Adenosine deaminase base editors and methods of using same to modify a nucleobase in a target sequence
The disclosure provides compositions comprising novel adenosine base editors (e.g., ABE8) that have increased efficiency and methods of using these adenosine deaminase variants for editing a target sequence.
1. A fusion protein comprising a polynucleotide programmable DNA binding domain and at least one adenosine deaminase variant comprising a serine (S) at amino acid position 82 of the following sequence and having at least 85% identity to the following sequence
MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAI
GLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHS
RIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLC
YFFRMPRQVFNAQKKAQSST (SEQ ID NO: 4).
2. The fusion protein of claim 1 , wherein the adenosine deaminase variant further comprises an alteration at amino acid position 166.
3. The fusion protein of claim 1 , wherein the adenosine deaminase variant further comprises a T166R alteration.
4. The fusion protein of claim 1 , wherein the adenosine deaminase variant further comprises one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, and Q154R.
5. The fusion protein of claim 1 , wherein the adenosine deaminase variant further comprises an alteration or combination of alterations selected from the group consisting of:
Y147T and Q154R;
Y147T and Q154S;
Y147R and Q154S;
Q154S;
Y147R;
Q154R;
Y123H;
I76Y;
Y123H, and Y147T;
Y123H, and Y147R;
Y123H, and Q154R;
Y147R, Q154R, and Y123H;
Y147R, Q154R, and I76Y;
Y147R, Q154R, and T166R;
Y123H, Y147R, Q154R, and I76Y;
Y123H, Y147R, and Q154R; and
I76Y, Y123H, Y147R, and Q154R.
6. The fusion protein of claim 1 , wherein the fusion protein further comprises a second adenosine deaminase variant or a second adenosine deaminase.
7. The fusion protein of claim 1 , wherein the fusion protein is ABE8.5-m, ABE8.14-m, ABE8.15-m, ABE8.16-m, ABE8.17-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.22-m, ABE8.23-m, or ABE8.24-m.
8. The fusion protein of claim 6 , wherein the second adenosine deaminase comprises a wild-type adenosine deaminase.
9. The fusion protein of claim 8 , wherein the fusion protein is ABE8.5-d, ABE8.14-d, ABE8.15-d, ABE8.16-d, ABE8.17-d, ABE8.18-d, ABE8.19-d, ABE8.20-d, ABE8.22-d, ABE8.23-d, or ABE8.24-d.
10. The fusion protein of claim 1 , wherein the adenosine deaminase variant is TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.22, TadA*8.23, or TadA*8.24.
11. The fusion protein of claim 1 , wherein the adenosine deaminase variant comprises a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 N-terminal or C-terminal amino acid residues relative to the full-length adenosine deaminase.
12. The fusion protein of claim 1 , wherein the polynucleotide programmable DNA binding domain comprises the following sequence:
EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDK
GRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD
WDPKKYGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF
EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKFLQKG
NELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQ
ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP
RAFKYFDTTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD GG
SGGSGGSGGSGGSGGSGGM DKKYSIGLAIGTNSVGWAVITDEYKVPSKK
FKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC
YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYH
EKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDN
SDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIA
QLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD
NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRY
DEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYK
FIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAIL
RRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEET
ITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVY
NELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK
KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDI
VLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN
GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG
DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE
NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYY
LQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNR
GKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK
AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL
VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYG
DYKVYDVRKMIAKSEQ EGADKRTADGSEFESPKKKRKV
(SEQ ID NO: 12).
13. The fusion protein of claim 1 , wherein the polynucleotide programmable DNA binding domain comprises a Cas9 domain.
14. The fusion protein of claim 1 , wherein the polynucleotide programmable DNA binding domain comprises the following amino acid sequence:
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA
LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR
LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD
LRLIYLALAHMIKERGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP
INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP
NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI
LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI
FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY
YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD
LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI
IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ
LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGEANRNFMQLIHDD
SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV
MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP
VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD
SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL
TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI
REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK
YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI
TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV
QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE
KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK
YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE
DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK
PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
SITGLYETRIDLSQLGGD (SEQ ID NO: 1).
15. The fusion protein of claim 1 , wherein the Cas9 domain is a Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), a Streptococcus pyogenes Cas9 (SpCas9), or variants thereof.
16. The fusion protein of claim 1 , wherein the polynucleotide programmable DNA binding domain is a nuclease inactive or nickase variant.
17. The fusion protein of claim 1 , comprising a linker between the polynucleotide programmable DNA binding domain and the adenosine deaminase variant.
18. The fusion protein of claim 1 , comprising one or more nuclear localization signals.
19. The fusion protein of claim 1 , wherein the adenosine deaminase variant has at least 90% identity to the sequence.
20. The fusion protein of claim 1 , wherein the adenosine deaminase variant has at least 95% identity to the sequence.
21. A polynucleotide encoding the fusion protein of claim 1 .
22. An expression vector comprising the polynucleotide of claim 21 .
23. The expression vector of claim 22 , wherein the vector is a viral vector selected from the group consisting of adeno-associated virus (AAV), retroviral vector, adenoviral vector, lentiviral vector, Sendai virus vector, and herpesvirus vector.
24. A cell comprising the fusion protein of claim 1 .
25. A base editor comprising the fusion polypeptide of claim 1 in a complex with one or more guide polynucleotides.
26. A method for base editing comprising contacting a polynucleotide sequence with the fusion protein of claim 1 , wherein the adenosine deaminase variant deaminates a nucleobase in the polynucleotide, thereby editing the polynucleotide sequence.
27. A method of correcting a genetic defect in a subject, the method comprising administering to the subject
(i) a base editor comprising or consisting essentially of the fusion protein of claim 1 , or
(ii) a polynucleotide encoding said base editor and one or more guide polynucleotides that direct the base editor to deaminate a target nucleobase in a target nucleotide sequence of the subject, thereby correcting the genetic defect.
28. The method of claim 27 , comprising delivering the base editor, or polynucleotide encoding said base editor, and one or more guide polynucleotides to a cell of the subject.
29. The method of claim 27 , wherein the subject is a mammal or a human.
30. The method of claim 27 , wherein the deamination of the target nucleobase replaces the target nucleobase with a wild type nucleobase.