Granted Patent B2

US 12,553,065 · App. 18/106,039 · Granted Feb 17, 2026

Inventors: Feng Zhang (Cambridge, MA); Patrick Hsu (Cambridge, MA); Chie-yu Lin (Cambridge, MA); Fei Ran (Cambridge, MA)

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

C12N15/907A61K48/005C12N9/22C12N15/102C12N15/1082C12N15/63C12N2800/80

Abstract

Claims (27)

1 . An engineered CRISPR-Cas system for modifying a genomic locus of interest in a eukaryotic cell, comprising:

a Cas9 protein or a polynucleotide encoding the Cas9 protein, wherein the Cas9 protein comprises at least one mutation in a catalytic domain and is a nickase, wherein the Cas9 protein is fused to at least one nuclear localization signal (NLS);

a first CRISPR-Cas system chimeric RNA engineered to hybridize to a first target sequence at the genomic locus of interest, wherein the first chimeric RNA is capable of forming a first CRISPR complex with the Cas9 protein and directing sequence-specific binding of the first CRISPR complex to the first target sequence in the nucleus of the eukaryotic cell, thereby allowing the Cas9 protein to cleave a first DNA strand of the genomic locus of interest to produce a first nick; and

a second CRISPR-Cas system chimeric RNA engineered to hybridize to a second target sequence at the genomic locus of interest, wherein the second chimeric RNA is capable of forming a second CRISPR complex with the Cas9 protein and directing sequence-specific binding of the second CRISPR complex to the second target sequence in the nucleus of the eukaryotic cell, thereby allowing the Cas9 protein to cleave a second DNA strand of the genomic locus of interest to produce a second nick, wherein the first nick is located 26-200 nucleotides 5′ of the second nick.

2 . The engineered CRISPR-Cas system of claim 1 , wherein the Cas9 protein is fused to at least two NLSs.

3 . The engineered CRISPR-Cas system of claim 1 , wherein the Cas9 protein comprises at least one mutation in RuvC domain.

4 . The engineered CRISPR-Cas system of claim 3 , wherein the Cas9 protein comprises at least one mutation selected from the group consisting of D10A, E762A and D986A.

5 . The engineered CRISPR-Cas system of claim 3 , wherein the Cas9 protein comprises D10A mutation.

6 . The engineered CRISPR-Cas system of claim 1 , wherein the Cas9 protein comprises at least one mutation in HNH domain.

7 . The engineered CRISPR-Cas system of claim 6 , wherein the Cas9 protein comprises at least one mutation selected from the group consisting of H840A, N854A and N863A.

8 . The engineered CRISPR-Cas system of claim 6 , wherein the Cas9 protein comprises H840A mutation.

9 . The engineered CRISPR-Cas system of claim 1 , wherein the Cas9 protein is S. pyogenes Cas9.

10 . The engineered CRISPR-Cas system of claim 1 , wherein the Cas9 protein is S. aureus Cas9.

11 . The engineered CRISPR-Cas system of claim 1 , wherein the Cas9 protein is fused to at least one heterologous protein domain.

12 . The engineered CRISPR-Cas system of claim 11 , wherein the heterologous protein domain is a methylase, a demethylase, a transcriptional activator, a transcriptional repressor, a recombinase, a transposase, a histone remodeler, a DNA methyltransferase, or a cryptochrome.

13 . The engineered CRISPR-Cas system of claim 1 , wherein the first nick is located 26-100 nucleotides 5′ of the second nick.

14 . The engineered CRISPR-Cas system of claim 1 , wherein the first nick is located 30-200 nucleotides 5′ of the second nick.

15 . The engineered CRISPR-Cas system of claim 1 , wherein the first nick is located 30-100 nucleotides 5′ of the second nick.

16 . The engineered CRISPR-Cas system of claim 1 , wherein the first nick is about 34-50 nucleotides 5′ of the second nick.

17 . The engineered CRISPR-Cas system of claim 1 , wherein the engineered CRISPR-Cas system comprises a viral vector encoding the Cas9.

18 . The engineered CRISPR-Cas system of claim 1 , wherein the engineered CRISPR-Cas system comprises an AAV vector encoding the Cas9.

19 . The engineered CRISPR-Cas system of claim 1 , wherein the engineered CRISPR-Cas system comprises an mRNA encoding the Cas9.

20 . The engineered CRISPR-Cas system of claim 1 , wherein the genomic locus of interest encodes a gene product.

21 . The engineered CRISPR-Cas system of claim 20 , wherein the gene product is a protein.

22 . The engineered CRISPR-Cas system of claim 1 , wherein the eukaryotic cell is a mammalian cell.

23 . The engineered CRISPR-Cas system of claim 1 , further comprising an exogenous recombination template for targeted integration into the genomic locus of interest.

24 . The engineered CRISPR-Cas system of claim 23 , wherein the exogenous recombination template is at least 1,000 nucleotides in length.

Assignments (5)

Continuity (9)

References Cited (400)