Nucleobase editors and uses thereof
Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing proteins or protein domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing proteins or domains, are provided.
1. A fusion protein comprising:
(i) a Cas9 domain, wherein the Cas9 domain when in conjunction with a bound guide RNA (gRNA) specifically binds to a target nucleic acid sequence;
(ii) a cytidine deaminase domain, wherein the cytidine deaminase domain deaminates a cytosine base in a single-stranded portion of the target nucleic acid sequence when in conjunction with the Cas9 domain and the gRNA; and
(iii) an uracil glycosylase inhibitor (UGI) domain, wherein the UGI domain inhibits a uracil-DNA glycosylase.
2. The fusion protein of claim 1 , wherein the Cas9 domain comprises the amino acid sequence provided in SEQ ID NO: 674.
3. The fusion protein of claim 1 , wherein the Cas9 domain is a Cas9 nickase (nCas9) domain that cuts a nucleotide target strand of a nucleotide duplex, wherein the nucleotide target strand is the strand that binds to the gRNA.
4. The fusion protein of claim 1 , wherein the Cas9 domain is a Cas9 nickase (nCas9) domain that comprises a D10A mutation in the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260.
5. The fusion protein of claim 1 , wherein the Cas9 domain is a Cas9 nickase (nCas9) domain that comprises one or more of N497A, R661A, Q695A, or Q926A mutations in the amino acid sequence provided in SEQ ID NO: 10, or one or more corresponding mutations in any of the amino acid sequences provided in SEQ ID NOs: 11-260.
6. The fusion protein of claim 1 , wherein the cytidine deaminase domain is a deaminase from an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase.
7. The fusion protein of claim 6 , wherein the APOBEC family deaminase is selected from the group consisting of APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and APOBEC3H deaminase.
8. The fusion protein of claim 1 , wherein the cytidine deaminase domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 266-284, 607-610, 5724-5736, and 5738-5741.
9. The fusion protein of claim 1 , wherein the cytidine deaminase domain is a rat APOBEC1 (rAPOBEC1) deaminase comprising one or more mutations selected from the group consisting of W90Y, R126E, and R132E in SEQ ID NO: 284, or one or more corresponding mutations in another APOBEC deaminase.
10. The fusion protein of claim 1 , wherein the cytidine deaminase domain is a human APOBEC1 (hAPOBEC1) deaminase comprising one or more mutations selected from the group consisting of W90Y, Q126E, and R132E in SEQ ID NO: 5724, or one or more corresponding mutations in another APOBEC deaminase.
11. The fusion protein of claim 1 , wherein the cytidine deaminase domain is a human APOBEC3G (hAPOBEC3G) deaminase comprising one or more mutations selected from the group consisting of W285Y, R320E, and R326E in SEQ ID NO: 275, or one or more corresponding mutations in another APOBEC deaminase.
12. The fusion protein of claim 1 , wherein the cytidine deaminase domain is an activation-induced deaminase (AID).
13. The fusion protein of claim 1 , wherein the cytidine deaminase domain is a cytidine deaminase 1 from Petromyzon marinus (pmCDA1).
14. The fusion protein of claim 1 , wherein the UGI domain comprises the amino acid sequence as set forth in SEQ ID NO: 600.
15. The fusion protein of claim 1 , wherein the fusion protein comprises the structure:
NH 2 -[cytidine deaminase domain]-[Cas9 domain]-[UGI domain]-COOH, and wherein each instance of “-” comprises an optional linker.
16. The fusion protein of claim 1 , wherein the cytidine deaminase domain of (ii) and the Cas9 domain of (i) are linked via a linker comprising the amino acid sequence (GGGS) n (SEQ ID NO: 265) n , (GGGGS) n (SEQ ID NO: 5) n , (G) n , (EAAAK) n (SEQ ID NO: 6) n , (GGS) n , (SGGS) n (SEQ ID NO: 4288) n , SGSETPGTSESATPES (SEQ ID NO: 7) n , or (XP) n motif, or a combination thereof, wherein n is independently an integer between 1 and 30, inclusive, and wherein X is any amino acid.
17. The fusion protein of claim 1 , wherein the cytidine deaminase domain of (ii) and the Cas9 domain of (i) are linked via a linker comprising the amino acid sequence: SGSETPGTSESATPES (SEQ ID NO: 7).
18. The fusion protein of claim 1 further comprising a nuclear localization sequence (NLS).
19. The fusion protein of claim 18 , wherein the NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 741) or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 742).
20. The fusion protein of claim 18 , wherein the fusion protein comprises the structure: NH 2 -[cytidine deaminase domain]-[Cas9 domain]-[UGI domain]-[NLS]-COOH, and wherein each instance of “-” comprises an optional linker.
21. The fusion protein of claim 18 , wherein the UGI domain and the NLS are linked via a linker comprising the amino acid sequence: SGGS (SEQ ID NO: 4288), or wherein the Cas9 domain and the UGI domain are linked via a linker comprising the amino acid sequence: SGGS (SEQ ID NO: 4288).
22. The fusion protein of claim 1 , wherein the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 594.
23. A complex comprising the fusion protein of claim 1 and the guide RNA bound to the Cas9 domain of the fusion protein.