Granted Patent B2

US 12,435,320 · App. 17/081,387 · Granted Oct 7, 2025

Inventors: Feng Zhang (Cambridge, MA); Bernd Zetsche (Cambridge, MA); Amit Choudhary (Cambridge, MA)

Assignees: THE BROAD INSTITUTE, INC.; MASSACHUSETTS INSTITUTE OF TECHNOLOGY; PRESIDENT AND FELLOWS OF HARVARD COLLEGE

C12N9/22A01K67/0275A61K48/005C07K14/721C12N9/003C12N15/102C12N15/907C12Y105/01003A01K2207/10A01K2217/05A01K2227/105C07K2319/00C07K2319/095

Abstract

Claims (33)

1. A composition comprising:

(a) a non-naturally occurring or engineered Cas9 attached to at least two destabilization domains (DD); wherein a first DD is attached to the N-terminus of the Cas9 and a second DD is attached to the C-terminus of the Cas9, the first and second DDs being the same or different; wherein the Cas9 is attached to the at least two DDs via fusion, tether, or non-covalent bond;

(b) a guide RNA that forms a CRISPR complex with the Cas9; and

(c) at least one stabilizing ligand that binds to at least one of the destabilization domains;

wherein binding of the at least one stabilizing ligand to at least one of the destabilization domains inhibits degradation of the Cas9 by proteasome, and wherein the guide RNA directs sequence-specific binding of the CRISPR complex to a target DNA.

2. The composition of claim 1 , wherein the Cas9 is an Sp Cas9, an Sa Cas9, an St Cas9, or an Fn Cas9.

3. The composition of claim 1 , wherein the Cas9 comprises a Rec2 or HD2 truncation.

4. The composition of claim 3 , wherein the truncation comprises removal or replacement with a linker.

5. The composition of claim 4 , wherein the linker comprises a branch or otherwise allows for tethering of the at least two DDs and/or a functional domain.

6. The composition of claim 1 , wherein the at least two DDs are attached to the Cas9 by fusion with said Cas9.

7. The composition of claim 6 , wherein the fusion comprises a linker between at least one of the DDs and the Cas9.

8. The composition of claim 7 , wherein the linker comprises a GlySer linker or a localization signal.

9. The composition of claim 1 , further comprising at least one Nuclear Export Signal (NES) or at least one Nuclear Localization Signal (NLS).

10. The composition of claim 9 , wherein the Cas9 comprises two or more NESs.

11. The composition of claim 1 , wherein at least one of the DDs comprises ER50 or DHFR50.

12. The composition of claim 1 , wherein the Cas9 comprises at least one mutation.

13. The composition of claim 12 , wherein the Cas9 is a nickase.

14. The composition of claim 12 , wherein the Cas9 has substantially no nuclease activity due to the mutation(s).

15. The composition of claim 1 , wherein the Cas9 is a split Cas9.

16. The composition of claim 1 , wherein the Cas9 comprises a functional domain.

17. The composition of claim 1 , wherein the Cas9 is fused to a first ER50 domain at its N-terminus and a second ER50 domain at its N-terminus.

18. The composition of claim 1 , wherein the Cas9 is fused to a first DHFR domain at its N-terminus and a second DHFR domain at its N-terminus.

19. The composition of claim 1 , wherein the first DD comprises SEQ ID NO: 50.

20. The composition of claim 19 , wherein the second DD comprises SEQ ID NO:51.

21. The composition of claim 1 , wherein at least one of the at least two DDs comprises SEQ ID NO:51.

22. The composition of claim 1 , wherein the stabilizing ligan is trimethoprim (TMP), 4-hydroxytamoxifen (4HT), or CMP8.

23. A composition comprising a non-naturally occurring or engineered Cas9 attached to at least two destabilization domains (DD); wherein a first DD is attached to the N-terminus of the Cas9 and a second DD is attached to the C-terminus of the Cas9, the first and second DDs being the same or different; wherein the Cas9 is attached to the at least two DDs via fusion, tether, or non-covalent bond; and wherein the Cas9 attached to the at least two DDs comprises an amino acid sequence selected from SEQ ID NOs: 56-61.

24. The composition of claim 23 , further comprising a guide polynucleotide capable of forming a CRISPR-Cas complex with the Cas9.

25. The composition of claim 23 , wherein the Cas9 attached to the at least two DDs comprises SEQ ID NO:61.

26. A composition comprising a non-naturally occurring or engineered split Cas9 attached to at least one destabilization domain (DD) via fusion, tether, or non-covalent bond, wherein the Cas9 is a Staphylococcus aureus Cas9 (SaCas9) split into an N-terminus portion (Cas9 (N)) and a C-terminus portion (Cas9 (C)).

27. The composition of claim 26 , wherein the DD occurs between Cas9 (N) and Cas9 (C).

28. The composition of claim 27 , wherein the at least one DD comprises a linker between Cas9 (N) and/or Cas9 (C).

29. A composition comprising a non-naturally occurring or engineered Cas9 attached to at least one destabilization domain (DD) via fusion, tether, or non-covalent bond, wherein the Cas9 is a Staphylococcus aureus Cas9 (SaCas9) having N580A mutation and is a nickase.

Assignments (4)

Continuity (5)

References Cited (400)