        NH2-[adenosine deaminase]-[napDNAbp domain]-[cytidine deaminase]-NLS-COOH;
        NH2-[adenosine deaminase][cytidine deaminase]-[napDNAbp domain]-NLS-COOH;
        NH2-[cytidine deaminase]-[adenosine deaminase]-[napDNAbp domain]-NLS-COOH;
        NH2-[napDNAbp domain]-[adenosine deaminase]-[cytidine deaminase]-NLS-COOH; or
        NH2-[napDNAbp domain]-[cytidine deaminase]-[adenosine deaminase]-NLS-COOH. In some embodiments, the NLS is present in a linker or the NLS is flanked by linkers, for example described herein. A bipartite NLS comprises two basic amino acid clusters, which are separated by a relatively short spacer sequence (hence bipartite—2 parts, while monopartite NLSs are not). The NLS of nucleoplasmin, KR[PAATKKAGQA]KKKK (SEQ ID NO: 85), is the prototype of the ubiquitous bipartite signal: two clusters of basic amino acids, separated by a spacer of about 10 amino acids. The sequence of an exemplary bipartite NLS follows: PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 83)
    
    


A vector that encodes a CRISPR enzyme comprising one or more nuclear localization sequences (NLSs) can be used. For example, there can be or be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs used. A CRISPR enzyme can comprise the NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs at or near the carboxy-terminus, or any combination thereof (e.g., one or more NLS at the amino-terminus and one or more NLS at the carboxy terminus). When more than one NLS is present, each can be selected independently of others, such that a single NLS can be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies.
CRISPR enzymes used in the methods can comprise about 6 NLSs. An NLS is considered near the N- or C-terminus when the nearest amino acid to the NLS is within about 50 amino acids along a polypeptide chain from the N- or C-terminus, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 amino acids.
Additional Domains
A base editor described herein can include any domain which helps to facilitate the nucleobase editing, modification or altering of a nucleobase of a polynucleotide. In some embodiments, a base editor comprises a polynucleotide programmable nucleotide binding domain (e.g., Cas9), a nucleobase editing domain (e.g., deaminase domain), and one or more additional domains. In some embodiments, the additional domain can facilitate enzymatic or catalytic functions of the base editor, binding functions of the base editor, or be inhibitors of cellular machinery (e.g., enzymes) that could interfere with the desired base editing result. In some embodiments, a base editor can comprise a nuclease, a nickase, a recombinase, a deaminase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain.
In some embodiments, a base editor can comprise an uracil glycosylase inhibitor (UGI) domain. In some embodiments, cellular DNA repair response to the presence of U: G heteroduplex DNA can be responsible for a decrease in nucleobase editing efficiency in cells. In such embodiments, uracil DNA glycosylase (UDG) can catalyze removal of U from DNA in cells, which can initiate base excision repair (BER), mostly resulting in reversion of the U:G pair to a C:G pair. In such embodiments, BER can be inhibited in base editors comprising one or more domains that bind the single strand, block the edited base, inhibit UGI, inhibit BER, protect the edited base, and/or promote repairing of the non-edited strand. Thus, this disclosure contemplates a base editor fusion protein comprising a UGI domain.
In some embodiments, a base editor comprises as a domain all or a portion of a double-strand break (DSB) binding protein. For example, a DSB binding protein can include a Gam protein of bacteriophage Mu that can bind to the ends of DSBs and can protect them from degradation. See Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), the entire content of which is hereby incorporated by reference.
Additionally, in some embodiments, a Gam protein can be fused to an N terminus of a base editor. In some embodiments, a Gam protein can be fused to a C terminus of a base editor. The Gam protein of bacteriophage Mu can bind to the ends of double strand breaks (DSBs) and protect them from degradation. In some embodiments, using Gam to bind the free ends of DSB can reduce indel formation during the process of base editing. In some embodiments, 174-residue Gam protein is fused to the N terminus of the base editors. See Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017). In some embodiments, a mutation or mutations can change the length of a base editor domain relative to a wild type domain. For example, a deletion of at least one amino acid in at least one domain can reduce the length of the base editor. In another case, a mutation or mutations do not change the length of a domain relative to a wild type domain. For example, substitutions in any domain does not change the length of the base editor.
Non-limiting examples of such base editors, where the length of all the domains is the same as the wild type domains, can include:

    
    
        NH2-[nucleobase editing domain]-Linker1-[APOBEC1]-Linker2-[nucleobase editing domain]-COOH;
        NH2-[nucleobase editing domain]-Linker1-[APOBEC1]-[nucleobase editing domain]-COOH;
        NH2-[nucleobase editing domain]-[APOBEC1]-Linker2-[nucleobase editing domain]-COOH;
        NH2-[nucleobase editing domain]-[APOBEC1]-[nucleobase editing domain]-COOH;
        NH2-[nucleobase editing domain]-Linker1-[APOBEC1]-Linker2-[nucleobase editing domain]-[UGI]-COOH;
        NH2-[nucleobase editing domain]-Linker1-[APOBEC1]-[nucleobase editing domain]-[UGI]-COOH;
        NH2-[nucleobase editing domain]-[APOBEC1]-Linker2-[nucleobase editing domain]-[UGI]-COOH;
        NH2-[nucleobase editing domain]-[APOBEC1]-[nucleobase editing domain]-[UGI]-COOH;
        NH2-[UGI]-[nucleobase editing domain]-Linker1-[APOBEC1]-Linker2-[nucleobase editing domain]-COOH;
        NH2-[UGI]-[nucleobase editing domain]-Linker1-[APOBEC1]-[nucleobase editing domain]-COOH;
        NH2-[UGI]-[nucleobase editing domain]-[APOBEC1]-Linker2-[nucleobase editing domain]-COOH; or
        NH2-[UGI]-[nucleobase editing domain]-[APOBEC1]-[nucleobase editing domain]-COOH.

Base Editor System

    
    


Provided herein are systems, compositions, and methods for editing a nucleobase using a base editor system. In some embodiments, the base editor system comprises (1) a base editor (BE) comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain (e.g., a deaminase domain) for editing the nucleobase; and (2) a guide polynucleotide (e.g., guide RNA) in conjunction with the polynucleotide programmable nucleotide binding domain. In some embodiments, the base editor system is a cytidine base editor (CBE). In some embodiments, the base editor system is an adenosine base editor (ABE). In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the nucleobase editing domain is a deaminase domain. In some embodiments, a deaminase domain can be a cytidine deaminase or an cytosine deaminase. In some embodiments, a deaminase domain can be an adenine deaminase or an adenosine deaminase. In some embodiments, the adenosine base editor can deaminate adenine in DNA. In some embodiments, the base editor is capable of deaminating a cytidine in DNA.
In some embodiments, a base editing system as provided herein provides a new approach to genome editing that uses a fusion protein containing a catalytically defective Streptococcus pyogenes Cas9, a deaminase (e.g., cytidine or adenosine deaminase), and an inhibitor of base excision repair to induce programmable, single nucleotide (C→T or A→G) changes in DNA without generating double-strand DNA breaks, without requiring a donor DNA template, and without inducing an excess of stochastic insertions and deletions.
Details of nucleobase editing proteins are described in International PCT Application Nos. PCT/2017/045381 (WO2018/027078) and PCT/US2016/058344 (WO2017/070632), each of which is incorporated herein by reference for its entirety. Also see Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), the entire contents of which are hereby incorporated by reference.
Use of the base editor system provided herein comprises the steps of (a) contacting a target nucleotide sequence of a polynucleotide (e.g., double- or single stranded DNA or RNA) of a subject with a base editor system comprising a nucleobase editor (e.g., an adenosine base editor or a cytidine base editor) and a guide polynucleic acid (e.g., gRNA), wherein the target nucleotide sequence comprises a targeted nucleobase pair; (b) inducing strand separation of said target region; (c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase; and (d) cutting no more than one strand of said target region, where a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase. It should be appreciated that in some embodiments, step (b) is omitted. In some embodiments, said targeted nucleobase pair is a plurality of nucleobase pairs in one or more genes. In some embodiments, the base editor system provided herein is capable of multiplex editing of a plurality of nucleobase pairs in one or more genes. In some embodiments, the plurality of nucleobase pairs is located in the same gene. In some embodiments, the plurality of nucleobase pairs is located in one or more genes, wherein at least one gene is located in a different locus.
In some embodiments, the cut single strand (nicked strand) is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase. In some embodiments, the base editor comprises a Cas9 domain. In some embodiments, the first base is adenine, and the second base is not a G, C, A, or T. In some embodiments, the second base is inosine.
In some embodiments, a single guide polynucleotide may be utilized to target a deaminase to a target nucleic acid sequence. In some embodiments, a single pair of guide polynucleotides may be utilized to target different deaminases to a target nucleic acid sequence.
The nucleobase components and the polynucleotide programmable nucleotide binding component of a base editor system may be associated with each other covalently or non-covalently. For example, in some embodiments, the deaminase domain can be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain. In some embodiments, a polynucleotide programmable nucleotide binding domain can be fused or linked to a deaminase domain. In some embodiments, a polynucleotide programmable nucleotide binding domain can target a deaminase domain to a target nucleotide sequence by non-covalently interacting with or associating with the deaminase domain. For example, in some embodiments, the nucleobase editing component, e.g., the deaminase component can comprise an additional heterologous portion or domain that is capable of interacting with, associating with, or capable of forming a complex with an additional heterologous portion or domain that is part of a polynucleotide programmable nucleotide binding domain. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may be a K Homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a steril alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.
A base editor system may further comprise a guide polynucleotide component. It should be appreciated that components of the base editor system may be associated with each other via covalent bonds, noncovalent interactions, or any combination of associations and interactions thereof. In some embodiments, a deaminase domain can be targeted to a target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the nucleobase editing component of the base editor system, e.g., the deaminase component, can comprise an additional heterologous portion or domain (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) that is capable of interacting with, associating with, or capable of forming a complex with a portion or segment (e.g., a polynucleotide motif) of a guide polynucleotide. In some embodiments, the additional heterologous portion or domain (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) can be fused or linked to the deaminase domain. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may be a K Homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.
In some embodiments, a base editor system can further comprise an inhibitor of base excision repair (BER) component. It should be appreciated that components of the base editor system may be associated with each other via covalent bonds, noncovalent interactions, or any combination of associations and interactions thereof. The inhibitor of BER component may comprise a base excision repair inhibitor. In some embodiments, the inhibitor of base excision repair can be a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the inhibitor of base excision repair can be an inosine base excision repair inhibitor. In some embodiments, the inhibitor of base excision repair can be targeted to the target nucleotide sequence by the polynucleotide programmable nucleotide binding domain. In some embodiments, a polynucleotide programmable nucleotide binding domain can be fused or linked to an inhibitor of base excision repair. In some embodiments, a polynucleotide programmable nucleotide binding domain can be fused or linked to a deaminase domain and an inhibitor of base excision repair. In some embodiments, a polynucleotide programmable nucleotide binding domain can target an inhibitor of base excision repair to a target nucleotide sequence by non-covalently interacting with or associating with the inhibitor of base excision repair. For example, in some embodiments, the inhibitor of base excision repair component can comprise an additional heterologous portion or domain that is capable of interacting with, associating with, or capable of forming a complex with an additional heterologous portion or domain that is part of a polynucleotide programmable nucleotide binding domain. In some embodiments, the inhibitor of base excision repair can be targeted to the target nucleotide sequence by the guide polynucleotide. For example, in some embodiments, the inhibitor of base excision repair can comprise an additional heterologous portion or domain (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) that is capable of interacting with, associating with, or capable of forming a complex with a portion or segment (e.g., a polynucleotide motif) of a guide polynucleotide. In some embodiments, the additional heterologous portion or domain of the guide polynucleotide (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) can be fused or linked to the inhibitor of base excision repair. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may be a K Homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.
In some embodiments, the base editor inhibits base excision repair (BER) of the edited strand. In some embodiments, the base editor protects or binds the non-edited strand. In some embodiments, the base editor comprises UGI activity. In some embodiments, the base editor comprises a catalytically inactive inosine-specific nuclease. In some embodiments, the base editor comprises nickase activity. In some embodiments, the intended edit of base pair is upstream of a PAM site. In some embodiments, the intended edit of base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edit of base-pair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site.
In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker or a spacer. In some embodiments, the linker or spacer is 1-25 amino acids in length. In some embodiments, the linker or spacer is 5-20 amino acids in length. In some embodiments, the linker or spacer is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.
In some embodiments, the base editing fusion proteins provided herein need to be positioned at a precise location, for example, where a target base is placed within a defined region (e.g., a “deamination window”). In some embodiments, a target can be within a 4 base region. In some embodiments, such a defined target region can be approximately 15 bases upstream of the PAM. See Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), the entire contents of which are hereby incorporated by reference.
In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edit of base pair is within the target window. In some embodiments, the target window comprises the intended edit of base pair. In some embodiments, the method is performed using any of the base editors provided herein. In some embodiments, a target window is a deamination window. A deamination window can be the defined region in which a base editor acts upon and deaminates a target nucleotide. In some embodiments, the deamination window is within a 2, 3, 4, 5, 6, 7, 8, 9, or 10 base regions. In some embodiments, the deamination window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases upstream of the PAM.
The base editors of the present disclosure can comprise any domain, feature or amino acid sequence which facilitates the editing of a target polynucleotide sequence. For example, in some embodiments, the base editor comprises a nuclear localization sequence (NLS). In some embodiments, an NLS of the base editor is localized between a deaminase domain and a polynucleotide programmable nucleotide binding domain. In some embodiments, an NLS of the base editor is localized C-terminal to a polynucleotide programmable nucleotide binding domain.
Other exemplary features that can be present in a base editor as disclosed herein are localization sequences, such as cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. In some embodiments, the fusion protein comprises one or more His tags.
In some embodiments, non-limiting exemplary cytidine base editors (CBE) include BE1 (APOBEC1-XTEN-dCas9), BE2 (APOBEC1-XTEN-dCas9-UGI), BE3 (APOBEC1-XTEN-dCas9(A840H)-UGI), BE3-Gam, saBE3, saBE4-Gam, BE4, BE4-Gam, saBE4, or saB4E-Gam. BE4 extends the APOBEC1-Cas9n(D1OA) linker to 32 amino acids and the Cas9n-UGI linker to 9 amino acids, and appends a second copy of UGI to the C-terminus of the construct with another 9-amino acid linker into a single base editor construct. The base editors saBE3 and saBE4 have the S. pyogenes Cas9n(D1OA) replaced with the smaller S. aureus Cas9n(D1OA). BE3-Gam, saBE3-Gam, BE4-Gam, and saBE4-Gam have 174 residues of Gam protein fused to the N-terminus of BE3, saBE3, BE4, and saBE4 via the 16 amino acid XTEN linker.
In some embodiments, the adenosine base editor (ABE) can deaminate adenine in DNA. In some embodiments, ABE is generated by replacing APOBEC1 component of BE3 with natural or engineered E. coli TadA, human ADAR2, mouse ADA, or human ADAT2. In some embodiments, ABE comprises evolved TadA variant. In some embodiments, the ABE is ABE 1.2 (TadA*-XTEN-nCas9-NLS). In some embodiments, TadA* comprises A106V and D108N mutations.
In some embodiments, the ABE is a second-generation ABE. In some embodiments, the ABE is ABE2.1, which comprises additional mutations D147Y and E155V in TadA* (TadA*2.1). In some embodiments, the ABE is ABE2.2, ABE2.1 fused to catalytically inactivated version of human alkyl adenine DNA glycosylase (AAG with E125Q mutation). In some embodiments, the ABE is ABE2.3, ABE2.1 fused to catalytically inactivated version of E. coli Endo V (inactivated with D35A mutation). In some embodiments, the ABE is ABE2.6 which has a linker twice as long (32 amino acids, (SGGS)2 (SEQ ID NO: 1425)-XTEN-(SGGS)2 (SEQ ID NO: 1425)) as the linker in ABE2.1. In some embodiments, the ABE is ABE2.7, which is ABE2.1 tethered with an additional wild-type TadA monomer. In some embodiments, the ABE is ABE2.8, which is ABE2.1 tethered with an additional TadA*2.1 monomer. In some embodiments, the ABE is ABE2.9, which is a direct fusion of evolved TadA (TadA*2.1) to the N-terminus of ABE2.1. In some embodiments, the ABE is ABE2.10, which is a direct fusion of wild-type TadA to the N-terminus of ABE2.1. In some embodiments, the ABE is ABE2.11, which is ABE2.9 with an inactivating E59A mutation at the N-terminus of TadA* monomer. In some embodiments, the ABE is ABE2.12, which is ABE2.9 with an inactivating E59A mutation in the internal TadA* monomer.
In some embodiments, the ABE is a third generation ABE. In some embodiments, the ABE is ABE3.1, which is ABE2.3 with three additional TadA mutations (L84F, H123Y, and 1156F).
In some embodiments, the ABE is a fourth generation ABE. In some embodiments, the ABE is ABE4.3, which is ABE3.1 with an additional TadA mutation A142N (TadA*4.3).
In some embodiments, the ABE is a fifth generation ABE. In some embodiments, the ABE is ABE5.1, which is generated by importing a consensus set of mutations from surviving clones (H36L, R51L, S146C, and K157N) into ABE3.1. In some embodiments, the ABE is ABE5.3, which has a heterodimeric construct containing wild-type E. coli TadA fused to an internal evolved TadA*. In some embodiments, the ABE is ABE5.2, ABE5.4, ABE5.5, ABE5.6, ABE5.7, ABE5.8, ABE5.9, ABE5.10, ABE5.11, ABE5.12, ABE5.13, or ABE5.14, as shown in Table 12 below. In some embodiments, the ABE is a sixth generation ABE. In some embodiments, the ABE is ABE6.1, ABE6.2, ABE6.3, ABE6.4, ABE6.5, or ABE6.6, as shown in Table 12 below. In some embodiments, the ABE is a seventh generation ABE. In some embodiments, the ABE is ABE7.1, ABE7.2, ABE7.3, ABE7.4, ABE7.5, ABE7.6, ABE7.7, ABE7.8, ABE 7.9, or ABE7.10, as shown in Table 12 below.







TABLE 12







Genotypes of ABEs






























23
26
36
37
48
49
51
72
84
87
105
108
123
125
142
145
147
152
155
156
157
161

































ABE0.1
W
R
H
N
P

R
N
L
S
A
D
H
G
A
S
D
R
E
I
K
K


ABE0.2
W
R
H
N
P

R
N
L
S
A
D
H
G
A
S
D
R
E
I
K
K


ABE1.1
W
R
H
N
P

R
N
L
S
A
N
H
G
A
S
D
R
E
I
K
K


ABE1.2
W
R
H
N
P

R
N
L
S
V
N
H
G
A
S
D
R
E
I
K
K


ABE2.1
W
R
H
N
P

R
N
L
S
V
N
H
G
A
S
Y
R
V
I
K
K


ABE2.2
W
R
H
N
P

R
N
L
S
V
N
H
G
A
S
Y
R
V
I
K
K


ABE2.3
W
R
H
N
P

R
N
L
S
V
N
H
G
A
S
Y
R
V
I
K
K


ABE2.4
W
R
H
N
P

R
N
L
S
V
N
H
G
A
S
Y
R
V
I
K
K


ABE2.5
W
R
H
N
P

R
N
L
S
V
N
H
G
A
S
Y
R
V
I
K
K


ABE2.6
W
R
H
N
P

R
N
L
S
V
N
H
G
A
S
Y
R
V
I
K
K


ABE2.7
W
R
H
N
P

R
N
L
S
V
N
H
G
A
S
Y
R
V
I
K
K


ABE2.8
W
R
H
N
P

R
N
L
S
V
N
H
G
A
S
Y
R
V
I
K
K


ABE2.9
W
R
H
N
P

R
N
L
S
V
N
H
G
A
S
Y
R
V
I
K
K


ABE2.10
W
R
H
N
P

R
N
L
S
V
N
H
G
A
S
Y
R
V
I
K
K


ABE2.11
W
R
H
N
P

R
N
L
S
V
N
H
G
A
S
Y
R
V
I
K
K


ABE2.12
W
R
H
N
P

R
N
L
S
V
N
H
G
A
S
Y
R
V
I
K
K


ABE3.1
W
R
H
N
P

R
N
F
S
V
N
Y
G
A
S
Y
R
V
F
K
K


ABE3.2
W
R
H
N
P

R
N
F
S
V
N
Y
G
A
S
Y
R
V
F
K
K


ABE3.3
W
R
H
N
P

R
N
F
S
V
N
Y
G
A
S
Y
R
V
F
K
K


ABE3.4
W
R
H
N
P

R
N
F
S
V
N
Y
G
A
S
Y
R
V
F
K
K


ABE3.5
W
R
H
N
P

R
N
F
S
V
N
Y
G
A
S
Y
R
V
F
K
K


ABE3.6
W
R
H
N
P

R
N
F
S
V
N
Y
G
A
S
Y
R
V
F
K
K


ABE3.7
W
R
H
N
P

R
N
F
S
V
N
Y
G
A
S
Y
R
V
F
K
K


ABE3.8
W
R
H
N
P

R
N
F
S
V
N
Y
G
A
S
Y
R
V
F
K
K


ABE4.1
W
R
H
N
P

R
N
L
S
V
N
H
G
N
S
Y
R
V
I
K
K


ABE4.2
W
G
H
N
P

R
N
L
S
V
N
H
G
N
S
Y
R
V
I
K
K


ABE4.3
W
R
H
N
P

R
N
F
S
V
N
Y
G
N
S
Y
R
V
F
K
K


ABE5.1
W
R
L
N
P

L
N
F
S
V
N
Y
G
A
C
Y
R
V
F
N
K


ABE5.2
W
R
H
S
P

R
N
F
S
V
N
Y
G
A
S
Y
R
V
F
K
T


ABE5.3
W
R
L
N
P

L
N
I
S
V
N
Y
G
A
C
Y
R
V
I
N
K


ABE5.4
W
R
H
S
P

R
N
F
S
V
N
Y
G
A
S
Y
R
V
F
K
T


ABE5.5
W
R
L
N
P

L
N
F
S
V
N
Y
G
A
C
Y
R
V
F
N
K


ABE5.6
W
R
L
N
P

L
N
F
S
V
N
Y
G
A
C
Y
R
V
F
N
K


ABE5.7
W
R
L
N
P

L
N
F
S
V
N
Y
G
A
C
Y
R
V
F
N
K


ABE5.8
W
R
L
N
P

L
N
F
S
V
N
Y
G
A
C
Y
R
V
F
N
K


ABE5.9
W
R
L
N
P

L
N
F
S
V
N
Y
G
A
C
Y
R
V
F
N
K


ABE5.10
W
R
L
N
P

L
N
F
S
V
N
Y
G
A
C
Y
R
V
F
N
K


ABE5.11
W
R
L
N
P

L
N
F
S
V
N
Y
G
A
C
Y
R
V
F
N
K


ABE5.12
W
R
L
N
P

L
N
F
S
V
N
Y
G
A
C
Y
R
V
F
N
K


ABE5.13
W
R
H
N
P

L
D
F
S
V
N
Y
A
A
S
Y
R
V
F
K
K


ABE5.14
W
R
H
N
S

L
N
F
C
V
N
Y
G
A
S
Y
R
V
F
K
K


ABE6.1
W
R
H
N
S

L
N
F
S
V
N
Y
G
N
S
Y
R
V
F
K
K


ABE6.2
W
R
H
N
T
V
L
N
F
S
V
N
Y
G
N
S
Y
R
V
F
N
K


ABE6.3
W
R
L
N
S

L
N
F
S
V
N
Y
G
A
C
Y
R
V
F
N
K


ABE6.4
W
R
L
N
S

L
N
F
S
V
N
Y
G
N
C
Y
R
V
F
N
K


ABE6.5
W
R
L
N
T
V
L
N
F
S
V
N
Y
G
A
C
Y
R
V
F
N
K


ABE6.6
W
R
L
N
T
V
L
N
F
S
V
N
Y
G
N
C
Y
R
V
F
N
K


ABE7.1
W
R
L
N
A

L
N
F
S
V
N
Y
G
A
C
Y
R
V
F
N
K


ABE7.2
W
R
L
N
A

L
N
F
S
V
N
Y
G
N
C
Y
R
V
F
N
K


ABE7.3
L
R
L
N
A

L
N
F
S
V
N
Y
G
A
C
Y
R
V
F
N
K


ABE7.4
R
R
L
N
A

L
N
F
S
V
N
Y
G
A
C
Y
R
V
F
N
K


ABE7.5
W
R
L
N
A

L
N
F
S
V
N
Y
G
A
C
Y
H
V
F
N
K


ABE7.6
W
R
L
N
A

L
N
I
S
V
N
Y
G
A
C
Y
P
V
F
N
K


ABE7.7
L
R
L
N
A

L
N
F
S
V
N
Y
G
A
C
Y
P
V
F
N
K


ABE7.8
L
R
L
N
A

L
N
F
S
V
N
Y
G
N
C
Y
R
V
F
N
K


ABE7.9
L
R
L
N
A

L
N
F
S
V
N
Y
G
N
C
Y
P
V
F
N
K


ABE7.10
R
R
L
N
A

L
N
F
S
V
N
Y
G
A
C
Y
P
V
F
N
K









In some embodiments, the base editor is an eighth generation ABE (ABE8). In some embodiments, the ABE8 contains a TadA*8 variant. In some embodiments, the ABE8 has a monomeric construct containing a TadA*8 variant (“ABE8.x-m”). In some embodiments, the ABE8 is ABE8.1-m, which has a monomeric construct containing TadA*7.10 with a YT47T mutation (TadA*8.1). In some embodiments, the ABE8 is ABE8.2-m, which has a monomeric construct containing TadA*7.10 with a Y147R mutation (TadA*8.2). In some embodiments, the ABE8 is ABE8.3-m, which has a monomeric construct containing TadA*7.10 with a Q154S mutation (TadA*8.3). In some embodiments, the ABE8 is ABE8.4-m, which has a monomeric construct containing TadA*7.10 with a Y123H mutation (TadA*8.4). In some embodiments, the ABE8 is ABE8.5-m, which has a monomeric construct containing TadA*7.10 with a V82S mutation (TadA*8.5). In some embodiments, the ABE8 is ABE8.6-m, which has a monomeric construct containing TadA*7.10 with a T166R mutation (TadA*8.6). In some embodiments, the ABE8 is ABE8.7-m, which has a monomeric construct containing TadA*7.10 with a Q154R mutation (TadA*8.7). In some embodiments, the ABE8 is ABE8.8-m, which has a monomeric construct containing TadA*7.10 with Y147R, Q154R, and Y123H mutations (TadA*8.8). In some embodiments, the ABE8 is ABE8.9-m, which has a monomeric construct containing TadA*7.10 with Y147R, Q154R and 176Y mutations (TadA*8.9). In some embodiments, the ABE8 is ABE8.10-m, which has a monomeric construct containing TadA*7.10 with Y147R, Q154R, and T166R mutations (TadA*8.10). In some embodiments, the ABE8 is ABE8.11-m, which has a monomeric construct containing TadA*7.10 with Y147T and Q154R mutations (TadA*8.11). In some embodiments, the ABE8 is ABE8.12-m, which has a monomeric construct containing TadA*7.10 with Y147T and Q154S mutations (TadA*8.12).
In some embodiments, the ABE8 is ABE8.13-m, which has a monomeric construct containing TadA*7.10 with Y123H (Y123H reverted from H123Y), Y147R, Q154R and I76Y mutations (TadA*8.13). In some embodiments, the ABE8 is ABE8.14-m, which has a monomeric construct containing TadA*7.10 with I76Y and V82S mutations (TadA*8.14). In some embodiments, the ABE8 is ABE8.15-m, which has a monomeric construct containing TadA*7.10 with V82S and Y147R mutations (TadA*8.15). In some embodiments, the ABE8 is ABE8.16-m, which has a monomeric construct containing TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y) and Y147R mutations (TadA*8.16). In some embodiments, the ABE8 is ABE8.17-m, which has a monomeric construct containing TadA*7.10 with V82S and Q154R mutations (TadA*8.17). In some embodiments, the ABE8 is ABE8.18-m, which has a monomeric construct containing TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y) and Q154R mutations (TadA*8.18). In some embodiments, the ABE8 is ABE8.19-m, which has a monomeric construct containing TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y), Y147R and Q154R mutations (TadA*8.19). In some embodiments, the ABE8 is ABE8.20-m, which has a monomeric construct containing TadA*7.10 with I76Y, V82S, Y123H (Y123H reverted from H123Y), Y147R and Q154R mutations (TadA*8.20). In some embodiments, the ABE8 is ABE8.21-m, which has a monomeric construct containing TadA*7.10 with Y147R and Q154S mutations (TadA*8.21). In some embodiments, the ABE8 is ABE8.22-m, which has a monomeric construct containing TadA*7.10 with V82S and Q154S mutations (TadA*8.22). In some embodiments, the ABE8 is ABE8.23-m, which has a monomeric construct containing TadA*7.10 with V82S and Y123H (Y123H reverted from H123Y) mutations (TadA*8.23). In some embodiments, the ABE8 is ABE8.24-m, which has a monomeric construct containing TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y), and Y147T mutations (TadA*8.24).
In some embodiments, the ABE8 has a heterodimeric construct containing wild-type E. coli TadA fused to a TadA*8 variant (“ABE8.x-d”). In some embodiments, the ABE8 is ABE8.1-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with a Y147T mutation (TadA*8.1). In some embodiments, the ABE8 is ABE8.2-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with a Y147R mutation (TadA*8.2). In some embodiments, the ABE8 is ABE8.3-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with a Q154S mutation (TadA*8.3). In some embodiments, the ABE8 is ABE8.4-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with a Y123H mutation (TadA*8.4). In some embodiments, the ABE8 is ABE8.5-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with a V82S mutation (TadA*8.5). In some embodiments, the ABE8 is ABE8.6-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with a T166R mutation (TadA*8.6). In some embodiments, the ABE8 is ABE8.7-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with a Q154R mutation (TadA*8.7). In some embodiments, the ABE8 is ABE8.8-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with Y147R, Q154R, and Y123H mutations (TadA*8.8). In some embodiments, the ABE8 is ABE8.9-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with Y147R, Q154R and I76Y mutations (TadA*8.9). In some embodiments, the ABE8 is ABE8.10-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with Y147R, Q154R, and T166R mutations (TadA*8.10). In some embodiments, the ABE8 is ABE8.11-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with Y147T and Q154R mutations (TadA*8.11). In some embodiments, the ABE8 is ABE8.12-d, which has heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with Y147T and Q154S mutations (TadA*8.12). In some embodiments, the ABE8 is ABE8.13-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with Y123H (Y123H reverted from H123Y), Y147R, Q154R and I76Y mutations (TadA*8.13). In some embodiments, the ABE8 is ABE8.14-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with I76Y and V82S mutations (TadA*8.14). In some embodiments, the ABE8 is ABE8.15-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with V82S and Y147R mutations (TadA*8.15). In some embodiments, the ABE8 is ABE8.16-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y) and Y147R mutations (TadA*8.16). In some embodiments, the ABE8 is ABE8.17-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with V82S and Q154R mutations (TadA*8.17). In some embodiments, the ABE8 is ABE8.18-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y) and Q154R mutations (TadA*8.18). In some embodiments, the ABE8 is ABE8.19-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y), Y147R and Q154R mutations (TadA*8.19). In some embodiments, the ABE8 is ABE8.20-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with I76Y, V82S, Y123H (Y123H reverted from H123Y), Y147R and Q154R mutations (TadA*8.20). In some embodiments, the ABE8 is ABE8.21-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with Y147R and Q154S mutations (TadA*8.21). In some embodiments, the ABE8 is ABE8.22-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with V82S and Q154S mutations (TadA*8.22). In some embodiments, the ABE8 is ABE8.23-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with V82S and Y123H (Y123H reverted from H123Y) mutations (TadA*8.23). In some embodiments, the ABE8 is ABE8.24-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y), and Y147T mutations (TadA*8.24).
In some embodiments, the ABE8 has a heterodimeric construct containing TadA*7.10 fused to a TadA*8 variant (“ABE8.x-7”). In some embodiments, the ABE8 is ABE8.1-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with a Y147T mutation (TadA*8.1). In some embodiments, the ABE8 is ABE8.2-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with a Y147R mutation (TadA*8.2). In some embodiments, the ABE8 is ABE8.3-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with a Q154S mutation (TadA*8.3). In some embodiments, the ABE8 is ABE8.4-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with aY123H mutation (TadA*8.4). In some embodiments, the ABE8 is ABE8.5-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with a V82S mutation (TadA*8.5). In some embodiments, the ABE8 is ABE8.6-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with a T166R mutation (TadA*8.6). In some embodiments, the ABE8 is ABE8.7-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with a Q154R mutation (TadA*8.7). In some embodiments, the ABE8 is ABE8.8-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147R, Q154R, and Y123H mutations (TadA*8.8). In some embodiments, the ABE8 is ABE8.9-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147R, Q154R and I76Y mutations (TadA*8.9). In some embodiments, the ABE8 is ABE8.10-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147R, Q154R, and T166R mutations (TadA*8.10). In some embodiments, the ABE8 is ABE8.11-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147T and Q154R mutations (TadA*8.11). In some embodiments, the ABE8 is ABE8.12-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147T and Q154S mutations (TadA*8.12). In some embodiments, the ABE8 is ABE8.13-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y123H (Y123H reverted from H123Y), Y147R, Q154R and I76Y mutations (TadA*8.13). In some embodiments, the ABE8 is ABE8.14-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with I76Y and V82S mutations (TadA*8.14). In some embodiments, the ABE8 is ABE8.15-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S and Y147R mutations (TadA*8.15). In some embodiments, the ABE8 is ABE8.16-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y) and Y147R mutations (TadA*8.16). In some embodiments, the ABE8 is ABE8.17-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S and Q154R mutations (TadA*8.17). In some embodiments, the ABE8 is ABE8.18-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y) and Q154R mutations (TadA*8.18). In some embodiments, the ABE8 is ABE8.19-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y), Y147R and Q154R mutations (TadA*8.19). In some embodiments, the ABE8 is ABE8.20-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with I76Y, V82S, Y123H (Y123H reverted from H123Y), Y147R and Q154R mutations (TadA*8.20). In some embodiments, the ABE8 is ABE8.21-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147R and Q154S mutations (TadA*8.21). In some embodiments, the ABE8 is ABE8.22-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S and Q154S mutations (TadA*8.22). In some embodiments, the ABE8 is ABE8.23-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S and Y123H (Y123H reverted from H123Y) mutations (TadA*8.23). In some embodiments, the ABE8 is ABE8.24-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y), and Y147T mutations (TadA*8.24
In some embodiments, the ABE is ABE8.1-m, ABE8.2-m, ABE8.3-m, ABE8.4-m, ABE8.5-m, ABE8.6-m, ABE8.7-m, ABE8.8-m, ABE8.9-m, ABE8.10-m, ABE8.11-m, ABE8.12-m, ABE8.13-m, ABE8.14-m, ABE8.15-m, ABE8.16-m, ABE8.17-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.21-m, ABE8.22-m, ABE8.23-m, ABE8.24-m, ABE8.1-d, ABE8.2-d, ABE8.3-d, ABE8.4-d, ABE8.5-d, ABE8.6-d, ABE8.7-d, ABE8.8-d, ABE8.9-d, ABE8.10-d, ABE8.11-d, ABE8.12-d, ABE8.13-d, ABE8.14-d, ABE8.15-d, ABE8.16-d, ABE8.17-d, ABE8.18-d, ABE8.19-d, ABE8.20-d, ABE8.21-d, ABE8.22-d, ABE8.23-d, or ABE8.24-d as shown in Table 13 below.







