IP Library › Granted Patent US 12,398,406
Granted Patent B2
US 12,398,406 · App. 18/069,898 · Granted Aug 26, 2025

CAS9 proteins including ligand-dependent inteins

Inventors: David R. Liu (Cambridge, MA); Kevin Davis (Cambridge, MA)
Assignee: President and Fellows of Harvard College
C12N15/907C07K14/35C07K14/721C12N9/0071C12N9/22C12N9/78C12N15/63C12P19/34C12Y114/11C12Y305/04C07K2319/92
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Quick Facts
Patent No.
US 12,398,406
App. No.
18/069,898
Granted
Aug 26, 2025
Kind
B2
Abstract

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity of RNA-programmable endonucleases, such as Cas9, or for controlling the activity of proteins comprising a Cas9 variant fused to a functional effector domain, such as a nuclease, nickase, recombinase, deaminase, transcriptional activator, transcriptional repressor, or epigenetic modifying domain. For example, the inventive proteins provided comprise a ligand-dependent intein, the presence of which inhibits one or more activities of the protein (e.g., gRNA binding, enzymatic activity, target DNA binding). The binding of a ligand to the intein results in self-excision of the intein, restoring the activity of the protein.

Claims (41)

1. A polynucleotide encoding a hybrid protein comprising an intein flanked by two extein sequences of a Cas9 protein, wherein the two extein sequences ligate to form the Cas9 protein upon excision of the intein.

2. The polynucleotide of claim 1 , wherein an activity of the Cas9 protein is disrupted by the intein, and wherein the disrupted activity is restored upon excision of the intein from the Cas9 protein.

3. The polynucleotide of claim 1 , wherein the Cas9 protein is capable of binding a guide RNA (gRNA) prior to excision of the intein.

4. The polynucleotide of claim 1 , wherein the Cas9 protein has no or minimal gRNA binding activity prior to excision of the intein.

5. The polynucleotide of claim 1 , wherein the intein is a ligand-dependent intein.

6. The polynucleotide of claim 1 , wherein the intein comprises a ligand-binding domain.

7. The polynucleotide of claim 6 , wherein the ligand-binding domain comprises an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-14.

8. The polynucleotide of claim 1 , wherein the Cas9 protein comprises a nuclease-inactivated Cas9 (dCas9) domain.

9. The polynucleotide of claim 1 , wherein the Cas9 protein comprises a Cas9 nickase.

10. The polynucleotide of claim 1 , wherein the hybrid protein further comprises a nucleic acid-editing domain.

11. The polynucleotide of claim 10 , wherein the nucleic acid-editing domain comprises a heterologous nuclease domain, a recombinase domain, or a deaminase domain.

12. The polynucleotide of claim 11 , wherein the deaminase domain comprises a cytidine deaminase domain.

13. The polynucleotide of claim 11 , wherein the deaminase domain comprises an adenosine deaminase domain.

14. The polynucleotide of claim 11 , wherein the intein is a ligand-dependent intein.

15. The polynucleotide of claim 11 , wherein the Cas9 protein comprises a nuclease-inactivated Cas9 (dCas9) domain.

16. The polynucleotide of claim 11 , wherein the Cas9 protein comprises a Cas9 nickase.

17. The polynucleotide of claim 11 , wherein the Cas9 protein and the deaminase domain are separated by a linker.

18. The polynucleotide of claim 17 , wherein the linker comprises a peptide linker.

19. The polynucleotide of claim 17 , wherein the linker comprises a non-peptide linker.

20. The polynucleotide of claim 1 , wherein (a) the intein is a ligand-dependent intein, (b) the Cas9 protein comprises a dCas9 domain or is a Cas9 nickase, and (c) the Cas9 protein further comprises a deaminase domain.

21. The polynucleotide of claim 20 , wherein the deaminase domain comprises a cytidine deaminase domain.

22. The polynucleotide of claim 20 , wherein the deaminase domain comprises an adenosine deaminase domain.

23. The polynucleotide of claim 1 , wherein the Cas9 protein comprises a Cas9 domain having the amino acid sequence selected from the group consisting of SEQ ID NO: 2 (Cas9), SEQ ID NO: 4 (Cas9 nickase) and SEQ ID NO: 5 (dCas9).

24. The polynucleotide of claim 1 , wherein the intein is inserted at or replaces residue Cys80, Ala127, Thr146, Ser219, Thr333, Thr519, Cys574, Thr622, Ser701, Ala728, Thr995, Ser1006, Ser1154, Ser1159, or Ser1274 of a Cas9 protein having the amino acid sequence set forth as SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5.

25. The polynucleotide of claim 1 , wherein the hybrid protein comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 27-41.

26. The polynucleotide of claim 17 , wherein linker is a peptide linker.

27. The polynucleotide of claim 17 , wherein linker is a non-peptide linker.

28. A method for site-specific DNA cleavage, comprising:

a) introducing the polynucleotide of claim 1 into a cell under conditions where the hybrid protein is expressed;

b) contacting the hybrid protein with a ligand, wherein binding of the ligand to the intein induces self-excision of the intein; and

c) contacting a DNA with the Cas9 protein, wherein the Cas9 protein is associated with a gRNA,

wherein self-excision of the intein from the hybrid protein in step (b) allows the Cas9 protein to cleave the DNA, thereby producing cleaved DNA.

29. A method of DNA editing, the method comprising:

a) introducing the polynucleotide of claim 10 into a cell under conditions where the hybrid protein is expressed, wherein the intein is a ligand-dependent intein, and wherein an activity of the Cas9 protein selected from the group consisting of nuclease activity, target nucleic acid binding activity, and gRNA binding activity is disrupted by the intein;

b) contacting the hybrid protein with a gRNA capable of targeting the Cas9 protein to a target nucleotide sequence in a DNA molecule;

c) contacting the hybrid protein with a ligand that binds to the ligand-dependent intein and induces self-excision of the ligand-dependent intein, thereby restoring the activity of the Cas9 protein; and

d) contacting the target nucleotide sequence with the restored Cas9 protein, thereby editing the DNA molecule.

30. The method of DNA editing of claim 29 , wherein the nucleic acid-editing domain comprises a cytidine deaminase domain.

31. The method of DNA editing of claim 29 , wherein the nucleic acid-editing domain comprises an adenosine deaminase domain.

32. The polynucleotide of claim 6 , wherein the ligand-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-14.

33. The polynucleotide of claim 1 , wherein the Cas9 protein comprises an amino acid sequence having at least about 90% identical to a sequence selected form the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5.

Assignments (3)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 25, 2023
From: LIU, DAVID R.
To: HOWARD HUGHES MEDICAL INSTITUTE
Reel/Frame 063769/0034 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 25, 2023
From: HOWARD HUGHES MEDICAL INSTITUTE
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 063769/0037 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 25, 2023
From: DAVIS, KEVIN
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 063769/0054 →
Continuity (6)
Continuation 16888646 · May 29, 2020
Continuation 16132276 · Sep 14, 2018
Continuation 15329925
Provisional Application 62135629 · Mar 19, 2015
Provisional Application 62030943 · Jul 30, 2014
Related Publication 20230279443A1 · Sep 7, 2023
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