IP Library › Granted Patent US 12,215,365
Granted Patent B2
US 12,215,365 · App. 17/408,306 · Granted Feb 4, 2025

Cas variants for gene editing

Inventors: David R. Liu (Cambridge, MA); Alexis Christine Komor (Cambridge, MA)
Assignee: President and Fellows of Harvard College
C12N9/22A61K38/465A61K38/50A61K47/61C12N9/6472C12N9/78C12N15/01C12N15/102C12P19/34C12Q1/6883C12Y301/00C12Y304/22062C12Y305/04C12Y305/04001C12Y305/04004C12Y305/04005C07K2319/00C07K2319/80C12Q2600/156C12Y301/22
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Quick Facts
Patent No.
US 12,215,365
App. No.
17/408,306
Granted
Feb 4, 2025
Kind
B2
Abstract

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 enzymes or enzyme 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 enzymes or domains, are provided.

Claims (42)

1. A multi-molecular complex comprising:

(a) a cytidine deaminase;

(b) a Cas9 variant protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 37, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 37, wherein the Cas9 variant comprises one or more nuclease inactivating mutations within a RuvC1 subdomain and/or a HNH subdomain; and

(c) a single guide RNA (sgRNA) comprising a region complementary to a strand of the target nucleic acid molecule;

wherein the cytidine deaminase, the target nucleic acid molecule, the Cas9 variant, and the sgRNA associate in a multi-molecular complex; and

wherein the multi-molecular complex deaminates a cytidine in the target nucleic acid molecule.

2. The complex of claim 1 , wherein the Cas9 variant protein comprises the nuclease inactivating mutation within the RuvC1 subdomain.

3. The complex of claim 1 , wherein the nuclease inactivating mutation within the RuvC1 subdomain corresponds to a mutation at D10 in SEQ ID NO: 2 or SEQ ID NO: 37.

4. The complex of claim 1 , wherein the Cas9 variant protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2.

5. The complex of claim 1 , wherein the Cas9 variant protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 2.

6. The complex of claim 1 , wherein the Cas9 variant protein comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 2.

7. The complex of claim 1 , wherein the nuclease inactivating mutation within the RuvC1 subdomain corresponds to a D10A mutation in SEQ ID NO: 2 or SEQ ID NO: 37.

8. The complex of claim 1 , wherein the Cas9 variant protein comprises the nuclease inactivating mutation within the HNH subdomain.

9. The complex of claim 1 , wherein the nuclease inactivating mutation within the HNH subdomain corresponds to a mutation at position H839 of SEQ ID NO: 2.

10. The complex of claim 1 , wherein the Cas9 variant protein comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 37.

11. The complex of claim 1 , wherein the Cas9 variant protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 37.

12. The complex of claim 1 , wherein the Cas9 variant protein comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 37.

13. The complex of claim 1 , wherein the nuclease inactivating mutation within the HNH subdomain corresponds to an H839A mutation in SEQ ID NO: 2 or an H840A mutation in SEQ ID NO: 37.

14. The complex of claim 1 , wherein the Cas9 variant protein is derived from Corynebacterium ulcerous, Corynebacterium diphtheria, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus iniae, Belliella baltica, Psychroflexus torquisl, Streptococcus thermophilus, Listeria innocua, Campylobacter jejuni , or Neisseria meningitidis.

15. The complex of claim 14 , wherein the Cas9 variant protein is derived from Neisseria meningitidis.

16. The complex of claim 1 , wherein the cytidine deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase.

17. The complex of claim 1 , wherein the cytidine deaminase is an APOBEC1 family deaminase.

18. The complex of claim 1 , wherein the cytidine deaminase is an activation-induced cytidine deaminase (AID).

19. The complex of claim 1 , wherein the target nucleic acid molecule comprises a sequence associated with a disease or disorder.

20. The complex of claim 19 , wherein the sequence associated with the disease or disorder encodes a protein, and wherein the deamination introduces a stop codon into the sequence associated with the disease or disorder, resulting in a truncation of the encoded protein.

21. The complex of claim 19 , wherein the deamination corrects a point mutation in the target nucleic acid molecule, wherein the point mutation is associated with the disease or disorder.

22. The complex of claim 21 , wherein the target nucleic acid molecule comprises a T to C point mutation, and wherein the deamination of the mutant C base results in a nucleic acid sequence that is not associated with the disease or disorder.

23. The complex of claim 21 , wherein the target nucleic acid molecule comprises an A to G point mutation, and wherein the deamination of the C complementary to the mutant G base results in a nucleic acid sequence that is not associated with the disease or disorder.

24. The complex of claim 1 , wherein the cytidine deaminase comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 6 (human AID), SEQ ID NO: 7 (mouse AID), SEQ ID NO: 10 (mouse APOBEC-3), SEQ ID NO: 15 (human APOBEC-3), SEQ ID NO: 16 (human APOBEC-3F), SEQ ID NO: 17 (human APOBEC-3B), SEQ ID NO: 18 (human APOBEC-3C), SEQ ID NO: 19 (human APOBEC-3A), SEQ ID NO: 20 (human APOBEC-3H), SEQ ID NO: 21 (human APOBEC-3D), SEQ ID NO: 22 (human APOBEC-1), SEQ ID NO: 23 (mouse APOBEC-1), and SEQ ID NO: 24 (rat APOBEC-1).