TABLE 13







Adenosine Deaminase Base Editor 8 (ABE8) Variants










Adenosine



ABE8
Deaminase
Adenosine Deaminase Description





ABE8.1-m
TadA*8.1
Monomer_TadA*7.10 + Y147T


ABE8.2-m
TadA*8.2
Monomer_TadA*7.10 + Y147R


ABE8.3-m
TadA*8.3
Monomer_TadA*7.10 + Q154S


ABE8.4-m
TadA*8.4
Monomer_TadA*7.10 + Y123H


ABE8.5-m
TadA*8.5
Monomer_TadA*7.10 + V82S


ABE8.6-m
TadA*8.6
Monomer_TadA*7.10 + T166R


ABE8.7-m
TadA*8.7
Monomer_TadA*7.10 + Q154R


ABE8.8-m
TadA*8.8
Monomer_TadA*7.10 + Y147R_Q154R_Y123H


ABE8.9-m
TadA*8.9
Monomer_TadA*7.10 + Y147R_Q154R_I76Y


ABE8.10-m
TadA*8.10
Monomer_TadA*7.10 + Y147R_Q154R_T166R


ABE8.11-m
TadA*8.11
Monomer_TadA*7.10 + Y147T_Q154R


ABE8.12-m
TadA*8.12
Monomer_TadA*7.10 + Y147T_Q154S


ABE8.13-m
TadA*8.13
Monomer_TadA*7.10 + Y123H_Y147R_Q154R_I76Y


ABE8.14-m
TadA*8.14
Monomer_TadA*7.10 + I76Y_V82S


ABE8.15-m
TadA*8.15
Monomer_TadA*7.10 + V82S_Y147R


ABE8.16-m
TadA*8.16
Monomer_TadA*7.10 + V82S_Y123H_Y147R


ABE8.17-m
TadA*8.17
Monomer_TadA*7.10 + V82S_Q154R


ABE8.18-m
TadA*8.18
Monomer_TadA*7.10 + V82S_Y123H_Q154R


ABE8.19-m
TadA*8.19
Monomer_TadA*7.10 + V82S_Y123H_Y147R_Q154R


ABE8.20-m
TadA*8.20
Monomer_TadA*7.10 + I76Y_V82S_Y123H_Y147R_Q154R


ABE8.21-m
TadA*8.21
Monomer_TadA*7.10 + Y147R_Q154S


ABE8.22-m
TadA*8.22
Monomer_TadA*7.10 + V82S_Q154S


ABE8.23-m
TadA*8.23
Monomer_TadA*7.10 + V82S_Y123H


ABE8.24-m
TadA*8.24
Monomer_TadA*7.10 + V82S_Y123H_Y147T


ABE8.1-d
TadA*8.1
Heterodimer_(WT) + (TadA*7.10 + Y147T)


ABE8.2-d
TadA*8.2
Heterodimer_(WT) + (TadA*7.10 + Y147R)


ABE8.3-d
TadA*8.3
Heterodimer_(WT) + (TadA*7.10 + Q154S)


ABE8.4-d
TadA*8.4
Heterodimer_(WT) + (TadA*7.10 + Y123H)


ABE8.5-d
TadA*8.5
Heterodimer_(WT) + (TadA*7.10 + V82S)


ABE8.6-d
TadA*8.6
Heterodimer_(WT) + (TadA*7.10 + T166R)


ABE8.7-d
TadA*8.7
Heterodimer_(WT) + (TadA*7.10 + Q154R)


ABE8.8-d
TadA*8.8
Heterodimer_(WT) + (TadA*7.10 + Y147R_Q154R_Y123H)


ABE8.9-d
TadA*8.9
Heterodimer_(WT) + (TadA*7.10 + Y147R_Q154R_I76Y)


ABE8.10-d
TadA*8.10
Heterodimer_(WT) + (TadA*7.10 + Y147R_Q154R_T166R)


ABE8.11-d
TadA*8.11
Heterodimer_(WT) + (TadA*7.10 + Y147T_Q154R)


ABE8.12-d
TadA*8.12
Heterodimer_(WT) + (TadA*7.10 + Y147T_Q154S)


ABE8.13-d
TadA*8.13
Heterodimer_(WT) + (TadA*7.10 + Y123H_Y147T_Q154R_I76Y)


ABE8.14-d
TadA*8.14
Heterodimer_(WT) + (TadA*7.10 + I76Y_V82S)


ABE8.15-d
TadA*8.15
Heterodimer_(WT) + (TadA*7.10 + V82S_Y147R)


ABE8.16-d
TadA*8.16
Heterodimer_(WT) + (TadA*7.10 + V82S_Y123H_Y147R)


ABE8.17-d
TadA*8.17
Heterodimer_(WT) + (TadA*7.10 + V82S_Q154R)


ABE8.18-d
TadA*8.18
Heterodimer_(WT) + (TadA*7.10 + V82S_Y123H_Q154R)


ABE8.19-d
TadA*8.19
Heterodimer_(WT) + (TadA*7.10 + V82S_Y123H_Y147R_Q154R)


ABE8.20-d
TadA*8.20
Heterodimer_(WT) + (TadA*7.10 + I76Y_V82S_Y123H_Y147R_Q154R)


ABE8.21-d
TadA*8.21
Heterodimer_(WT) + (TadA*7.10 + Y147R_Q154S)


ABE8.22-d
TadA*8.22
Heterodimer_(WT) + (TadA*7.10 + V82S_Q154S)


ABE8.23-d
TadA*8.23
Heterodimer_(WT) + (TadA*7.10 + V82S_Y123H)


ABE8.24-d
TadA*8.24
Heterodimer_(WT) + (TadA*7.10 + V82S_Y123H_Y147T)









In some embodiments, the ABE8 is ABE8a-m, which has a monomeric construct containing TadA*7.10 with R26C, A109S, T111R, D119N, H122N, Y147D, F149Y, T1661, and D167N mutations (TadA*8a). In some embodiments, the ABE8 is ABE8b-m, which has a monomeric construct containing TadA*7.10 with V88A, A109S, T111R, D119N, H122N, F149Y, T166I, and D167N mutations (TadA*8b). In some embodiments, the ABE8 is ABE8c-m, which has a monomeric construct containing TadA*7.10 with R26C, A109S, T111R, D119N, H122N, F149Y, T166I, and D167N mutations (TadA*8c). In some embodiments, the ABE8 is ABE8d-m, which has a monomeric construct containing TadA*7.10 with V88A, T111R, D119N, and F149Y mutations (TadA*8d). In some embodiments, the ABE8 is ABE8e-m, which has a monomeric construct containing TadA*7.10 with A109S, T111R, D119N, H122N, Y147D, F149Y, T166I, and D167N mutations (TadA*8e).
In some embodiments, the ABE8 is ABE8a-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with R26C, A109S, T111R, D119, H122N, Y147D, F149Y, T166I, and D167N mutations (TadA*8a). In some embodiments, the ABE8 is ABE8b-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with V88A, A109S, T111R, D119N, H122N, F149Y, T1661, and D167N mutations (TadA*8b). In some embodiments, the ABE8 is ABE8c-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with R26C, A109S, T111R, D119N, H122N, F149Y, T166I, and D167N mutations (TadA*8c). In some embodiments, the ABE8 is ABE8d-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with V88A, T111R, D119N, and F149Y mutations (TadA*8d). In some embodiments, the ABE8 is ABE8e-d, which has a heterodimeric construct containing wild-type E. coli TadA fused to TadA*7.10 with A109S, T111R, D119N, H122N, Y147D, F149Y, T1661, and D167N mutations (TadA*8e).
In some embodiments, the ABE8 is ABE8a-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with R26C, A109S, T111R, D119, H122N, Y147D, F149Y, T166I, and D167N mutations (TadA*8a). In some embodiments, the ABE8 is ABE8b-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V88A, A109S, T111R, D119N, H122N, F149Y, T166I, and D167N mutations (TadA*8b). In some embodiments, the ABE8 is ABE8c-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with R26C, A109S, T111R, D119N, H122N, F149Y, T1661, and D167N mutations (TadA*8c). In some embodiments, the ABE8 is ABE8d-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V88A, T111R, Di 19N, and F149Y mutations (TadA*8d). In some embodiments, the ABE8 is ABE8e-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with A109S, T111R, D119N, H122N, Y147D, F149Y, T166I, and D167N mutations (TadA*8e). In some embodiments, the ABE is ABE8a-m, ABE8b-m, ABE8c-m, ABE8d-m, ABE8e-m, ABE8a-d, ABE8b-d, ABE8c-d, ABE8d-d, or ABE8e-d, as shown in Table 14 below. In some embodiments, the ABE is ABE8e-m or ABE8e-d. ABE8e shows efficient adenine base editing activity and low indel formation when used with Cas homologues other than SpCas9, for example, SaCas9, SaCas9-KKH, Cas12a homologues, e.g., LbCas12a, enAs-Cas12a, SpCas9-NG and circularly permuted CP1028-SpCas9 and CP1041-SpCas9. In addition to the mutations shown for ABE8e in Table 14, off-target RNA and DNA editing were reduced by introducing a V106W substitution into the TadA domain (as described in M. Richter et al., 2020, Nature Biotechnology, doi.org/10.1038/s41587-020-0453-z, the entire contents of which are incorporated by reference herein).







TABLE 14







Additional Adenosine Deaminase Base Editor 8 Variants









ABE8 Base
Adenosine



Editor
Deaminase
Adenosine Deaminase Description





ABE8a-m
TadA*8a
Monomer_TadA*7.10 + R26C + A109S + T111R + D119N +




H122N + Y147D + F149Y + T166I + D167N


ABE8b-m
TadA*8b
Monomer_TadA*7.10 + V88A + A109S + T111R + D119N +




H122N + F149Y + T166I + D167N


ABE8c-m
TadA*8c
Monomer_TadA*7.10 + R26C + A109S + T111R + D119N +




H122N + F149Y + T166I + D167N


ABE8d-m
TadA*8d
Monomer_TadA*7.10 + V88A + T111R + D119N + F149Y


ABE8e-m
TadA*8e
Monomer_TadA*7.10 + A109S + T111R + D119N + H122N +




Y147D + F149Y + T166I + D167N


ABE8a-d
TadA*8a
Heterodimer_(WT) + (TadA*7.10 + R26C + A109S + T111R +




D119N + H122N + Y147D + F149Y + T166I + D167N)


ABE8b-d
TadA*8b
Heterodimer_(WT) + (TadA*7.10 + V88A + A109S + T111R +




D119N + H122N + F149Y + T166I + D167N)


ABE8c-d
TadA*8c
Heterodimer_(WT) + (TadA*7.10 + R26C + A109S + T111R +




D119N + H122N + F149Y + T166I + D167N)


ABE8d-d
TadA*8d
Heterodimer_(WT) + (TadA*7.10 + V88A + T111R + D119N +




F149Y)


ABE8e-d
TadA*8e
Heterodimer_(WT) + (TadA*7.10 + A109S + T111R +




D119N + H122N + Y147D + F149Y + T166I + D167N)









In some embodiments, base editors (e.g., ABE8) are generated by cloning an adenosine deaminase variant (e.g., TadA*8) into a scaffold that includes a circular permutant Cas9 (e.g. CP5 or CP6) and a bipartite nuclear localization sequence. In some embodiments, the base editor (e.g., ABE7.9, ABE7.10, or ABE8) is an NGC PAM CP5 variant (S. pyogenes Cas9 or spVRQR Cas9). In some embodiments, the base editor (e.g., ABE7.9, ABE7.10, or ABE8) is an AGA PAM CP5 variant (S. pyogenes Cas9 or spVRQR Cas9). In some embodiments, the base editor (e.g., ABE7.9, ABE7.10, or ABE8) is an NGC PAM CP6 variant (S. pyogenes Cas9 or spVRQR Cas9). In some embodiments, the base editor (e.g. ABE7.9, ABE7.10, or ABE8) is an AGA PAM CP6 variant (S. pyogenes Cas9 or spVRQR Cas9).
In some embodiments, the ABE has a genotype as shown in Table 15 below.







TABLE 15







Genotypes of ABEs






























23
26
36
37
48
49
51
72
84
87
105
108
123
125
142
145
147
152
155
156
157
161

































ABE7.9
L
R
L
N
A

L
N
F
S
V
N
Y
G
N
C
Y
P
V
F
N
K


ABE7.10
R
R
L
N
A

L
N
F
S
V
N
Y
G
A
C
Y
P
V
F
N
K









As shown in Table 16 below, genotypes of 40 ABE8s are described. Residue positions in the evolved E. coli TadA portion of ABE are indicated. Mutational changes in ABE8 are shown when distinct from ABE7.10 mutations. In some embodiments, the ABE has a genotype of one of the ABEs as shown in Table 16 below.








TABLE 16







Residue Identity in Evolved TadA


























23
36
48
51
76
82
84
106
108
123
146
147
152
154
155
156
157
166





























ABE7.10
R
L
A
L
I
V
F
V
N
Y
C
Y
P
Q
V
F
N
T


ABE8.1-m











T


ABE8.2-m











R


ABE8.3-m













S


ABE8.4-m









H


ABE8.5-m





S


ABE8.6-m

















R


ABE8.7-m













R


ABE8.8-m









H

R

R


ABE8.9-m




Y






R

R


ABE8.10-m











R

R



R


ABE8.11-m











T

R


ABE8.12-m











T

S


ABE8.13-m




Y




H

R

R


ABE8.14-m




Y
S


ABE8.15-m





S





R


ABE8.16-m





S



H

R


ABE8.17-m





S







R


ABE8.18-m





S



H



R


ABE8.19-m





S



H

R

R


ABE8.20-m




Y
S



H

R

R


ABE8.21-m











R

S


ABE8.22-m





S







S


ABE8.23-m





S



H


ABE8.24-m





S



H

T


ABE8.1-d











T


ABE8.2-d











R


ABE8.3-d













S


ABE8.4-d









H


ABE8.5-d





S


ABE8.6-d

















R


ABE8.7-d













R


ABE8.8-d









H

R

R


ABE8.9-d




Y






R

R


ABE8.10-d











R

R



R


ABE8.11-d











T

R


ABE8.12-d











T

S


ABE8.13-d




Y




H

R

R


ABE8.14-d




Y
S


ABE8.15-d





S





R


ABE8.16-d





S



H

R


ABE8.17-d





S







R


ABE8.18-d





S



H



R


ABE8.19-d





S



H

R

R


ABE8.20-d




Y
S



H

R

R


ABE8.21-d











R

S


ABE8.22-d





S







S


ABE8.23-d





S



H


ABE8.24-d





S



H

T









In some embodiments, the base editor is ABE8.1, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:







ABE8.1_Y147T_CP5_NGC PAM_monomer


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG


 


LHDPTAHAEIMALRQGGLVMQNYRIIDATLYVTFEPCVMCAGAMIHSRIG


 


RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFR


 


MPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSE


 


IGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGR


 


DFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP


 


KKYGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP


 


IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKFLQKGNELAL


 


PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSK


 


RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFD


 


TTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGSG


 


GSGGSGGSGGMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR


 


HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM


 


AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRK


 


KLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQ


 


TYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN


 


LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLF


 


LAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR


 


QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELL


 


VKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI


 


EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQS


 


FIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL


 


SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNAS


 


LGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA


 


HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN


 


RNEMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQT


 


VKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE


 


LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH


 


IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK


 


LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMN


 


TKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN


 


AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEGADKRTADGSEF


 


ESPKKKRKV*






In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, and the underlined sequence denotes a bipartite nuclear localization sequence.
In some embodiments, the base editor is ABE8.1, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:







pNMG-B335 ABE8.1_Y147T_CP5_NGC PAM_monomer


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG


 


LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG


 


RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFR


 


MPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSE


 


IGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGR


 


DFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP


 


KKYGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP


 


IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKFLQKGNELAL


 


PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSK


 


RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFD


 


TTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGSG


 


GSGGSGGSGGMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR


 


HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM


 


AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRK


 


KLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQ


 


TYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN


 


LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLF


 


LAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR


 


QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELL


 


VKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI


 


EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQS


 


FIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL


 


SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNAS


 


LGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA


 


HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN


 


RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQT


 


VKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE


 


LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH


 


IVPQSFLKDDSIDNKVLTRSDKSRGKSDNVPSEEVVKKMKNYWRQLLNAK


 


LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMN


 


TKYDENDKL1REVKVLTLKSKLVSDHRKDEQEYKVREINNYHHAHDAYLN


 


AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEGADKRTADGSEF


 


ESPKKKRKV*






In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, and the underlined sequence denotes a bipartite nuclear localization sequence.
In some embodiments, the base editor is ABE8.14, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:







pNMG-357_ABE8.14 with NGC PAM CPS


MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG


 


RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG


 


RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR


 


MRRQEIKAQKKAQSSTDGGSSGGSSGSETPGTSESATPESSGGSSGGSMS


 


EVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH


 


DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV


 


VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMP


 


RQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSEIG


 


KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF


 


ATVRKVLSMPQVNIVKKTEVQTGGESKESILPKRNSDKLIARKKDWDPKK


 


YGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPID


 


FLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKFLQKGNELALPS


 


KYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV


 


ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTT


 


IARKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGSGGS


 


GGSGGSGGMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS


 


IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAK


 


VDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL


 


VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY


 


NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI


 


ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLA


 


AKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQ


 


LPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK


 


LNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK


 


ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFI


 


EFMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSG


 


EQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLG


 


TYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL


 


FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKOSGKTILDFLKSDGFANRN


 


FMQLIHDDSLTFKEDIQKAOVSGQGDSLHEHIANLAGSPATKKGILQTVK


 


VVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG


 


SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV


 


PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLI


 


TQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTK


 


YDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAV


 


VGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEGADKRTADGSEFES


 


PKKKRKV*






In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, and the underlined sequence denotes a bipartite nuclear localization sequence.
In some embodiments, the base editor is ABE8.8-m, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:







ABE8.8-m


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG


 


LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG


 


RVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFR


 


MPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD


 


KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL


 


FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE


 


ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR


 


LIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN


 


ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNF


 


KSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL


 


SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFF


 


DQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ


 


RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV


 


GPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNL


 


PNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL


 


FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK


 


DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLK


 


RRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL


 


TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMG


 


RHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE


 


NTQLQNEKLYLYYLONGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSI


 


DNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK


 


AERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE


 


VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP


 


KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL


 


ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT


 


GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG


 


KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS


 


LFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDN


 


EQKQLFVEQHKHYLDEIIEQTSEFSKRVILADANLDKVLSAYNKHRDKPI


 


REQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI


 


TGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV*






In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations.
In some embodiments, the base editor is ABE8.8-d, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:







ABE8.8-d


MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG


 


RHDPTAHAEIMALRQGGLVMQNYRIIDATLYVTLEPCVMCAGAMIHSRIG


 


RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR


 


MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS


 


EVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH


 


DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV


 


VFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMP


 


RRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK


 


YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD


 


SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES


 


FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI


 


YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS


 


GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS


 


NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD


 


ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ


 


SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT


 


FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP


 


LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN


 


EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK


 


TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK


 


DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR


 


RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF


 


KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH


 


KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT


 


QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN


 


KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE


 


RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL


 


ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN


 


GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG


 


FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS


 


KKLKSVKELLGITIMERSSEEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF


 


ELENGRKRMLASAGELQKGNELALPSKYVNELYLASHYEKLKGSPEDNEQ


 


KQLEVEQHKHYLDEIIEQISEESKRVILADANLDKVLSAYNKHRDKPIRE


 


QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVIDATLIHQSITG


 


LYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV*






In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations.
In some embodiments, the base editor is ABE8.13-m, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:







ABE8.13-m


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG


 


LHDPTAHAEIMALRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIG


 


RVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFR


 


MPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD


 


KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL


 


FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE


 


ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR


 


LIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN


 


ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNF


 


KSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL


 


SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFF


 


DQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ


 


RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV


 


GPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNL


 


PNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL


 


FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK


 


DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLK


 


RRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL


 


TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMG


 


RHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE


 


NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSI


 


DNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK


 


AERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE


 


VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP


 


KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL


 


ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT


 


GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG


 


KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS


 


LFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDN


 


EQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI


 


REQAENITHLFTLTNLGAPAAFKYEDTTTDRKRYTSTKEVLDATLIHQSI


 


TGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV*






In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations.
In some embodiments, the base editor is ABE8.13-d, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:







ABE8.13-d


MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG


 


RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG


 


RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR


 


MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS


 


EVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH


 


DPTAHAEIMALRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRV


 


VFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMP


 


RRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK


 


YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD


 


SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES


 


FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI


 


YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS


 


GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS


 


NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD


 


ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ


 


SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT


 


FDNGSIPHQIHLGELHALLRRQEDFYPFLKDNREKIEKILTFRIPYYVGP


 


LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN


 


EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK


 


TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK


 


DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR


 


RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF


 


KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH


 


KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT


 


QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN


 


KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE


 


RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL


 


ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN


 


GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG


 


FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS


 


KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF


 


ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ


 


KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE


 


QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG


 


LYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV*






In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations.
In some embodiments, the base editor is ABE8.17-m, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:







ABE8.17-m


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG


 


LHDPTAHAEIMALRQGGLVMQNYRIIDATLYSTFEPCVMCAGAMIHSRIG


 


RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR


 


MPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD


 


KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL


 


FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE


 


ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR


 


LIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN


 


ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNF


 


KSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL


 


SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFF


 


DQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ


 


RTFDNGSTPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV


 


GPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNL


 


PNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL


 


FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK


 


DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLK


 


RRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL


 


TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMG


 


RHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE


 


NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSI


 


DNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK


 


AERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE


 


VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP


 


KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL


 


ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT


 


GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG


 


KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS


 


LFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDN


 


EQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI


 


REQAENITHLFTLTNLGAPAAFKYEDTTTDRKRYTSTKEVLDATLIHQSI


 


TGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV*






In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations.
In some embodiments, the base editor is ABE8.17-d, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:







ABE8.17-d


MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG


 


RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG


 


RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR


 


MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS


 


EVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH


 


DPTAHAEIMALRQGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRV


 


VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMP


 


RRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK


 


YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD


 


SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES


 


FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI


 


YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS


 


GVDAKATLSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS


 


NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD


 


ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ


 


SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT


 


FDNGSIPHQIHLGELHALLRRQEDFYPFLKDNREKIEKILTFRIPYYVGP


 


LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN


 


EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK


 


TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK


 


DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR


 


RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF


 


KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH


 


KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT


 


QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN


 


KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE


 


RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL


 


ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN


 


GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG


 


FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS


 


KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF


 


ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ


 


KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE


 


QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG


 


LYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV*






In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations.
In some embodiments, the base editor is ABE8.20-m, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:







ABE8.20-m


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG


 


LHDPTAHAEIMALRQGGLVMQNYRLYDATLYSTFEPCVMCAGAMIHSRIG


 


RVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFR


 


MPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD


 


KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL


 


FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE


 


ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR


 


LIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN


 


ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNF


 


KSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL


 


SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFF


 


DQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ


 


RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV


 


GPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNL


 


PNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL


 


FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK


 


DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLK


 


RRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL


 


TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMG


 


RHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE


 


NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSI


 


DNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK


 


AERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE


 


VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP


 


KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL


 


ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT


 


GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG


 


KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS


 


LFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDN


 


EQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI


 


REQAENITHLFTLTNLGAPAAFKYEDTTTDRKRYTSTKEVLDATLIHQSI


 


TGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV*






In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations.
In some embodiments, the base editor is ABE8.20-d, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity:







ABE8.20-d


MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG


 


RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG


 


RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR


 


MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS


 


EVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH


 


DPTAHAEIMALRQGGLVMQNYRLYDATLYSTFEPCVMCAGAMIHSRIGRV


 


VFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMP


 


RRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK


 


YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD


 


SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES


 


FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI


 


YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPTNAS


 


GVDAKATLSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS


 


NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD


 


ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ


 


SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT


 


FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP


 


LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN


 


EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK


 


TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK


 


DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR


 


RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF


 


KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH


 


KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT


 


QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN


 


KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE


 


RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL


 


ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN


 


GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG


 


FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS


 


KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF


 


ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ


 


KQLEVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE


 


QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG


 


LYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV*






In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations.
In some embodiments, an ABE8 of the invention is selected from the following sequences:








01. monoABE8.1_bpNLS + Y147T



MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR


 


QGGLVMQNYRI1DATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH


 


RVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS


 


GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSTKKNLIGALLFDSGET


 


AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI


 


VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI


 


QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQTGDQYADLFLAAKNLSDAILLSDILRVNTEITK


 


APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP


 


ILEKMDGTEELLVKLNREDLLRKQRTFDNGSTPHQIHLGELHAILRRQEDFYPFLKDNREKIEK


 


ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK


 


VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK


 


KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE


 


RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH


 


DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE


 


MARENQTTQKGQKNSRERMKRIEEGTKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ


 


ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK


 


LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV


 


RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT


 


VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV


 


AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR


 


KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS


 


EFSKRVILADANLDKVLSAYNKHRDKPIREQAENITHLFTLTNLGAPAAFKYFDTTIDRKQYRS


 


TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV


 


02. monoABE8.1_bpNLS + Y147R


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR


 


QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH


 


RVEITEGILADECAALLCRFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS


 


GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET


 


AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI


 


VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI


 


QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK


 


APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP


 


ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK


 


ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDKNLPNEK


 


VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK


 


KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE


 


RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLTNGTRDKOSGKTJLDFTKSDGFANRNFMOTTH


 


DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE


 


MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ


 


ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK


 


LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV


 


RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT


 


VRKVLSMPQVNIVKKTEVOTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV


 


AKVEKGKSKKLKSVKELLGITIMERSSEEKNPLDFLEAKGYKEVKKDLIIKLPKYSLFELENGR


 


KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS


 


EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS


 


TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV


 


03. monoABE8.1_bpNLS + Q154S


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR


 


QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH


 


RVEITEGILADECAALLCYFFRMPRSVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS


 


GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET


 


AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI


 


VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI


 


QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK


 


APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP


 


ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK


 


ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK


 


VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK


 


KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE


 


RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH


 


DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE


 


MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLONGRDMYVDQ


 


ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK


 


LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV


 


RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT


 


VRKVLSMPQVNIVKKTEVOTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV


 


AKVEKGKSKKLKSVKELLGITIMERSSEEKNPLDFLEAKGYKEVKKDLLLKLPKYSLFELENGR


 


KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS


 


EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS


 


TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV


 


04. monoABE8.1_bpNLS + Y123H


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR


 


QGGLVMQNYRIIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNH


 


RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS


 


GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET


 


AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI


 


VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI


 


QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK


 


APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP


 


ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK


 


TLTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDKNLPNEK


 


VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK


 


KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE


 


RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH


 


DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE


 


MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLONGRDMYVDQ


 


ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK


 


LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV


 


RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT


 


VRKVLSMPQVNIVKKTEVOTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV


 


AKVEKGKSKKLKSVKELLGITIMERSSEEKNPLDFLEAKGYKEVKKDLLLKLPKYSLFELENGR


 


KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS


 


EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS


 


TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV


 


05. monoABE8.1_bpNLS + V82S


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR


 


QGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH


 


RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS


 


GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET


 


AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI


 


VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI


 


QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK


 


APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP


 


ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK


 


ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK


 


VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK


 


KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE


 


RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH


 


DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE


 


MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ


 


ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK


 


LITQRKFDNLTKAERGGLSELDKAGFTKRQLVETRQITKHVAQTLDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV


 


RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT


 


VRKVLSMPQVNIVKKTEVOTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV


 


AKVEKGKSKKLKSVKELLGITIMERSSEEKNPLDFLEAKGYKEVKKDLLLKLPKYSLFELENGR


 


KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS


 


EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS


 


TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV


 


06. monoABE8.1_bpNLS + T166R


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR


 


QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH


 


RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSRDSGGSSGGSSGSETPGTSESATPESS


 


GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET


 


AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI


 


VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI


 


QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK


 


APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP


 


ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK


 


ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDKNLPNEK


 


VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK


 


KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE


 


RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH


 


DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE


 


MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLONGRDMYVDQ


 


ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK


 


LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV


 


RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT


 


VRKVLSMPQVNIVKKTEVOTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV


 


AKVEKGKSKKLKSVKELLGITIMERSSEEKNPLDFLEAKGYKEVKKDLLLKLPKYSLFELENGR


 


KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS


 


EFSKRVILADANLDKVLSAYNKHRDKPIREQAENITHLFTLTNLGAPAAFKYFDTTIDRKQYRS


 


TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV


 


07. monoABE8.1bpNLS + Q154R


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR


 


QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH


 


RVEITEGILADECAALLCYFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS


 


GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET


 


AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI


 


VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI


 


QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK


 


APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP


 


ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK


 


ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDKNLPNEK


 


VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK


 


KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE


 


RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLTNGTRDKOSGKTJLDFTKSDGFANRNFMOTTH


 


DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE


 


MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ


 


ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK


 


LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV


 


RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT


 


VRKVLSMPQVNIVKKTEVOTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV


 


AKVEKGKSKKLKSVKELLGITIMERSSEEKNPLDFLEAKGYKEVKKDLLLKLPKYSLFELENGR


 


KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS


 


EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS


 


TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV


 


08. monoABE8.1_bpNLS + Y147R_Q154R_Y123H


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR


 


QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNH


 


RVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS


 


GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET


 


AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI


 


VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI


 


QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK


 


APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP


 


ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK


 


ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK


 


VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK


 


KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE


 


RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH


 


DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE


 


MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLONGRDMYVDQ


 


ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK


 


LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV


 


RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT


 


VRKVLSMPQVNIVKKTEVOTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV


 


AKVEKGKSKKLKSVKELLGITIMERSSEEKNPLDFLEAKGYKEVKKDLLLKLPKYSLFELENGR


 


KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS


 


EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS


 


TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV


 


09. monoABE8.1_bpNLS + Y147R_Q154R_I76Y


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR


 


QGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH


 


RVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS


 


GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET


 


AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI


 


VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI


 


QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK


 


APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP


 


ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK


 


TLTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDKNLPNEK


 


VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK


 


KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE


 


RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH


 


DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE


 


MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLONGRDMYVDQ


 


ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK


 


LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV


 


RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT


 


VRKVLSMPQVNIVKKTEVOTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV


 


AKVEKGKSKKLKSVKELLGITIMERSSEEKNPLDFLEAKGYKEVKKDLLLKLPKYSLFELENGR


 


KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS


 


EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS


 


TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV


 


10. monoABE8.1_bpNLS + Y147R_Q154R_T166R


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR


 


QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH


 


RVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSRDSGGSSGGSSGSETPGTSESATPESS


 


GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET


 


AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI


 


VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI


 


QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK


 


APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP


 


ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK


 


ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK


 


VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK


 


KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE


 


RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH


 


DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE


 


MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ


 


ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK


 


LITQRKFDNLTKAERGGLSELDKAGFTKRQLVETRQITKHVAQTLDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV


 


RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT


 


VRKVLSMPQVNIVKKTEVOTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV


 


AKVEKGKSKKLKSVKELLGITIMERSSEEKNPLDFLEAKGYKEVKKDLLLKLPKYSLFELENGR


 


KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS


 


EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS


 


TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV


 


11. monoABE8.1_bpNLS + Y147T_Q151R


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR


 


QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH


 


RVEITEGILADECAALLCTFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS


 


GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET


 


AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI


 


VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI


 


QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK


 


APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP


 


ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK


 


ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDKNLPNEK


 


VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK


 


KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE


 


RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH


 


DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE


 


MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLONGRDMYVDQ


 


ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK


 


LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV


 


RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT


 


VRKVLSMPQVNIVKKTEVOTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV


 


AKVEKGKSKKLKSVKELLGITIMERSSEEKNPLDFLEAKGYKEVKKDLLLKLPKYSLFELENGR


 


KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS


 


EFSKRVILADANLDKVLSAYNKHRDKPIREQAENITHLFTLTNLGAPAAFKYFDTTIDRKQYRS


 


TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV


 


12. monoABE8.1_bpNLS + Y147T_Q154S


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR


 


QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH


 


RVEITEGILADECAALLCTFFRMPRSVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS


 


GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET


 


AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI


 


VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI


 


QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK


 


APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP


 


ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK


 


ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDKNLPNEK


 


VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK


 


KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE


 


RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLTNGTRDKOSGKTJLDFTKSDGFANRNFMOTTH


 


DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE


 


MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ


 


ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK


 


LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV


 


RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT


 


VRKVLSMPQVNIVKKTEVOTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV


 


AKVEKGKSKKLKSVKELLGITIMERSSEEKNPLDFLEAKGYKEVKKDLLLKLPKYSLFELENGR


 


KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS


 


EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS


 


TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV


 


13. monoABE8.1_bpNLS + H123Y123H_Y147R_Q154R_I76Y


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR


 


QGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNH


 


RVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS


 


GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET


 


AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI


 


VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI


 


QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK


 


APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP


 


ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK


 


ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK


 


VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK


 


KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE


 


RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH


 


DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE


 


MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLONGRDMYVDQ


 


ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK


 


LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV


 


RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT


 


VRKVLSMPQVNIVKKTEVOTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV


 


AKVEKGKSKKLKSVKELLGITIMERSSEEKNPLDFLEAKGYKEVKKDLLLKLPKYSLFELENGR


 


KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS


 


EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS


 


TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV


 


14. monoABE8.1_bpNLS + V82S + Q154R


MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR


 


QGGLVMQNYRIIDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH


 


RVEITEGILADECAALLCYFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS


 


GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET


 


AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI


 


VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI


 


QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP


 


NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK


 


APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP


 


ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK


 


TLTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFTERMTNFDKNLPNEK


 


VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK


 


KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE


 


RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH


 


DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE


 


MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ


 


ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK


 


LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK


 


VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV


 


RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT


 


VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV


 


AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR


 


KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS


 


EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS


 


TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRRV






In some embodiments, the base editor is a ninth generation ABE (ABE9). In some embodiments, the ABE9 contains a TadA*9 variant. ABE9 base editors include an adenosine deaminase variant comprising an amino acid sequence, which contains alterations relative to an ABE 7*10 reference sequence, as described herein. The term “monomer” as used in Table 19 refers to a monomeric form of TadA*7.10 comprising the alterations described in Table 19. The term “heterodimer” as used in Table 19 refers to the specified wild-type E. coli TadA adenosine deaminase fused to a TadA*7.10 comprising the alterations described in Table 19 as described herein. Details of ABE9 base editors are described in International PCT Application No. PCT/2020/049975, which is incorporated herein by reference for its entirety.







