Granted Patent B2

US 12,571,005 · App. 17/489,308 · Granted Mar 10, 2026

Inventors: Feng Zhang (Cambridge, MA); Omar O. Abudayyeh (Cambridge, MA); James E. Dahlman (Cambridge, MA); Patrick Hsu (Cambridge, MA); David A. Scott (Cambridge, MA)

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

C12N15/907A01K67/0275C07K14/4702C12N9/22C12N9/96C12N15/11C12N15/111C12Y301/21004A01K2217/07A01K2227/105C12N2310/20

Abstract

Claims (28)

1 . A method of modifying a eukaryotic cell, comprising introducing into the eukaryotic cell an engineered composition comprising:

I. a Cas9 protein or a nucleic acid encoding the Cas9, wherein the Cas9 comprises one or more mutations in a catalytic domain and is a nickase;

II. a first CRISPR-Cas system chimeric RNA or a nucleic acid encoding the first chimeric RNA, wherein the first chimeric RNA comprises a first guide sequence capable of hybridizing to a first target DNA sequence at a genomic locus of interest in the eukaryotic cell and directing sequence-specific binding of a first CRISPR complex to the first target DNA sequence, a tracr-mate sequence, a tracr sequence capable of hybridizing to the tracr-mate sequence, and a protector RNA comprising at least 8 contiguous nucleotides that are complementary to the guide sequence; and

III. a second CRISPR-Cas system chimeric RNA or a nucleic acid encoding the second chimeric RNA, wherein the second chimeric RNA comprises a second guide sequence capable of hybridizing to a second target DNA sequence at the genomic locus of interest in the eukaryotic cell and directing sequence-specific binding of a second CRISPR complex to the second target DNA sequence, a tracr-mate sequence, a tracr sequence capable of hybridizing to the tracr-mate sequence, and a protector RNA comprising at least 8 contiguous nucleotides that are complementary to the guide sequence,

wherein the first CRISPR complex cleaves one DNA strand of the genomic locus of interest to produce a first nick, and the second CRISPR complex cleaves the opposite DNA strand of the genomic locus of interest to produce a second nick.

2 . The method of claim 1 , wherein the composition comprises a vector encoding the chimeric RNA and the Cas9.

3 . The method of claim 2 , wherein the vector is a viral vector.

4 . The method of claim 2 , wherein the vector is an AAV vector.

5 . The method of claim 1 , wherein the composition comprises the chimeric RNA and the Cas9.

6 . The method of claim 1 , wherein the composition comprises the chimeric RNA and an mRNA encoding the Cas9.

7 . The method of claim 1 , wherein the Cas9 is S. pyogenes Cas9 or S. aureus Cas9.

8 . The method of claim 1 , wherein the Cas9 comprises one or more mutations in a RuvC domain selected from the group consisting of D10A, E762A and D986A.

9 . The method of claim 8 , wherein the Cas9 comprises a D10A mutation.

10 . The method of claim 1 , wherein the Cas9 comprises one or more mutations in a HNH domain selected from the group consisting of H840A, N854A and N863A.

11 . The method of claim 10 , wherein the Cas9 comprises a H840A mutation.

12 . The method of claim 1 , wherein the Cas9 is fused to a heterologous protein domain.

13 . The method of claim 12 , wherein the heterologous protein domain has one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.

14 . The method of claim 1 , wherein the guide sequence comprises 15-25 nucleotides in length.

15 . The method of claim 1 , wherein the guide sequence comprises 20 nucleotides in length.

16 . The method of claim 1 , wherein the target DNA sequence is located within the nucleus or mitochondrion of the eukaryotic cell.

17 . The method of claim 1 , wherein the eukaryotic cell is a mammalian cell.

18 . The method of claim 1 , wherein the eukaryotic cell is a human cell.

19 . The method of claim 1 , wherein the protector RNA comprises at least 10 contiguous nucleotides that are complementary to the guide sequence.

20 . The method of claim 1 , wherein the protector RNA comprises at least 12 contiguous nucleotides that are complementary to the guide sequence.

21 . The method of claim 1 , wherein the protector RNA comprises 8 to 18 contiguous nucleotides that are complementary to the guide sequence.

22 . The method of claim 1 , wherein the protector RNA comprises 10 to 16 contiguous nucleotides that are complementary to the guide sequence.

23 . The method of claim 1 , further comprising introducing into the eukaryotic cell an exogenous recombination template for targeted integration into the genomic locus of interest.

24 . The method of claim 23 , wherein the exogenous recombination template is at least 1,000 nucleotides in length.

Assignments (5)

Continuity (7)

References Cited (400)