Cytosine to guanine base editor
Some aspects of this disclosure provide compositions, 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 capable of inducing a cytosine (C) to guanine (G) change in a nucleic acid (e.g., genomic DNA) are provided. In some embodiments, fusion proteins of a nucleic acid programmable DNA binding protein (e.g., Cas9) and nucleic acid editing proteins or protein domains, e.g., deaminase domains, polymerase domains, and/or base excision enzymes 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 a nucleic acid programmable DNA binding protein (e.g., Cas9), and nucleic acid editing proteins or domains, are provided.
1. A fusion protein comprising (i) a nucleic acid programmable DNA binding protein (napDNAbp) domain, wherein the napDNAbp domain when in association with a guide RNA (gRNA) specifically binds a target nucleic acid molecule; (ii) a cytidine deaminase domain, wherein the cytidine deaminase domain deaminates a cytosine base in the target nucleic acid molecule; and (iii) a uracil binding protein (UBP), wherein the UBP is a uracil DNA glycosylase (UDG) or a uracil base excision enzyme.
2. The fusion protein of claim 1 , wherein the uracil binding protein is a uracil base excision enzyme.
3. The fusion protein of claim 1 , wherein the uracil binding protein is a uracil DNA glycosylase (UDG).
4. The fusion protein of claim 1 , wherein the uracil binding protein comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 48 (UDG), SEQ ID NO: 49 (UdgX), SEQ ID NO: 50 (UdgX*), SEQ ID NO: 51 (UdgX_On), or SEQ ID NO: 53 (SMUG1).
5. The fusion protein of claim 4 , wherein the uracil binding protein comprises the amino acid sequence of SEQ ID NO: 49 (UdgX), SEQ ID NO: 48 (UDG), SEQ ID NO: 50 (UdgX*), SEQ ID NO: 51 (UdgX_On), or SEQ ID NO: 53 (SMUG1).
6. The fusion protein of claim 1 , wherein the fusion protein comprises the structure:
NH 2 -[cytidine deaminase domain]-[napDNAbp domain]-[UBP]-COOH,
wherein each instance of “]-[” comprises an optional linker.
7. The fusion protein of claim 1 , wherein at least one of (i) the cytidine deaminase domain and the napDNAbp domain, and (ii) the napDNAbp domain and the UBP are fused via a linker.
8. The fusion protein of claim 1 , wherein the fusion protein further comprises (iv) a nucleic acid polymerase domain (NAP).
9. The fusion protein of claim 8 , wherein the nucleic acid polymerase domain has translesion polymerase activity.
10. The fusion protein of claim 8 , wherein the nucleic acid polymerase domain is from Rev7, Rev1 complex, polymerase iota, polymerase kappa, or polymerase eta.
11. The fusion protein of claim 8 , wherein the nucleic acid polymerase domain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 54-64.
12. The fusion protein of claim 8 , wherein the fusion protein comprises the structure:
NH 2 -[cytidine deaminase domain]-[napDNAbp domain]-[UBP]-[NAP]-COOH;
NH 2 -[cytidine deaminase domain]-[napDNAbp domain]-[NAP]-[UBP]-COOH;
NH 2 -[cytidine deaminase domain]-[NAP]-[napDNAbp domain]-[UBP]-COOH; or
NH 2 -[NAP]-[cytidine deaminase domain]-[napDNAbp domain]-[UBP]-COOH;
wherein each instance of “]-[” comprises an optional linker.
13. The fusion protein of claim 1 , wherein the napDNAbp domain comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 4-26.
14. The fusion protein of claim 13 , wherein the napDNAbp domain comprises the amino acid sequence of any one of SEQ ID NOs: 4-26.
15. The fusion protein of claim 1 , wherein the napDNAbp domain is a Cas9 nickase (nCas9) or a nuclease inactive Cas9 (dCas9).
16. The fusion protein of claim 1 , wherein the cytidine deaminase domain is a deaminase from the apolipoprotein B mRNA-editing complex (APOBEC) family.
17. The fusion protein of claim 16 , wherein the 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.
18. The fusion protein of claim 1 , wherein the cytidine deaminase domain comprises (i) an amino acid sequence that is at least 85% identical to an amino acid sequence of any one of SEQ ID NOs: 67-101.
19. 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 of SEQ ID NO: 93.
20. A fusion protein comprising:
(i) a first domain comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 4-40;
(ii) a second domain comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 67-101; and
(iii) a third domain comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 48-53.
21. A complex comprising a nucleic acid molecule and the fusion protein of claim 1 .
22. A pharmaceutical composition comprising the fusion protein of claim 1 and a pharmaceutically acceptable excipient.
23. A method comprising contacting a nucleic acid molecule with the fusion protein of claim 1 .
24. A method of editing a nucleobase pair of a double-stranded DNA sequence, the method comprising:
contacting a target region of the double-stranded DNA sequence with a complex comprising the fusion protein of claim 1 and a guide nucleic acid, wherein the target region comprises a target nucleobase pair; and thereby:
inducing strand separation of said target region; and
excising a cytosine or a thymine in a single strand of the target region.
25. A method of treating a subject having or suspected of having a disease or disorder comprising administering the fusion protein of claim 1 ex vivo to a cell from the subject.
26. The fusion protein of claim 20 , wherein:
(i) the first domain comprises the amino acid sequence of any one of SEQ ID NOs: 4-40;
(ii) the second domain comprises the amino acid sequence of any one of SEQ ID NOs: 67-101; and
(iii) the third domain comprises the amino acid sequence of any one of SEQ ID NOs: 48-53.
27. The fusion protein of claim 1 , wherein the uracil binding protein comprises the amino acid sequence of SEQ ID NO: 49 (UdgX).
28. The fusion protein of claim 1 , wherein the uracil binding protein comprises a UdgX or UdgX*.
29. The fusion protein of claim 7 , wherein the linker comprises the amino acid sequence of any one of SEQ ID NOs: 102-109, 120, and 123.
30. The fusion protein of claim 18 , wherein the fusion protein comprises an amino acid sequence of any one of SEQ ID NOs: 67-101.
31. A cell comprising the fusion protein of claim 1 .