TABLE 17







Adenosine Deaminase Base Editor 9 (ABE9) Variants








ABE9 Description
Alterations





ABE9.1_monomer
E25F, V82S, Y123H, T133K, Y147R, Q154R


ABE9.2_monomer
E25F, V82S, Y123H, Y147R, Q154R


ABE9.3 _monomer
V82S, Y123H, P124W, Y147R, Q154R


ABE9.4_monomer
L51W, V82S, Y123H, C146.R, Y147R, Q154R


ABE9.5_monomer
P54C, V82S, Y123H, Y147R, Q154R


ABE9.6_monomer
Y73S, V82S, Y123H, Y147R, Q154R


ABE9.7_monomer
N38G, V82T, Y123H, Y147R, Q154R


ABE9.8_monomer
R23H, V82S, Y123H, Y147R, Q154R


ABE9.9_monomer
R21N, V82S, Y123H, Y147R, Q154R


ABE9.10_monomer
V82S, Y123H, Y147R, Q154R, A158K


ABE9.11_monomer
N72K, V82S, Y123H, D139L, Y147R, Q154R,


ABE9.12_monomer
E25F, V82S, Y123H, D139M, Y147R, Q154R


ABE9.13 monomer
M70V. V82S, M94V, Y123H, Y147R, Q154R


ABE9.14_monomer
Q71M, V82S, Y123H, Y147R, Q154R


ABE9.15_heterodimer
E25F, V82S, Y123H, T133K, Y147R, Q154R


ABE9.16_heterodimer
E25F, V82S, Y123H, Y147R, Q154R


ABE9.17_heterodimer
V82S, Y123H, P124W, Y147R, Q154R


ABE9.18_heterodimer
L51W, V82S, Y123H, C146R, Y147R, Q154R


ABE9.19_heterodimer
P54C, V82S, Y123H, Y147R, Q154R


ABE9.2 _heterodimer
Y73S, V82S, Y123H, Y147R, Q154R


ABE9.21_heterodimer
N38G, V82T, Y123H, Y147R, Q154R


ABE9.22_heterodimer
R23H, V82S, Y123H, Y147R, Q154R


ABE9.23_heterodimer
R21N, V82S, Y123H, Y147R, Q154R


ABE9.24_heterodimer
V82S, Y123H, Y147R, Q154R, A158K


ABE9.25_heterodimer
N72K, V82S, Y123H, D139L, Y147R, Q154R,


ABE9.26_heterodimer
E25F, V82S, Y123H, D139M, Y147R, Q154R


ABE9.27_heterodimer
M70V, V82S, M94V, Y123H, Y147R, Q154R


ABE9.28_heterodimer
Q71M, V82S, Y123H, Y147R, Q154R


ABE9.29_monomer
E25F_I76Y_V82S_Y123H_Y147R_Q154R


ABE9.30_monomer
I76Y_V82T_Y123H_Y147R_Q154R


ABE9.31_monomer
N38G_I76Y_V82S_Y123H_Y147R_Q154R


ABE9.32_monomer
N38G_I76Y_V82T_Y123H_Y147R_Q154R


ABE9.33_monomer
R23H_I76Y_V82S_Y123H_Y147R_Q154R


ABE9.34_monomer
P54C_I76Y_V82S_Y123H_Y147R_Q154R


ABE9.35_monomer
R21N_I76Y_V82S_Y123H_Y147R_Q154R


ABE9.36_monomer
I76Y_V82S_Y123H_D138M_Y147R_Q154R


ABE9.37_monomer
Y72S_I76Y_V82S_Y123H_Y147R_Q154R


ABE9.38_heterodimer
E25F_I76Y_V82S_Y123H_Y147R_Q154R


ABE9.39_heterodimer
I76Y_V82T_Y123H_Y147R_Q154R


ABE9.40_heterodimer
N38G_I76Y_V82S_Y123H_Y147R_Q154R


ABE9.41_heterodimer
N38G_I76Y_V82T_Y123H_Y147R_Q154R


ABE9.42_heterodimer
R23H_I76Y_V82S_Y123H_Y147R_Q154R


ABE9.43_heterodimer
P54C_I76Y_V82S_Y123H_Y147R_Q154R


ABE9.44_heterodimer
R21N_I76Y_V82S_Y123H_Y147R_Q154R


ABE9.45_heterodimer
I76Y_V82S_Y123H_D138M_Y147R_Q154R


ABE9.46_heterodimer
Y72S_I76Y_V82S_Y123H_Y147R_Q154R


ABE9.47_monomer
N72K_V82S, Y123H, Y147R, Q154R


ABE9.48_monomer
Q71M_V82S, Y123H, Y147R, Q154R


ABE9.49_monomer
M70V, V82S, M94V, Y123H, Y147R, Q154R


ABE9.50_monomer
V82S, Y123H, T133K, Y147R, Q154R


ABE9.51_monomer
V82S, Y123H, T133K, Y147R, Q154R,



A158K


ABE9.52_monomer
M70V, Q71M, N72K, V82S, Y123H, Y147R,



Q154R


ABE9.53_heterodimer
N72K_V82S, Y123H, Y147R, Q154R


ABE9.54_heterodimer
Q71M_V82S, Y123H, Y147R, Q154R


ABE9.55_heterodimer
M70V, V82S, M94V, Y123H, Y147R, Q154R


ABE9.56_heterodimer
V82S, Y123H, T133K, Y147R, Q154R


ABE9.57_heterodimer
V82S, Y123H, T133K, Y147R, Q154R,



A158K


ABE9.58_heterodimer
M70V, Q71M, N72K, V82S, Y123H, Y147R,



Q154R









In some embodiments, the base editor is a fusion protein comprising a polynucleotide programmable nucleotide binding domain (e.g., Cas9-derived domain) fused to a nucleobase editing domain (e.g., all or a portion of a deaminase domain). In certain embodiments, the fusion proteins provided herein comprise one or more features that improve the base editing activity of the fusion proteins. For example, any of the fusion proteins provided herein may comprise a Cas9 domain that has reduced nuclease activity. In some embodiments, any of the fusion proteins provided herein may have a Cas9 domain that does not have nuclease activity (dCas9), or a Cas9 domain that cuts one strand of a duplexed DNA molecule, referred to as a Cas9 nickase (nCas9).
In some embodiments, the base editor further comprises a domain comprising all or a portion of a uracil glycosylase inhibitor (UGI). In some embodiments, the base editor comprises a domain comprising all or a portion of a uracil binding protein (UBP), such as a uracil DNA glycosylase (UDG). In some embodiments, the base editor comprises a domain comprising all or a portion of a nucleic acid polymerase. In some embodiments, a base editor can comprise as a domain all or a portion of a nucleic acid polymerase (NAP). For example, a base editor can comprise all or a portion of a eukaryotic NAP. In some embodiments, a NAP or portion thereof incorporated into a base editor is a DNA polymerase. In some embodiments, a NAP or portion thereof incorporated into a base editor has translesion polymerase activity. In some embodiments, a NAP or portion thereof incorporated into a base editor is a translesion DNA polymerase. In some embodiments, a NAP or portion thereof incorporated into a base editor is a Rev7, Rev1 complex, polymerase iota, polymerase kappa, or polymerase eta. In some embodiments, a NAP or portion thereof incorporated into a base editor is a eukaryotic polymerase alpha, beta, gamma, delta, epsilon, gamma, eta, iota, kappa, lambda, mu, or nu component. In some embodiments, a NAP or portion thereof incorporated into a base editor comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a nucleic acid polymerase (e.g., a translesion DNA polymerase). In some embodiments, a nucleic acid polymerase or portion thereof incorporated into a base editor is a translesion DNA polymerase.
In some embodiments, a domain of the base editor can comprise multiple domains. For example, the base editor comprising a polynucleotide programmable nucleotide binding domain derived from Cas9 can comprise an REC lobe and an NUC lobe corresponding to the REC lobe and NUC lobe of a wild-type or natural Cas9. In another example, the base editor can comprise one or more of a RuvCI domain, BH domain, REC1 domain, REC2 domain, RuvCII domain, L1 domain, HNH domain, L2 domain, RuvCIII domain, WED domain, TOPO domain or CTD domain. In some embodiments, one or more domains of the base editor comprise a mutation (e.g., substitution, insertion, deletion) relative to a wild-type version of a polypeptide comprising the domain. For example, an HNH domain of a polynucleotide programmable DNA binding domain can comprise an H840A substitution. In another example, a RuvCI domain of a polynucleotide programmable DNA binding domain can comprise a D10A substitution.
Different domains (e.g., adjacent domains) of the base editor disclosed herein can be connected to each other with or without the use of one or more linker domains (e.g., an XTEN linker domain). In some embodiments, a linker domain can be a bond (e.g., covalent bond), chemical group, or a molecule linking two molecules or moieties, e.g., two domains of a fusion protein, such as, for example, a first domain (e.g., Cas9-derived domain) and a second domain (e.g., an adenosine deaminase domain or a cytidine deaminase domain). In some embodiments, a linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-hetero atom bond, etc.). In certain embodiments, a linker is a carbon nitrogen bond of an amide linkage. In certain embodiments, a linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, a linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, a linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In some embodiments, a linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In some embodiments, a linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, a linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, a linker comprises a polyethylene glycol moiety (PEG). In certain embodiments, a linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. A linker can include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile can be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates. In some embodiments, a linker joins a gRNA binding domain of an RNA-programmable nuclease, including a Cas9 nuclease domain, and the catalytic domain of a nucleic acid editing protein. In some embodiments, a linker joins a dCas9 and a second domain (e.g. UGI, etc.).
Linkers
In certain embodiments, linkers may be used to link any of the peptides or peptide domains of the invention. The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polypeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.
Typically, a linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, a linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, a linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, a linker is 2-100 amino acids in length, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. In some embodiments, the linker is about 3 to about 104 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100) amino acids in length. Longer or shorter linkers are also contemplated.
In some embodiments, any of the fusion proteins provided herein, comprise a cytidine or adenosine deaminase and a Cas9 domain that are fused to each other via a linker. Various linker lengths and flexibilities between the cytidine or adenosine deaminase and the Cas9 domain can be employed (e.g., ranging from very flexible linkers of the form (GGGS)n (SEQ ID NO: 1308), (GGGGS)n (SEQ ID NO: 109), and (G)n to more rigid linkers of the form (EAAAK)n (SEQ ID NO: 1309), (SGGS)n (SEQ ID NO: 57), SGSETPGTSESATPES (SEQ ID NO: 56) (see, e.g., Guilinger J P, et al. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents are incorporated herein by reference) and (XP)n) in order to achieve the optimal length for activity for the cytidine or adenosine deaminase nucleobase editor. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS)n motif, wherein n is 1, 3, or 7 (SEQ ID NO: 1450). In some embodiments, cytidine deaminase or adenosine deaminase and the Cas9 domain of any of the fusion proteins provided herein are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 56), which can also be referred to as the XTEN linker. In some embodiments, a linker comprises a plurality of proline residues and is 5-21, 5-14, 5-9, 5-7 amino acids in length, e.g., PAPAP (SEQ ID NO: 65), PAPAPA (SEQ ID NO: 66), PAPAPAP (SEQ ID NO: 67), PAPAPAPA (SEQ ID NO: 68), P(AP)4 (SEQ ID NO: 69), P(AP)7 (SEQ ID NO: 70), P(AP)10 (SEQ ID NO: 71) (see, e.g., Tan J, Zhang F, Karcher D, Bock R. Engineering of high-precision base editors for site-specific single nucleotide replacement. Nat Commun. 2019 Jan. 25; 10(1):439; the entire contents are incorporated herein by reference). Such proline-rich linkers are also termed “rigid” linkers.
In another embodiment, the base editor system comprises a component (protein) that interacts non-covalently with a deaminase (DNA deaminase), e.g., an adenosine or a cytidine deaminase, and transiently attracts the adenosine or cytidine deaminase to the target nucleobase in a target polynucleotide sequence for specific editing, with minimal or reduced bystander or target-adjacent effects. Such a non-covalent system and method involving deaminase-interacting proteins serves to attract a DNA deaminase to a particular genomic target nucleobase and decouples the events of on-target and target-adjacent editing, thus enhancing the achievement of more precise single base substitution mutations. In an embodiment, the deaminase-interacting protein binds to the deaminase (e.g., adenosine deaminase or cytidine deaminase) without blocking or interfering with the active (catalytic) site of the deaminase from engaging the target nucleobase (e.g., adenosine or cytidine, respectively). Such as system, termed “MagnEdit,” involves interacting proteins tethered to a Cas9 and gRNA complex and can attract a co-expressed adenosine or cytidine deaminase (either exogenous or endogenous) to edit a specific genomic target site, and is described in McCann, J. et al., 2020, “MagnEdit—interacting factors that recruit DNA-editing enzymes to single base targets,” Life-Science-Alliance, Vol. 3, No. 4 (e201900606), (doi 10.26508/Isa.201900606), the contents of which are incorporated by reference herein in their entirety. In an embodiment, the DNA deaminase is an adenosine deaminase variant (e.g., TadA*8) as described herein.
In another embodiment, a system called “Suntag,” involves non-covalently interacting components used for recruiting protein (e.g., adenosine deaminase or cytidine deaminase) components, or multiple copies thereof, of base editors to polynucleotide target sites to achieve base editing at the site with reduced adjacent target editing, for example, as described in Tanenbaum, M. E. et al., “A protein tagging system for signal amplification in gene expression and fluorescence imaging,” Cell. 2014 October 23; 159(3): 635-646. doi:10.1016/j.cell.2014.09.039; and in Huang, Y.-H. et al., 2017, “DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A,” Genome Biol 18: 176. doi:10.1186/s13059-017-1306-z, the contents of each of which are incorporated by reference herein in their entirety. In an embodiment, the DNA deaminase is an adenosine deaminase variant (e.g., TadA*8) as described herein.
Nucleic Acid Programmable DNA Binding Proteins with Guide RNAs
Provided herein are compositions and methods for base editing in host cells, e.g. immune cells (e.g., T- or NK-cells). Further provided herein are compositions comprising a guide polynucleic acid sequence, e.g. a guide RNA sequence, or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more guide RNAs as provided herein. In some embodiments, a composition for base editing as provided herein further comprises a polynucleotide that encodes a base editor, e.g. a C-base editor or an A-base editor. For example, a composition for base editing may comprise a mRNA sequence encoding a BE, a BE4, an ABE, and a combination of one or more guide RNAs as provided. A composition for base editing may comprise a base editor polypeptide and a combination of one or more of any guide RNAs provided herein. Such a composition may be used to effect base editing in an immune cell through different delivery approaches, for example, electroporation, nucleofection, viral transduction or transfection. In some embodiments, the composition for base editing comprises an mRNA sequence that encodes a base editor and a combination of one or more guide RNA sequences provided herein for electroporation.
Some aspects of this disclosure provide complexes comprising any of the fusion proteins provided herein, and a guide RNA bound to a nucleic acid programmable DNA binding protein (napDNAbp) domain (e.g., a Cas9 (e.g., a dCas9, a nuclease active Cas9, or a Cas9 nickase) or Cas12) of the fusion protein. These complexes are also termed ribonucleoproteins (RNPs). In some embodiments, the guide nucleic acid (e.g., guide RNA) is from 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In some embodiments, the guide RNA comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is an RNA sequence. In some embodiments, the target sequence is a sequence in the genome of a bacteria, yeast, fungi, insect, plant, or animal. In some embodiments, the target sequence is a sequence in the genome of a human. In some embodiments, the 3′ end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3′ end of the target sequence is immediately adjacent to a non-canonical PAM sequence (e.g., a sequence listed in Table 5 or 5′-NAA-3′). In some embodiments, the guide nucleic acid (e.g., guide RNA) is complementary to a sequence in a gene of interest (e.g., a gene associated with a disease or disorder).
Some aspects of this disclosure provide methods of using the fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule with any of the fusion proteins provided herein, and with at least one guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the 3′ end of the target sequence is immediately adjacent to an AGC, GAG, TTT, GTG, or CAA sequence. In some embodiments, the 3′ end of the target sequence is immediately adjacent to an NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, NGTN, NGTN, or 5′ (TTTV) sequence. In some embodiments, the 3′ end of the target sequence is immediately adjacent to an e.g., TTN, DTTN, GTTN, ATTN, ATTC, DTTNT, WTTN, HATY, TTTN, TTTV, TTTC, TG, RTR, or YTN PAM site.
It will be understood that the numbering of the specific positions or residues in the respective sequences depends on the particular protein and numbering scheme used. Numbering might differ, e.g., in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues.
It will be apparent to those of skill in the art that in order to target any of the fusion proteins disclosed herein, to a target site, e.g., a site comprising a mutation to be edited, it is typically necessary to co-express the fusion protein together with a guide RNA. As explained in more detail elsewhere herein, a guide RNA typically comprises a tracrRNA framework allowing for napDNAbp (e.g., Cas9 or Cas12) binding, and a guide sequence, which confers sequence specificity to the napDNAbp:nucleic acid editing enzyme/domain fusion protein. Alternatively, the guide RNA and tracrRNA may be provided separately, as two nucleic acid molecules. In some embodiments, the guide RNA comprises a structure, wherein the guide sequence comprises a sequence that is complementary to the target sequence. The guide sequence is typically 20 nucleotides long. The sequences of suitable guide RNAs for targeting napDNAbp:nucleic acid editing enzyme/domain fusion proteins to specific genomic target sites will be apparent to those of skill in the art based on the instant disclosure. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to a nucleic sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting any of the provided fusion proteins to specific target sequences are provided herein.
Distinct portions of sgRNA are predicted to form various features that interact with Cas9 (e.g., SpyCas9) and/or the DNA target. Six conserved modules have been identified within native crRNA:tracrRNA duplexes and single guide RNAs (sgRNAs) that direct Cas9 endonuclease activity (see Briner et al., Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality Mol Cell. 2014 Oct. 23; 56(2):333-339). The six modules include the spacer responsible for DNA targeting, the upper stem, bulge, lower stem formed by the CRISPR repeat:tracrRNA duplex, the nexus, and hairpins from the 3′ end of the tracrRNA. The upper and lower stems interact with Cas9 mainly through sequence-independent interactions with the phosphate backbone. In some embodiments, the upper stem is dispensable. In some embodiments, the conserved uracil nucleotide sequence at the base of the lower stem is dispensable. The bulge participates in specific side-chain interactions with the Rec domain of Cas9. The nucleobase of U44 interacts with the side chains of Tyr 325 and His 328, while G43 interacts with Tyr 329. The nexus forms the core of the sgRNA:Cas9 interactions and lies at the intersection between the sgRNA and both Cas9 and the target DNA. The nucleobases of A51 and A52 interact with the side chain of Phe 1105; U56 interacts with Arg 457 and Asn 459; the nucleobase of U59 inserts into a hydrophobic pocket defined by side chains of Arg 74, Asn 77, Pro 475, Leu 455, Phe 446, and Ile 448; C60 interacts with Leu 455, Ala 456, and Asn 459, and C61 interacts with the side chain of Arg 70, which in turn interacts with C15. In some embodiments, one or more of these mutations are made in the bulge and/or the nexus of a sgRNA for a Cas9 (e.g., spyCas9) to optimize sgRNA:Cas9 interactions.
Moreover, the tracrRNA nexus and hairpins are critical for Cas9 pairing and can be swapped to cross orthogonality barriers separating disparate Cas9 proteins, which is instrumental for further harnessing of orthogonal Cas9 proteins. In some embodiments, the nexus and hairpins are swapped to target orthogonal Cas9 proteins. In some embodiments, a sgRNA is dispensed of the upper stem, hairpin 1, and/or the sequence flexibility of the lower stem to design a guide RNA that is more compact and conformationally stable. In some embodiments, the modules are modified to optimize multiplex editing using a single Cas9 with various chimeric guides or by concurrently using orthogonal systems with different combinations of chimeric sgRNAs. Details regarding guide functional modules and methods thereof are described, for example, in Briner et al., Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality Mol Cell. 2014 Oct. 23; 56(2):333-339, the contents of which is incorporated by reference herein in its entirety.
The domains of the base editor disclosed herein can be arranged in any order. Non-limiting examples of a base editor comprising a fusion protein comprising e.g., a polynucleotide-programmable nucleotide-binding domain (e.g., Cas9 or Cas12) and a deaminase domain (e.g., cytidine or adenosine deaminase) can be arranged as follows:

    
    
        NH2-[nucleobase editing domain]-Linker1-[nucleobase editing domain]-COOH;
        NH2-[deaminase]-Linker1-[nucleobase editing domain]-COOH;
        NH2-[deaminase]-Linker1-[nucleobase editing domain]-Linker2-[UGI]-COOH;
        NH2-[deaminase]-Linker1-[nucleobase editing domain]-COOH;
        NH2-[adenosine deaminase]-Linker1-[nucleobase editing domain]-COOH;
        NH2-[nucleobase editing domain]-[deaminase]-COOH;
        NH2-[deaminase]-[nucleobase editing domain]-[inosine BER inhibitor]-COOH;
        NH2-[deaminase]-[inosine BER inhibitor]-[nucleobase editing domain]-COOH;
        NH2-[inosine BER inhibitor]-[deaminase]-[nucleobase editing domain]-COOH;
        NH2-[nucleobase editing domain]-[deaminase]-[inosine BER inhibitor]-COOH;
        NH2-[nucleobase editing domain]-[inosine BER inhibitor]-[deaminase]-COOH;
        NH2-[inosine BER inhibitor]-[nucleobase editing domain]-[deaminase]-COOH;
        NH2-[nucleobase editing domain]-Linker1-[deaminase]-Linker2-[nucleobase editing domain]-COOH;
        NH2-[nucleobase editing domain]-Linker1-[deaminase]-[nucleobase editing domain]-COOH;
        NH2-[nucleobase editing domain]-[deaminase]-Linker2-[nucleobase editing domain]-COOH;
        NH2-[nucleobase editing domain]-[deaminase]-[nucleobase editing domain]-COOH;
        NH2-[nucleobase editing domain]-Linker1-[deaminase]-Linker2-[nucleobase editing domain]-[inosine BER inhibitor]-COOH;
        NH2-[nucleobase editing domain]-Linker1-[deaminase]-[nucleobase editing domain]-[inosine BER inhibitor]-COOH;
        NH2-[nucleobase editing domain]-[deaminase]-Linker2-[nucleobase editing domain]-[inosine BER inhibitor]-COOH;
        NH2-[nucleobase editing domain]-[deaminase]-[nucleobase editing domain]-[inosine BER inhibitor]-COOH;
        NH2-[inosine BER inhibitor]-[nucleobase editing domain]-Linker1-[deaminase]-Linker2-[nucleobase editing domain]-COOH;
        NH2-[inosine BER inhibitor]-[nucleobase editing domain]-Linker1-[deaminase]-[nucleobase editing domain]-COOH;
        NH2-[inosine BER inhibitor]-[nucleobase editing domain]-[deaminase]-Linker2-[nucleobase editing domain]-COOH; or
        NH2-[inosine BER inhibitor]NH2-[nucleobase editing domain]-[deaminase]-[nucleobase editing domain]-COOH.
    
    


In some embodiments, the base editing fusion proteins provided herein need to be positioned at a precise location, for example, where a target base is placed within a defined region (e.g., a “deamination window”). In some embodiments, a target can be within a 4-base region. In some embodiments, such a defined target region can be approximately 15 bases upstream of the PAM. See Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), the entire contents of which are hereby incorporated by reference.
A defined target region can be a deamination window. A deamination window can be the defined region in which a base editor acts upon and deaminates a target nucleotide. In some embodiments, the deamination window is within a 2, 3, 4, 5, 6, 7, 8, 9, or 10 base regions. In some embodiments, the deamination window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases upstream of the PAM.
The base editors of the present disclosure can comprise any domain, feature or amino acid sequence which facilitates the editing of a target polynucleotide sequence. For example, in some embodiments, the base editor comprises a nuclear localization sequence (NLS). In some embodiments, an NLS of the base editor is localized between a deaminase domain and a napDNAbp domain. In some embodiments, an NLS of the base editor is localized C-terminal to a napDNAbp domain.
Non-limiting examples of protein domains which can be included in the fusion protein include a deaminase domain (e.g., adenosine deaminase or cytidine deaminase), a uracil glycosylase inhibitor (UGI) domain, epitope tags, reporter gene sequences, and/or protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, gene silencing activity, chromatin modifying activity, epigenetic modifying activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Additional domains can be a heterologous functional domain. Such heterologous functional domains can confer a function activity, such as modification of a target polypeptide associated with target DNA (e.g., a histone, a DNA binding protein, etc.), leading to, for example, histone methylation, histone acetylation, histone ubiquitination, and the like. Other functions and/or activities conferred can include transposase activity, integrase activity, recombinase activity, ligase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, or any combination of the above.
Other functions conferred can include methyltransferase activity, demethylase activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, remodeling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthetase activity, and demyristoylation activity, or any combination thereof.
A domain may be detected or labeled with an epitope tag, a reporter protein, other binding domains. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). Additional protein sequences can include amino acid sequences that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.
Methods of Using Fusion Proteins Comprising a Cytidine or Adenosine Deaminase and a Cas9 Domain
Some aspects of this disclosure provide methods of using the fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule with any of the fusion proteins provided herein, and with at least one guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the 3′ end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3′ end of the target sequence is not immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3′ end of the target sequence is immediately adjacent to an AGC, GAG, TTT, GTG, or CAA sequence. In some embodiments, the 3′ end of the target sequence is immediately adjacent to an NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, NGTN, NGTN, or 5′ (TTTV) sequence.
In some embodiments, a fusion protein of the invention is used for mutagenizing a target of interest. In particular, a cytidine deaminase or adenosine deaminase nucleobase editor described herein is capable of making multiple mutations within a target sequence. These mutations may affect the function of the target. For example, when a cytidine deaminase or adenosine deaminase nucleobase editor is used to target a regulatory region the function of the regulatory region is altered and the expression of the downstream protein is reduced or eliminated.
It will be understood that the numbering of the specific positions or residues in the respective sequences depends on the particular protein and numbering scheme used. Numbering might be different, e.g., in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues.
It will be apparent to those of skill in the art that in order to target any of the fusion proteins comprising a Cas9 domain and a cytidine or adenosine deaminase, as disclosed herein, to a target site, e.g., a site comprising a mutation to be edited, it is typically necessary to co-express the fusion protein together with a guide RNA, e.g., an sgRNA. As explained in more detail elsewhere herein, a guide RNA typically comprises a tracrRNA framework allowing for Cas9 binding, and a guide sequence, which confers sequence specificity to the Cas9:nucleic acid editing enzyme/domain fusion protein. Alternatively, the guide RNA and tracrRNA may be provided separately, as two nucleic acid molecules. In some embodiments, the guide RNA comprises a structure, wherein the guide sequence comprises a sequence that is complementary to the target sequence. The guide sequence is typically 20 nucleotides long. The sequences of suitable guide RNAs for targeting Cas9:nucleic acid editing enzyme/domain fusion proteins to specific genomic target sites will be apparent to those of skill in the art based on the instant disclosure. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to a nucleic sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting any of the provided fusion proteins to specific target sequences are provided herein.
Base Editor Efficiency
In some embodiments, the purpose of the methods provided herein is to disrupt the normal function of a gene product via gene editing. The nucleobase editing proteins provided herein can be validated for gene editing-based human therapeutics in vitro. It will be understood by the skilled artisan that the nucleobase editing proteins provided herein, e.g., the fusion proteins comprising a polynucleotide programmable nucleotide binding domain (e.g., Cas9) and a nucleobase editing domain (e.g., an adenosine deaminase domain or a cytidine deaminase domain) can be used to edit a nucleotide from A to G or C to T.
CRISPR-Cas9 nucleases have been widely used to mediate targeted genome editing. In most genome editing applications, Cas9 forms a complex with a guide polynucleotide (e.g., single guide RNA (sgRNA)) and induces a double-stranded DNA break (DSB) at the target site specified by the sgRNA sequence. Cells primarily respond to this DSB through the non-homologous end-joining (NHEJ) repair pathway, which results in stochastic insertions or deletions (indels) that can cause frameshift mutations that disrupt the gene. In the presence of a donor DNA template with a high degree of homology to the sequences flanking the DSB, gene correction can be achieved through an alternative pathway known as homology directed repair (HDR). Unfortunately, under most non-perturbative conditions, HDR is inefficient, dependent on cell state and cell type, and dominated by a larger frequency of indels. As most of the known genetic variations associated with human disease are point mutations, methods that can more efficiently and cleanly make precise point mutations are needed. Base editing systems as provided herein provide a new way to provide genome editing without generating double-strand DNA breaks, without requiring a donor DNA template, and without inducing an excess of stochastic insertions and deletions.
In some embodiments, the present disclosure provides base editors that efficiently generate an intended mutation, such as a STOP codon, in a nucleic acid (e.g., a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations. In some embodiments, an intended mutation is a mutation that is generated by a specific base editor (e.g., adenosine base editor or cytidine base editor) bound to a guide polynucleotide (e.g., gRNA), specifically designed to generate the intended mutation. In some embodiments, the intended mutation is in a gene associated with a target antigen associated with a disease or disorder, e.g., T- or NK-cell malignancy. In some embodiments, the intended mutation is an adenine (A) to guanine (G) point mutation (e.g., SNP) in a gene associated with a target antigen associated with a disease or disorder, e.g., T- or NK-cell malignancy. In some embodiments, the intended mutation is an adenine (A) to guanine (G) point mutation within the coding region or non-coding region of a gene (e.g., regulatory region or element). In some embodiments, the intended mutation is a cytosine (C) to thymine (T) point mutation (e.g., SNP) in a gene associated with a target antigen associated with a disease or disorder, e.g., T- or NK-cell malignancy. In some embodiments, the intended mutation is a cytosine (C) to thymine (T) point mutation within the coding region or non-coding region of a gene (e.g., regulatory region or element). In some embodiments, the intended mutation is a point mutation that generates a STOP codon, for example, a premature STOP codon within the coding region of a gene. In some embodiments, the intended mutation is a mutation that eliminates a stop codon.
The base editors of the invention advantageously modify a specific nucleotide base encoding a protein without generating a significant proportion of indels. An “indel”, as used herein, refers to the insertion or deletion of a nucleotide base within a nucleic acid. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene. In some embodiments, it is desirable to generate base editors that efficiently modify (e.g. mutate) a specific nucleotide within a nucleic acid, without generating a large number of insertions or deletions (i.e., indels) in the nucleic acid. In some embodiments, it is desirable to generate base editors that efficiently modify (e.g. mutate or methylate) a specific nucleotide within a nucleic acid, without generating a large number of insertions or deletions (i.e., indels) in the nucleic acid. In certain embodiments, any of the base editors provided herein can generate a greater proportion of intended modifications (e.g., methylations) versus indels. In certain embodiments, any of the base editors provided herein can generate a greater proportion of intended modifications (e.g., mutations) versus indels.
In some embodiments, the base editors provided herein are capable of generating a ratio of intended mutations to indels (i.e., intended point mutations:unintended point mutations) that is greater than 1:1. In some embodiments, the base editors provided herein are capable of generating a ratio of intended mutations to indels that is at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, or at least 1000:1, or more. The number of intended mutations and indels may be determined using any suitable method.
In some embodiments, the base editors provided herein can limit formation of indels in a region of a nucleic acid. In some embodiments, the region is at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor. In some embodiments, any of the base editors provided herein can limit the formation of indels at a region of a nucleic acid to less than T %, less than 1.5%, less than 2%, less than 2.5%, less than 3%, less than 3.5%, less than 4%, less than 4.5%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, or less than 20%. The number of indels formed at a nucleic acid region may depend on the amount of time a nucleic acid (e.g., a nucleic acid within the genome of a cell) is exposed to a base editor. In some embodiments, a number or proportion of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a nucleic acid (e.g., a nucleic acid within the genome of a cell) to a base editor.
Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of efficiently generating an intended mutation in a nucleic acid (e.g. a nucleic acid within a genome of a subject) without generating a considerable number of unintended mutations (e.g., spurious off-target editing or bystander editing). In some embodiments, an intended mutation is a mutation that is generated by a specific base editor bound to a gRNA, specifically designed to generate the intended mutation. In some embodiments, the intended mutation is a mutation that generates a stop codon, for example, a premature stop codon within the coding region of a gene. In some embodiments, the intended mutation is a mutation that eliminates a stop codon. In some embodiments, the intended mutation is a mutation that alters the splicing of a gene. In some embodiments, the intended mutation is a mutation that alters the regulatory sequence of a gene (e.g., a gene promotor or gene repressor). In some embodiments, any of the base editors provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended mutations:unintended mutations) that is greater than 1:1. In some embodiments, any of the base editors provided herein are capable of generating a ratio of intended mutations to unintended mutations that is at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 150:1, at least 200:1, at least 250:1, at least 500:1, or at least 1000:1, or more. It should be appreciated that the characteristics of the base editors described herein may be applied to any of the fusion proteins, or methods of using the fusion proteins provided herein.
Base editing is often referred to as a “modification”, such as, a genetic modification, a gene modification and modification of the nucleic acid sequence and is clearly understandable based on the context that the modification is a base editing modification. A base editing modification is therefore a modification at the nucleotide base level, for example as a result of the deaminase activity discussed throughout the disclosure, which then results in a change in the gene sequence, and may affect the gene product. In essence therefore, the gene editing modification described herein may result in a modification of the gene, structurally and/or functionally, wherein the expression of the gene product may be modified, for example, the expression of the gene is knocked out; or conversely, enhanced, or, in some circumstances, the gene function or activity may be modified. Using the methods disclosed herein, a base editing efficiency may be determined as the knockdown efficiency of the gene in which the base editing is performed, wherein the base editing is intended to knockdown the expression of the gene. A knockdown level may be validated quantitatively by determining the expression level by any detection assay, such as assay for protein expression level, for example, by flow cytometry; assay for detecting RNA expression such as quantitative RT-PCR, northern blot analysis, or any other suitable assay such as pyrosequencing; and may be validated qualitatively by nucleotide sequencing reactions.
In some embodiments, the modification, e.g., single base edit results in at least 10% reduction of the gene targeted expression. In some embodiments, the base editing efficiency may result in at least 10% reduction of the gene targeted expression. In some embodiments, the base editing efficiency may result in at least 20% reduction of the gene targeted expression. In some embodiments, the base editing efficiency may result in at least 30% reduction of the gene targeted expression. In some embodiments, the base editing efficiency may result in at least 40% reduction of the gene targeted expression. In some embodiments, the base editing efficiency may result in at least 50% reduction of the gene targeted expression. In some embodiments, the base editing efficiency may result in at least 60% reduction of the targeted gene expression. In some embodiments, the base editing efficiency may result in at least 70% reduction of the targeted gene expression. In some embodiments, the base editing efficiency may result in at least 80% reduction of the targeted gene expression. In some embodiments, the base editing efficiency may result in at least 90% reduction of the targeted gene expression. In some embodiments, the base editing efficiency may result in at least 91% reduction of the targeted gene expression. In some embodiments, the base editing efficiency may result in at least 92% reduction of the targeted gene expression. In some embodiments, the base editing efficiency may result in at least 93% reduction of the targeted gene expression. In some embodiments, the base editing efficiency may result in at least 94% reduction of the targeted gene expression. In some embodiments, the base editing efficiency may result in at least 95% reduction of the targeted gene expression. In some embodiments, the base editing efficiency may result in at least 96% reduction of the targeted gene expression. In some embodiments, the base editing efficiency may result in at least 97% reduction of the targeted gene expression. In some embodiments, the base editing efficiency may result in at least 98% reduction of the targeted gene expression. In some embodiments, the base editing efficiency may result in at least 99% reduction of the targeted gene expression. In some embodiments, the base editing efficiency may result in knockout (100% knockdown of the gene expression) of the gene that is targeted.
In some embodiments, any of base editor systems provided herein result in less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% indel formation in the target polynucleotide sequence.
In some embodiments, targeted modifications, e.g., single base editing, are used simultaneously to target at least 4, 5, 6, 7, 8, 9, 10, 11, 12 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 different endogenous sequences for base editing with different guide RNAs. In some embodiments, targeted modifications, e.g. single base editing, are used to sequentially target at least 4, 5, 6, 7, 8, 9, 10, 11, 12 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 50, or more different endogenous gene sequences for base editing with different guide RNAs.
Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of efficiently generating an intended mutation, such as a point mutation, in a nucleic acid (e.g., a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations (i.e., mutation of bystanders). In some embodiments, any of the base editors provided herein are capable of generating at least 0.01% of intended mutations (i.e., at least 0.01% base editing efficiency). In some embodiments, any of the base editors provided herein are capable of generating at least 0.01%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of intended mutations.
In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described herein result in less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% indel formation in the target polynucleotide sequence. In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described herein result in less than 0.8% indel formation in the target polynucleotide sequence. In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described herein result in at most 0.8% indel formation in the target polynucleotide sequence. In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described herein result in less than 0.3% indel formation in the target polynucleotide sequence. In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described results in lower indel formation in the target polynucleotide sequence compared to a base editor system comprising one of ABE7 base editors. In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described herein results in lower indel formation in the target polynucleotide sequence compared to a base editor system comprising an ABE7.10.
In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described herein has reduction in indel frequency compared to a base editor system comprising one of the ABE7 base editors. In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described herein has at least 0.01%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% reduction in indel frequency compared to a base editor system comprising one of the ABE7 base editors. In some embodiments, a base editor system comprising one of the ABE8 base editor variants described herein has at least 0.01%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% reduction in indel frequency compared to a base editor system comprising an ABE7.10.
The invention provides adenosine deaminase variants (e.g., ABE8 variants) that have increased efficiency and specificity. In particular, the adenosine deaminase variants described herein are more likely to edit a desired base within a polynucleotide, and are less likely to edit bases that are not intended to be altered (e.g., “bystanders”).
In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced bystander editing or mutations. In some embodiments, an unintended editing or mutation is a bystander mutation or bystander editing, for example, base editing of a target base (e.g., A or C) in an unintended or non-target position in a target window of a target nucleotide sequence. In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced bystander editing or mutations compared to a base editor system comprising an ABE7 base editor, e.g., ABE7.10. In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced bystander editing or mutations by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to a base editor system comprising an ABE7 base editor, e.g., ABE7.10. In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced bystander editing or mutations by at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, or at least 3.0 fold compared to a base editor system comprising an ABE7 base editor, e.g., ABE7.10.
In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced spurious editing. In some embodiments, an unintended editing or mutation is a spurious mutation or spurious editing, for example, non-specific editing or guide independent editing of a target base (e.g., A or C) in an unintended or non-target region of the genome. In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced spurious editing compared to a base editor system comprising an ABE7 base editor, e.g., ABE7.10. In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced spurious editing by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to a base editor system comprising an ABE7 base editor, e.g., ABE7.10. In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced spurious editing by at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, or at least 3.0 fold compared to a base editor system comprising an ABE7 base editor, e.g., ABE7.10.
In some embodiments, any of the ABE8 base editor variants described herein have at least 0.01%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% base editing efficiency. In some embodiments, the base editing efficiency may be measured by calculating the percentage of edited nucleobases in a population of cells. In some embodiments, any of the ABE8 base editor variants described herein have base editing efficiency of at least 0.01%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% as measured by edited nucleobases in a population of cells.
In some embodiments, any of the ABE8 base editor variants described herein has higher base editing efficiency compared to the ABE7 base editors. In some embodiments, any of the ABE8 base editor variants described herein have at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 155%, at least 160%, at least 165%, at least 170%, at least 175%, at least 180%, at least 185%, at least 190%, at least 195%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 450%, or at least 500% higher base editing efficiency compared to an ABE7 base editor, e.g., ABE7.10.
In some embodiments, any of the ABE8 base editor variants described herein has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3.0 fold, at least 3.1 fold, at least 3.2, at least 3.3 fold, at least 3.4 fold, at least 3.5 fold, at least 3.6 fold, at least 3.7 fold, at least 3.8 fold, at least 3.9 fold, at least 4.0 fold, at least 4.1 fold, at least 4.2 fold, at least 4.3 fold, at least 4.4 fold, at least 4.5 fold, at least 4.6 fold, at least 4.7 fold, at least 4.8 fold, at least 4.9 fold, or at least 5.0 fold higher base editing efficiency compared to an ABE7 base editor, e.g., ABE7.10.
In some embodiments, any of the ABE8 base editor variants described herein have at least 0.01%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% on-target base editing efficiency. In some embodiments, any of the ABE8 base editor variants described herein have on-target base editing efficiency of at least 0.01%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% as measured by edited target nucleobases in a population of cells.
In some embodiments, any of the ABE8 base editor variants described herein has higher on-target base editing efficiency compared to the ABE7 base editors. In some embodiments, any of the ABE8 base editor variants described herein have at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 155%, at least 160%, at least 165%, at least 170%, at least 175%, at least 180%, at least 185%, at least 190%, at least 195%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 450%, or at least 500% higher on-target base editing efficiency compared to an ABE7 base editor, e.g., ABE7.10.
In some embodiments, any of the ABE8 base editor variants described herein has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3.0 fold, at least 3.1 fold, at least 3.2 fold, at least 3.3 fold, at least 3.4 fold, at least 3.5 fold, at least 3.6 fold, at least 3.7 fold, at least 3.8 fold, at least 3.9 fold, at least 4.0 fold, at least 4.1 fold, at least 4.2 fold, at least 4.3 fold, at least 4.4 fold, at least 4.5 fold, at least 4.6 fold, at least 4.7 fold, at least 4.8 fold, at least 4.9 fold, or at least 5.0 fold higher on-target base editing efficiency compared to an ABE7 base editor, e.g., ABE7.10.
The ABE8 base editor variants described herein may be delivered to a host cell via a plasmid, a vector, a LNP complex, or an mRNA. In some embodiments, any of the ABE8 base editor variants described herein is delivered to a host cell as an mRNA. In some embodiments, an ABE8 base editor delivered via a nucleic acid based delivery system, e.g., an mRNA, has on-target editing efficiency of at least at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% as measured by edited nucleobases. In some embodiments, an ABE8 base editor delivered by an mRNA system has higher base editing efficiency compared to an ABE8 base editor delivered by a plasmid or vector system. In some embodiments, any of the ABE8 base editor variants described herein has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 155%, at least 160%, at least 165%, at least 170%, at least 175%, at least 180%, at least 185%, at least 190%, at least 195%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300% higher, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 450%, or at least 500% on-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system. In some embodiments, any of the ABE8 base editor variants described herein has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3.0 fold, at least 3.1 fold, at least 3.2 fold, at least 3.3 fold, at least 3.4 fold, at least 3.5 fold, at least 3.6 fold, at least 3.7 fold, at least 3.8 fold, at least 3.9 fold, at least 4.0 fold, at least 4.1 fold, at least 4.2 fold, at least 4.3 fold, at least 4.4 fold, at least 4.5 fold, at least 4.6 fold, at least 4.7 fold, at least 4.8 fold, at least 4.9 fold, or at least 5.0 fold higher on-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system.
In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described herein result in less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% off-target editing in the target polynucleotide sequence.
In some embodiments, any of the ABE8 base editor variants described herein has lower guided off-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system. In some embodiments, any of the ABE8 base editor variants described herein has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% lower guided off-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system. In some embodiments, any of the ABE8 base editor variants described herein has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, or at least 3.0 fold lower guided off-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system. In some embodiments, any of the ABE8 base editor variants described herein has at least about 2.2 fold decrease in guided off-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system.
In some embodiments, any of the ABE8 base editor variants described herein has lower guide-independent off-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system. In some embodiments, any of the ABE8 base editor variants described herein has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% lower guide-independent off-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system. In some embodiments, any of the ABE8 base editor variants described herein has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3.0 fold, at least 5.0 fold, at least 10.0 fold, at least 20.0 fold, at least 50.0 fold, at least 70.0 fold, at least 100.0 fold, at least 120.0 fold, at least 130.0 fold, or at least 150.0 fold lower guide-independent off-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system. In some embodiments, ABE8 base editor variants described herein has 134.0 fold decrease in guide-independent off-target editing efficiency (e.g., spurious RNA deamination) when delivered by an mRNA system compared to when delivered by a plasmid or vector system. In some embodiments, ABE8 base editor variants described herein does not increase guide-independent mutation rates across the genome.
In some embodiments, a single gene delivery event (e.g., by transduction, transfection, electroporation or any other method) can be used to target base editing of 5 sequences within a cell's genome. In some embodiments, a single gene delivery event can be used to target base editing of 6 sequences within a cell's genome. In some embodiments, a single gene delivery event can be used to target base editing of 7 sequences within a cell's genome. In some embodiments, a single electroporation event can be used to target base editing of 8 sequences within a cell's genome. In some embodiments, a single gene delivery event can be used to target base editing of 9 sequences within a cell's genome. In some embodiments, a single gene delivery event can be used to target base editing of 10 sequences within a cell's genome. In some embodiments, a single gene delivery event can be used to target base editing of 20 sequences within a cell's genome. In some embodiments, a single gene delivery event can be used to target base editing of 30 sequences within a cell's genome. In some embodiments, a single gene delivery event can be used to target base editing of 40 sequences within a cell's genome. In some embodiments, a single gene delivery event can be used to target base editing of 50 sequences within a cell's genome.
In some embodiments, the method described herein, for example, the base editing methods has minimum to no off-target effects.
In some embodiments, the base editing method described herein results in at least 50% of a cell population that have been successfully edited (i.e., cells that have been successfully engineered). In some embodiments, the base editing method described herein results in at least 55% of a cell population that have been successfully edited. In some embodiments, the base editing method described herein results in at least 60% of a cell population that have been successfully edited. In some embodiments, the base editing method described herein results in at least 65% of a cell population that have been successfully edited. In some embodiments, the base editing method described herein results in at least 70% of a cell population that have been successfully edited. In some embodiments, the base editing method described herein results in at least 75% of a cell population that have been successfully edited. In some embodiments, the base editing method described herein results in at least 80% of a cell population that have been successfully edited. In some embodiments, the base editing method described herein results in at least 85% of a cell population that have been successfully edited. In some embodiments, the base editing method described herein results in at least 90% of a cell population that have been successfully edited. In some embodiments, the base editing method described herein results in at least 95% of a cell population that have been successfully edited. In some embodiments, the base editing method described herein results in about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of a cell population that have been successfully edited.
In some embodiments, the live cell recovery following a base editing intervention is greater than at least 60%, 70%, 80%, 90% of the starting cell population at the time of the base editing event. In some embodiments, the live cell recovery as described above is about 70%. In some embodiments, the live cell recovery as described above is about 75%. In some embodiments, the live cell recovery as described above is about 80%. In some embodiments, the live cell recovery as described above is about 85%. In some embodiments, the live cell recovery as described above is about 90%, or about 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99%, or 100% of the cells in the population at the time of the base editing event.
In some embodiments the engineered cell population can be further expanded in vitro by about 2 fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, or about 100-fold.
The number of intended mutations and indels can be determined using any suitable method, for example, as described in International PCT Application Nos. PCT/2017/045381 (WO2018/027078) and PCT/US2016/058344 (WO2017/070632); Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017); the entire contents of which are hereby incorporated by reference.
In some embodiments, to calculate indel frequencies, sequencing reads are scanned for exact matches to two 10-bp sequences that flank both sides of a window in which indels can occur. If no exact matches are located, the read is excluded from analysis. If the length of this indel window exactly matches the reference sequence the read is classified as not containing an indel. If the indel window is two or more bases longer or shorter than the reference sequence, then the sequencing read is classified as an insertion or deletion, respectively. In some embodiments, the base editors provided herein can limit formation of indels in a region of a nucleic acid. In some embodiments, the region is at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor.
The number of indels formed at a target nucleotide region can depend on the amount of time a nucleic acid (e.g., a nucleic acid within the genome of a cell) is exposed to a base editor. In some embodiments, the number or proportion of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing the target nucleotide sequence (e.g., a nucleic acid within the genome of a cell) to a base editor. It should be appreciated that the characteristics of the base editors as described herein can be applied to any of the fusion proteins, or methods of using the fusion proteins provided herein.
Details of base editor efficiency are described in International PCT Application Nos. PCT/2017/045381 (WO 2018/027078) and PCT/US2016/058344 (WO 2017/070632), each of which is incorporated herein by reference for its entirety. Also see Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), the entire contents of which are hereby incorporated by reference. In some embodiments, editing of a plurality of nucleobase pairs in one or more genes using the methods provided herein results in formation of at least one intended mutation. In some embodiments, said formation of said at least one intended mutation results in the disruption the normal function of a gene. In some embodiments, said formation of said at least one intended mutation results decreases or eliminates the expression of a protein encoded by a gene. It should be appreciated that multiplex editing can be accomplished using any method or combination of methods provided herein.
Multiplex Editing
In some embodiments, the base editor system provided herein is capable of multiplex editing of a plurality of nucleobase pairs in one or more genes. In some embodiments, the plurality of nucleobase pairs is located in the same gene. In some embodiments, the plurality of nucleobase pairs is located in one or more gene, wherein at least one gene is located in a different locus. In some embodiments, the multiplex editing can comprise one or more guide polynucleotides. In some embodiments, the multiplex editing can comprise one or more base editor systems. In some embodiments, the multiplex editing can comprise one or more base editor systems with a single guide polynucleotide. In some embodiments, the multiplex editing can comprise one or more base editor systems with a plurality of guide polynucleotides. In some embodiments, the multiplex editing can comprise one or more guide polynucleotides with a single base editor system. In some embodiments, the multiplex editing can comprise at least one guide polynucleotide that does not require a PAM sequence to target binding to a target polynucleotide sequence. In some embodiments, multiplex editing can comprise at least one guide polynucleotide that requires a PAM sequence to target binding to a target polynucleotide sequence. In some embodiments, the multiplex editing can comprise a mix of at least one guide polynucleotide that does not require a PAM sequence to target binding to a target polynucleotide sequence and at least one guide polynucleotide that require a PAM sequence to target binding to a target polynucleotide sequence. It should be appreciated that the characteristics of the multiplex editing using any of the base editors as described herein can be applied to any combination of methods using any base editor provided herein. It should also be appreciated that the multiplex editing using any of the base editors as described herein can comprise a sequential editing of a plurality of nucleobase pairs.
In some embodiments, the plurality of nucleobase pairs are in one more genes. In some embodiments, the plurality of nucleobase pairs is in the same gene. In some embodiments, at least one gene in the one more genes is located in a different locus.
In some embodiments, the editing is editing of the plurality of nucleobase pairs in at least one protein coding region. In some embodiments, the editing is editing of the plurality of nucleobase pairs in at least one protein non-coding region. In some embodiments, the editing is editing of the plurality of nucleobase pairs in at least one protein coding region and at least one protein non-coding region.
In some embodiments, the editing is in conjunction with one or more guide polynucleotides. In some embodiments, the base editor system can comprise one or more base editor system. In some embodiments, the base editor system can comprise one or more base editor systems in conjunction with a single guide polynucleotide. In some embodiments, the base editor system can comprise one or more base editor system in conjunction with a plurality of guide polynucleotides. In some embodiments, the editing is in conjunction with one or more guide polynucleotide with a single base editor system. In some embodiments, the editing is in conjunction with at least one guide polynucleotide that does not require a PAM sequence to target binding to a target polynucleotide sequence. In some embodiments, the editing is in conjunction with at least one guide polynucleotide that require a PAM sequence to target binding to a target polynucleotide sequence. In some embodiments, the editing is in conjunction with a mix of at least one guide polynucleotide that does not require a PAM sequence to target binding to a target polynucleotide sequence and at least one guide polynucleotide that require a PAM sequence to target binding to a target polynucleotide sequence. It should be appreciated that the characteristics of the multiplex editing using any of the base editors as described herein can be applied to any of combination of the methods of using any of the base editors provided herein. It should also be appreciated that the editing can comprise a sequential editing of a plurality of nucleobase pairs.
In some embodiments, the base editor system capable of multiplex editing of a plurality of nucleobase pairs in one or more genes comprises one of ABE9 base editors. In some embodiments, the base editor system capable of multiplex editing of a plurality of nucleobase pairs in one or more genes comprises one of the ABE8 base editor variants described herein. In some embodiments, the base editor system capable of multiplex editing of a plurality of nucleobase pairs in one or more genes comprises one of ABE7 base editors. In some embodiments, the base editor system capable of multiplex editing comprising one of the ABE8 base editor variants described herein has higher multiplex editing efficiency compared the base editor system capable of multiplex editing comprising one of ABE7 base editors. In some embodiments, the base editor system capable of multiplex editing comprising one of the ABE8 base editor variants described herein has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 155%, at least 160%, at least 165%, at least 170%, at least 175%, at least 180%, at least 185%, at least 190%, at least 195%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300% higher, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 450%, or at least 500% higher multiplex editing efficiency compared the base editor system capable of multiplex editing comprising one of ABE7 base editors. In some embodiments, the base editor system capable of multiplex editing comprising one of the ABE8 base editor variants described herein has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3.0 fold, at least 3.1 fold, at least 3.2 fold, at least 3.3 fold, at least 3.4 fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, or at least 6.0 fold higher multiplex editing efficiency compared the base editor system capable of multiplex editing comprising one of ABE7 base editors.
Delivery System
The suitability of nucleobase editors to target one or more nucleotides in a gene (e.g., CD2) is evaluated as described herein. In one embodiment, a single cell of interest is transfected, transduced, or otherwise modified with a nucleic acid molecule or molecules encoding a base editing system described herein together with a small amount of a vector encoding a reporter (e.g., GFP). These cells can be any cell line known in the art, including immune cells (e.g., T- or NK-cells), or immortalized human cell lines, such as 293T, K562 or U20S. Alternatively, primary cells (e.g., human) may be used. Cells may also be obtained from a subject or individual, such as from tissue biopsy, surgery, blood, plasma, serum, or other biological fluid. Such cells may be relevant to the eventual cell target.
Delivery may be performed using a viral vector. In one embodiment, transfection may be performed using lipid transfection (such as Lipofectamine or Fugene) or by electroporation. Following transfection, expression of a reporter (e.g., GFP) can be determined either by fluorescence microscopy or by flow cytometry to confirm consistent and high levels of transfection. These preliminary transfections can comprise different nucleobase editors to determine which combinations of editors give the greatest activity. The system can comprise one or more different vectors. In one embodiment, the base editor is codon optimized for expression of the desired cell type, preferentially a eukaryotic cell, preferably a mammalian cell or a human cell.
The activity of the nucleobase editor is assessed as described herein, i.e., by sequencing the genome of the cells to detect alterations in a target sequence. For Sanger sequencing, purified PCR amplicons are cloned into a plasmid backbone, transformed, miniprepped and sequenced with a single primer. Sequencing may also be performed using next generation sequencing (NGS) techniques. When using next generation sequencing, amplicons may be 300-500 bp with the intended cut site placed asymmetrically. Following PCR, next generation sequencing adapters and barcodes (for example Illumina multiplex adapters and indexes) may be added to the ends of the amplicon, e.g., for use in high throughput sequencing (for example on an Illumina MiSeq). The fusion proteins that induce the greatest levels of target specific alterations in initial tests can be selected for further evaluation.
In particular embodiments, the nucleobase editors are used to target polynucleotides of interest. In one embodiment, a nucleobase editor of the invention is delivered to cells (e.g., immune cells (e.g., T- or NK-cells)) in conjunction with one or more guide RNAs that are used to target one or more nucleic acid sequences of interest within the genome of a cell, thereby altering the target gene(s) (e.g., a CD2). In some embodiments, a base editor is targeted by one or more guide RNAs to introduce one or more edits to the sequence of one or more genes of interest (e.g., CD2, TRAC, B2M, CIITA, TRBC1, TRBC2, PD-1, CD52). In some embodiments, the one or more edits to the sequence of one or more genes of interest decrease or eliminate expression of the protein encoded by the gene in the host cell (e.g., immune cells (e.g., T- or NK-cells)). In some embodiments, expression of one or more proteins encoded by one or more genes of interest (e.g., CD2) is completely knocked out or eliminated in the host cell (e.g., immune cells (e.g., T- or NK-cells)).
In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human cell. In some embodiments, the host cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a NK cell. In some embodiments, the one or more edits are introduced into one or more genes selected from CD2, CD3, CD5, CD7, CD52, B2M, CIITA, TRBC1, TRBC2, TRAC, and PD-1, or combinations thereof. In some embodiments, the one or more edits are introduced into the CD2 gene. In some embodiments, the one or more edits are introduced into the CD5 gene. In some embodiments, the one or more edits are introduced into the CD7 gene. In some embodiments, the one or more edits are introduced into the CD2, CD52, TRAC, and PD-1 genes. In some embodiments, the one or more edits are introduced into the CD5, CD52, TRAC, and PD-1 genes. In some embodiments, the one or more edits are introduced into the CD7, CD3, CD52, and PD-1 genes.

