Granted Patent B2

US 12,637,698 · App. 18/317,465 · Granted May 26, 2026

Inventors: David Frendewey (New York, NY); Ka-Man Venus Lai (Seattle, WA); Wojtek Auerbach (Ridgewood, NJ); Gustavo Droguett (New City, NY); Anthony Gagliardi (Hopewell Junction, NY); David M. Valenzuela (Yorktown Heights, NY); Vera Voronina (Sleepy Hollow, NY); Lynn Macdonald (Harrison, NY); Andrew J. Murphy (Croton-on-Hudson, NY); George D. Yancopoulos (Yorktown Heights, NY)

Assignee: Regeneron Pharmaceuticals, Inc.

C12N15/907A01K67/0275C12N9/22C12N15/102C12N15/8509C12Q1/6888A01K2227/105

Abstract

Claims (38)

1 . A method for producing an F0 generation mouse or rat, comprising:

(a) producing a genetically modified mouse or rat embryonic stem(ES) cell with a biallelic genomic modification, comprising contacting the genome of a mouse or rat ES cell with:

(i) a Cas protein;

(ii) a first guide RNA that hybridizes to a first CRISPR RNA recognition sequence within a genomic target locus, wherein the first guide RNA comprises a first tracrRNA and a first CRISPR RNA;

(iii) a second guide RNA that hybridizes to a second CRISPR RNA recognition sequence within the genomic target locus, wherein the second guide RNA comprises a second tracrRNA and a second CRISPR RNA; and

(iv) a targeting vector comprising a nucleic acid insert flanked by a 5′ homology arm that hybridizes to a 5′ target sequence and a 3′ homology arm that hybridizes to a 3′ target sequence,

wherein the genome comprises a pair of first and second homologous chromosomes comprising the genomic target locus, and

wherein the Cas protein cleaves at least one of the first and second CRISPR RNA recognition sequences to generate at least one double-strand break in at least one of the first and second homologous chromosomes to produce the genetically modified mouse or rat ES cell;

(b) introducing the genetically modified mouse or rat ES cell into a mouse or rat host embryo; and

(c) gestating the mouse or rat host embryo in a surrogate mother,

wherein the surrogate mother produces the F0 generation mouse or rat comprising the biallelic genomic modification.

2 . The method of claim 1 , wherein step (a) further comprises introducing into the mouse or rat ES cell:

(v) a third guide RNA that hybridizes to a third CRISPR RNA recognition sequence within the genomic target locus, wherein the third guide RNA comprises a third tracrRNA and a third CRISPR RNA; and

(vi) a fourth guide RNA that hybridizes to a fourth CRISPR RNA recognition sequence within the genomic target locus, wherein the fourth guide RNA comprises a fourth tracrRNA and a fourth CRISPR RNA.

3 . The method of claim 2 , wherein:

(a) the first CRISPR RNA recognition sequence and the third CRISPR RNA recognition sequence are separated by about 25 bp to about 1 kb; and

(b) the second CRISPR RNA recognition sequence and the fourth CRISPR RNA recognition sequence are separated by about 25 bp to about 1 kb.

4 . The method of claim 2 , wherein the first and third CRISPR RNA recognition sequences are a first pair of CRISPR RNA recognition sequences, and the second and fourth CRISPR RNA recognition sequences are a second pair of CRISPR RNA recognition sequences, wherein the first pair and second pair are separated by about 25 bp to about 100 Mb.

5 . The method of claim 1 , wherein introducing both the first and second guide RNAs results in increased biallelic modification efficiency compared to introducing either the first guide RNA or the second guide RNA alone.

6 . The method of claim 1 , wherein the nucleic acid insert is inserted between the 5′ and 3′ target sequences.

7 . The method of claim 1 , wherein the biallelic genomic modification comprises a deletion between the first and second CRISPR RNA recognition sequences in both the first and second homologous chromosomes.

8 . The method of claim 7 , wherein the deletion is at least 20 kb.

9 . The method of claim 7 , wherein the biallelic genomic modification further comprises insertion of the nucleic acid insert between the 5′ and 3′ target sequences in both the first and second homologous chromosomes.

10 . The method of claim 1 , wherein the first and second CRISPR RNA recognition sequences are separated by at least 1 kb.

11 . The method of claim 1 , wherein the mouse or rat ES cell is the mouse ES cell.

12 . The method of claim 1 , wherein the targeting vector is a large targeting vector (LTVEC), wherein:

(a) the LTVEC is at least 10 kb;

(b) the sum total of the 5′ and 3′ homology arms of the LTVEC is at least 10 kb;

(c) the LTVEC is from about 50 kb to about 300 kb; or

(d) the sum total of the 5′ and 3′ homology arms of the LTVEC is from about 10 kb to about 200 kb.

13 . The method of claim 1 , wherein the Cas protein is Cas9 and has nuclease activity on both strands of double-stranded DNA.

14 . The method of claim 1 , wherein the first guide RNA comprises the first CRISPR RNA and the first tracrRNA fused together, and the second guide RNA comprises the second CRISPR RNA and the second tracrRNA fused together.

15 . The method of claim 1 , wherein the first guide RNA comprises the first CRISPR RNA and the first tracrRNA, wherein the first CRISPR RNA and the first tracrRNA are separate RNA molecules, and the second guide RNA comprises the second CRISPR RNA and the second tracrRNA, wherein the second CRISPR RNA and the second tracrRNA are separate RNA molecules.

16 . The method of claim 1 , wherein:

(a) the Cas protein is introduced into the mouse or rat ES cell in the form of a protein, a messenger RNA (mRNA) encoding the Cas protein, or a DNA encoding the Cas protein; and/or

(b) the first guide RNA is introduced into the mouse or rat ES cell in the form of an RNA or in the form of a DNA encoding the first guide RNA; and/or

(c) the second guide RNA is introduced into the mouse or rat ES cell in the form of an RNA or in the form of a DNA encoding the second guide RNA.

17 . The method of claim 1 , wherein the first and second CRISPR RNA recognition sequences flank all or part of a coding sequence for a gene.

Assignments (1)

Continuity (6)

References Cited (400)