25. An in vitro or ex vivo method of nucleic acid editing, the method comprising contacting a target nucleic acid molecule with the complex of claim 1 , whereby the method results in deamination of a cytidine in the target nucleic acid molecule.

26. The method of claim 25 , wherein the target nucleic acid molecule comprises a sequence associated with a disease or disorder, and wherein the deamination of the cytidine results in a sequence that is not associated with the disease or disorder.

27. The method of claim 26 , wherein the target nucleic acid molecule comprises a point mutation associated with a disease or disorder, and wherein the deamination corrects the point mutation.

28. The method of claim 27 , wherein the target nucleic acid molecule comprises a T to C point mutation, and wherein the deamination of the mutant C base results in a nucleic acid sequence that is not associated with the disease or disorder.

29. The method of claim 26 , wherein the target nucleic acid molecule associated with the disease or disorder encodes a protein, and wherein the deamination introduces a stop codon into the sequence associated with the disease or disorder, resulting in a truncation of the encoded protein.

30. A cell comprising the complex of claim 1 .

31. The complex of claim 1 , wherein the cytidine deaminase comprises an amino acid sequence having at least 90% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 6 (human AID), SEQ ID NO: 7 (mouse AID), SEQ ID NO: 10 (mouse APOBEC-3), SEQ ID NO: 15 (human APOBEC-3), SEQ ID NO: 16 (human APOBEC-3F), SEQ ID NO: 17 (human APOBEC-3B), SEQ ID NO: 18 (human APOBEC-3C), SEQ ID NO: 19 (human APOBEC-3A), SEQ ID NO: 20 (human APOBEC-3H), SEQ ID NO: 21 (human APOBEC-3D), SEQ ID NO: 22 (human APOBEC-1), SEQ ID NO: 23 (mouse APOBEC-1), and SEQ ID NO: 24 (rat APOBEC-1).

32. The complex of claim 1 , wherein the cytidine deaminase comprises an amino acid sequence having at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 6 (human AID), SEQ ID NO: 7 (mouse AID), SEQ ID NO: 10 (mouse APOBEC-3), SEQ ID NO: 15 (human APOBEC-3), SEQ ID NO: 16 (human APOBEC-3F), SEQ ID NO: 17 (human APOBEC-3B), SEQ ID NO: 18 (human APOBEC-3C), SEQ ID NO: 19 (human APOBEC-3A), SEQ ID NO: 20 (human APOBEC-3H), SEQ ID NO: 21 (human APOBEC-3D), SEQ ID NO: 22 (human APOBEC-1), SEQ ID NO: 23 (mouse APOBEC-1), and SEQ ID NO: 24 (rat APOBEC-1).

33. The complex of claim 1 , wherein the cytidine deaminase comprises an amino acid sequence having at least 99% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 6 (human AID), SEQ ID NO: 7 (mouse AID), SEQ ID NO: 10 (mouse APOBEC-3), SEQ ID NO: 15 (human APOBEC-3), SEQ ID NO: 16 (human APOBEC-3F), SEQ ID NO: 17 (human APOBEC-3B), SEQ ID NO: 18 (human APOBEC-3C), SEQ ID NO: 19 (human APOBEC-3A), SEQ ID NO: 20 (human APOBEC-3H), SEQ ID NO: 21 (human APOBEC-3D), SEQ ID NO: 22 (human APOBEC-1), SEQ ID NO: 23 (mouse APOBEC-1), and SEQ ID NO: 24 (rat APOBEC-1).

34. The complex of claim 1 , wherein the nuclease inactivating mutation within the HNH subdomain corresponds to a mutation at position H840 of SEQ ID NO: 37.

35. The complex of claim 1 , wherein the cytidine deaminase is an APOBEC3 family deaminase.

36. The method of claim 26 , wherein the sequence associated with the disease or disorder encodes a protein, and wherein the deamination introduces a stop codon into the sequence associated with the disease or disorder, resulting in a truncation of the encoded protein.

37. The method of claim 27 , wherein the target nucleic acid molecule comprises an A to G point mutation, and wherein the deamination of the C complementary to the mutant G base results in a nucleic acid sequence that is not associated with the disease or disorder.

Assignments (3)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 9, 2022
From: LIU, DAVID R.
To: HOWARD HUGHES MEDICAL INSTITUTE
Reel/Frame 059874/0623 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 9, 2022
From: HOWARD HUGHES MEDICAL INSTITUTE
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 059874/0630 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 9, 2022
From: KOMOR, ALEXIS CHRISTINE
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 059874/0639 →
Continuity (19)
Continuation 16374634 · Apr 3, 2019
Continuation 15103608
Continuation 14325815 · Jul 8, 2014
Continuation 14326109 · Jul 8, 2014
Continuation 14326140 · Jul 8, 2014
Continuation 14326269 · Jul 8, 2014
Continuation 14326290 · Jul 8, 2014
Continuation 14326318 · Jul 8, 2014
Continuation 14326303 · Jul 8, 2014
Continuation 14325815 · Jul 8, 2014
Continuation 14326109 · Jul 8, 2014
Continuation 14326140 · Jul 8, 2014
Continuation 14326290 · Jul 8, 2014
Continuation 14326318 · Jul 8, 2014
Continuation 14326303 · Jul 8, 2014
Continuation 14326269 · Jul 8, 2014
Provisional Application 61980333 · Apr 16, 2014
Provisional Application 61915386 · Dec 12, 2013
Related Publication 20220119785A1 · Apr 21, 2022
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