Nucleic Acid-Based Delivery of Nucleobase Editors and gRNAs

Nucleic acids encoding a cytidine or adenosine deaminase nucleobase editor according to the present disclosure can be administered to subjects or delivered into cells in vitro or in vivo by art-known methods or as described herein. For example, cytidine or adenosine deaminase nucleobase editors can be delivered by, e.g., vectors (e.g., viral or non-viral vectors), non-vector based methods (e.g., using naked DNA, DNA complexes, lipid nanoparticles), or a combination thereof.
Nucleic acids encoding cytidine or adenosine deaminase nucleobase editors can be delivered directly to cells (e.g., immune cells) as naked DNA or RNA, for instance by means of transfection or electroporation, or can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by the target cells. Nucleic acid vectors, such as the vectors described herein can also be used. In particular embodiments, a polynucleotide, e.g. a mRNA encoding a base editor or a functional component thereof may be co-electroporated with a combination of multiple guide RNAs as described herein.
Nucleic acid vectors can comprise one or more sequences encoding a domain of a fusion protein described herein. A vector can also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization), associated with (e.g., inserted into or fused to) a sequence coding for a protein. As one example, a nucleic acid vectors can include a Cas9 coding sequence that includes one or more nuclear localization sequences (e.g., a nuclear localization sequence from SV40), and one or more deaminases.
The nucleic acid vector can also include any suitable number of regulatory/control elements, e.g., promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, or internal ribosome entry sites (IRES). These elements are well known in the art.
Nucleic acid vectors according to this disclosure include recombinant viral vectors. Exemplary viral vectors are set forth herein above. Other viral vectors known in the art can also be used. In addition, viral particles can be used to deliver genome editing system components in nucleic acid and/or peptide form. For example, “empty” viral particles can be assembled to contain any suitable cargo. Viral vectors and viral particles can also be engineered to incorporate targeting ligands to alter target tissue specificity.
In addition to viral vectors, non-viral vectors can be used to deliver nucleic acids encoding genome editing systems according to the present disclosure. One important category of non-viral nucleic acid vectors are nanoparticles, which can be organic or inorganic. Nanoparticles are well known in the art. Any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components. For instance, organic (e.g. lipid and/or polymer) nanoparticles can be suitable for use as delivery vehicles in certain embodiments of this disclosure. Exemplary lipids for use in nanoparticle formulations, and/or gene transfer are shown in Table 18 (below).







TABLE 18







Lipids Used for Gene Transfer









Lipid
Abbreviation
Feature





1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine
DOPC
Helper


1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine
DOPE
Helper


Cholesterol

Helper


N-[1-(2,3-Dioleyloxy)prophyl]N,N,N-trimethylammonium
DOTMA
Cationic


chloride


1,2-Dioleoyloxy-3-trimethylammonium-propane
DOTAP
Cationic


Dioctadecylamidoglycylspermine
DOGS
Cationic


N-(3-Aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-
GAP-DLRIE
Cationic


propanaminium bromide


Cetyltrimethylammonium bromide
CTAB
Cationic


6-Lauroxyhexyl ornithinate
LHON
Cationic


1-(2,3-Dioleoyloxypropyl)-2,4,6-trimethylpyridinium
2Oc
Cationic


2,3-Dioleyloxy-N-[2(sperminecarboxamido-ethyl]-N,N-
DOSPA
Cationic


dimethyl-1-propanaminium trifluoroacetate


1,2-Dioleyl-3-trimethylammonium-propane
DOPA
Cationic


N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-
MDRIE
Cationic


propanaminium bromide


Dimyristooxypropyl dimethyl hydroxyethyl ammonium bromide
DMRI
Cationic


3β-[N-(N′,N′-Dimethylaminoethane)-carbamoyl]cholesterol
DC-Chol
Cationic


Bis-guanidium-tren-cholesterol
BGTC
Cationic


1,3-Diodeoxy-2-(6-carboxy-spermyl)-propylamide
DOSPER
Cationic


Dimethyloctadecylammonium bromide
DDAB
Cationic


Dioctadecylamidoglicylspermidin
DSL
Cationic


rac-[(2,3-Dioctadecyloxypropyl)(2-hydroxyethyl)]-
CLIP-1
Cationic


dimethylammonium chloride


rac-[2(2,3-Dihexadecyloxypropyl-
CLIP-6
Cationic


oxymethyloxy)ethyl]trimethylammoniun bromide


Ethyldimyristoylphosphatidylcholine
EDMPC
Cationic


1,2-Distearyloxy-N,N-dimethyl-3-aminopropane
DSDMA
Cationic


1,2-Dimyristoyl-trimethylammonium propane
DMTAP
Cationic


O,O′-Dimyristyl-N-lysyl aspartate
DMKE
Cationic


1,2-Distearoyl-sn-glycero-3-ethylphosphocholine
DSEPC
Cationic


N-Palmitoyl D-erythro-sphingosyl carbamoyl-spermine
CCS
Cationic


N-t-Butyl-N0-tetradecyl-3-tetradecylaminopropionamidine
diC14-amidine
Cationic


Octadecenolyoxy[ethyl-2-heptadecenyl-3 hydroxyethyl]
DOTIM
Cationic


imidazolinium chloride


N1-Cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine
CDAN
Cationic


2-(3-[Bis(3-amino-propyl)-amino]propylamino)-N-
RPR209120
Cationic


ditetradecylcarbamoylme-ethyl-acetamide


1,2-dilinoleyloxy-3-dimethylaminopropane
DLinDMA
Cationic


2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane
DLin-KC2-DMA
Cationic


dilinoleyl-methyl-4-dimethylaminobutyrate
DLin-MC3-DMA
Cationic









Table 19 lists exemplary polymers for use in gene transfer and/or nanoparticle formulations.







TABLE 19







Polymers Used for Gene Transfer










Polymer
Abbreviation







Poly(ethylene)glycol
PEG



Polyethylenimine
PEI



Dithiobis (succinimidylpropionate)
DSP



Dimethyl-3,3′-dithiobispropionimidate
DTBP



Poly(ethylene imine)biscarbamate
PEIC



Poly(L-lysine)
PLL



Histidine modified PLL



Poly(N-vinylpyrrolidone)
PVP



Poly(propylenimine)
PPI



Poly(amidoamine)
PAMAM



Poly(amidoethylenimine)
SS-PAEI



Triethylenetetramine
TETA



Poly(β-aminoester)



Poly(4-hydroxy-L-proline ester)
PHP



Poly(allylamine)



Poly(α-[4-aminobutyl]-L-glycolic acid)
PAGA



Poly(D,L-lactic-co-glycolic acid)
PLGA



Poly(N-ethyl-4-vinylpyridinium bromide)



Poly(phosphazene)s
PPZ



Poly(phosphoester)s
PPE



Poly(phosphoramidate)s
PPA



Poly(N-2-hydroxypropylmethacrylamide)
pHPMA



Poly (2-(dimethylamino)ethyl methacrylate)
pDMAEMA



Poly(2-aminoethyl propylene phosphate)
PPE-EA



Chitosan



Galactosylated chitosan



N-Dodacylated chitosan



Histone



Collagen



Dextran-spermine
D-SPM










Table 20 summarizes delivery methods for a polynucleotide encoding a fusion protein described herein.












TABLE 20







Delivery into


Type of




Non-Dividing
Duration of
Genome
Molecule


Delivery
Vector/Mode
Cells
Expression
Integration
Delivered







Physical
(e.g.,
YES
Transient
NO
Nucleic Acids



electroporation,



and Proteins



particle gun,



Calcium



Phosphate



transfection


Viral
Retrovirus
NO
Stable
YES
RNA



Lentivirus
YES
Stable
YES/NO with
RNA






modification



Adenovirus
YES
Transient
NO
DNA



Adeno-
YES
Stable
NO
DNA



Associated



Virus (AAV)



Vaccinia Virus
YES
Very
NO
DNA





Transient



Herpes Simplex
YES
Stable
NO
DNA



Virus


Non-Viral
Cationic
YES
Transient
Depends on
Nucleic Acids



Liposomes


what is
and Proteins






delivered



Polymeric
YES
Transient
Depends on
Nucleic Acids



Nanoparticles


what is
and Proteins






delivered


Biological
Attenuated
YES
Transient
NO
Nucleic Acids


Non-Viral
Bacteria


Delivery
Engineered
YES
Transient
NO
Nucleic Acids


Vehicles
Bacteriophages
YES
Transient
NO
Nucleic Acids



Mammalian



Virus-like



Particles



Biological
YES
Transient
NO
Nucleic Acids



liposomes:



Erythrocyte



Ghosts and



Exosomes









In another aspect, the delivery of genome editing system components or nucleic acids encoding such components, for example, a nucleic acid binding protein such as, for example, Cas9 or variants thereof, and a gRNA targeting a genomic nucleic acid sequence of interest, may be accomplished by delivering a ribonucleoprotein (RNP) to cells. The RNP comprises the nucleic acid binding protein, e.g., Cas9, in complex with the targeting gRNA. RNPs may be delivered to cells using known methods, such as electroporation, nucleofection, or cationic lipid-mediated methods, for example, as reported by Zuris, J. A. et al., 2015, Nat. Biotechnology, 33(1):73-80. RNPs are advantageous for use in CRISPR base editing systems, particularly for cells that are difficult to transfect, such as primary cells. In addition, RNPs can also alleviate difficulties that may occur with protein expression in cells, especially when eukaryotic promoters, e.g., CMV or EF1A, which may be used in CRISPR plasmids, are not well-expressed. Advantageously, the use of RNPs does not require the delivery of foreign DNA into cells. Moreover, because an RNP comprising a nucleic acid binding protein and gRNA complex is degraded over time, the use of RNPs has the potential to limit off-target effects. In a manner similar to that for plasmid based techniques, RNPs can be used to deliver binding protein (e.g., Cas9 variants) and to direct homology directed repair (HDR).
A promoter used to drive base editor coding nucleic acid molecule expression can include AAV ITR. This can be advantageous for eliminating the need for an additional promoter element, which can take up space in the vector. The additional space freed up can be used to drive the expression of additional elements, such as a guide nucleic acid or a selectable marker. ITR activity is relatively weak, so it can be used to reduce potential toxicity due to over expression of the chosen nuclease.
Any suitable promoter can be used to drive expression of the base editor and, where appropriate, the guide nucleic acid. For ubiquitous expression, promoters that can be used include CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains, etc. For brain or other CNS cell expression, suitable promoters can include: SynapsinI for all neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. For liver cell expression, suitable promoters include the Albumin promoter. For lung cell expression, suitable promoters can include SP-B. For endothelial cells, suitable promoters can include ICAM. For hematopoietic cells suitable promoters can include IFNbeta or CD45. For Osteoblasts suitable promoters can include OG-2.
In some embodiments, a base editor of the present disclosure is of small enough size to allow separate promoters to drive expression of the base editor and a compatible guide nucleic acid within the same nucleic acid molecule. For instance, a vector or viral vector can comprise a first promoter operably linked to a nucleic acid encoding the base editor and a second promoter operably linked to the guide nucleic acid.
The promoter used to drive expression of a guide nucleic acid can include: Pol III promoters such as U6 or H1 Use of Pol II promoter and intronic cassettes to express gRNA Adeno Associated Virus (AAV).
In particular embodiments, a fusion protein of the invention is encoded by a polynucleotide present in a viral vector (e.g., adeno-associated virus (AAV), AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAV10, and variants thereof), or a suitable capsid protein of any viral vector. Thus, in some aspects, the disclosure relates to the viral delivery of a fusion protein. Examples of viral vectors include retroviral vectors (e.g. Maloney murine leukemia virus, MML-V), adenoviral vectors (e.g. AD100), lentiviral vectors (HIV and FIV-based vectors), herpesvirus vectors (e.g. HSV-2).
In some aspects, the methods described herein for editing specific genes in an immune cell can be used to genetically modify a CAR-T cell. Such CAR-T cells, and methods to produce such CAR-T cells are described in International Application Nos. PCT/US2016/060736, PCT/US2016/060734, PCT/US2016/034873, PCT/US2015/040660, PCT/EP2016/055332, PCT/IB2015/058650, PCT/EP2015/067441, PCT/EP2014/078876, PCT/EP2014/059662, PCT/IB2014/061409, PCT/US2016/019192, PCT/US2015/059106, PCT/US2016/052260, PCT/US2015/020606, PCT/US2015/055764, PCT/CN2014/094393, PCT/US2017/059989, PCT/US2017/027606, and PCT/US2015/064269, the contents of each is hereby incorporated in its entirety.
Viral Vectors
A base editor described herein can therefore be delivered with viral vectors. In some embodiments, a base editor disclosed herein can be encoded on a nucleic acid that is contained in a viral vector. In some embodiments, one or more components of the base editor system can be encoded on one or more viral vectors. For example, a base editor and guide nucleic acid can be encoded on a single viral vector. In other embodiments, the base editor and guide nucleic acid are encoded on different viral vectors. In either case, the base editor and guide nucleic acid can each be operably linked to a promoter and terminator. The combination of components encoded on a viral vector can be determined by the cargo size constraints of the chosen viral vector.
The use of RNA or DNA viral based systems for the delivery of a base editor takes advantage of highly evolved processes for targeting a virus to specific cells in culture or in the host and trafficking the viral payload to the nucleus or host cell genome. Viral vectors can be administered directly to cells in culture, patients (in vivo), or they can be used to treat cells in vitro, and the modified cells can optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
Viral vectors can include lentivirus (e.g., HIV and FIV-based vectors), Adenovirus (e.g., AD100), Retrovirus (e.g., Maloney murine leukemia virus, MML-V), herpesvirus vectors (e.g., HSV-2), and Adeno-associated viruses (AAVs), 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 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 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 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. The viral vectors can be injected into the tissue of interest. For cell-type specific base editing, the expression of the base editor and optional guide nucleic acid can be driven by a cell-type specific promoter.
The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (See, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700).
Retroviral vectors, especially lentiviral vectors, can require polynucleotide sequences smaller than a given length for efficient integration into a target cell. For example, retroviral vectors of length greater than 9 kb can result in low viral titers compared with those of smaller size. In some aspects, a base editor of the present disclosure is of sufficient size so as to enable efficient packaging and delivery into a target cell via a retroviral vector. In some embodiments, a base editor is of a size so as to allow efficient packing and delivery even when expressed together with a guide nucleic acid and/or other components of a targetable nuclease system.
Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and psi.2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, Adeno-associated virus (“AAV”) vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA can be packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line can also be infected with adenovirus as a helper. The helper virus can promote replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid in some cases is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.
In applications where transient expression is preferred, adenoviral based systems can be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (“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, and 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). 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).
AAV is a small, single-stranded DNA dependent virus belonging to the parvovirus family. The 4.7 kb wild-type (wt) AAV genome is made up of two genes that encode four replication proteins and three capsid proteins, respectively, and is flanked on either side by 145-bp inverted terminal repeats (ITRs). The virion is composed of three capsid proteins, Vp1, Vp2, and Vp3, produced in a 1:1:10 ratio from the same open reading frame but from differential splicing (Vp1) and alternative translational start sites (Vp2 and Vp3, respectively). Vp3 is the most abundant subunit in the virion and participates in receptor recognition at the cell surface defining the tropism of the virus. A phospholipase domain, which functions in viral infectivity, has been identified in the unique N terminus of Vp1.
Similar to wt AAV, recombinant AAV (rAAV) utilizes the cis-acting 145-bp ITRs to flank vector transgene cassettes, providing up to 4.5 kb for packaging of foreign DNA. Subsequent to infection, rAAV can express a fusion protein of the invention and persist without integration into the host genome by existing episomally in circular head-to-tail concatemers. Although there are numerous examples of rAAV success using this system, in vitro and in vivo, the limited packaging capacity has limited the use of AAV-mediated gene delivery when the length of the coding sequence of the gene is equal or greater in size than the wt AAV genome.
Viral vectors can be selected based on the application. For example, for in vivo gene delivery, AAV can be advantageous over other viral vectors. In some embodiments, AAV allows low toxicity, which can be due to the purification method not requiring ultra-centrifugation of cell particles that can activate the immune response. In some embodiments, AAV allows low probability of causing insertional mutagenesis because it doesn't integrate into the host genome. Adenoviruses are commonly used as vaccines because of the strong immunogenic response they induce. Packaging capacity of the viral vectors can limit the size of the base editor that can be packaged into the vector.
AAV has a packaging capacity of about 4.5 Kb or 4.75 Kb including two 145 base inverted terminal repeats (ITRs). This means disclosed base editor as well as a promoter and transcription terminator can fit into a single viral vector. Constructs larger than 4.5 or 4.75 Kb can lead to significantly reduced virus production. For example, SpCas9 is quite large, the gene itself is over 4.1 Kb, which makes it difficult for packing into AAV. Therefore, embodiments of the present disclosure include utilizing a disclosed base editor which is shorter in length than conventional base editors. In some examples, the base editors are less than 4 kb. Disclosed base editors can be less than 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. In some embodiments, the disclosed base editors are 4.5 kb or less in length.
An AAV can be AAV1, AAV2, AAV5 or any combination thereof. One can select the type of AAV with regard to the cells to be targeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereof for targeting brain or neuronal cells; and one can select AAV4 for targeting cardiac tissue. AAV8 is useful for delivery to the liver. A tabulation of certain AAV serotypes as to these cells can be found in Grimm, D. et al, J. Virol. 82: 5887-5911 (2008)).
In some embodiments, lentiviral vectors are used to transduce a modified immune cell (e.g., T- or NK-cell) with a chimeric antigen receptor. Lentiviruses are complex retroviruses that have the ability to infect and express their genes in both mitotic and post-mitotic cells. The most commonly known lentivirus is the human immunodeficiency virus (HIV), which uses the envelope glycoproteins of other viruses to target a broad range of cell types.
Lentiviruses can be prepared as follows. After cloning pCasES10 (which contains a lentiviral transfer plasmid backbone), HEK293FT at low passage (p=5) were seeded in a T-75 flask to 50% confluence the day before transfection in DMEM with 10% fetal bovine serum and without antibiotics. After 20 hours, media is changed to OptiMEM (serum-free) media and transfection was done 4 hours later. Cells are transfected with 10 μg of lentiviral transfer plasmid (pCasES10) and the following packaging plasmids: 5 μg of pMD2.G (VSV-g pseudotype), and 7.5 pg of psPAX2 (gag/pol/rev/tat). Transfection can be done in 4 mL OptiMEM with a cationic lipid delivery agent (50 pl Lipofectamine 2000 and 100 pl Plus reagent). After 6 hours, the media is changed to antibiotic-free DMEM with 10% fetal bovine serum. These methods use serum during cell culture, but serum-free methods are preferred.
Lentivirus can be purified as follows. Viral supernatants are harvested after 48 hours. Supernatants are first cleared of debris and filtered through a 0.45 μm low protein binding (PVDF) filter. They are then spun in an ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets are resuspended in 50 μl of DMEM overnight at 4° C. They are then aliquoted and immediately frozen at −80° C.
In another embodiment, minimal non-primate lentiviral vectors based on the equine infectious anemia virus (EIAV) are also contemplated. In another embodiment, RetinoStat®, an equine infectious anemia virus-based lentiviral gene therapy vector that expresses angiostatic proteins endostatin and angiostatin that is contemplated to be delivered via a subretinal injection. In another embodiment, use of self-inactivating lentiviral vectors are contemplated.
Any RNA of the systems, for example a guide RNA or a base editor-encoding mRNA, can be delivered in the form of RNA. Base editor-encoding mRNA can be generated using in vitro transcription. For example, nuclease mRNA can be synthesized using a PCR cassette containing the following elements: T7 promoter, optional kozak sequence (GCCACC), nuclease sequence, and 3′ UTR such as a 3′ UTR from beta globin-polyA tail. The cassette can be used for transcription by T7 polymerase. Guide polynucleotides (e.g., gRNA) can also be transcribed using in vitro transcription from a cassette containing a T7 promoter, followed by the sequence “GG”, and guide polynucleotide sequence.
To enhance expression and reduce possible toxicity, the base editor-coding sequence and/or the guide nucleic acid can be modified to include one or more modified nucleoside e.g. using pseudo-U or 5-Methyl-C.
The small packaging capacity of AAV vectors makes the delivery of a number of genes that exceed this size and/or the use of large physiological regulatory elements challenging. These challenges can be addressed, for example, by dividing the protein(s) to be delivered into two or more fragments, wherein the N-terminal fragment is fused to a split intein-N and the C-terminal fragment is fused to a split intein-C. These fragments are then packaged into two or more AAV vectors. As used herein, “intein” refers to a self-splicing protein intron (e.g., peptide) that ligates flanking N-terminal and C-terminal exteins (e.g., fragments to be joined). The use of certain inteins for joining heterologous protein fragments is described, for example, in Wood et al., J. Biol. Chem. 289(21); 14512-9 (2014). For example, when fused to separate protein fragments, the inteins IntN and IntC recognize each other, splice themselves out and 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. Other suitable inteins will be apparent to a person of skill in the art.
A fragment of a fusion protein of the invention can vary in length. In some embodiments, a protein fragment ranges from 2 amino acids to about 1000 amino acids in length. In some embodiments, a protein fragment ranges from about 5 amino acids to about 500 amino acids in length. In some embodiments, a protein fragment ranges from about 20 amino acids to about 200 amino acids in length. In some embodiments, a protein fragment ranges from about 10 amino acids to about 100 amino acids in length. Suitable protein fragments of other lengths will be apparent to a person of skill in the art.
In one embodiment, dual AAV vectors are generated by splitting a large transgene expression cassette in two separate halves (5′ and 3′ ends, or head and tail), where 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 is then achieved upon co-infection of the same cell by both dual AAV vectors followed by: (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); or (3) a combination of these two mechanisms (dual AAV hybrid vectors). The use of dual AAV vectors in vivo results in the expression of full-length proteins. The use of the dual AAV vector platform represents an efficient and viable gene transfer strategy for transgenes of >4.7 kb in size.
Inteins
Inteins (intervening protein) are auto-processing domains found in a variety of diverse organisms, which carry out a process known as protein splicing. Protein splicing is a multi-step biochemical reaction comprised of both the cleavage and formation of peptide bonds. While the endogenous substrates of protein splicing are proteins found in intein-containing organisms, inteins can also be used to chemically manipulate virtually any polypeptide backbone.
In protein splicing, the intein excises itself out of a precursor polypeptide by cleaving two peptide bonds, thereby ligating the flanking extein (external protein) sequences via the formation of a new peptide bond. This rearrangement occurs post-translationally (or possibly co-translationally). Intein-mediated protein splicing occurs spontaneously, requiring only the folding of the intein domain.
About 5% of inteins are split inteins, which are transcribed and translated as two separate polypeptides, the N-intein and C-intein, each fused to one extein. Upon translation, the intein fragments spontaneously and non-covalently assemble into the canonical intein structure to carry out protein splicing in trans. The mechanism of protein splicing entails a series of acyl-transfer reactions that result in the cleavage of two peptide bonds at the intein-extein junctions and the formation of a new peptide bond between the N- and C-exteins. This process is initiated by activation of the peptide bond joining the N-extein and the N-terminus of the intein. Virtually all inteins have a cysteine or serine at their N-terminus that attacks the carbonyl carbon of the C-terminal N-extein residue. This N to O/S acyl-shift is facilitated by a conserved threonine and histidine (referred to as the TXXH motif), along with a commonly found aspartate, which results in the formation of a linear (thio)ester intermediate. Next, this intermediate is subject to trans-(thio)esterification by nucleophilic attack of the first C-extein residue (+1), which is a cysteine, serine, or threonine. The resulting branched (thio)ester intermediate is resolved through a unique transformation: cyclization of the highly conserved C-terminal asparagine of the intein. This process is facilitated by the histidine (found in a highly conserved HNF motif) and the penultimate histidine and may also involve the aspartate. This succinimide formation reaction excises the intein from the reactive complex and leaves behind the exteins attached through a non-peptidic linkage. This structure rapidly rearranges into a stable peptide bond in an intein-independent fashion.
In some embodiments, a portion or fragment of a nuclease (e.g., Cas9) 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, an N-terminal fragment of a base editor (e.g., ABE, CBE) is fused to a split intein-N and a C-terminal fragment is fused to a split intein-C. These fragments are then packaged into two or more AAV vectors. 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 one embodiment, inteins are utilized to join fragments or portions of a cytidine or adenosine deaminase base editor protein that is grafted onto an AAV capsid protein. The use of certain inteins for joining heterologous protein fragments is described, for example, in Wood et al., J. Biol. Chem. 289(21); 14512-9 (2014). For example, when fused to separate protein fragments, the inteins IntN and IntC recognize each other, splice themselves out and 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. Other suitable inteins will be apparent to a person of skill in the art.
In some embodiments, an ABE was split into N- and C-terminal fragments at Ala, Ser, Thr, or Cys residues within selected regions of SpCas9. These regions correspond to loop regions identified by Cas9 crystal structure analysis. The N-terminus of each fragment is fused to an intein-N and the C-terminus of each fragment is fused to an intein C at amino acid positions S303, T310, T313, S355, A456, S460, A463, T466, S469, T472, T474, C574, S577, A589, and S590, which are indicated in bold capital letters in the sequence below.









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 301 llSdilrvnT eiTkaplsas mikrydehhq



 



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 361 gyidggasqe efykfikpil ekmdgteell



 



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 421 ailrrqedfy pflkdnreki ekiltfripy



 



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 481 vvdkgasaqs fiermtnfdk nlpnekvlpk



 



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 541 sgeqkkaivd Ilfktnrkvt vkqlkedyfk



 



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 601 ikdkdfldne enedilediv ltltlfedre



 



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 661 rlsrklingi rdkqsgktil dfiksdgfan



 



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 721 hehianlags paikkgiiqt vkvvdelvkv



 



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 781 mkrieegike lgsqilkehp ventqlqnek



 



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 841 ivpqsflkdd sidnkvltrs dknrgksdnv



 



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 901 tkaergglse ldkagfikrq lvetrqitkh



 



     vaqildsrmn tkydendkli revkvitlks



 



 961 klvsdfrkdf qfykvreinn yhhahdayln



 



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1021 miakseqeig katakytfys nimnffktei



 



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1081 atvrkvlsmp qvnivkktev qtggfskesi



 



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1141 ysvlvvakve kgkskklksy kellgitime



 



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1201 yslfelengr krmlasagel qkgnelalps



 



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1261 qhkhyldeii eqisefskry iladanldkv



 



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Use of Nucleobase Editors

Target antigens associated with neoplastic cells may also be expressed on healthy immune cells. Accordingly, using modified immune cells (e.g. CAR-T) cells to target the antigen on neoplastic cells may also result in the killing of healthy immune cells also expressing that antigen, otherwise known as fratricide or self-killing. The use of modified immune cells in combination with base editors that can decrease or eliminate the expression of a target antigen (e.g., CD2) on the modified immune cell to provide fratricide resistance provides new strategies for using gene correction with applications in therapeutics and basic research.
The present disclosure provides methods for producing a fratricide resistant modified immune cell. In some embodiments, a method is provided that comprises editing an immune cell (e.g., T- or NK-cell) obtained from a healthy subject or donor with a nucleobase editor (e.g., an adenosine deaminase base editor or a cytidine deaminase base editor) and one or more guide RNAs to decrease or eliminate the expression of a target antigen (e.g., CD2) on the immune cell (e.g., T- or NK-cell). The immune cell is then transduced with a chimeric antigen receptor directed to the target antigen (e.g., CD2). The resulting modified immune cell (e.g., CAR-T) is fratricide resistant and is able to target neoplastic cells without targeting healthy immune cells. In some embodiments, the fratricide resistant modified immune cell is produced as provided in Example 2. In some embodiments, a method is provided that produces a fratricide resistant CD2 CAR-T cell.
The present disclosure also provides methods for the treatment of a subject diagnosed with a disease (e.g., T- or NK-cell malignancy) using a modified immune cell as provided herein. In some embodiments, a method is provided that comprises administering an effective amount of a modified immune cell produced as provided herein to a subject having or having a propensity of having a disease (e.g., T- or NK-cell malignancy). The present disclosure provides methods for the treatment of T- or NK-cell malignancies that are associated with a target antigen that can be corrected by deaminase mediated gene editing. Suitable diseases that can be treated with the strategies and modified immune cells as provided herein will be apparent to those of skill in the art based on the instant disclosure.
Provided herein are methods of using a base editor or base editor system for editing a nucleobase in a target nucleotide sequence. In some embodiments, the activity of the base editor system (e.g., comprising a deaminase, a Cas9 domain, and one or more guide RNAs) results in the disruption of a splice site or results in introducing a premature STOP codon into the target nucleotide sequence.
In some embodiments, the deaminases (e.g., adenosine deaminase or cytidine deaminase) provided herein are capable of deaminating a deoxyadenosine residue of DNA. Other aspects of the disclosure provide fusion proteins that comprise a deaminase (e.g., an adenosine deaminase or cytidine deaminase) and a domain (e.g., a Cas9 or a Cpf1 protein) capable of binding to a specific nucleotide sequence. For example, adenosine can be converted to an inosine residue, which typically base pairs with a cytosine residue. Such fusion proteins are useful inter alia for targeted editing of nucleic acid sequences. Such fusion proteins can be used for targeted editing of DNA in vitro, e.g., for the generation of mutant cells or animals; for the introduction of targeted mutations, e.g., for the correction of genetic defects in cells ex vivo, e.g., in cells obtained from a subject that are subsequently re-introduced into the same or another subject; and for the introduction of targeted mutations in vivo, e.g., the correction of genetic defects or the introduction of deactivating mutations in disease-associated genes in a G to A, or a T to C to mutation can be treated using the nucleobase editors provided herein. The present disclosure provides modified immune cells, fusion proteins, nucleic acids, vectors, compositions, methods, kits, systems, etc. that utilize the deaminases and nucleobase editors.
Methods for Editing Nucleic Acids
Some aspects of the disclosure provide methods for editing a nucleic acid. In some embodiments, the method is a method for editing a nucleobase of a nucleic acid (e.g., a base pair of a double-stranded DNA sequence). In some embodiments, the method comprises the steps of: a) contacting a target region of a nucleic acid (e.g., a double-stranded DNA sequence) with a complex comprising a base editor (e.g., a Cas9 domain fused to a cytidine or adenosine deaminase) and a guide nucleic acid (e.g., gRNA), wherein the target region comprises a targeted nucleobase pair, b) inducing strand separation of said target region, c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase, and d) cutting no more than one strand of said target region, where a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase. In some embodiments, the method results in less than 20% indel formation in the nucleic acid. It should be appreciated that in some embodiments, step b is omitted. In some embodiments, the method results in less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2%, or less than 0.1% indel formation. In some embodiments, the method further comprises replacing the second nucleobase with a fifth nucleobase that is complementary to the fourth nucleobase, thereby generating an intended edited base pair (e.g., C·G to T·A). In some embodiments, at least 5% of the intended base pairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended base pairs are edited.
In some embodiments, the ratio of intended products to unintended products in the target nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 200:1, or more. In some embodiments, the ratio of intended mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, or more. In some embodiments, the cut single strand (nicked strand) is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase. In some embodiments, the base editor comprises a Cas9 domain. In some embodiments, the base editor protects or binds the non-edited strand. In some embodiments, the base editor comprises nickase activity. In some embodiments, the intended edited base pair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edited base pair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some embodiments, linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In one embodiment, the linker is 32 amino acids in length. In another embodiment, a “long linker” is at least about 60 amino acids in length. In other embodiments, the linker is between about 3-100 amino acids in length. In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edited base pair is within the target window. In some embodiments, the target window comprises the intended edited base pair. In some embodiments, the method is performed using any of the base editors provided herein. In some embodiments, a target window is a methylation window.
In some embodiments, the disclosure provides methods for editing a nucleotide. In some embodiments, the disclosure provides a method for editing a nucleobase pair of a double-stranded DNA sequence. In some embodiments, the method comprises a) contacting a target region of the double-stranded DNA sequence with a complex comprising a base editor and a guide nucleic acid (e.g., gRNA), where the target region comprises a target nucleobase pair, b) inducing strand separation of said target region, c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase, d) cutting no more than one strand of said target region, wherein a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase, and the second nucleobase is replaced with a fifth nucleobase that is complementary to the fourth nucleobase, thereby generating an intended edited base pair, wherein the efficiency of generating the intended edited base pair is at least 5%. It should be appreciated that in some embodiments, step b is omitted. In some embodiments, at least 5% of the intended base pairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended base pairs are edited. In some embodiments base editing by a method described herein may have a base conversion efficiency of at least 10% at any particular gene site. In some embodiments, base editing by a method described herein may have a base conversion efficiency of at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% at least 55% or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, 96%, 97%, 98% or at least 99% at any particular gene site. In some embodiments base editing by a method described herein may have a base conversion efficiency of at least 70% at any particular gene site. In some embodiments base editing by a method described herein may have a base conversion efficiency of at least 80% at any particular gene site. In some embodiments base editing by a method described herein may have a base conversion efficiency of at least 90% at any particular gene site.
In some embodiments, the method causes less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2%, or less than 0.1% indel formation. In some embodiments, the ratio of intended product to unintended products at the target nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 200:1, or more. In some embodiments, the ratio of intended mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, or more. In some embodiments, the cut single strand is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase. In some embodiments, the nucleobase editor comprises nickase activity. In some embodiments, the intended edited base pair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edited base pair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some embodiments, the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. e.g., In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edited base pair occurs within the target window. In some embodiments, the target window comprises the intended edited base pair. In some embodiments, the nucleobase editor is any one of the base editors provided herein.
Pharmaceutical Compositions
In some aspects, the present invention provides a pharmaceutical composition comprising any of the genetically modified immune cells, base editors, fusion proteins, or the fusion protein-guide polynucleotide complexes described herein. More specifically, provided herein are pharmaceutical compositions comprising a genetically modified immune cell, or a population of such immune cells, expressing a chimeric antigen receptor (CAR), wherein said modified immune cell, or a population thereof, has at least one edited gene to provide fratricide resistance, enhance the function of the modified immune cell or to reduce immunosuppression or inhibition of the modified immune cell, wherein expression of the edited gene is either knocked out or knocked down. In some embodiments the at least one edited gene is CD3, CD5, CD7, CD33, CD123, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, PDCD1/PD1, CBLB, TGFBR2, ZAP70, NFATc1, TET2, or combination thereof. In some embodiments, CD3, CD5, CD7, CD33, or CD123 is edited. In some embodiments, CD3, CD5, CD7, CD33, or CD123 is edited in combination with one or more genes selected from TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PDCD1/PD1.
In some embodiments, the pharmaceutical composition comprises a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD5 and expresses a CD5 chimeric antigen receptor. In some embodiments, the pharmaceutical composition comprises a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD7 and expresses a CD7 chimeric antigen receptor. In some embodiments, the pharmaceutical composition comprises a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD33 and expresses a CD33 chimeric antigen receptor. In some embodiments, the pharmaceutical composition comprises a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD3 and expresses a CD3 chimeric antigen receptor. In some embodiments, the pharmaceutical composition comprises a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD123 and expresses a CD123 chimeric antigen receptor.
In some embodiments, the pharmaceutical composition comprises a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD5 and at least one edited gene selected from TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PDCD1/PD1, and expresses a CD5 chimeric antigen receptor. In some embodiments, the pharmaceutical composition comprises a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD7 and at least one edited gene selected from TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and expresses a CD7 chimeric antigen receptor. In some embodiments, the pharmaceutical composition comprises a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD33 and at least one edited gene selected from TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and expresses a CD33 chimeric antigen receptor. In some embodiments, the pharmaceutical composition comprises a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD3 and at least one edited gene selected from TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and expresses a CD3 chimeric antigen receptor. In some embodiments, the pharmaceutical composition comprises a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD123 and at least one edited gene selected from TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and expresses a CD123 chimeric antigen receptor.
The pharmaceutical compositions of the present invention can be prepared in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (21st ed. 2005). In general, the immune cell, or population thereof is admixed with a suitable carrier prior to administration or storage, and in some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers generally comprise inert substances that aid in administering the pharmaceutical composition to a subject, aid in processing the pharmaceutical compositions into deliverable preparations, or aid in storing the pharmaceutical composition prior to administration. Pharmaceutically acceptable carriers can include agents that can stabilize, optimize or otherwise alter the form, consistency, viscosity, pH, pharmacokinetics, solubility of the formulation. Such agents include buffering agents, wetting agents, emulsifying agents, diluents, encapsulating agents, and skin penetration enhancers. For example, carriers can include, but are not limited to, saline, buffered saline, dextrose, arginine, sucrose, water, glycerol, ethanol, sorbitol, dextran, sodium carboxymethyl cellulose, and combinations thereof.
Some nonlimiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.
Pharmaceutical compositions can comprise one or more pH buffering compounds to maintain the pH of the formulation at a predetermined level that reflects physiological pH, such as in the range of about 5.0 to about 8.0. The pH buffering compound used in the aqueous liquid formulation can be an amino acid or mixture of amino acids, such as histidine or a mixture of amino acids such as histidine and glycine. Alternatively, the pH buffering compound is preferably an agent which maintains the pH of the formulation at a predetermined level, such as in the range of about 5.0 to about 8.0, and which does not chelate calcium ions. Illustrative examples of such pH buffering compounds include, but are not limited to, imidazole and acetate ions. The pH buffering compound may be present in any amount suitable to maintain the pH of the formulation at a predetermined level.
Pharmaceutical compositions can also contain one or more osmotic modulating agents, i.e., a compound that modulates the osmotic properties (e.g., tonicity, osmolality, and/or osmotic pressure) of the formulation to a level that is acceptable to the blood stream and blood cells of recipient individuals. The osmotic modulating agent can be an agent that does not chelate calcium ions. The osmotic modulating agent can be any compound known or available to those skilled in the art that modulates the osmotic properties of the formulation. One skilled in the art may empirically determine the suitability of a given osmotic modulating agent for use in the inventive formulation. Illustrative examples of suitable types of osmotic modulating agents include, but are not limited to: salts, such as sodium chloride and sodium acetate; sugars, such as sucrose, dextrose, and mannitol; amino acids, such as glycine; and mixtures of one or more of these agents and/or types of agents. The osmotic modulating agent(s) may be present in any concentration sufficient to modulate the osmotic properties of the formulation.
In addition to the modified immune cell, or population thereof, and the carrier, the pharmaceutical compositions of the present invention can include at least one additional therapeutic agent useful in the treatment of disease. For example, some embodiments of the pharmaceutical composition described herein further comprises a chemotherapeutic agent. In some embodiments, the pharmaceutical composition further comprises a cytokine peptide or a nucleic acid sequence encoding a cytokine peptide. In some embodiments, the pharmaceutical compositions comprising the modified immune cell or population thereof can be administered separately from an additional therapeutic agent.
In some embodiments, the at least one additional therapeutic agent is one or more additional modified immune cells, or one or more populations of modified immune cells thereof. In some embodiments, the one or more additional modified immune effector cells comprise at least one edited gene to knockout or knockdown expression of the edited gene. In some embodiments, the at least one edited gene is CD3, CD5, CD7, CD33, CD123, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, PD1, or combinations thereof. In some embodiments, CD3, CD5, CD7, CD33, or CD123 is edited. In some embodiments, CD3, CD5, CD7, CD33, or CD123 is edited in combination with one or more genes selected from TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and/or PD1.
In some embodiments, the pharmaceutical compositions comprises two or more populations of modified immune cells thereof. In some embodiments, the pharmaceutical composition comprises 1) a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD5 and at least one edited gene selected from TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and expresses a CD5 chimeric antigen receptor; and 2) a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD7 and at least one edited gene selected from TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and expresses a CD7 chimeric antigen receptor.
In some embodiments, the pharmaceutical composition comprises 1) a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD33 and at least one edited gene selected from TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and expresses a CD33 chimeric antigen receptor; and 2) a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD123 and at least one edited gene selected from TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and expresses a CD123 chimeric antigen receptor.
The pharmaceutical compositions of the present invention can be used to treat any disease or condition that is responsive to autologous or allogeneic immune cell immunotherapy. For example, the pharmaceutical compositions, in some embodiments are useful in the treatment of neoplasia. In some embodiments, the neoplasia is a solid tumor. In other embodiments, the neoplasia is a liquid tumor. In some embodiments, the neoplasia is a hematological cancer. In some embodiments, the hematological cancer is a B cell cancer, and in some embodiments, the B cell cancer is multiple myeloma. In some embodiments, the B cell cancer is relapsed of relapsed/refractory multiple myeloma. In some embodiments, the hematological cancer is leukemia, myeloma, and/or lymphoma. In some cases, the leukemia is an acute leukemia. Acute leukemias include, for example, an acute myeloid leukemia (AML). Acute leukemias also include, for example, an acute lymphoid leukemia or an acute lymphocytic leukemia (ALL); ALL includes B-lineage ALL; T-lineage ALL; and T-cell acute lymphocytic leukemia (T-ALL).
Nonlimiting examples of neoplasia include T-cell acute lymphoblastic leukemia (T-ALL), mycosis fungoides (MF), Sézary syndrome (SS), Peripheral T/NK-cell lymphoma, Anaplastic large cell lymphoma ALK+, Primary cutaneous T-cell lymphoma, T-cell large granular lymphocytic leukemia, Angioimmunoblastic T/NK-cell lymphoma, Hepatosplenic T-cell lymphoma, Primary cutaneous CD30+ lymphoproliferative disorders, Extranodal NK/T-cell lymphoma, Adult T-cell leukemia/lymphoma, T-cell prolymphocytic leukemia, Subcutaneous panniculitis-like T-cell lymphoma, Primary cutaneous gamma-delta T-cell lymphoma, Aggressive NK-cell leukemia, and Enteropathy-associated T-cell lymphoma. In some embodiments, the neoplasia is T-cell acute lymphoblastic leukemia (T-ALL). In some embodiments, the neoplasia is acute myelogenous leukemia (AML).
In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., leukemia) is administered with an effective amount of one or more of 1) a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD5 and at least one edited gene selected from TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and PDCD1/PD1, and expresses a CD5 chimeric antigen receptor; 2) a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD7 and at least one edited gene selected from TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and expresses a CD7 chimeric antigen receptor; 3) a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD33 and at least one edited gene selected from TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and expresses a CD33 chimeric antigen receptor; and 4) a modified immune effector cell (e.g., CAR-T cell) or population thereof that lacks or has reduced levels of CD123 and at least one edited gene selected from TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and expresses a CD123 chimeric antigen receptor.
One consideration concerning the therapeutic use of genetically modified immune cells of the invention is the quantity of cells necessary to achieve an optimal or satisfactory effect. The quantity of cells to be administered may vary for the subject being treated. In one embodiment, between 104 to 1010, between 105 to 109, or between 106 and 108 genetically modified immunoresponsive cells of the invention are administered to a human subject. In some embodiments, at least about 1×108, 2×108, 3×108, 4×108, and 5×108 genetically modified immune cells of the invention are administered to a human subject. Determining the precise effective dose may be based on factors for each individual subject, including their size, age, sex, weight, and condition. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art.
The skilled artisan can readily determine the number of cells and amount of optional additives, vehicles, and/or carriers in compositions and to be administered in methods of the invention. Typically, additives (in addition to the active immune cell(s)) are present in an amount of 0.001 to 50% (weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about 1 wt %, still more preferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and still more preferably about 0.05 to about 5 wt %. Of course, for any composition to be administered to an animal or human, and for any particular method of administration, it is preferred to determine therefore: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model (e.g., a rodent such as a mouse); and, the dosage of the composition(s), concentration of components therein, and the timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations can be ascertained without undue experimentation.
In one embodiment, the method and compositions described herein may be used in generating engineered T cells that express a CAR and may have one or more base edited modifications, such that the engineered T cell can mount a specific immune response against the target. The CAR may be specifically directed towards an antigen target, the antigen may be presented by a cell in a host. In some embodiments, the immune response encompasses cytotoxicity. In some embodiments, the engineered T cell has enhanced cytotoxic response against its target. In some embodiments, the engineered T cell induces an enhanced cytotoxic response against its target as compared to a non-engineered T cell. In some embodiments, the engineered T cell exhibits an enhanced cytotoxic response by at least 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or more compared to a non-engineered cell. In some embodiments, the engineered T cell can kill 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 100%, at least 200%, at least 500% or at least 1000% more target cells than a non-engineered cell. In some embodiments, the T cell can induce higher memory response. In some embodiments, the T cell can induce lower levels of inflammatory cytokines than a non-engineered cell, that is, the engineered cell does not cause a cytokine storm response.
In some embodiments, the engineered T cell is administered to an allogenic host, wherein the engineered T cell has no rejection by the host. In some embodiments, the allogenic T cell induces negligible or minimum rejection by the host. In some embodiments, the engineered T cell has fratricide resistance.
In some embodiments, the pharmaceutical composition is formulated for delivery to a subject. Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration.
In some embodiments, the pharmaceutical composition described herein is administered locally to a diseased site (e.g., tumor site). In some embodiments, the pharmaceutical composition described herein is administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.
In other embodiments, the pharmaceutical composition described herein is delivered in a controlled release system. In one embodiment, a pump can be used (see, e.g., Langer, 1990, Science 249: 1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71: 105.) Other controlled release systems are discussed, for example, in Langer, supra.
In some embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human. In some embodiments, pharmaceutical composition for administration by injection are solutions in sterile isotonic use as solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
A pharmaceutical composition for systemic administration can be a liquid, e.g., sterile saline, lactated Ringer's or Hank's solution. In addition, the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated. The pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration. The particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein. Compounds can be entrapped in “stabilized plasmid-lipid particles” (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol %) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating (Zhang Y. P. et al., Gene Ther. 1999, 6: 1438-47). Positively charged lipids such as N-[l-(2,3-dioleoyloxi)propyl]-N,N,N-trimethyl-amoniummethylsulfate, or “DOTAP,” are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Pat. Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of which is incorporated herein by reference.
The pharmaceutical composition described herein can be administered or packaged as a unit dose, for example. The term “unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
Further, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a compound of the invention in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile used for reconstitution or dilution of the lyophilized compound of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
In another aspect, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and can have a sterile access port. For example, the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is a compound of the invention. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
In some embodiments, any of the fusion proteins, gRNAs, and/or complexes described herein are provided as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises any of the fusion proteins provided herein. In some embodiments, the pharmaceutical composition comprises any of the complexes provided herein. In some embodiments, the pharmaceutical composition comprises a ribonucleoprotein complex comprising an RNA-guided nuclease (e.g., Cas9) that forms a complex with a gRNA and a cationic lipid. In some embodiments pharmaceutical composition comprises a gRNA, a nucleic acid programmable DNA binding protein, a cationic lipid, and a pharmaceutically acceptable excipient. Pharmaceutical compositions can optionally comprise one or more additional therapeutically active substances.
In some embodiments, compositions provided herein are administered to a subject, for example, to a human subject, in order to effect a targeted genomic modification within the subject. In some embodiments, cells are obtained from the subject and contacted with any of the pharmaceutical compositions provided herein. In some embodiments, cells removed from a subject and contacted ex vivo with a pharmaceutical composition are re-introduced into the subject, optionally after the desired genomic modification has been effected or detected in the cells. Methods of delivering pharmaceutical compositions comprising nucleases are known, and are described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which are incorporated by reference herein in their entireties. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals or organisms of all sorts, for example, for veterinary use.
Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, domesticated animals, pets, and commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys.
Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient(s) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit. Pharmaceutical formulations can additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated in its entirety herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. See also PCT application PCT/US2010/055131 (Publication number WO2011/053982 A8, filed Nov. 2, 2010), incorporated in its entirety herein by reference, for additional suitable methods, reagents, excipients and solvents for producing pharmaceutical compositions comprising a nuclease.
Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.
The compositions, as described above, can be administered in effective amounts. The effective amount will depend upon the mode of administration, the particular condition being treated, and the desired outcome. It may also depend upon the stage of the condition, the age and physical condition of the subject, the nature of concurrent therapy, if any, and like factors well-known to the medical practitioner. For therapeutic applications, it is that amount sufficient to achieve a medically desirable result.
In some embodiments, compositions in accordance with the present disclosure can be used for treatment of any of a variety of diseases, disorders, and/or conditions.
Methods of Treatment
Some aspects of the present invention provide methods of treating a subject in need, the method comprising administering to a subject in need an effective therapeutic amount of a pharmaceutical composition as described herein. More specifically, the methods of treatment comprise administering to a subject in need thereof one or more pharmaceutical compositions comprising a population of modified immune cells expressing a chimeric receptor (CAR) and having at least one edited gene, wherein the at least one edited gene provides fratricide resistance, enhances the function or reduces the immunosuppression or inhibition of the modified immune cell, and wherein expression of the at least one edited gene is either knocked out or knocked down. In some embodiments, the method of treatment is an autologous immune cell therapy. In other embodiments, the method of treatment is an allogeneic immune cell therapy.
In certain embodiments, the specificity of an immune cell is redirected to a marker expressed on the surface of a diseased or altered cell in a subject by genetically modifying the immune cell to express a chimeric antigen receptor contemplated herein. In some embodiments, the method of treatment comprises administering to a subject an immune cell as described herein, wherein the immune cell has been genetically modified to redirect its specificity to a marker expressed on a neoplastic cell. In some embodiments, the neoplasia is a B cell cancer; for example, a B cell cancer such as a lymphoma, leukemia, or a myeloma, for example, multiple myeloma. In some embodiments, the neoplasia is a solid tumor. In other embodiments, the neoplasia is a liquid tumor. In some embodiments, the neoplasia is a hematological cancer. In some embodiments, the hematological cancer is leukemia, myeloma, and/or lymphoma. In some cases, the leukemia comprises a pre-leukemia. In some cases, the leukemia is an acute leukemia. Acute leukemias include, for example, an acute myeloid leukemia (AML). Acute leukemias also include, for example, an acute lymphoid leukemia or an acute lymphocytic leukemia (ALL); ALL includes B-lineage ALL; T-lineage ALL; and T-cell acute lymphocytic leukemia (T-ALL).
Nonlimiting examples of neoplasia include T-cell acute lymphoblastic leukemia (T-ALL), mycosis fungoides (MF), Sézary syndrome (SS), Peripheral T/NK-cell lymphoma, Anaplastic large cell lymphoma ALK+, Primary cutaneous T-cell lymphoma, T-cell large granular lymphocytic leukemia, Angioimmunoblastic T/NK-cell lymphoma, Hepatosplenic T-cell lymphoma, Primary cutaneous CD30+ T lymphoproliferative disorders, Extranodal NK/T-cell lymphoma, Adult T-cell leukemia/lymphoma, T-cell prolymphocytic leukemia, Subcutaneous panniculitis-like T-cell lymphoma, Primary cutaneous gamma-delta T-cell lymphoma, Aggressive NK-cell leukemia, and Enteropathy-associated T-cell lymphoma. In some embodiments, the neoplasia is T-cell acute lymphoblastic leukemia (T-ALL). In some embodiments, the neoplasia is acute myelogenous leukemia (AML). Thus, some embodiments of the present disclosure provide a method of treating a neoplasia in a subject.
In some embodiments, the methods of treatment comprise administering to a subject having or having a propensity to develop a neoplasia (e.g., leukemia) an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of functional T Cell Receptor Alpha Constant (TRAC), Cluster of Differentiation 33 (CD33), Cluster of Differentiation 3 (CD3), Cluster of Differentiation 5 (CD5), Cluster of Differentiation 7 (CD7), Cluster of Differentiation 52 (CD52), Cluster of Differentiation 123 (CD123), Programmed Cell Death 1 (PD-1), Beta-2 Microglobulin (B2M), Fas cell surface death receptor (FAS), Lymphocyte-activation gene 3 (LAG-3), Class II, Major Histocompatibility Complex, Transactivator (CIITA), T Cell Receptor Beta Constant 1 (TRBC1), T Cell Receptor Beta Constant 2 (TRBC2), or combinations thereof. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD123 and expresses a CD123 chimeric antigen receptor. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD5 and expresses a CD5 chimeric antigen receptor. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD7 and expresses a CD7 chimeric antigen receptor. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD33 and expresses a CD33 chimeric antigen receptor. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD3 and expresses a CD3 chimeric antigen receptor.
In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of functional TRAC. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of functional B2M. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of functional FAS. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of functional LAG-3. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of functional TRBC1 and/or TRBC2 In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of functional CIITA. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD52. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of PD-1.
In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., leukemia) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) or a population thereof that lacks or has reduced levels of CD123 and lacks or has reduced levels of TRAC, LAG-3, FAS, CD52, B2M, CIITA, TRBC1, TRBC2, and/or PD-1, or combinations thereof and expresses a CD123 chimeric antigen receptor containing. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., leukemia) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD5 and lacks or has reduced levels of TRAC, LAG-3, FAS, CD52, B2M, CIITA, TRBC1, TRBC2, and/or PD-1, or combinations thereof and expresses a CD5 chimeric antigen receptor. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., leukemia) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD7 and lacks or has reduced levels of TRAC, LAG-3, FAS, CD52, B2M, CIITA, TRBC1, TRBC2, and/or PD-1, or combinations thereof and expresses a CD7 chimeric antigen receptor. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., leukemia) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD33 and lacks or has reduced levels of TRAC, LAG-3, FAS, CD52, B2M, CIITA, TRBC1, TRBC2, and/or PD-1, or combinations thereof and expresses a CD33 chimeric antigen receptor. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., leukemia) is administered an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD3 and lacks or has reduced levels of TRAC, LAG-3, FAS, CD52, B2M, CIITA, TRBC1, TRBC2, and/or PD-1, or combinations thereof and expresses a CD3 chimeric antigen receptor.
In some embodiments of the methods of treating a neoplasia in a subject comprise administering to the subject an immune cell as described herein and one or more additional therapeutic agents. For example, the immune cell of the present invention can be co-administered with one or more additional modified immune cells or a population thereof. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is co-administered with an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD123 and expresses a CD123 chimeric antigen receptor and an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD33 and expresses a CD33 chimeric antigen receptor. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is co-administered with an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD5 and expresses a CD5 chimeric antigen receptor and an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD7 and expresses a CD7 chimeric antigen receptor. In some embodiments, any of the immune cells further comprises a mutation in one or more of CD3, CD5, CD7, CD33, CD123, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and/or PD1, or a combination thereof. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is co-administered with an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD3 and expresses a CD3 chimeric antigen receptor and an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD7 and expresses a CD7 chimeric antigen receptor. In some embodiments, a subject having or having a propensity to develop a neoplasia (e.g., T- or NK-cell malignancy) is co-administered with an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD3 and expresses a CD3 chimeric antigen receptor and an effective amount of a modified immune effector cell (e.g., CAR-T cell) that lacks or has reduced levels of CD123 and expresses a CD123 chimeric antigen receptor.
In some embodiments, the immune cell of the present invention can be co-administered with a cytokine. In some embodiments, the cytokine is IL-2, IFN-α, IFN-γ, or a combination thereof. In some embodiments, the immune cell is co-administered with a chemotherapeutic agent. The chemotherapeutic can be cyclophosphamide, doxorubicin, vincristine, prednisone, or rituximab, or a combination thereof. Other chemotherapeutics include obinutuzumab, bendamustine, chlorambucil, cyclophosphamide, ibrutinib, methotrexate, cytarabine, dexamethasone, cisplatin, bortezomib, fludarabine, idelalisib, acalabrutinib, lenalidomide, venetoclax, cyclophosphamide, ifosfamide, etoposide, pentostatin, melphalan, carfilzomib, ixazomib, panobinostat, daratumumab, elotuzumab, thalidomide, lenalidomide, or pomalidomide, or a combination thereof. “Co-administered” refers to administering two or more therapeutic agents or pharmaceutical compositions during a course of treatment. Such co-administration can be simultaneous administration or sequential administration. Sequential administration of a later-administered therapeutic agent or pharmaceutical composition can occur at any time during the course of treatment after administration of the first pharmaceutical composition or therapeutic agent.
In some embodiments of the present invention, an administered immune cell proliferates in vivo and can persist in the subject for an extended period of time. Immune cells of the present invention, in some embodiments can mature into memory immune cells and remain in circulation within the subject, thereby generating a population of cells able to actively respond to recurrence of a diseased or altered cell expressing the marker recognized by the chimeric antigen receptor.
Provided herein is a method of treating a neoplasia or a liquid cancer with a modified immune cell (e.g., an immune cell obtained from a healthy subject, or a T cell such as, for example, a cytotoxic T cell, a regulatory T cell, or a T helper cell) produced by a method of expressing or introducing in an immune cell a nucleobase editor polypeptide and contacting the cell with two or more guide RNAs capable of targeting a nucleic acid molecule encoding at least one polypeptide, wherein a first polypeptide is Cbl Proto-Oncogene B (CBLB) and a second polypeptide is selected from the group consisting of a T Cell Receptor Alpha Constant (TRAC), beta-2 microglobulin (B2M), and programmed cell death protein 1 (PD1) polypeptide, wherein the nucleobase editor polypeptide comprises a nucleic acid programmable DNA binding protein (napDNAbp) and a cytidine deaminase. In one instance of such method, three guide RNAs are expressed in or contact the cell, each targeting a B2M, TRAC, PDCD1 or CBLB polynucleotide. In another instance of such method, four guide RNAs are expressed in or contact the cell, each targeting one of a B2M, TRAC, PDCD1, and CBLB polynucleotide. In one instance, two guide RNAs are expressed in or contact the cell, each targeting a B2M or TRAC polynucleotide. In another instance, three guide RNAs are expressed in or contact the cell, each targeting a B2M, TRAC, PDCD1 or CBLB polynucleotide. In another instance, four guide RNAs are expressed in or contact the cell, each targeting one of a B2M, TRAC, PDCD1, and CBLB polynucleotide. In another instance, three guide RNAs are expressed in or contact the cell, each targeting a B2M, TRAC, and PDCD1 polynucleotide. In another instance, the guide RNA targets a TRAC exon 4 splice acceptor site, B2M exon 1 splice donor site, or PDCD1 exon 1 splice donor site. In another embodiment, the method of producing the modified immune cell further comprises expressing in the cell a guide RNA that targets a cytotoxic T-lymphocyte associated protein 4 (CTLA4) polynucleotide.
Provided herein is a method of treating a neoplasia or a liquid cancer with a modified immune cell (e.g., an immune cell obtained from a healthy subject, or a T cell such as, for example, a cytotoxic T cell, a regulatory T cell, or a T helper cell) produced by a method of expressing or introducing in an immune cell a nucleobase editor polypeptide and contacting the cell with two or more guide RNAs capable of targeting a nucleic acid molecule encoding at least one polypeptide selected from the group consisting of a Cbl Proto-Oncogene B (CBLB), T Cell Receptor Alpha Constant (TRAC), beta-2 microglobulin (B2M), and programmed cell death protein 1 (PD1) polypeptide, wherein the nucleobase editor polypeptide comprises a nucleic acid programmable DNA binding protein (napDNAbp) and an adenosine deaminase. In one instance, two guide RNAs are expressed in or contact the cell, each targeting a B2M or TRAC polynucleotide. In another instance, three guide RNAs are expressed in or contact the cell, each targeting a B2M, TRAC, PDCD1 or CBLB polynucleotide. In another instance, four guide RNAs are expressed in or contact the cell, each targeting one of a B2M, TRAC, PDCD1, and CBLB polynucleotide. In another instance, three guide RNAs are expressed in or contact the cell, each targeting a B2M, TRAC, and PDCD1 polynucleotide. In another instance, the guide RNA targets a TRAC exon 4 splice acceptor site, B2M exon 1 splice donor site, or PDCD1 exon 1 splice donor site. In another embodiment, the method of producing the modified immune cell further comprises expressing in the cell a guide RNA that targets a cytotoxic T-lymphocyte associated protein 4 (CTLA4) polynucleotide.
Provided herein is a method of treating a neoplasia or a liquid cancer with a modified immune cell (e.g., an immune cell obtained from a healthy subject, or a T cell such as, for example, a cytotoxic T cell, a regulatory T cell, or a T helper cell) produced by a method of expressing or introducing in an immune cell a nucleobase editor polypeptide and contacting the cell with two or more guide RNAs capable of targeting a nucleic acid molecule encoding at least one polypeptide selected from the group consisting of a V-Set Immunoregulatory Receptor (VISTA), T Cell Immunoglobulin Mucin 3 (Tim-3), T Cell Immunoreceptor With Ig and ITIM Domains (TIGIT), Transforming Growth Factor Beta Receptor II (TGFbRII), Regulatory Factor X Associated Ankyrin Containing Protein (RFXANK), PVR Related Immunoglobulin Domain Containing (PVRIG), Lymphocyte-Activation Gene 3 (Lag3), Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4), Chitinase 3 Like 1 (Chi31), Cluster of Differentiation 96 (CD96), B and T Lymphocyte Associated (BTLA), Tet Methylcytosine Dioxygenase 2 (TET2), Sprouty RTK Signaling Antagonist 1 (Spry1), Sprouty RTK Signaling Antagonist 2 (Spry2), Class II Major Histocompatibility Complex Transactivator (CIITA), Cluster of Differentiation 3 (CD3), Cluster of Differentiation 7 (CD7), Cluster of Differentiation 33 (CD33), Cluster of Differentiation 52 (CD52), Cluster of Differentiation 123 (CD123), T Cell Receptor Beta Constant 1 (TRBC1), T Cell Receptor Beta Constant 2 (TRBC2), Cytokine Inducible SH2 Containing Protein (CISH), Acetyl-CoA Acetyltransferase 1 (ACAT1), Cytochrome P450 Family 11 Subfamily A Member 1 (Cyp11a1), GATA Binding Protein 3 (GATA3), Nuclear Receptor Subfamily 4 Group A Member 1 (NR4A1), Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2), Nuclear Receptor Subfamily 4 Group A Member 3 (NR4A3), Methylation-Controlled J Protein (MCJ), Fas Cell Surface Death Receptor (FAS), and Selectin P Ligand/P-Selectin Glycoprotein Ligand-1 (SELPG/PSGL1) polypeptide, wherein the nucleobase editor polypeptide comprises a nucleic acid programmable DNA binding protein (napDNAbp) and an adenosine or cytidine deaminase. In one embodiment, the method of producing the cell further comprises contacting the cell with two or more guide RNAs capable of targeting a nucleic acid molecule encoding at least one polypeptide selected from the group consisting of a T Cell Receptor Alpha Constant (TRAC), beta-2 microglobulin (B2M), and programmed cell death protein 1 (PD1) polypeptide. In another embodiment, the method of producing the modified immune cell further comprises expressing in the cell a guide RNA that targets a cytotoxic T-lymphocyte associated protein 4 (CTLA4) polynucleotide.
Provided herein is a method of treating a neoplasia or a liquid cancer as described herein with modified immune cell (e.g., an immune cell obtained from a healthy subject, or a T cell such as, for example, a cytotoxic T cell, a regulatory T cell, or a T helper cell) having anti-neoplasia activity produced by a method comprising expressing in an immune cell isolated from a subject having a neoplasia one or more guide RNAs that target a polynucleotide encoding a polypeptide selected from the group consisting of CBLB, PD1, CTLA4, TGFBR2, TIGIT, or a minor histocompatibility polypeptide and a nucleobase editor polypeptide comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a cytidine deaminase or an adenosine deaminase. In one instance, the guide RNAs each target a CBLB and a PDCD1 polynucleotide. In one instance, the guide RNAs each target a PDCD1 and a CTLA4 polynucleotide. In one instance, the guide RNAs each target a CBLB, PDCD1, and CTLA4 polynucleotide. In one instance, the guide RNAs each target a CBLB, PD1, and CTLA4 polynucleotide.
In any of such methods of producing a modified immune cell as described above, in one instance, the guide RNA targets a splice acceptor site or a splice donor site in a target polynucleotide. In any of such methods of producing a modified immune cell as described above, in one instance, the nucleobase editor polypeptide generates a stop codon in a target polynucleotide. In any of such methods of producing a modified immune cell as described above, in one instance, the nucleobase editor polypeptide generates a stop codon in a PDCD1 exon 2. In any of such methods of producing a modified immune cell as described above, in one instance, the nucleobase editor polypeptide further comprises expressing one or more Nuclear Localization Signals (NLS) such as, for example, a bipartite NLS, an N-terminal NLS, a C-terminal NLS, or one or more uracil glycosylase inhibitors.
In any of such methods of producing a modified immune cell as described above, in one instance, the napDNAbp is selected from the group consisting of Cas9, CasX, CasY, Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12j/CasΦ, and BvCas12b or active fragments thereof. In any of such methods of producing a modified immune cell as described above, in one instance, the napDNAbp domain comprises a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9. In any of such methods of producing a modified immune cell as described above, in one instance, the cytidine deaminase is Petromyzon marinus cytosine deaminase 1 (pmCDA1), or Activation-induced cytidine deaminase (AICDA) and the adenosine deaminase is TadA.
In any of such methods of producing a modified immune cell as described above, in one instance, the method further comprises expressing in the immune cell one or more guide RNAs that target a TIGIT polynucleotide, TGFBR2 polynucleotide, ZAP70 polynucleotide, NFATcl polynucleotide, and TET2 polynucleotide.
It will be understood that, in some instances, the modified immune cell to be administered to the subject comprises no detectable translocations.
Provided herein is a method of treating a neoplasia or a liquid cancer with a modified immune cell (e.g., an immune cell obtained from a healthy subject, or a T cell such as, for example, a cytotoxic T cell, a regulatory T cell, or a T helper cell) having reduced immunogenicity and increased anti-neoplasia activity, the modified immune cell comprising a mutation in a TRAC, B2M, CBLB, or PD1 polynucleotide, or in a combination thereof. In one instance, the modified immune cell comprises mutations in B2M and TRAC polynucleotides. In another instance, the modified immune cell comprises mutations in B2M, TRAC, and PDCD1 polynucleotides. In another instance, the modified immune cell comprises mutations in B2M, TRAC, and CBLB polynucleotides. In another instance, the modified immune cell comprises mutations in B2M, TRAC, CBLB, and PD1 polynucleotides. In another instance, the modified immune the cell comprises a mutation in one or more polynucleotides encoding TIGIT, TGFBR2, ZAP70, NFATc1, or TET2. In another instance, the modified immune cell comprises a mutation in one or more polynucleotides encoding V-Set Immunoregulatory Receptor (VISTA), T Cell Immunoglobulin Mucin 3 (Tim-3), T Cell Immunoreceptor With Ig and ITIM Domains (TIGIT), Transforming Growth Factor Beta Receptor II (TGFbRII), Regulatory Factor X Associated Ankyrin Containing Protein (RFXANK), PVR Related Immunoglobulin Domain Containing (PVRIG), Lymphocyte-Activation Gene 3 (Lag3), Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4), Chitinase 3 Like 1 (Chi311), Cluster of Differentiation 96 (CD96), B and T Lymphocyte Associated (BTLA), Tet Methylcytosine Dioxygenase 2 (TET2), Sprouty RTK Signaling Antagonist 1 (Spry1), Sprouty RTK Signaling Antagonist 2 (Spry2), Class II Major Histocompatibility Complex Transactivator (CIITA), Cluster of Differentiation 3 (CD3), Cluster of Differentiation 7 (CD7), Cluster of Differentiation 33 (CD33), Cluster of Differentiation 52 (CD52), Cluster of Differentiation 123 (CD123), T Cell Receptor Beta Constant 1 (TRBC1), T Cell Receptor Beta Constant 2 (TRBC2), Cytokine Inducible SH2 Containing Protein (CISH), Acetyl-CoA Acetyltransferase 1 (ACAT1), Cytochrome P450 Family 11 Subfamily A Member 1 (Cyp11a1), GATA Binding Protein 3 (GATA3), Nuclear Receptor Subfamily 4 Group A Member 1 (NR4A1), Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2), Nuclear Receptor Subfamily 4 Group A Member 3 (NR4A3), Methylation-Controlled J Protein (MCJ), Fas Cell Surface Death Receptor (FAS), or Selectin P Ligand/P-Selectin Glycoprotein Ligand-1 (SELPG/PSGL1). In another instance, the modified immune cell expresses a chimeric antigen receptor that comprises, for example, an extracellular domain having an affinity for a marker associated with neoplasia (e.g., a B cell maturation antigen (BCMA)).
Provided herein is a method of treating a neoplasia or a liquid cancer with a modified immune cell (e.g., an immune cell obtained from a healthy subject, or a T cell such as, for example, a cytotoxic T cell, a regulatory T cell, or a T helper cell) having reduced immunogenicity and increased anti-neoplasia activity, the modified immune cell comprising a mutation in one or more of a CD33, PD-1, CD52, TRAC polynucleotide, or in a combination thereof. In various embodiments, the modified immune cell comprises mutations in two or more of CD33, PD-1, CD52, TRAC, or a combination thereof. In various embodiments, the modified immune cell comprises mutations in three or more of CD33, PD-1, CD52, TRAC, or a combination thereof. In one instance, the modified immune cell comprises mutations in CD33, PD-1, CD52, and TRAC.
Provided herein is a method of treating a neoplasia or a liquid cancer with a modified immune cell (e.g., an immune cell obtained from a healthy subject, or a T cell such as, for example, a cytotoxic T cell, a regulatory T cell, or a T helper cell) having reduced immunogenicity and increased anti-neoplasia activity, the modified immune cell comprising a mutation in one or more of a CD3, PD-1, CD52, TRAC, CD7 polynucleotide, or a combination thereof. In various embodiments, the modified immune cell comprises mutations in two or more of CD3, PD-1, CD52, TRAC, CD7, or a combination thereof. In one instance, the modified immune cell comprises mutations in CD3 and CD7. In various embodiments, the modified immune cell comprises mutations in three or more of CD3, PD-1, CD52, TRAC, CD7, or a combination thereof. In one instance, the modified immune cell comprises mutations in CD3, PD-1, CD52, TRAC, and CD7.
Provided herein is a method of treating a neoplasia or a liquid cancer with a modified immune cell (e.g., an immune cell obtained from a healthy subject, or a T cell such as, for example, a cytotoxic T cell, a regulatory T cell, or a T helper cell) produced by a method of expressing or introducing in an immune cell a nucleobase editor polypeptide; two or more guide RNAs capable of targeting a nucleic acid molecule encoding at least one polypeptide, selected from the group consisting of beta-2 microglobulin (B2M) and programmed cell death protein 1 (PD1) polypeptide; a Cas12 polypeptide; a guide RNA capable of targeting a nucleic acid molecule encoding TRAC; and a DNA donor template encoding a chimeric antigen receptor (CAR) or T cell receptor, wherein the nucleobase editor polypeptide comprises a nucleic acid programmable DNA binding protein (napDNAbp) and a cytidine deaminase.
Provided herein is a method of treating a neoplasia or a liquid cancer with a modified immune cell (e.g., an immune cell obtained from a healthy subject, or a T cell such as, for example, a cytotoxic T cell, a regulatory T cell, or a T helper cell) having reduced immunogenicity and increased anti-neoplasia activity, the modified immune cell comprising a mutation in one or more of a B2M or PD1 polynucleotide, an insertion of a nucleic acid encoding a chimeric antigen receptor (CAR) at the TRAC locus, and expressing the CAR on the cell surface of the immune cell.
In some embodiments of the present invention, provided herein is a method of treating a liquid cancer in a subject in need thereof, comprising administering to the subject in need an effective therapeutic amount of a pharmaceutical composition as described herein. A liquid cancer to be treated by such methods include, but are not limited to, a lymphoma, a leukemia, a myeloma, or a combination thereof.
In one instance, provided herein is a method of treating a liquid cancer in a subject in need thereof, comprising administering to the subject in need an effective therapeutic amount of a pharmaceutical composition as described herein, wherein the liquid cancer comprises a lymphoma. A liquid lymphoma to be treated using the methods described herein includes, but is not limited to, malignant plasma cell neoplasm, Hodgkin's lymphoma; nodular lymphocyte predominant Hodgkin's lymphoma; polycythemia vera, Hodgkin lymphoma; small lymphocytic lymphoma (SLL); diffuse large B-cell lymphoma; immunoblastic large cell lymphoma; precursor B-lymphoblastic lymphoma; mantle cell lymphoma; marginal zone lymphoma; mycosis fungoides; anaplastic large cell lymphoma; Sézary syndrome; precursor T-lymphoblastic lymphoma; and B-cell lymphoma unclassified with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma; B-cell non-Hodgkin's lymphoma (NHL); follicular lymphoma; Burkitt's lymphoma; Burkitt-like lymphoma; marginal zone lymphoma; mantle cell lymphoma; diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma; large cell lymphoma; precursor B-lymphoblastic lymphoma; B-cell lymphoma unclassified with features intermediate between diffuse large B-cell lymphoma and Burkitt's lymphoma; marginal zone lymphoma; mucosa-associated lymphatic tissue lymphoma; small cell lymphocytic lymphoma; mantle cell lymphoma; primary mediastinal (thymic) large B-cell lymphoma; lymphoplasmacytic lymphoma (such as Waldenstrom's macroglobulinemia); nodal marginal zone B cell lymphoma; splenic marginal zone lymphoma; intravascular large B-cell lymphoma; primary effusion lymphoma; lymphomatoid granulomatosis; T cell/histiocyte-rich large B-cell lymphoma; primary central nervous system lymphoma; primary cutaneous diffuse large B-cell lymphoma (leg type); EBV positive diffuse large B-cell lymphoma of the elderly; diffuse large B-cell lymphoma associated with inflammation; intravascular large B-cell lymphoma; ALK-positive large B-cell lymphoma; plasmablastic lymphoma; large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease; mediastinal (thymic) large B cell lymphoma; intravascular large B cell lymphoma; primary effusion lymphoma; nasal type, enteropathy-type T cell lymphoma; hepatosplenic T cell lymphoma; blastic NK cell lymphoma; primary cutaneous anaplastic large cell lymphoma; lymphomatoid papulosis; angioimmunoblastic T cell lymphoma; peripheral T cell lymphoma; unspecified, anaplastic large cell lymphoma; classical Hodgkin lymphomas (nodular sclerosis, mixed cellularity, lymphocyte-rich, lymphocyte depleted or not depleted); nodular lymphocyte-predominant Hodgkin lymphoma; adult T cell lymphoma; a B-Cell Lymphoma-DLBCL, a B-Cell Lymphoma-DLBCL-germinal center-like; a B-Cell Lymphoma-DLBCL-activated B-cell-like; Non-Hodgkin lymphoma; primary central nervous system (CNS) lymphoma; T-cell lymphoma; peripheral T cell lymphomas, cutaneous T-cell lymphoma (CTCL); anaplastic large cell lymphoma (ALCL); AIDS-related lymphoma; enteropathy-type T cell lymphoma; hepatosplenic T cell lymphoma; blastic NK cell lymphoma; primary cutaneous anaplastic large cell lymphoma; lymphomatoid papulosis, angioimmunoblastic T cell lymphoma; peripheral T cell lymphoma; unspecified, anaplastic large cell lymphoma; classical Hodgkin lymphomas (nodular sclerosis, mixed cellularity, lymphocyte-rich, lymphocyte depleted or not depleted); and nodular lymphocyte-predominant Hodgkin lymphoma; other lymphomas; and a combination of such lymphomas.
In one instance, provided herein is a method of treating a liquid cancer in a subject in need thereof, comprising administering to the subject in need an effective therapeutic amount of a pharmaceutical composition as described herein, wherein the liquid cancer comprises a leukemia. In some cases, the leukemia comprises a pre-leukemia. Pre-leukemias include, for example, myelodysplastic syndrome (MDS). In some cases, the leukemia is an acute leukemia. Acute leukemias include, for example, an acute myeloid leukemia (AML). Acute leukemias also include, for example, an acute lymphoid leukemia or an acute lymphocytic leukemia (ALL); ALL includes B-lineage ALL; T-lineage ALL; and T-cell acute lymphocytic leukemia (T-ALL). Other leukemias to be treated with the methods described herein include, but are not limited to, hairy cell leukemia (HCL); chronic myeloid leukemia (CIVIL); chronic myelomonocytic leukemia (CMML); chronic lymphocytic leukemia (CLL); B-cell prolymphocytic leukemia, myeloid leukemia; Burkitt's leukemia; T cell prolymphocytic leukemia; T cell large granular lymphocytic leukemia; aggressive natural killer (NK) cell leukemia; adult T cell leukemia (ATL); acute promyeloid leukemia (APML); prolymphocytic leukemia (PLL); erythroblastic leukemia; acute megakaryoblastic leukemia; large granular lymphocytic leukemia (LGF); chronic myelogenous leukemia (CML); other leukemias; and a combination of such leukemias.
In one instance, provided herein is a method of treating a liquid cancer in a subject in need thereof, comprising administering to the subject in need an effective therapeutic amount of a pharmaceutical composition as described herein, wherein the liquid cancer comprises a myeloma. A myeloma to be treated using the methods described herein includes, but is not limited to, multiple myeloma; smoldering multiple myeloma; plasma cell myeloma; non-secretory myeloma, IgD myeloma; osteosclerotic myeloma; other myelomas; and a combination of such myelomas.
In one instance, provided herein is a method of treating a liquid cancer in a subject in need thereof, comprising administering to the subject in need an effective therapeutic amount of a pharmaceutical composition as described herein, wherein the liquid cancer comprises a Kahler's disease; Myelomatosis; plasmacytoma; solitary plasmacytoma of bone; extramedullary plasmacytomamycosis fungoides/Sezary Syndrome; primary cutaneous CD30-positive T cell lymphoproliferative disorders; hyper eosinophilia; chronic eosinophilia; and other hematopoietic cells related cancer.
In one embodiment, a subject is administered at least 0.1×105 cells, at least 0.5×105 cells, at least 1×105 cells, at least 5×105 cells, at least 1×106 cells, at least 0.5×107 cells, at least 1×107 cells, at least 0.5×108 cells, at least 1×108 cells, at least 0.5×109 cells, at least 1×109 cells, at least 2×109 cells, at least 3×109 cells, at least 4×109 cells, at least 5×109 cells, or at least 1×1010 cells. In particular embodiments, about 1×107 cells to about 1×109 cells, about 2×107 cells to about 0.9×109 cells, about 3×107 cells to about 0.8×109 cells, about 4×107 cells to about 0.7×109 cells, about 5×107 cells to about 0.6×109 cells, or about 5×107 cells to about 0.5×109 cells are administered to the subject.
In one embodiment, a subject is administered at least 0.1×104 cells/kg of bodyweight, at least 0.5×104 cells/kg of bodyweight, at least 1×104 cells/kg of bodyweight, at least 5×104 cells/kg of bodyweight, at least 1×105 cells/kg of bodyweight, at least 0.5×106 cells/kg of bodyweight, at least 1×106 cells/kg of bodyweight, at least 0.5×107 cells/kg of bodyweight, at least 1×107 cells/kg of bodyweight, at least 0.5×108 cells/kg of bodyweight, at least 1×108 cells/kg of bodyweight, at least 2×108 cells/kg of bodyweight, at least 3×108 cells/kg of bodyweight, at least 4×108 cells/kg of bodyweight, at least 5×108 cells/kg of bodyweight, or at least 1×109 cells/kg of bodyweight. In particular embodiments, about 1×106 cells/kg of bodyweight to about 1×108 cells/kg of bodyweight, about 2×106 T cells/kg of bodyweight to about 0.9×108 cells/kg of bodyweight, about 3×106 cells/kg of bodyweight to about 0.8×108 cells/kg of bodyweight, about 4×106 cells/kg of bodyweight to about 0.7×108 cells/kg of bodyweight, about 5×106 cells/kg of bodyweight to about 0.6×108 cells/kg of bodyweight, or about 5×106 cells/kg of bodyweight to about 0.5×108 cells/kg of bodyweight are administered to the subject.
One of ordinary skill in the art would recognize that multiple administrations of the pharmaceutical compositions contemplated in particular embodiments may be required to affect the desired therapy. For example, a composition may be administered to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5, years, 10 years, or more. In any of such methods, the methods may comprise administering to the subject an effective amount of the modified immune cells and the method increases or reduces an immune response. In any of such methods, the methods may comprise administering one or more doses of an effective amount of the modified immune cells per day. In any of such methods, the methods may comprise administering two or more doses of an effective amount of the modified immune cells per day. In any of such methods, the methods may comprise administering three or more doses of an effective amount of the modified immune cells per day. In any of such methods, the methods may comprise administering one or more doses of an effective amount of the modified immune cells per week. In any of such methods, the methods may comprise administering two or more doses of an effective amount of the modified immune cells per week. In any of such methods, the methods may comprise administering three or more doses of an effective amount of the modified immune cells per week. In any of such methods, the methods may comprise administering one or more doses of an effective amount of the modified immune cells per month. In any of such methods, the methods may comprise administering two or more doses of an effective amount of the modified immune cells per month. In any of such methods, the methods may comprise administering three or more doses of an effective amount of the modified immune cells per month.
In certain embodiments, it may be desirable to administer activated the pharmaceutical composition to the subject and then subsequently redraw blood (or have an apheresis performed), activate T cells therefrom, and reinfuse the patient with these activated and expanded T cells. This process can be carried out multiple times every few weeks. In certain embodiments, T cells can be activated from blood draws of from 10 cc to 400 cc. In certain embodiments, T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, 100 cc, 150 cc, 200 cc, 250 cc, 300 cc, 350 cc, or 400 cc or more.
Administration of the pharmaceutical compositions contemplated herein may be carried out using conventional techniques including, but not limited to, infusion, transfusion, or parenterally. In some embodiments, parenteral administration includes infusing or injecting intravascularly, intravenously, intramuscularly, intraarterially, intrathecally, intratumorally, intradermally, intraperitoneally, transtracheally, subcutaneously, subcuticularly, intraarticularly, subcapsularly, subarachnoidly and intrastemally.
The response in individual subjects can be characterized as a complete response, a partial response, stable disease, and progressive disease. In some embodiments, the response is complete response (CR). Complete response can be defined as disappearance of all circulating tumor cells (CTC) or a mononuclear blood cells (MNBC) i.e., any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm. In some examples (e.g., AML), complete response can be defined as the following: bone marrow blasts <5%; absence of blasts with Auer rods; absence of extramedullary disease; absolute neutrophil count >1.0×109/L (1000/μL); platelet count >100×109/L (100 000/μL); and independence of red cell transfusions. In certain embodiments, the response is a CR with Incomplete Recovery (CRi). CR with Incomplete Recovery, in some examples (e.g., AML), can be defined to include all CR criteria except for residual neutropenia (<1.0×109/L [1000/μL]) or thrombocytopenia (<100×109/L [100 000/μL]). In certain embodiments, the response is a morphologic leukemia free state. Morphologic leukemia free state, in some examples (e.g., AML), can be defined to include bone marrow blasts <5%; absence of blasts with Auer rods; absence of extramedullary disease; and no hematologic recovery required. In certain embodiments, the response is a partial response (PR). Partial response can be defined to mean at least 30% decrease in the sum of diameters of circulating tumor cells (CTC) or a mononuclear blood cells (MNBC), taking as reference the baseline sum diameters. In some examples (e.g., AML), partial response can be defined to include all hematologic criteria of CR; decrease of bone marrow blast percentage to 5% to 25%; and decrease of pretreatment bone marrow blast percentage by at least 50%. In certain embodiments, the response is a morphologic leukemia free state. Morphologic leukemia free state, in some examples (e.g., AML), can be defined to include bone marrow blasts <5%; absence of blasts with Auer rods; absence of extramedullary disease; and no hematologic recovery required. In certain embodiments, the response is a relapse.
Relapse, in some examples (e.g., AML), can be defined to include bone marrow blasts <5%; absence of blasts with Auer rods; absence of extramedullary disease; and no hematologic recovery required. In some embodiments, the response is progressive disease (PD). Progressive disease can be defined as at least a 20% increase in the number of circulating tumor cells (CTC) or a mononuclear blood cells (MNBC), taking as reference the smallest number on study (this includes the baseline number if that is the smallest) and an absolute increase of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, or at least 100 or more circulating tumor cells (CTC) or a mononuclear blood cells (MNBC).
The appearance of one or more new lesions can also be considered as progression. In some embodiments, the disease can be stable disease (SD). Stable disease can be characterized by neither sufficient decrease in liquid cancer cell number to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest number of CTCs and/or MNBCs while on study. In certain embodiments, the response is a pathological complete response. A pathological complete response, e.g., as determined by a pathologist following examination of tissue removed at the time of surgery or biopsy, generally refers to an absence of histological evidence of invasive and/or non-invasive liquid cancer cells in the surgical specimen.
In some embodiments, the pharmaceutical composition is administered two or three times a week. In another embodiment, the pharmaceutical composition is administered two times a week. In another embodiment, the pharmaceutical composition is administered once every 2 or 3 weeks. In another embodiment, the pharmaceutical composition is administered once every 1 or 2 weeks. In another embodiment, the pharmaceutical composition is administered on days 1, 4, 8, and 11 of a 21-day cycle. In another embodiment, the pharmaceutical composition is administered on days 1, 8, and 15 of a 28-day cycle.
In some embodiments, the amount of the compound administered is about 0.5-30 mg per kilogram body weight of the human subject. In another embodiment, the amount of the compound administered is about 0.5-20 mg per kilogram body weight of the human subject. In another embodiment, the amount of the compound administered is about 0.5-10 mg per kilogram body weight of the human subject. In another embodiment, the amount of the compound administered is about 0.04 mg, about 0.08 mg, about 0.16 mg, about 0.32 mg, about 0.64 mg, about 1.25 mg, about 1.28 mg, about 1.92 mg, about 2.5 mg, about 3.56 mg, about 3.75 mg, about 5.0 mg, about 7.12 mg, about 7.5 mg, about 10 mg, about 14.24 mg, about 15 mg, about 20 mg, or about 30 mg per kilogram body weight of the human subject. In another embodiment, the amount of the compound administered is about 1.92 mg, about 3.75 mg, about 7.5 mg, about 15.0 mg, or about 30.0 mg per kilogram body weight of the human subject and the compound is administered two times a week. In another embodiment, the amount of the compound administered is about 1.28 mg, about 2.56 mg, about 5.0 mg, about 10 mg, or about 20 mg per kilogram body weight of the human subject and the compound is administered two times a week. In another embodiment, the amount of the compound administered is about 1.92 mg, about 3.75 mg, about 7.5 mg, about 15.0 mg, or about 30.0 mg per kilogram body weight of the human subject and the compound is administered once a week. In another embodiment, the amount of the compound administered is about 1.28 mg, about 2.56 mg, about 5.0 mg, about 10 mg, or about 20 mg per kilogram body weight of the human subject and the compound is administered once a week. In another embodiment, the amount of the compound administered is about 1.92 mg, about 3.75 mg, about 7.5 mg, about 15.0 mg, or about 30.0 mg per kilogram body weight of the human subject and the compound is administered once a day three, five or seven times in a seven day period. In another embodiment, the compound is administered intravenously once a day, seven times in a seven day period. In another embodiment, the amount of the compound administered is about 1.28 mg, about 2.56 mg, about 5.0 mg, about 10 mg, or about 20 mg per kilogram body weight of the human subject and the compound is administered once a day three, five or seven times in a seven day period. In another embodiment, the compound is administered intravenously once a day, seven times in a seven day period.
In some embodiments, the compound is administered over a period of 0.25 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, or 12 h. In another embodiment, the compound is administered over a period of 0.25-2 h. In another embodiment, the compound is gradually administered over a period of 1 h. In another embodiment, the compound is gradually administered over a period of 2 h.
In some embodiments, the in vivo circulating half-life of the compound is about 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h or 12 h. In another embodiment, the in vivo circulating half-life of the compound is about 4 h. In another embodiment, in vivo circulating the half-life of the compound is about 6 h. In some embodiments, the biological tissue half-life of the compound is about 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h or 12 h. In another embodiment, the biological tissue half-life of the compound is about 10 h.
In some embodiments, the treatment results in about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% reduction in the number of liquid cancer cells within a period of 1 month after treatment initiation. In another embodiment, the treatment results in at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% reduction in the number of liquid cancer cells within a period of 1 month after treatment initiation. In another embodiment, the treatment results in about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% reduction in the number of liquid cancer cells within a period of 1 year after treatment initiation. In another embodiment, the treatment results in at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% reduction in the number of liquid cancer cells within a period of 1 year after treatment initiation. In another embodiment, the treatment results in about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% reduction the number of liquid cancer cells within a period of 6 months after treatment initiation. In another embodiment, the treatment results in at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% reduction in the number of liquid cancer cells within a period of 6 months after treatment initiation. In another embodiment, the treatment results in about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% reduction in the number of liquid cancer cells within a period of 3 months after treatment initiation. In another embodiment, the treatment results in at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% reduction in the number of liquid cancer cells within a period of 3 months after treatment initiation.
In some embodiments, the treatment results in an increased survival time of the human subject as compared to the expected survival time of the human subject if the human subject was not treated with the compound. In another embodiment, the increase in the survival time of the human subject is at least 30 days. In another embodiment, the increase in the survival time of the human subject is at least 3 months. In another embodiment, the increase in the survival time of the human subject is at least 6 months. In another embodiment, the increase in the survival time of the human subject is at least 1 year.
In some embodiments, the human subject is refractory and/or intolerant to one or more other treatment of the liquid cancer. In another embodiment, the human subject has had at least one unsuccessful prior treatment and/or therapy of the liquid cancer. In some embodiments, the human subject to be treated with the described methods is a child (e.g., 0-18 years of age). In other embodiments, the human subject to be treated with the described methods is an adult (e.g., 18+ years of age). Kits
The invention provides kits for the treatment of a neoplasia in a subject. In some embodiments, the kit is for the treatment of a solid tumor. In other embodiments, the kit is for the treatment of a liquid tumor. In some embodiments, the kit is for the treatment of a hematological cancer. In some embodiments, the hematological cancer is a B cell cancer. In some embodiments, the hematological cancer is leukemia, myeloma, and/or lymphoma. In some embodiments, the kit is for the treatment of a neoplasia selected from the group consisting of T-cell acute lymphoblastic leukemia (T-ALL), mycosis fungoides (MF), Sézary syndrome (SS), Peripheral T/NK-cell lymphoma, Anaplastic large cell lymphoma ALK+, Primary cutaneous T-cell lymphoma, T-cell large granular lymphocytic leukemia, Angioimmunoblastic T/NK-cell lymphoma, Hepatosplenic T-cell lymphoma, Primary cutaneous CD30+lymphoproliferative disorders, Extranodal NK/T-cell lymphoma, Adult T-cell leukemia/lymphoma, T-cell prolymphocytic leukemia, Subcutaneous panniculitis-like T-cell lymphoma, Primary cutaneous gamma-delta T-cell lymphoma, Aggressive NK-cell leukemia, and Enteropathy-associated T-cell lymphoma. In some embodiments, the kit is for the treatment of T-cell acute lymphoblastic leukemia (T-ALL). In some embodiments, the kit is for the treatment of acute myelogenous leukemia (AML). In some embodiments, the kit is for the treatment of a human subject.
In some embodiments, the kit comprises any of the chimeric antigen receptors as provided herein. In some embodiments, the kit comprises a nucleic acid encoding any of the chimeric antigen receptors as provided herein. In some embodiments, the kit comprises any of the modified immune cells as provided herein. In some embodiments, the kit includes a modified immune cell expressing a CD3 CAR, CD5 CAR, a CD7 CAR, a CD33 CAR and/or a CD123 CAR. In some embodiments, any of the immune cells further comprises a mutation in one or more of CD3, CD5, CD7, CD33, CD123, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and/or PDT, or a combination thereof.
In some embodiments, the kit includes a population of any of the modified immune cells provided herein. In some embodiments, the kit comprises any of the pharmaceutical compositions as provided herein. In some embodiments, the kit includes a population of CD123 modified immune cells or a pharmaceutical composition comprising a CD123 modified immune cell or population of modified immune cells. In some embodiments, the kit includes a population of CD5 modified immune cells or a pharmaceutical composition comprising a CD5 modified immune cell or population of modified immune cells. In some embodiments, the kit includes a population of CD7 modified immune cells or a pharmaceutical composition comprising a CD7 modified immune cell or population of modified immune cells. In some embodiments, the kit includes a population of CD33 modified immune cells or a pharmaceutical composition comprising a CD33 modified immune cell or population of modified immune cells. In some embodiments, the kit includes a population of CD3 modified immune cells or a pharmaceutical composition comprising a CD3 modified immune cell or population of modified immune cells.
In some embodiments, the kit further includes a base editor polypeptide or a polynucleotide encoding a base editor polypeptide, wherein the base editor polypeptide comprises a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase. In some embodiments, the napDNAbp is Cas9 or Cas12. In some embodiments, the polynucleotide encoding the base editor is a mRNA sequence. In some embodiments, the deaminase is a cytidine deaminase or an adenosine deaminase.
The invention provides kits comprising a nucleic acid construct comprising a nucleotide sequence encoding a cytidine or adenosine deaminase nucleobase editor at least one guide RNA, each guide RNA having a nucleic acid sequence at least 85% complementary to a nucleic acid sequence of gene encoding TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, PD1, CBLB, and/or CTLA4. In some embodiments, the nucleotide sequence encoding the cytidine or adenosine deaminase comprises a heterologous promoter that drives expression of the cytidine or adenosine deaminase nucleobase editor.
Some aspects of this disclosure provide kits comprising a nucleic acid construct, comprising a nucleotide encoding a nucleobase editor and a guide RNA. In some embodiments, the nucleic acid construct comprises a heterologous promoter that drives expression of the nucleobase editor. In some embodiments, this disclosure provides kits comprising a nucleic acid construct, comprising (a) a nucleotide sequence encoding (a) a Cas9 domain fused to a cytidine or adenosine deaminase as provided herein; and (b) a heterologous promoter that drives expression of the sequence of (a). In some embodiments, the Cas9 domain is fused to a cytidine deaminase. In some embodiments, the Cas9 domain is fused to an adenosine deaminase.
In some embodiments, the kit comprises a cytidine deaminase nucleobase editor and a guide RNA. In some embodiments, the kit comprises an adenosine deaminase nucleobase editor and a guide RNA. In some embodiments, the kit further one or more guide nucleic acid sequences. In some embodiments, the one of more guide nucleic acid sequences target CD3, CD5, CD7, CD33, or CD123. In some embodiments, the one or more guide nucleic acid sequences target each one of CD3, CD5, CD7, CD33, CD123, TRAC, LAG-3, FAS, CIITA, TRBC1, TRBC2, CD52, B2M, and/or PD1In some embodiments, the guide RNA has a nucleic acid sequence that is at least 85% complementary to a nucleic acid sequence of a gene encoding CD5. In some embodiments, the kit comprises a nucleobase editor and a CD5 guide RNA. In some embodiments, the guide RNA has a nucleic acid sequence that is at least 85% complementary to a nucleic acid sequence of a gene encoding CD7. In some embodiments, the kit comprises a nucleobase editor and a CD7 guide RNA. In some embodiments, the guide RNA has a nucleic acid sequence that is at least 85% complementary to a nucleic acid sequence of a gene encoding CD33. In some embodiments, the kit comprises a nucleobase editor and a CD33 guide RNA. In some embodiments, the guide RNA has a nucleic acid sequence that is at least 85% complementary to a nucleic acid sequence of a gene encoding CD3. In some embodiments, the kit comprises a nucleobase editor and a CD3 guide RNA. In some embodiments, the guide RNA has a nucleic acid sequence that is at least 85% complementary to a nucleic acid sequence of a gene encoding CD123. In some embodiments, the kit comprises a cytidine deaminase nucleobase editor and a CD123 guide RNA. In some embodiments, the kit may further include one or more additional guide RNAs, each guide RNA having a nucleic acid sequence at least 85% complementary to a nucleic acid sequence of gene encoding TRAC, LAG-3, FAS, PD1, B2M, CIITA, TRBC1, TRBC2, and/or CD52.
The neoplasia treatment kits may further comprise written instructions for using the modified immune cells in the treatment of the neoplasia. In other embodiments, the instructions include at least one of the following: precautions; warnings; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. In a further embodiment, a kit can comprise instructions in the form of a label or separate insert (package insert) for suitable operational parameters. In yet another embodiment, the kit can comprise one or more containers with appropriate positive and negative controls or control samples, to be used as standard(s) for detection, calibration, or normalization. The kit can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as (sterile) phosphate-buffered saline, Ringer's solution, or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
EXAMPLES
Example 1: Disruption of Splice Sites and Introduction of Stop Codons in Genes Expressed in Immune Cells
A nucleobase editor, BE4, was used to disrupt splice sites and insert stop codons into a subset of genes expressed in immune cells. A plasmid construct, pCMV_BE4max, encodes BE4, which comprises an APOBEC-1 cytidine deaminase domain having cytidine deaminase activity, a Cas9 domain comprising a D10A mutation and having nickase activity, and two uracil DNA glycosylase inhibitor (UGI) domains. UGI is an 83-amino acid residue protein derived from Bacillus subtilis bacteriophage PBS1 that potently blocks to edit the splice sites of certain genes expressed in immune cells. BE4 further comprises N-terminal and C-terminal nuclear localization signals (NLSs).









>pCMV_BE4max



ATATGCCAAGTACGCCCCCTATTGACGTCAATGAC



 



GGTAAATGGCCCGCCTGGCATTATGCCCAGTACAT



 



GACCTTATGGGACTTTCCTACTTGGCAGTACATCT



 



ACGTATTAGTCATCGCTATTACCATGGTGATGCGG



 



TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTT



 



TGACTCACGGGGATTTCCAAGTCTCCACCCCATTG



 



ACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAA



 



CGGGACTTTCCAAAATGTCGTAACAACTCCGCCCC



 



ATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGG



 



AGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGT



 



CAGATCCGCTAGAGATCCGCGGCCGCTAATACGAC



 



TCACTATAGGGAGAGCCGCCACCATGAAACGGACA



 



GCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAA



 



GCGGAAAGTCTCCTCAGAGACTGGGCCTGTCGCCG



 



TCGATCCAACCCTGCGCCGCCGGATTGAACCTCAC



 



GAGTTTGAAGTGTTCTTTGACCCCCGGGAGCTGAG



 



AAAGGAGACATGCCTGCTGTACGAGATCAACTGGG



 



GAGGCAGGCACTCCATCTGGAGGCACACCTCTCAG



 



AACACAAATAAGCACGTGGAGGTGAACTTCATCGA



 



GAAGTTTACCACAGAGCGGTACTTCTGCCCCAATA



 



CCAGATGTAGCATCACATGGTTTCTGAGCTGGTCC



 



CCTTGCGGAGAGTGTAGCAGGGCCATCACCGAGTT



 



CCTGTCCAGATATCCACACGTGACACTGTTTATCT



 



ACATCGCCAGGCTGTATCACCACGCAGACCCAAGG



 



AATAGGCAGGGCCTGCGCGATCTGATCAGCTCCGG



 



CGTGACCATCCAGATCATGACAGAGCAGGAGTCCG



 



GCTACTGCTGGCGGAACTTCGTGAATTATTCTCCT



 



AGCAACGAGGCCCACTGGCCTAGGTACCCACACCT



 



GTGGGTGCGCCTGTACGTGCTGGAGCTGTATTGCA



 



TCATCCTGGGCCTGCCCCCTTGTCTGAATATCCTG



 



CGGAGAAAGCAGCCCCAGCTGACCTTCTTTACAAT



 



CGCCCTGCAGTCTTGTCACTATCAGAGGCTGCCAC



 



CCCACATCCTGTGGGCCACAGGCCTGAAGTCTGGA



 



GGATCTAGCGGAGGATCCTCTGGCAGCGAGACACC



 



AGGAACAAGCGAGTCAGCAACACCAGAGAGCAGTG



 



GCGGCAGCAGCGGCGGCAGCGACAAGAAGTACAGC



 



ATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTG



 



GGCCGTGATCACCGACGAGTACAAGGTGCCCAGCA



 



AGAAATTCAAGGTGCTGGGCAACACCGACCGGCAC



 



AGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTT



 



CGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGA



 



AGAGAACCGCCAGAAGAAGATACACCAGACGGAAG



 



AACCGGATCTGCTATCTGCAAGAGATCTTCAGCAA



 



CGAGATGGCCAAGGTGGACGACAGCTTCTTCCACA



 



GACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAG



 



AAGCACGAGCGGCACCCCATCTTCGGCAACATCGT



 



GGACGAGGTGGCCTACCACGAGAAGTACCCCACCA



 



TCTACCACCTGAGAAAGAAACTGGTGGACAGCACC



 



GACAAGGCCGACCTGCGGCTGATCTATCTGGCCCT



 



GGCCCACATGATCAAGTTCCGGGGCCACTTCCTGA



 



TCGAGGGCGACCTGAACCCCGACAACAGCGACGTG



 



GACAAGCTGTTCATCCAGCTGGTGCAGACCTACAA



 



CCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCG



 



GCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTG



 



AGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCA



 



GCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAA



 



ACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAAC



 



TTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAA



 



ACTGCAGCTGAGCAAGGACACCTACGACGACGACC



 



TGGACAACCTGCTGGCCCAGATCGGCGACCAGTAC



 



GCCGACCTGTTTCTGGCCGCGAAGAACCTGTCCGA



 



CGCCATCCTGCTGAGCGACATCCTGAGAGTGAACA



 



CCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATG



 



ATCAAGAGATACGACGAGCACCACCAGGACCTGAC



 



CCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTG



 



AGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAG



 



AACGGCTACGCCGGCTACATTGACGGCGGAGCCAG



 



CCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCC



 



TGGAAAAGATGGACGGCACCGAGGAACTGCTCGTG



 



AAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCG



 



GACCTTCGACAACGGCAGCATCCCCCACCAGATCC



 



ACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAG



 



GAAGATTTTTACCCATTCCTGAAGGACAACCGGGA



 



AAAGATCGAGAAGATCCTGACCTTCCGCATCCCCT



 



ACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGA



 



TTCGCCTGGATGACCAGAAAGAGCGAGGAAACCAT



 



CACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGG



 



GCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACC



 



AACTTCGATAAGAACCTGCCCAACGAGAAGGTGCT



 



GCCCAAGCACAGCCTGCTGTACGAGTACTTCACCG



 



TGTATAACGAGCTGACCAAAGTGAAATACGTGACC



 



GAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGA



 



GCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGA



 



CCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAG



 



GACTACTTCAAGAAAATCGAGTGCTTCGACTCCGT



 



GGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCT



 



CCCTGGGCACATACCACGATCTGCTGAAAATTATC



 



AAGGACAAGGACTTCCTGGACAATGAGGAAAACGA



 



GGACATTCTGGAAGATATCGTGCTGACCCTGACAC



 



TGTTTGAGGACAGAGAGATGATCGAGGAACGGCTG



 



AAAACCTATGCCCACCTGTTCGACGACAAAGTGAT



 



GAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGG



 



GCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGG



 



GACAAGCAGTCCGGCAAGACAATCCTGGATTTCCT



 



GAAGTCCGACGGCTTCGCCAACAGAAACTTCATGC



 



AGCTGATCCACGACGACAGCCTGACCTTTAAAGAG



 



GACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGA



 



TAGCCTGCACGAGCACATTGCCAATCTGGCCGGCA



 



GCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTG



 



AAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCG



 



GCACAAGCCCGAGAACATCGTGATCGAAATGGCCA



 



GAGAGAACCAGACCACCCAGAAGGGACAGAAGAAC



 



AGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCAT



 



CAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACC



 



CCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTG



 



TACCTGTACTACCTGCAGAATGGGCGGGATATGTA



 



CGTGGACCAGGAACTGGACATCAACCGGCTGTCCG



 



ACTACGATGTGGACCATATCGTGCCTCAGAGCTTT



 



CTGAAGGACGACTCCATCGACAACAAGGTGCTGAC



 



CAGAAGCGACAAGAACCGGGGCAAGAGCGACAACG



 



TGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAAC



 



TACTGGCGGCAGCTGCTGAACGCCAAGCTGATTAC



 



CCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGA



 



GAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTC



 



ATCAAGAGACAGCTGGTGGAAACCCGGCAGATCAC



 



AAAGCACGTGGCACAGATCCTGGACTCCCGGATGA



 



ACACTAAGTACGACGAGAATGACAAGCTGATCCGG



 



GAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGT



 



GTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAG



 



TGCGCGAGATCAACAACTACCACCACGCCCACGAC



 



GCCTACCTGAACGCCGTCGTGGGAACCGCCCTGAT



 



CAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGT



 



ACGGCGACTACAAGGTGTACGACGTGCGGAAGATG



 



ATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTAC



 



CGCCAAGTACTTCTTCTACAGCAACATCATGAACT



 



TTTTCAAGACCGAGATTACCCTGGCCAACGGCGAG



 



ATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGA



 



AACCGGGGAGATCGTGTGGGATAAGGGCCGGGATT



 



TTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAA



 



GTGAATATCGTGAAAAAGACCGAGGTGCAGACAGG



 



CGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGA



 



ACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGG



 



GACCCTAAGAAGTACGGCGGCTTCGACAGCCCCAC



 



CGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGG



 



AAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAA



 



GAGCTGCTGGGGATCACCATCATGGAAAGAAGCAG



 



CTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCA



 



AGGGCTACAAAGAAGTGAAAAAGGACCTGATCATC



 



AAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAA



 



CGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAAC



 



TGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAA



 



TATGTGAACTTCCTGTACCTGGCCAGCCACTATGA



 



GAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGA



 



AACAGCTGTTTGTGGAACAGCACAAGCACTACCTG



 



GACGAGATCATCGAGCAGATCAGCGAGTTCTCCAA



 



GAGAGTGATCCTGGCCGACGCTAATCTGGACAAAG



 



TGCTGTCCGCCTACAACAAGCACCGGGATAAGCCC



 



ATCAGAGAGCAGGCCGAGAATATCATCCACCTGTT



 



TACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCA



 



AGTACTTTGACACCACCATCGACCGGAAGAGGTAC



 



ACCAGCACCAAAGAGGTGCTGGACGCCACCCTGAT



 



CCACCAGAGCATCACCGGCCTGTACGAGACACGGA



 



TCGACCTGTCTCAGCTGGGAGGTGACAGCGGCGGG



 



AGCGGCGGGAGCGGGGGGAGCACTAATCTGAGCGA



 



CATCATTGAGAAGGAGACTGGGAAACAGCTGGTCA



 



TTCAGGAGTCCATCCTGATGCTGCCTGAGGAGGTG



 



GAGGAAGTGATCGGCAACAAGCCAGAGTCTGACAT



 



CCTGGTGCACACCGCCTACGACGAGTCCACAGATG



 



AGAATGTGATGCTGCTGACCTCTGACGCCCCCGAG



 



TATAAGCCTTGGGCCCTGGTCATCCAGGATTCTAA



 



CGGCGAGAATAAGATCAAGATGCTGAGCGGAGGAT



 



CCGGAGGATCTGGAGGCAGCACCAACCTGTCTGAC



 



ATCATCGAGAAGGAGACAGGCAAGCAGCTGGTCAT



 



CCAGGAGAGCATCCTGATGCTGCCCGAAGAAGTCG



 



AAGAAGTGATCGGAAACAAGCCTGAGAGCGATATC



 



CTGGTCCATACCGCCTACGACGAGAGTACCGACGA



 



AAATGTGATGCTGCTGACATCCGACGCCCCAGAGT



 



ATAAGCCCTGGGCTCTGGTCATCCAGGATTCCAAC



 



GGAGAGAACAAAATCAAAATGCTGTCTGGCGGCTC



 



AAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCA



 



AGAAGAAGAGGAAAGTCTAACCGGTCATCATCACC



 



ATCACCATTGAGTTTAAACCCGCTGATCAGCCTCG



 



ACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTG



 



CCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTG



 



CCACTCCCACTGTCCTTTCCTAATAAAATGAGGAA



 



ATTGCATCGCATTGTCTGAGTAGGTGTCATTCTAT



 



TCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGG



 



AGGATTGGGAAGACAATAGCAGGCATGCTGGGGAT



 



GCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAAC



 



CAGCTGGGGCTCGATACCGTCGACCTCTAGCTAGA



 



GCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTG



 



TGAAATTGTTATCCGCTCACAATTCCACACAACAT



 



ACGAGCCGGAAGCATAAAGTGTAAAGCCTAGGGTG



 



CCTAATGAGTGAGCTAACTCACATTAATTGCGTTG



 



CGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTC



 



GTGCCAGCTGCATTAATGAATCGGCCAACGCGCGG



 



GGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCT



 



TCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCG



 



GCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGG



 



TAATACGGTTATCCACAGAATCAGGGGATAACGCA



 



GGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGC



 



CAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTT



 



TCCATAGGCTCCGCCCCCCTGACGAGCATCACAAA



 



AATCGACGCTCAAGTCAGAGGTGGCGAAACCCGAC



 



AGGACTATAAAGATACCAGGCGTTTCCCCCTGGAA



 



GCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCG



 



CTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGG



 



AAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGT



 



A7CTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTG



 



GGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCG



 



CTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCA



 



ACCCGGTAAGACACGACTTATCGCCACTGGCAGCA



 



GCCACTGGTAACAGGATTAGCAGAGCGAGGTATGT



 



AGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTA



 



ACTACGGCTACACTAGAAGAACAGTATTTGGTATC



 



TGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAG



 



AGTTGGTAGCTCTTGATCCGGCAAACAAACCACCG



 



CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAG



 



ATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCC



 



TTTGATCTTTTCTACGGGGTCTGACACTCAGTGGA



 



ACGAAAACTCACGTTAAGGGATTTTGGTCATGAGA



 



TTATCAAAAAGGATCTTCACCTAGATCCTTTTAAA



 



TTAAAAATGAAGTTTTAAATCAATCTAAAGTATAT



 



ATGAGTAAACTTGGTCTGACAGTTACCAATGCTTA



 



ATCAGTGAGGCACCTATCTCAGCGATCTGTCTATT



 



TCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGT



 



AGATAACTACGATACGGGAGGGCTTACCATCTGGC



 



CCCAGTGCTGCAATGATACCGCGAGACCCACGCTC



 



ACCGGCTCCAGATTTATCAGCAATAAACCAGCCAG



 



CCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACT



 



TTATCCGCCTCCATCCAGTCTATTAATTGTTGCCG



 



GGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTT



 



TGCGCAACGTTGTTGCCATTGCTACAGGCATCGTG



 



GTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAG



 



CTCCGGTTCCCAACGATCAAGGCGAGTTACATGAT



 



CCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTC



 



GGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGC



 



AGTGTTATCACTCATGGTTATGGCAGCACTGCATA



 



ATTCTCTTACTGTCATGCCATCCGTAAGATGCTTT



 



TCTGTGACTGGTGAGTACTCAACCAAGTCATTCTG



 



AGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCC



 



CGGCGTCAATACGGGATAATACCGCGCCACATAGC



 



AGAACTTTAAAAGTGCTCATCATTGGAAAACGTTC



 



TTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGT



 



TGAGATCCAGTTCGATGTAACCCACTCGTGCACCC



 



AACTGATCTTCAGCATCTTTTACTTTCACCAGCGT



 



TTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCG



 



CAAAAAAGGGAATAAGGGCGACACGGAAATGTTGA



 



ATACTCATACTCTTCCTTTTTCAATATTATTGAAG



 



CATTTATCAGGGTTATTGTCTCATGAGCGGATACA



 



TATTTGAATGTATTTA.GAAAAATAAACAAATAGG



 



GGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTG



 



ACGTCGACGGATCGGGAGATCGATCTCCCGATCCC



 



CTAGGGTCGACTCTCAGTACAATCTGCTCTGATGC



 



CGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTG



 



TGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATT



 



TAAGCTACAACAAGGCAAGGCTTGACCGACAATTG



 



CATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGC



 



TGCTTCGCGATGTACGGGCCAGATATACGCGTTGA



 



CATTGATTATTGACTAGTTATTAATAGTAATCAAT



 



TACGGGGTCATTAGTTCATAGCCCATATATGGAGT



 



TCCGCGTTACATAACTTACGGTAAATGGCCCGCCT



 



GGCTGACCGCCCAACGACCCCCGCCCATTGACGTC



 



AATAATGACGTATGTTCCCATAGTAACGCCAATAG



 



GGACTTTCCATTGACGTCAATGGGTGGAGTATTTA



 



CGGTAAACTGCCCACTTGGCAGTACATCAAGTGTA



 



TC






To ascertain the effectiveness of BE4 in knocking down or out protein expression in immune cells, a first population of immune cells was co-transfected with mRNA encoding BE4 and an sgRNA that targeted the C base complementary to the G base of the donor or acceptor splice site of TRAC exon 2, TRAC exon 4, or B2M exon 1, depending on the specific target site. mRNA was produced by in vitro transcription, (TriLin Biotechnologies). Briefly, 4 g of BE4 mRNA and 2 μg of synthetic gRNA were electroporated into 1M CD3+ T cells (Nucleofector™ Platform, Lonza Bioscience). The cells were then cultured for 3 days to allow sufficient time for base-editing. For comparison, a second population of immune cells was co-transfected with mRNA encoding a Cas9 nuclease and sgRNA that target the G base of the donor splice site of B2M exon 1. No discernible difference between BE4 editing and the Cas9 editing was observed, and the knockdown for each edited gene was greater than 90%, whereas unelectroporated control cells had no significant knockdown (FIG. 2).
It was hypothesized that the genetic modifications responsible for the observed knockdown of the targeted genes would differ if the cells were transfected with mRNA encoding BE4, which catalyzes single strand nicks, or with the Cas9 nuclease that catalyzes double-strand breaks. To test this hypothesis, immune cells were co-transfected with either 2 g BE4/1 μg sgRNA (medium) or 4 μg BE4/2 μg sgRNA (high) RNA encoding the BE4 base editor and sgRNA that target the G base of the donor splice site of the B2M exon 1. After incubation for 3, 5, and 7 days, DNA was collected and sequenced. Referring to FIG. 3, the majority of base edits revealed disruption of only the splice site and in the manner expected (i.e., C to T transition in the antisense strand was incorporated, resulting in a G to A transition in the sense strand). These results contrasted with those obtained from cells transfected with a Cas9 nuclease, which show that most edits in the Cas9 transfected cells were indels (FIG. 3).
Disruption of splice site and the introduction of stop codons can be effective in knocking down expression of a target gene. BE4-mediated editing of the splice acceptor in TRAC exon 4 and the splice donor in B2M exon 1 and PDCD1 exon 1 resulted in reduced expression of the full-length proteins (FIGS. 4 and 5). The BE4-mediated changes observed in the splice site were C to T transitions, although indels and C to G transversions were also observed. Insertion of an ochre stop codon into exon 2 of the PDCD1 gene, in which consecutive cytidine residues in the exon were targeted and edited to thymidine residues, also resulted in significant knock down of gene expression, albeit a lesser reduction than that seen for the TRAC and B2M genes (FIG. 4). These results further suggest that BE4-mediated single or consecutive cytidine base editing of genes expressed in immune cells results in efficient reduction of gene expression.
Example 2: In Silico Analysis of Spice Site Disruption and Stop Codon Insertion
To determine if designed gRNA would bind to off-site targets, the nucleic acid sequences of the gRNAs were analyzed using CAS-OFFinder. Referring to FIG. 6, an “X” bulge type indicates that the gRNA aligns with the genomic DNA and any discrepancy is a mismatch. As the number of mismatches increases from one to four, the potential off-site binding increases. For example, results for the TRAC exon 4 splice acceptor show that when there are three mismatches, there are 26 offsite binding possibilities, while there are 164 with four mismatches.
If the gRNA has a bulge, wherein the gRNA has twenty base pairs, but aligns with nineteen base pairs of genomic DNA, a bulge results. Again, referring to FIG. 6, when the TRAC exon 4 splice acceptor gRNA has a bulge of one base pair, the number of offsite binding possibilities increases with increasing mismatches; however, the number of possibilities is significantly lower than when there is no bulge (i.e., when the bulge size is zero).
Example 3: Multiplex Base Editing in Immune Cells
To determine if BE4 could mediate base editing of multiple genes to generate a multi-knockdown cell, immune cells were co-transfected with mRNA encoding a BE4 base editor along with sgRNA that target specific sites in B2M, TRAC, PD1, or in combinations thereof. Referring to FIG. 7, the BE4 system elicited effective knockdown, as measured by flow cytometry, to identify the percentage of cells with decreased protein production in single, double, and triple gene edits. The cells were gated on B2M and CD3 expression, with CD3 expression serving as a proxy for TRAC expression. Because PD1 staining is inefficient, direct measurement of cells expressing this protein was not performed. No differences were observed between cell populations with single, double, and triple gene edits, and immune cells modified to knock-down expression of B2M, TRAC, and PD1 (a triple gene edit) are detectably distinct from non-modified control immune cell (FIG. 8).
The modifications to the genes responsible for the decreased protein expression are summarized in FIG. 9. Specifically, and similarly to the mechanism resulting in decreased expression in single gene modification described in Example 1, C to T transitions constitute the vast number of edits observed in the modified B2M single modified gene cell population and in the B2M+PD1, B2M+TRAC, and B2M+TRAC+PD1 multiple modified genes cell populations. Indels and transversions constitute an insignificant minority of observed genetic changes in the edited genes.
Thus, concurrent modification of three genetic loci by base editing produced highly efficient gene knockouts with no detectable translocation events as assessed by Uni-Directional Targeted Sequencing (UDiTaS; Giannoukos et al., BMC Genomics. 2018 Mar. 21; 19(1):212. doi: 10.1186/s12864-018-4561-9). Additionally, translocations were not detected in BE4-edited genes. A droplet digital polymerase chain reaction (ddPCR) strategy (FIG. 10) was employed to detect translocations between the B2M, TRAC, and PD1 BE4-edited genes. DNA extracted from cells modified with BE4 or Cas9 to generate B2M+TRAC+PD1 edits was analyzed with next generation sequencing (NGS) using a QX200 droplet digital instrument (Bio-Rad) to determine the exact sequence of the BE4 and Cas9 edits. As shown on the left panel of FIG. 11, the B2M, TRAC, and PD1 genes were modified in most cells. ddPCR analysis showed that translocations were not present in the BE4-edited cells, but were observed in approximately 1.7% of the Cas9-edited cells (FIG. 11, right panel). Table 21 further illustrates that translocations were not observed in the BE4-edited cells.












TABLE 21









Control
Experimental





amplicon
amplicon



Base Editor
Translocation
droplets
droplets





















Cas9 nuclease
B2M-TRAC
61,206
585




B2M-PDCD1
55,970
291




PDCD1-TRAC
59,600
112



BE4
B2M-TRAC
90,717
0




B2M-PDCD1
89,028
0




PDCD1-TRAC
83,501
0










Example 4: BE4-Mediated Editing of Cbl Proto-Oncogene B (CBLB)
Cbl-b is a T cell receptor (TCR) signaling protein that negatively regulates TCR complex signaling (FIG. 12). Because T cells have a lower activation threshold when Cbl-b signaling is inhibited, knocking out or down this gene could significantly improve the effectiveness of a T cell or a T cell expressing a CAR. To determine if the Cbl-b gene was susceptible cytidine deamination mediated modification, cells were co-transfected with mRNA encoding a BE4 and sgRNA that target the splice site acceptor of exon 8 and 16, the splice site donor of exons 8, 10, 11, and 12, or that would promote the insertion of a STOP codon in exons 1, 4, and 8. Resulting cells were analyzed with flow cytometry.
Referring to FIG. 13, disruption of the splice site donor of exon 12 and the splice site acceptor of exon 8 resulted in the greatest reduction of Cbl-b expression (67.2% and 57.4%, respectively). And of the cells transfected with the exon 8 splice site acceptor and the exon 12 splice site donor sgRNAs, slightly more than 60% of the cells were edited successfully (FIG. 13, bar graph).
Example 5: Cas12b Nuclease Characterization in Immune Cells
Cas12b/c2c1 site specifically targets and cleaves both strands of a double stranded nucleic acid molecule. Two different Cas12b/c2c1 proteins, BhCas12b and BvCas12b, were characterized by determining the propensity the enzymes for mediating indels in the target nucleic acid molecule. mRNA encoding the Cas12b/c2c1 proteins was electroporated into T cells along with guide RNAs specific for a locus in the GRIN2B gene and for a locus in the DNMT1 gene. The cells were cultured for 3-5 days, followed by isolation of cellular DNA. Indel rates were determined by Next Generation Sequencing. Referring to FIG. 14, DNA isolated from cells treated with the BhCas12b protein had a much higher percentage (approximately 75%) of indels in the GRIN2B gene than did the DNA isolated from cells treated with the BvCas12b protein (approximately 20%). Indels in the DNMT1 gene were also observed at a higher rate in the DNA isolated from cells treated with BhCas12b (approximately 20%) than observed in the DNA isolated from cells treated with BvCas12b (approximately 0%).
The BhCas12b (V4) protein was used to disrupt the TRAC gene. T cells were transduced via electroporation with the mRNA encoding the BhCas12b (V4) protein along with guide RNAs specific for loci in the GRIN2B, DNMT1, and TRAC genes. 96 hours post-electroporation, cells were assessed using fluorescence assisted cell sorting (FACS) analysis, with cells being gated for CD3 (a proxy for TRAC). Referring to FIG. 15, approximately 95% of T cells transduced with a plasmid encoding GFP or with BhCas12b (V4) and guide RNAs specific for GRIN2B or DNMT1 were CD3+. Those cells transduced to express BhCas12b (V4) and guide RNAs specific for loci in the TRAC gene were less likely to be CD3+(approximately 2% to approximately 50%, depending on the guide RNA used). Three of the eleven TRAC guide RNAs tested led to approximately 100% BhCas12b (V4)-mediated indels.
Example 6: CAR-P2A-mCherry Lentivirus Expression Characterization
Cells were transduced to express a chimeric antigen receptor (CAR) using the CAR-P2A-mCherry lentivirus and analyzed for CAR expression using fluorescence assisted cell sorting (FACS). Cells were unstained, incubated with a BCMA protein conjugated to R-phycoerythrin (PE) or fluorescein isothiocyanate (FITC). Because BCMA is the CAR's target antigen, cells expressing the CAR will bind dye-labeled BCMA. Referring to FIG. 16, for cells that were not stained, FACS analysis only detected the presence of mCherry in the transduced sample, with some spillover into the PE channel. The BCMA-PE channel shows a highly positive signal beyond what was seen in the spillover, and these results were confirmed in cells incubated with BCMA-FITC. The dye-labeled BCMA protein detection results suggest almost identical expression of the CAR as that seen for mCherry. Referring to FIG. 17, 85% CAR expression was detected via FACS analysis in cells transduced with a poly(1,8-octanediol citrate) (POC) lentiviral vector.
Example 7: BE4 Produces Efficient, Durable Gene Knockout with High Product Purity
BE4 mediates base editing of multiple genes to generate a multi-knockdown cell. Immune cells were co-transfected with mRNA encoding a BE4 base editor along with sgRNA that target specific sites in B2M, TRAC, PD1, or in combinations thereof. As shown by sequencing data, base editing was efficient at modifying cells and durable up to at least 7 days (FIG. 18). High product purity was observed, as C to T transitions constituted the vast number of edits observed. Indels and C-to-G and C-to-A transversions constituted an insignificant minority of observed genetic changes in the edited genes. Base editing was also as efficient as spCas9 nuclease at generating desired modifications.
The BE4 system elicited effective knockdown as measured by flow cytometry, which identifies the percentage of cells with decreased surface expression (FIG. 19A). Cells gated on B2M expression displayed loss of B2M protein on the cell surface. As measured by flow cytometry, base editing was also as efficient as spCas9 nuclease at generating B2M protein knockout.
Example 8: Orthogonal Translocation Detection Assay Cannot Detect BE4-Induced Rearrangements in Triple-Edited T Cells
Immune cells were co-transfected with mRNA encoding a BE4 base editor along with sgRNAs that targeted specific sites in B2M, TRAC, and PD1. The triple-edited T cells were evaluated using a translocation detection assay that was capable of detecting specific translocations that were undesirable between B2M, TRAC, and PD1 target genes (FIG. 20). Notably, none of these specific translocations were detected in any of the BE4-edited genes (Table 22). In contrast, Cas9-treated cells displayed low, but detectable levels of the translocations. Thus, multiplex editing of T cells using the BE4 base editor did not generate translocations in contrast to multiplex editing using Cas9 nuclease.










TABLE 22





Type
Mock (%)
BE4-treated (%)
Cas9-treated (%)


















On-target
0
89.9/97.9/89.1
53.0/77.2/55.2


modification


(B2M/TRAC/PDCD1)


B2M-A/TRAC-A
0
0
0.925


B2M-A/TRAC-B
0
0
0.353


B2M-A/PDCD1-A
0
0
1.647


B2M-A/PDCD1-B
0
0
0.508


B2M-B/TRAC-A*
0
0
0.505





LLoDBE4 = 0.1%


*B2M-B only measurable in this experiment if translocation includes a local rearrangement at the B2M locus






Example 9: Multiplexed Base Editing does not Significantly Impair Cell Expansion
An extensive guide screen was performed across B2M, TRAC, and PD1 targets with both BE4 and spCas9 sgRNAs. Guides were selected for high editing efficiency and expansion based on single-plex test. Final cell yields compared between 1, 2 and 3 edits using BE4 and spCas9 and were normalized to electroporation only control. BE4 edited cells with the desired edits displayed high yields when up to 3 edits were made (FIG. 21). In contrast, spCas9 edited cells showed reduced yields when increasing numbers of multiplex edits were made. Thus, multiplexed base edited cells maintained high cell expansion even when up to 3 edits were being made. Thus, BE4 generated multiplex-edited T cells with no detectable genomic rearrangements while also maintaining high cell expansion compared to spCas9 treated samples.
Example 10: BE4 Generated Triple-Edited T Cells with Similar On-Target Editing Efficiency and Cellular Phenotype as spCas9
T cells were co-transfected with mRNA encoding a BE4 base editor along with sgRNAs that target specific sites in B2M, TRAC, and PD1. As shown by sequencing data, base editing was efficient at modifying cells at all three sites (FIG. 22). Modification of the genes by base editing was similar to that using spCas9 nuclease. Flow cytometry also showed decreased surface expression of B2M and CD3 (FIG. 23, upper panel). Compared to electroporation only control cells, BE4 and Cas9 multiplex edited cells displayed significant reductions of B2M and CD3 protein on the cell surface (>95% CD3-/B2M-). Although PD1 staining is less efficient, significant reductions (˜90%) in PD1 were observed in BE4 and Cas9 multiplex edited cells compared to electroporation only control cells (FIG. 23, lower panel).
Example 11: BE4 Editing does not Alter CAR Expression or Antigen-Dependent Cell Killing
T cells were co-transfected with mRNA encoding a BE4 base editor along with sgRNAs that target specific sites in B2M, TRAC, and PD1. A chimeric antigen receptor (CAR) targeting BCMA was introduced by integration of a lentiviral vector encoding the anti-BCMA CAR. CAR expression was observed by flow cytometry in BE4 and Cas9 edited cells (FIG. 24), compared to untreated cells that did not receive the lentiviral vector. The CAR-T cells were evaluated for cell killing by nuclear staining of the cells expressing BCMA and detecting loss of nuclear staining, indicating cell death. Antigen dependent cell killing was observed in cells transduced with the vector and expressing the CAR, including BE4 and Cas9 edited T cells (FIG. 25). In contrast, untreated cells that were not transduced with the vector did not display cell killing activity. Thus, BE4-generated CAR-T cells demonstrated comparable gene disruption, cell phenotype, and antigen-dependent cell killing compared to their nuclease-only counterparts.
Example 12: Cas12b and BE4 can be Paired for Highly Efficient Multiplex Editing in T Cells
CD3−, B2M− T cells were generated using BE4 only or using BE4 and Cas12b. For T cells generated using BE4 only, T cells were co-transfected with mRNA encoding a BE4 base editor along with sgRNAs that target specific sites in B2M and TRAC. For T cells generated using BE4 and Cas12b, T cells were co-transfected with mRNA encoding a BE4 base editor, and an sgRNA that targets a specific site in B2M, mRNA encoding BhCas12b (V4), and a Cas12b sgRNA that targets exon 4 of the TRAC gene, which was used to disrupt the TRAC gene. The resulting T cells were assessed using fluorescence assisted cell sorting (FACS) analysis to detect B2M and CD3 cell surface expression. Knockouts using BE4 only displayed a similar profile to those using BE4 and Cas12b. In particular, a high percentage of the T cells were CD3−, B2M−: 86% (BE4 only) and 88% (BE4+Cas12b), while the other possible phenotypes CD3−, B2M−; CD3+, B2M+ T cells; and CD3+, B2M− were represented less in the cell population (FIG. 26). In contrast, electroporation only control showed a population having a high percentage (97.8%) of CD3+ B2M+ cells and a very low percentage of CD3−, B2M− cells.
Cas12b was used to generate CD3−, CAR+ T cells. T cells were co-transfected with mRNA encoding BhCas12b (V4), a Cas12b sgRNA that targets exon 4 of the TRAC gene, and a double-stranded DNA (dsDNA) donor template encoding an anti-BCMA CAR. T cells were assessed using fluorescence assisted cell sorting (FACS) analysis to detect CD3 and BCMA cell surface expression. When increasing amounts of Cas12b were introduced into the cell in the presence of the sgRNA, CD3 expression decreased, as seen by a shift in the cell population to the CD3− quadrant (FIG. 27). When increasing amounts of donor template and were introduced in the cells under the same conditions, a shift to CD3−, CAR+ quadrant was observed in the cell population.
Thus, Cas12b can be paired with BE4 to generate multiplex-edited T cells, minimizing genomic rearrangements caused by multiple double-strand breaks.
Example 13: In Vivo Efficacy in Animal Models
To determine the anti-oncogenic activity of the compounds described herein in vivo, the compounds are, for example, given alone (IP, IV, PO, by inhalation or nasal routes) or in combination with sub-optimal doses of relevant chemotherapy (e.g., cyclophosphamide, doxorubicin, etoposide). In one example, 5×106 RS4; 11 cells (established from the bone marrow of a subject with acute lymphoblastic leukemia) that stably express luciferase are injected by tail vein in NOD-SCID mice 3 hours (hrs) after they have been subjected to total body irradiation. If left untreated, this form of leukemia is fatal in 3 weeks in this model. The leukemia is readily monitored, for example, by injecting the mice with D-luciferin (60 mg/kg) and imaging the anesthetized animals (e.g., Xenogen In Vivo Imaging System, Caliper Life Sciences, Hopkinton, MA). Total body bioluminescence is quantified by integration of photonic flux (photons/sec) by Living Image Software (Caliper Life Sciences, Hopkinton, MA). Compounds described herein alone or in combination with sub-optimal doses of relevant chemotherapeutics agents are, for example, administered to leukemic mice (10 days after injection/day 1 of experiment, in bioluminescence range of 14-16) by tail vein or IP routes at doses ranging from 0.1 mg/kg to 50 mg/kg for 7 to 21 days. Optionally, the mice are imaged throughout the experiment every other day and survival monitored daily for the duration of the experiment. Expired mice are optionally subjected to necropsy at the end of the experiment. Another animal model is implantation into NOD-SCID mice of DoHH2, a cell line derived from a human follicular lymphoma that stably expresses luciferase. These in vivo tests optionally generate preliminary pharmacokinetic, pharmacodynamic and toxicology data.
Example 14: Clinical Trials
To determine the suitability of the compounds described herein for treatment of humans, clinical trials are performed. For example, subjects diagnosed with liquid cancer and in need of treatment can be selected and separated in treatment and one or more control groups, wherein the treatment group is administered a compound described herein, while the control groups receive a placebo or a known anti-cancer drug. The treatment safety and efficacy of the compounds described herein can thus be evaluated by performing comparisons of the subject groups with respect to factors such as survival and quality-of-life. In this example, the subject group treated with a compound described herein can show improved long-term survival compared to a subject control group treated with a placebo.
Study Design
This study comprises a dose-escalation, 2-arm study designed to evaluate the safety, tolerability, PK, PD, and anti-liquid cancer cell effects of a compounds administered by IV infusion using 2 different dosing regimens of a 28- or 21-day cycle, in subjects with advanced liquid cancer (e.g., leukemia, myeloma, or liquid lymphoma) expressing WT p53 protein. For example, a compound described herein can be used in subjects with relapsed/refractory acute myeloid leukemia (AML) and/or acute lymphoid leukemia (ALL). Subjects receive a compound described herein either once weekly for three consecutive weeks for a 28-day cycle or twice weekly for two consecutive weeks for a 21-day cycle. Many subjects with a liquid lymphoma present circulating tumor cells (CTC) in peripheral blood, which can be detected and analyzed using flow cytometry. Thus, detection of study drug-specific target engagement in these cells is possible.
The study consists of a Dose Escalation Phase (DEP) and an Expansion Phase (EXP). The DEP is a “3+3” dose escalation design to establish the MTD or OBD of a compound described herein. The EXP enrolls up to 2 distinct groups of subjects with specific liquid cancers at the MTD or OBD to further investigate the clinical safety profile and potential efficacy of the dose level. The selection of subjects for the EXP is finalized based on results of the DEP, as well as data from additional nonclinical pharmacology studies. The later includes the investigation of multiple liquid cancer cell lines (e.g., leukemia, liquid lymphoma, myeloma, etc.) that facilitate the comparison of cell line sensitivity to a compound described herein across and within liquid cancer types.
Treatment of subjects in the dose escalation and the dose expansion phases of the study continues until documentation of disease progression, unacceptable toxicity, or subject or physician decision to discontinue therapy.
After the MTD or OBD is established, additional subjects can be enrolled in up to two separate expansion cohorts (approximately 30 subjects per expansion cohort to gain further experience at this dose level and in particular subject or liquid cancer cell types. Selection of subject or liquid cancer cell types is determined in part on the basis of observations made in the dose escalation portion of the study.
Safety is evaluated based on the incidence, severity, duration, causality, seriousness, and type of Adverse Event (AE), and changes in the subject's physical examination, vital signs and clinical laboratory results. Investigators use the NCI CTCAE version 4.0 to assess the severity of AEs.
Because the primary objectives of this study are based on safety and PK, statistical analyses are descriptive in nature and accounts for all doses studied and all observed responses, including subjects who achieve a complete response (CR) or partial response (PR) or who maintain stable disease (SD) based on IWG (2014) criteria. Subjects who receive at least one dose of a compound described herein constitute the safety population and are included in all safety analyses. Subjects who complete at least one cycle of a compound described herein and undergo a post-treatment objective disease assessment constitute the efficacy-evaluable subject population.
Example 15: Exemplary Methods of Identifying and Silencing Genes
In this Example, methods for gene silencing of targets for CAR-T are provided. FIG. 28A provides a diagram of genes to be targeted and considerations with respect to same. Targets include, for example, one or more of CD4, CD5, CD7, CS-1, TRAC, CAR, FAS, LAG-3, PD-1, β2M, CD52 and TCR.
There are two strategies for silencing with base editors. The first strategy assesses the target protein and then changes one or more of glutamine, arginine, or tryptophan to a stop codon to silence protein expression (see, item 1 of FIG. 28B). The second strategy involved splice disruption with CBE (see, item 2 of FIG. 28B) to silence protein expression.
Example 16: Multiple CAR-T Populations are Used to Address Clonality of Disease in AML or T-ALL
It is contemplated that leukemias become resistant or relapse in light of certain therapies due to the disease's evolution through reiterative process of clonal expansion and selection due to selection pressures in the tumor microenvironment. As an example, different clones of AML cancer cells can exhibit various antigens, such as CD33+ CD123−; CD33+CD123+, or CD33− CD123+. A therapeutic agent targeting CD33+ cancer cells would not address cells lacking in this particular antigen, such as CD33−CD123+. It is therefore contemplated that multiple CAR-T cell populations described herein address the clonality of disease.
In an exemplary method, CD33 and CD123 CAR-T cells are generated as illustrated in FIG. 29. The CD33 and CD123 CAR-T cells are administered to an AML patient having markers for CD33+CD123+ AML. The CD33 and CD123 CAR-T cells eliminate AML cancer cells in the patient that are CD33+ CD123−, CD33+CD123+, and CD33−CD123+.
In another exemplary method, CD3 and CD7 CAR-T cells are generated as illustrated in FIG. 30 by the methods described herein. The CD3 and CD7 CAR-T cells are administered to a T-ALL patient having markers for CD3+CD7+ T-ALL. The CD3 and CD7 CAR-T cells eliminate T-ALL cancer cells that are CD3+ CD7−, CD3+CD7+, and CD3−CD7+.
Example 17: Multiple CAR-T Populations are Modified to Eliminate Fratricide
A major limitation of traditional T cell and CAR-T therapies is that the activated cells may undergo self-killing or fratricide during cell manufacturing, storage, or when infused in patients. To avoid fratricide, the CAR-T cells described herein are modified to comprise a mutation in a polynucleotide encoding an antigen that the cell's CAR is specific to. As an example, CD7 CAR-T cells are modified to comprise a mutation in CD7 polynucleotide to prevent fratricide. As another example, CD3 CAR-T cells are modified to comprise a mutation in CD3 polynucleotide to prevent fratricide. In multiple CAR-T populations, additional modifications are performed to prevent cross-fratricide (i.e., mutations in both CD3 and CD7 polynucleotides in both CAR-T populations). Additional modifications include mutations to genes that may be involved in immunosuppression, graft versus host, host versus graft, and the like.
In an exemplary method, CD33 and CD123 CAR-T cells for the treatment of AML are generated as illustrated in FIG. 29. Both CD33 and CD123 CAR-T cells are modified to have a mutation in CD33 so that cells do not target themselves. Additional mutations in PD-1, CD52 and TRAC reduce or prevent immunosuppression, host versus graft and graft versus host, respectively.
In an exemplary method, CD3 and CD7 CAR-T cells for the treatment of T-ALL are generated as illustrated in FIG. 30. Both CD3 and CD7 CAR-T cells are modified to have mutations in CD3 (TRAC) and CD7 so that cells do not target themselves or the other population. Additional mutations in PD-1, CD52 and TRAC reduce or prevent immunosuppression, host versus graft and graft versus host, respectively.
Example 18: Four Simultaneous Base Edits for T-ALL CAR-T Cell does not Impact Yield Compared to Nuclease
The theoretical yield in the context of quad-T-ALL edits is determined in an assay in which the following components are tested. Controls are no electroporation (no EP), EP only, CBE Variant 1, CBE Variant 2, and Cas9. Results are shown in FIG. 31A and the corresponding viability results are shown in FIG. 31B. It was determined that four simultaneous base edits for T-ALL CAR-T does not impact yield compared to nuclease.
Example 19: Scaled-Up Lonza Electroporation Induces Highly Efficient, Multiplex T Cell Base Editing for T-ALL CAR-T Cell Program
T cells were cultured for 3 days post activation. 5×106 cells were electroporated with 10 μg mRNA for editor (CBE 1, CBE 2, or spCas9) and 5 μg of sgRNA for the T-ALL guides (TRAC, CD52, CD7, PD1). Suitable guides are provided in Table 23 below:







TABLE 23







Guide RNAs for T-ALL CAR-T









Guide RNA 
Guide Nucleic Acid 
SEQ ID 


(gRNA)
Sequence
NO:





TRAC gRNA
ususcsGuAuCUGUAAAACCAAGGU
1452



UUUAGAGCUAGAAAUAGCAAGUUAA




AAUAAGGCUAGUCCGUUAUCAACUU




GAAAAAGUGGCACCGAGUCGGUGCU




suSUSU



 


CD52 gRNA
ususasCCUGUACCAUAACCAGGGU
1453



UUUAGAGCUAGAAAUAGCAAGUUAA




AAUAAGGCUAGUCCGUUAUCAACUU




GAAAAAGUGGCACCGAGUCGGUGCU




sususu



 


CD52 gRNA
CSUscSUUACCUGUACCAUAACCGU
1454



UUUAGAGCUAGAAAUAGCAAGUUAA




AAUAAGGCUAGUCCGUUAUCAACUU




GAAAAAGUGGCACCGAGUCGGUGCU




sususu



 


CD7gRNA
usgscsACCUCUGGGGAGGACCUGU
1455



UUUAGAGCUAGAAAUAGCAAGUUAA




AAUAAGGCUAGUCCGUUAUCAACUU




GAAAAAGUGGCACCGAGUCGGUGCU




suSUSU



 


CD7 gRNA
cscsusACCUGUCACCAGGACCAGU
1456



UUUAGAGCUAGAAAUAGCAAGUUAA




AAUAAGGCUAGUCCGUUAUCAACUU




GAAAAAGUGGCACCGAGUCGGUGCU




suSUSU



 


PD1gRNA
csascsCuACCUAAGAACCAUCCGu
1457



UuUAGAGCUAGAAAUAGCAAGUUAA




AAUAAGGCUAGUCCGUUAUCAACUU




GAAAAAGUGGCACCGAGUCGGUGCU




SUSUSU









2′-O-methyl analogs and 3′ phosphorothioate internucleotide linkages are used at the first three 5′ and 3′ terminal RNA residues, chemical modifications at each end indicated by “s” following the nucleotide.
CBE 1 refers to rat-APOBEC-BE4, and CBE2 refers to orangutan “BE4” (ppAPOBEC). No EP and EP only were controls that either got no zap or a zap without editor/guide.
Theoretical yield was measured as follows: cells were counted daily, with viability measurements using AO/DAPI staining (with viability readouts also performed with the count-AML figure with hours post EP viability readout). Cells were split back each day to 0.5×106 cells/mL (theoretical because split and did not actually expand all cells). Calculations were then made to determine what the theoretical yield would have been if the cultures had not been backsplit. The percentage of gene modification was determined (FIG. 32).
Cells were taken through NGS and theoretical cell yield determined (FIG. 33).
Cell killing demonstrated by loss of fluorescence marker in model tumor cells (FIG. 34).
All of the CARs tested comprise a binder clone in either heavy to light (H2L) or light to heavy (L2H) orientations, followed by a CD8a Hinge, CD8 transmembrane, and a CD28z signaling domain.
Example 20: Exemplary CAR-T Cell Process Flow and Contract Manufacturing
Frozen apheresis leukopak from healthy donors are obtained and thawed using Pasmatherm according to manufacturer's instructions. CD4 and CD8 T-cells are isolated using, for example, CliniMACS Prodigy according to manufacturer's instructions. The T cells are then activated using TransAct according to manufacturer's instructions. Electroporation delivery of mRNA/RNP base editors occurs using Lonza 4D Nucleofactor according to manufacturer's instructions to knock out CD3, CD7, CD52 and/or PD1. Depletion of TCR a/0 is conducted using art-recognized techniques. The T-cells are expanded and lentivirus transduction of 3CAR and 7CAR occurs. CAR-T cells are cryopreserved with control rate freezers (CRF).
Example 21: Treatment of T-ALL with CAR-T Cells
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant neoplasm of the bone marrow. About 12-15% of ALL cases are diagnosed in children. Post-relapse 5 year survival is less than 25%. The standard of care is to use chemotherapy to induce a second remission followed by allogeneic hematopoietic stem cell transplantation (alloHSCT). However, many patients are refractory to chemotherapy or have high tumor burden and are not able to induce deep remission as a bridge for alloHSCT. Moreover, heavily pretreated patients are often not candidates for autologous CAR-T treatment. Accordingly, alternative treatment options are needed for the treatment of T-ALL.
To examine whether edited CAR-T cells can induce deep remission in patients in order to bridge treatment with alloHSCT, multiplex editing was used to create a fratricide resistant CAR− T cell. As shown in FIG. 35, a T-cell expressing a CD7 chimeric antigen receptor was edited to reduce or eliminate expression of CD7, TRAC, CD52, and PD-1 for targeting CD3− CD7+ or CD3+ CD7+ T-ALL tumor cells. As shown in FIG. 36, lymphodepletion was conducted in about 20-30 T-ALL patients with Cy/Flu/Campath at day −7. At day 0, patients were infused with CD7 CAR-T cells. The patients were then pre-conditioned with Cy/Flu/TBI/ATG at day 65. On day 70, patients received treatment with alloHSCT. It is expected that treatment with CD7 CAR-T cells induces T cell aplasia and allows T-ALL patients to be treated with alloHSCT without a bridge treatment.
Example 22: Producing CD7 CAR-T Cells
To produce TALL017 CD7 CAR-T cells as shown in FIG. 37A, frozen apheresis was received from healthy donors. The apheresis was then thawed with Plasmatherm (Barkey GmbH & Co. KG). CD4 and CD8 T-cells were isolated from the thawed apheresis using CliniMACS Prodigy (Miltenyi Biotec). On Day 2, mRNA base editors and guide RNA were delivered into the T-cells by electroporation using Lonza SD Nucleofector. Following base editing, a CD7 chimeric antigen receptor (CAR) was delivered into the T-cells via lentiviral transduction (7CAR8). The T-cells were then expanded in culture from Days 2-8. Following expansion, harvested CAR-T cells (>1B) were then cryopreserved. When thawed, the TALL017 process produced a heterogenous cell population.
Untransduced (UTD) and transduced TALL017 cells were compared 24 hours post-thaw. CAR activation was verified using flow cytometry by gating cells on CD2+ and CD4+ cells (FIG. 37C). As shown in FIG. 37C, mid-process fratricide induced by anti-CD7 CAR resulted in 7CAR-Ts that were still activated at the time of TALL017 cryopreservation. 7CAR-Ts that were cryopreserved mid-activation thawed poorly.
To produce TALL038 CD7 CAR-T cells as shown in FIG. 37B, frozen apheresis was received from healthy donors. The apheresis was then thawed with Plasmatherm (Barkey GmbH & Co. KG). CD4 and CD8 T-cells were isolated from the thawed apheresis using CliniMACS Prodigy (Miltenyi Biotec). On Day 2, mRNA base editors and guide RNA were delivered into the T-cells by electroporation using Lonza SD Nucleofector. Following base editing, a CD7 chimeric antigen receptor (CAR) was delivered into the T-cells via lentiviral transduction (7CAR8). The T-cells were then expanded in culture from Days 2-10. Following expansion, TCRα/β+ cells were depleted. The T-cells were then further expanded in culture from Days 10-12. The harvested CAR-T cells (>2B) were then cryopreserved on Day 12. When thawed, the TALL038 process produced a homogenous, monodisperse cell population.
Example 23: Evaluation of TALL017 and TALL038 CD7 CAR-T Cells
The editing efficiency in TALL017 and TALL038 CD7 CAR-T cells was compared using next generation sequencing (NGS). As shown in FIG. 38, editing of CD52, CD7, TRAC, and PD-1 in both types of CD7 CAR-T cells ranged from 80-99% efficiency. Untransduced (UTD) cells were used as a control.
24 hr post thaw protein expression of TCRα/β, CD52 and CD7 was examined in TALL017 and TALL038 CD7 CAR-T cells (FIGS. 39A and 39B). TALL038 cells demonstrated TCRα/β+ cells at <0.4%. Protein expression data matched pre-freeze metrics and post thaw NGS data for both TALL017 and TALL038 CD7 CAR-T cells. Untransduced (UTD) cells were used as a control.
In order to evaluate the identity of cells in a population of CD7 CAR-T cells, a gating scheme as shown in FIG. 40 was used. Cells were gated in the following order: FSC/SSC, Singlets, Live/Dead, CD45 (all leukocytes), CD19− (B cell exclusion), CD56−/CD14− (NK/monocyte exclusion), CD2+ (T cells), and CD4:CD8 of CD2+. As shown in FIG. 41, the identity of TALL017 and TALL038 CD7 CAR-T cell populations were a mix of CD2+/− and CD56+/− cells. There were no detectable mono or B cells.
Finally, CAR expression was evaluated. As shown in FIG. 42, both TALL017 and TALL038 CD7 CAR-T cell populations maintained CAR+ cell expression up to 48h post-thaw. Untransduced (UTD) cells were used as a control.
CD25 and CD69 expression was also evaluated 24 h and 48 post thaw in TALL017 and TALL038 CD7 CAR-T cells. As shown in FIG. 43, CD25 expression post thaw was lower in TALL038 cells compared to TALL017 cells. CD25 is a marker of mid-stage T cell activation. Unlike most CARs, anti-CD7 CAR induces mid-culture fratricide that further activates 7CARTs. Freezing TALL017 cells in an activated state was detrimental to cell health. TALL038 cells have dramatically lower CD25 expression compared to TALL017, which may suggest a higher resistance to freezing.
Example 24: In Vitro Characterization of TALL038 CD7 CAR-T Cells
In order to characterize TALL038 CD7 CAR-T cells in vitro, a bead based potency method was developed (FIG. 44). On Day 0, CD7 was coupled with Dynabeads and CD7 CAR-T cells and untranduced (UTD) cells were thawed. On Day 1, CD7 beads were purified and CD7 CAR-T cells were co-cultured, plated, and incubated at 37° C. On Day 2, the supernatant was collected and a readout was obtained using an enzyme-linked lectin assay (ELLA).
Using this assay, TALL038 CD7 CAR-T cells were evaluated for IFNγ, TNFα, IL-10, and IL-2 release. In response to CD7 antigen, TALL038 CD7 CAR-T cells released IFNγ, TNFα, and IL-2 (FIGS. 45A and 45B). Untransduced (UTD) cells were used as a control.
In order to further characterize TALL038 CD7 CAR-T cells in vitro, a co-culture method was developed to measure CAR directed T-cell killing of target tumor cells. FIG. 46 provides an overview of the assay. First, tumor cells are seeded. CAR-T cells are then added to the seeded tumor cells. The cells are then analyzed using IncuCyte® Scanning System. Finally, a reading is obtained using IncuCyte® Software.
TALL038 CD7 CAR-T cells were added to seeded CCRF-CEM GFP+ tumor cells. The T-Cell lots in Table 24 were tested in E:T ratios of 4:1 CAR:CCRF, 2:1 CAR:CCRF, or 1:1 CAR:CCRF:







TABLE 24







T-Cell Lots










Lot
% CAR+







TALL017 UTD
 0% CAR+



TALL017 7CAR8
~50% CAR+



TALL038 UTD
 0% CAR+



TALL038 7CAR8
~50% CAR+










As shown in FIGS. 47A and 47B, TALL038 CD7 CAR-T cells exhibited increased CCRF when re-challenged compared to TALL017 CAR T cells.
Example 25: In Vivo Characterization of TALL038 CD7 CAR-T Cells
Previous studies have shown that CCRF models showed a response to CD7-directed CAR-T therapy (see e.g., Gomes-Silva D., et al., CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies. Blood. 2017; 130:285-296).
In order to characterize TALL038 CD7 CAR-T cells in vivo, a study was developed to assess the efficacy of TALL038 drug product on in vivo growth in a CCRF model of T-ALL. Table 25 summarizes the study protocol.







TABLE 25







CCRF Model In Vivo Study
















CCRF-GFP+
N Mice Per

(T Cell) Cells

Tissue


Group
Tumor
Cells Per Mouse
Group
Treatment
Per Mouse*
Route
Collection





1
CCRF
1e5
10
DPBS
none
IV
Blood



GFP+


2
CCRF
1e5
10
UTD CAR
1.00E+07
IV
Blood



GFP+


3
CCRF
1e5
10
7CAR8
1.00E+07
IV
Blood



GFP+


4
CCRF
1e5
10
7CAR8
3.00E+06
IV
Blood



GFP+


5
CCRF
1e5
10
7CAR8
1.00E+06
IV
Blood



GFP+











6
CCRF
1e5
10
Excluded as outliers during randomization



GFP+









Ten 6-7 week old female NSG mice (Jackson Labs: Stock 005557) were implanted with CCRF-GFP+ cells (1e5) intravenously (IV). On day 11, the mice were transplanted with 250 μL total TALL038 1e7cells/mouse via IV. IVIS imaging measurements were taken 3x/week post injection for 2-3 weeks to monitor tumor burden. Whole blood was collected 1x/week for 2-3 weeks. 3×/week Bodyweights and Clinical Observations post implant were made.
FIG. 48 shows Bioluminescent Radiance Data at Day 10 Post CCRF Implant/Day −1 to CD7 CAR-T treatment (mean, SEM). At this timepoint, mice did not present with a measurable tumor burden needed for CD7 CAR-T treatment. FIGS. 49A and 49B show Bioluminescent Radiance Data at Day 19 Post CCRF Implant/Day 8 Post 7CAR8 Treatment (mean, SEM). At this timepoint, dose-dependent tumor growth inhibition was observed in response to CD7 CAR− T treatment. Mice did not lose weight in response to CD7 CAR-T treatment. FIGS. 50A and 50B show Bioluminescent Radiance Data at Day 38 Post CCRF Implant/Day 27 Post CD7 CAR-T treatment (mean, SEM). Following the date at which the first mouse in each group reached a humane endpoint (Flux>1e10), mean and standard error were no longer plotted. At this timepoint, sustained, dose-dependent inhibition was observed in each group prior to relapse. By using a logarithmic scale in FIG. 50B, the relapse in 1e7 of the CD7 CAR-T group was visualized. The Bioluminescent Radiance Data at Day 38 Post CCRF Implant/Day 27 Post CD7 CAR-T treatment are shown for individual mice in FIGS. 51A, 51B, 52A, and 52B. For the above experiments, UTD and vehicle were used as controls.
Example 26: CD5-Targeting CAR-Ts for T-ALL Treatment and Lymphoma Monotherapy
Previous studies have recognized the need for creating improved CAR-T products by targeting multiple cancer antigens (see e.g., Xinjie Xu, et al., Mechanisms of Relapse After CD19 CAR T-Cell Therapy for Acute Lymphoblastic Leukemia and Its Prevention and Treatment Strategies. Front Immunol. 2019; 10: 2664, Nov. 12, 2019; E. Mejstriková, et al., CD19-negative relapse of pediatric B-cell precursor acute lymphoblastic leukemia following blinatumomab treatment. Blood Cancer Journal, 7: 659 (2017); Hanren Dai, et al., Bispecific CAR-T cells targeting both CD19 and CD22 for therapy of adults with relapsed or refractory B cell acute lymphoblastic leukemia. J. Hematology & Oncology. 13:30 (2020); Robbie G. Majzner and Crystal L. Mackall, Tumor Antigen Escape from CAR T-cell Therapy. Cancer Discov. 2018 October; 8(10):1219-1226;
CD5 expression has been identified in T- and NK-cell malignancies, including but not limited to T-ALL, MF/SS, primary cutaneous T-cell lymphoma (NOS), T-cell large granular lymphocytic leukemia, angioimmunoblastic T/NK-cell lymphoma, Hepatosplenic T-cell lymphoma, primary cutaneous CD30+LPD, adult T-cell leukemia/lymphoma, and T-cell prolymphocytic leukemia. Combining CD5 with CD7 would unlock a greater potential for liquid cancer therapy for certain cancers, such as T-ALL, MF/SS, primary cutaneous T-cell lymphoma (NOS), angioimmunoblastic T/NK-cell lymphoma, and adult T-cell leukemia/lymphoma.
To reduce or eliminate expression of CD5 in T cells, 42 cytidine base editor single guide RNAs (sgRNAs) were generated (Agilent) as provided in Table 26 below.







TABLE 26







CDS Guide RNAs











Guide 

SEQ 


Guide Name
Grouping
Guide Sequence
ID NO:













RCM sgRNA 102
Ex1 SD
ACUCACCCAGCAUCCCCAGC
1458



(Pos 6)




 


RCM sgRNA 103
Ex2SA
AGCGACUGCAGAAAGAAGAG
1



(Pos6)




 


RCM sgRNA 104
Ex2 STOP
CAUACCAGCUGAGCCGUCCG
2



(pos 5/6)




 


RCM sgRNA 105
Ex3 SA
UGGAAAUCUGGGGGUCAGAA
1459



(Pos 8)




 


RCM sgRNA 106
Ex3 SD
GUUACCCACCUAAGCAGGUC
1460



(Pos 9)




 


RCM sgRNA 107
Ex3 STOP
CUGCCAGCGGCUGAACUGUG
1461



(pos 5)




 


RCM sgRNA 108
Ex3 STOP
CCUCCCACUGCUuGGAGCUC
1462



(Pos 5/6)




 


RCM sgRNA 109
Ex3 STOP
UCUGCCAGCGGCUGAACUGU
1463



(pos 6)




 


RCM sgRNA 110
Ex3 STOP
GUCUGCCAGCGGCUGAACUG
1464



(pos 7)




 


RCM sgRNA 111
Ex3 STOP
GAAGUGCCAGGGCCAGCUGG
1465



(Pos 8)




 


RCM sgRNA 112
Ex3 STOP
CCAUGUGCCAUCCGUCCUUG
1466



(Pos 8/9)




 


RCM sgRNA 113
Ex3 STOP
UUUGCAGCCAGAGCUGGGGC
1467



(Pos 9)




 


RCM sgRNA 114
Ex4 SA
GGUUCUGCAAUGAGACACUC
1468



(Pos 5)




 


RCM sgRNA 115
Ex5 SA
GAGCUAGGAGAGGAGAGAGC
1469



(Pos 5)




 


RCM sgRNA 116
Ex5 SD
UCACUUACCUGAGCAAAGGA
1470



(Pos 8)




 


RCM sgRNA 117
Ex5 SD
CUCACUUACCUGAGCAAAGG
1471



(Pos 9)




 


RCM sgRNA 118
Ex5 STOP
CUGCAGCUGGUGGCACAGUC
1472



(Pos 4)




 


RCM sgRNA 119
Ex5 STOP
CGGCCAGCACUGUGCCGGCG
1473



(Pos 5)




 


RCM sgRNA 120
Ex5 STOP
AUCUUCCAUUGGAUUGGCAA
1474



(Pos 6/7)




 


RCM sgRNA 121
Ex5 STOP
GAUCUUCCAUUGGAUUGGCA
1475



(Pos 7/8)




 


RCM sgRNA 122
Ex6 SA
AACCUGAGAGGGGAAGCAAU
1476



(Pos 4)




 


RCM sgRNA 123
Ex6 SA
AAACCUGAGAGGGGAAGCAA
1477



(Pos 5)




 


RCM sgRNA 124
Ex6 STOP
GUGCAGAGCCGUCUGGUGGG
1478



(Pos 4)




 


RCM sgRNA 125
Ex6 STOP
CUCCCACCGCAGCGAGCUCC
1479



(Pos 4/5)




 


RCM sgRNA 126
Ex6 STOP
CUCCCAAAGUUCGUGGCACU
1480



(Pos 4/5)




 


RCM sgRNA 127
Ex6 STOP
AGGUGCAGAGCCGUCUGGUG
1481



(Pos 5)




 


RCM sgRNA 128
Ex6 STOP
GGUGCAGAGCCGuCUGGUGG
1482



(Pos 5)




 


RCM sgRNA 129
Ex6 STOP
UCUCCCAAAGUUCGUGGCAC
1483



(Pos 5/6)




 


RCM sgRNA 130
Ex6 STOP
UGUCCCAGUGCCACGAACUU
1484



(Pos 6)




 


RCM sgRNA 131
Ex6 STOP
AAGGUGCAGAGCCGUCUGGU
1485



(Pos 7)




 


RCM sgRNA 132
Ex6 STOP
UCCUAUCGAGUGCUGGACGC
1486



(Pos 7)




 


RCM sgRNA 133
Ex6 STOP
CAAGGUGCAGAGCCGUCUGG
1487



(Pos 8)




 


RCM sgRNA 134
Ex6 STOP
GGUGCGCCAGGGGGCUCAGU
1488



(Pos 8)




 


RCM sgRNA 135
Ex6 STOP
GGGCUGCCCACUGAGCCCCC
1489



(Pos 8/9)




 


RCM sgRNA 136
Ex6 STOP
AGGUGCGCCAGGGGGCUCAG
1490



(Pos 9)




 


RCM sgRNA 137
Ex7 STOP
GGCCAGGAUCCAAACCCCGC
1491



(Pos 4)




 


RCM sgRNA 138
Ex8 STOP
CGCCAGUGGAUUGGCCCAAC
1492



(Pos 4)




 


RCM sgRNA 139
Ex8 STOP
AAGAAGCAGCGCCAGUGGAU
1493



(Pos 7)




 


RCM sgRNA 140
Ex9 SA
AAAGACACUGGGCAGAUGGU
1494



(Pos 8)




 


RCM sgRNA 141
Ex9 SD
GCUUACCUGGAUAAGCUGAC
1495



(Pos 6)




 


RCM sgRNA 142
Ex10 SA
UUCCAGAGCUGGGGAAAGAA
1496



(Pos 9)




 


RCM sgRNA 143
Ex10 STOP
AUGGGGCUCAGAGGCUGUAA
1371



(Pos 9)









CD5 editing was verified by screening the guides against a minimum of n=3 donors and a minimum of n=5 antibody clones 72 h post electroporation using flow cytometry, next generation sequencing (NGS), RNA, and Western blot analysis (FIG. 53). Electroporation only (EP) was used as a negative control. Use of cytidine base editors with the CD5 guide RNAs efficiently reduced or eliminated CD5 expression in T cells.
Two candidate guides, sgRNA 103 (splice disruption) and sgRNA 104 (STOP codon), were selected for further tested for base editing efficiency via NGS when used in combination with BE4. Both sgRNA 103 and sgRNA 104 demonstrated about 85% editing efficiency. Table 27 below provides the result from an in silico OT analysis performed using sgRNA 103 and sgRNA 104.







TABLE 27







In silico OT Analysis









bulge_type


















None
None
None
None
None
None
DNA
DNA
RNA
RNA









bulge_length


















0
0
0
0
0
0
1
1
1
1









num_mm



















target_site_name
pam
ot_category
0
2
3
4
5
6
1
2
1
2






















sgRCM103
NAA
Exon
0
1
5
46
323
1902
0
3
0
2


sgRCM103
NAA
None
0
3
52
647
5918
40065
0
8
0
80


sgRCM103
NAA
TS exon
0
0
0
3
27
94
0
0
0
0


sgRCM103
NAG
Exon
0
2
4
41
340
1883
0
0
0
2


sgRCM103
NAG
None
0
2
34
444
3633
24615
0
5
1
37


sgRCM103
NAG
TS exon
0
0
1
2
19
104
0
0
0
2


sgRCM103
NGA
Exon
0
0
0
25
200
1214
0
2
0
4


sgRCM103
NGA
None
0
0
23
381
3357
22500
2
5
0
29


sgRCM103
NGA
TS exon
0
0
0
0
18
60
0
1
0
0


sgRCM103
NGG
Exon
1
1
2
29
202
1202
0
1
0
2


sgRCM103
NGG
None
0
0
29
294
2710
18612
1
4
0
28


sgRCM103
NGG
TS exon
0
0
0
0
10
65
0
0
0
0


sgRCM104
NAA
Exon
0
0
0
4
26
256
0
0
0
1


sgRCM104
NAA
None
0
0
2
26
386
3483
0
0
0
3


sgRCM104
NAA
TS exon
0
0
0
0
1
7
0
0
0
0


sgRCM104
NAG
Exon
0
0
0
5
44
502
0
0
0
0


sgRCM104
NAG
None
0
0
2
36
461
5459
0
0
0
4


sgRCM104
NAG
TS exon
0
0
0
0
0
27
0
0
0
0


sgRCM104
NGA
Exon
0
0
0
2
38
364
0
0
0
1


sgRCM104
NGA
None
0
0
0
51
1101
9541
0
0
1
0


sgRCM104
NGA
TS exon
0
0
0
0
1
27
0
0
0
0


sgRCM104
NGG
Exon
1
0
0
2
70
506
0
0
0
0


sgRCM104
NGG
None
0
0
1
28
461
4267
0
2
0
1


sgRCM104
NGG
TS exon
0
0
0
0
2
34
0
0
0
0









Example 27: Production of CD5-Targeting CAR-Ts
In order to produce CD5-targeting CAR-T cells, a lentivirus vector (LVV) was engineered encoding a CD5 CAR (FIG. 55). Patient T cells were transduced with the CD5 CAR LVV. In vitro 7-14 day expansion of the CAR-T cells was conducted prior to patient re-infusion. FIG. 55 shows exemplary CD5 CAR construct designs. An exemplary CD5 CAR construct design includes an anti-CD5 scFv, hinge/spacer, transmembrane domain, signal 2 co-stimulatory domain, and a CD3(co-stimulatory domain. The hinge/spacer region may include CH3, CD8u or CD28. The transmembrane domain may include CD8u or CD28. The Co-stimulatory domain may include 41bb or CD28.
Thirty LVV were generated. Table 28 below provides the various LVV CD5 CAR constructs.







TABLE 28







CD5 CAR LVV Constructs








ID
Sequence





pCAR_BTx063
MND-5CAR-CH3-CD28TM-CD28-CD3Z-WPRE codon opt 1


pCAR_BTx064
MND-5CAR-CH3-CD28TM-CD28-CD3Z-WPRE codon opt 2


pCAR_BTx065
MND-5CAR-CH3-CD28TM-CD28-CD3Z-WPRE codon opt 3


pCAR_BTx066
MND-5CAR-CD8aH-CD28TM-CD28-CD3Z-WPRE codon opt 1


pCAR_BTx067
MND-5CAR-CD8aH-CD28TM-CD28-CD3Z-WPRE codon opt 2


pCAR_BTx068
MND-5CAR-CD8aH-CD28TM-CD28-CD3Z-WPRE codon opt 3


pCAR_BTx069
MND-5CAR-CD28H-CD28TM-CD28-CD3Z-WPRE codon opt 1


pCAR_BTx070
MND-5CAR-CD28H-CD28TM-CD28-CD3Z-WPRE codon opt 2


pCAR_BTx071
MND-5CAR-CD28H-CD28TM-CD28-CD3Z-WPRE codon opt 3


pCAR_BTx072
MND-5CAR-CH3-CD8aTM-41BB-CD3Z-WPRE codon opt 1


pCAR_BTx073
MND-5CAR-CH3-CD8aTM-41BB-CD3Z-WPRE codon opt 2


pCAR_BTx074
MND-5CAR-CH3-CD8aTM-41BB-CD3Z-WPRE codon opt 3


pCAR_BTx075
MND-5CAR-CD8aH-CD8aTM-41BB-CD3Z-WPRE codon opt 1


pCAR_BTx076
MND-5CAR-CD8aH-CD8aTM-41BB-CD3Z-WPRE codon opt 2


pCAR_BTx077
MND-5CAR-CD8aH-CD8aTM-41BB-CD3Z-WPRE codon opt 3


pCAR_BTx078
PGK-5CAR-CH3-CD28TM-CD28-CD3Z-WPRE codon opt 1


pCAR_BTx079
PGK-5CAR-CH3-CD28TM-CD28-CD3Z-WPRE codon opt 2


pCAR_BTx080
PGK-5CAR-CH3-CD28TM-CD28-CD3Z-WPRE codon opt 3


pCAR_BTx081
PGK-5CAR-CD8aH-CD28TM-CD28-CD3Z-WPRE codon opt 1


pCAR_BTx082
PGK-5CAR-CD8aH-CD28TM-CD28-CD3Z-WPRE codon opt 2


pCAR_BTx083
PGK-5CAR-CD8aH-CD28TM-CD28-CD3Z-WPRE codon opt 3


pCAR_BTx084
PGK-5CAR-CD28H-CD28TM-CD28-CD3Z-WPRE codon opt 1


pCAR_BTx085
PGK-5CAR-CD28H-CD28TM-CD28-CD3Z-WPRE codon opt 2


pCAR_BTx086
PGK-5CAR-CD28H-CD28TM-CD28-CD3Z-WPRE codon opt 3


pCAR_BTx087
PGK-5CAR-CH3-CD8aTM-41BB-CD3Z-WPRE codon opt 1


pCAR_BTx088
PGK-5CAR-CH3-CD8aTM-41BB-CD3Z-WPRE codon opt 2


pCAR_BTx089
PGK-5CAR-CH3-CD8aTM-41BB-CD3Z-WPRE codon opt 3


pCAR_BTx090
PGK-5CAR-CD8aH-CD8aTM-41BB-CD3Z-WPRE codon opt 1


pCAR_BTx091
PGK-5CAR-CD8aH-CD8aTM-41BB-CD3Z-WPRE codon opt 2


pCAR_BTx092
PGK-5CAR-CD28H-CD28TM-CD28-CD3Z-WPRE codon opt 1









Each of the CD5 CAR LVV constructs from Table 27 with either the MND promoter or the PGK promoter were screened to examine CAR expression in primary human T cells using either sgRNA 103 and sgRNA 104. Untransduced (UTD) cells were used as a control. As shown in FIG. 56, the CD5 CAR LVV constructs resulted in having CAR expression ranging from 0% to 82%. Twenty-four constructs showed detectable CAR expression on the surface of T cells at the end of the 10 day culture period.
To determine whether the hinge/spacer CAR construct sequence affected CAR expression, the CAR constructs in Table 29 containing different hinge/spacer sequences (in bold font) were examined in combination with either sgRNA 103 and sgRNA 104. Untransduced (UTD) cells were used as a control. As shown in FIG. 57, the hinge/spacer sequence did impact CAR expression.







TABLE 29







CD5 CAR Constructs








ID
Sequence





UTD
Untransduced (no lentivirus)


pCAR_BTx063
MND-5CAR-CH3-CD28TM-CD28-CD3Z-WPRE


pCAR_BTx067
MND-5CAR-CD8aH-CD28TM-CD28-CD3Z-WPRE


pCAR_BTx071
MND-5CAR-CD28H-CD28TM-CD28-CD3Z-WPRE









In order to further characterize CD5 LVV CAR-T cells in vitro, a live imaging cell killing assay was performed. CD5 LVV CAR-T cells (LV66, LV67, LV68, and LV69) were added to CCRF tumor cells. As shown in FIGS. 57A-57D, edited and unedited constructs were cytotoxic against CD5+CCRF cells.
CD5 CAR constructs (LV63-LV69) were evaluated in vitro for IFNγ production using either sgRNA 103 and sgRNA 104 in T cells alone or in CCRF cells (FIGS. 59A and 59B). All CD5 CAR constructs released IFNγ in the presence of CD5+ CCRF-CEM leukemia cell lines.
Example 28: Combination Therapy Using Multiple Modified Immune Effector Cells
To examine whether the use of multiple genetically modified CAR-T cells can enhance the treatment of patients with T- or NK-Cell malignancies, such as liquid cancers, patients were administered one or more modified CAR-T cell based on their immunophenotype. Immune cells from patient samples were immunophenotyped for the presence or absence of CD5, CD7, CD33, and CD123 using flow cytometry and/or sequence analysis. mRNA base editors and guide RNA were delivered into the immune cells by electroporation to edit either CD5, CD7, CD33, or CD123 in combination with one or more of TRAC, LAG-3, FAS, CD52, B2M, CIITA, TRBC1, TRBC2, and PD-1 to reduce and or eliminate expression of these genes in the immune cells. Following base editing, the CD5 CAR, CD7 CAR, CD33 CAR, or CD123 CAR was delivered into modified immune cells via lentiviral transduction to create CD5, CD7, CD33 and CD123 CAR-T cells, respectively.
Patients that were immunophenotyped as CD5+ CD7− were treated with a CD5 CAR-T cell or population thereof. Patients that were immunophenotyped as CD5− CD7+ were treated with a CD7 CAR-T cell or population thereof. Patients that were immunophenotyped as CD5+ CD7+ were treated with a CD5 CAR-T cell or population thereof and/or a CD7 CAR-T cell or population thereof. Patients that were immunophenotyped as CD33− CD123+ were treated with a CD123 CAR-T cell or population thereof. Patients that were immunophenotyped as CD33+ CD123− were treated with a CD33 CAR-T cell or population thereof. Patients that were immunophenotyped as CD33+ CD123+ were treated with a CD33 CAR-T cell or population thereof and/or a CD123 CAR-T cell or population thereof. Patients that were immunophenotyped as CD5+ CD7+ CD33+ CD123+ were treated with any combination of a CD33 CAR-T cell or population thereof, a CD5 CAR-T cell or population thereof, a CD7 CAR-T cell or population thereof, and/or a CD123 CAR-T cell or population thereof.
OTHER EMBODIMENTS
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Absent any indication otherwise, publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entireties.