Methods and compositions for the targeted modification of a mouse ES cell genome

Patent metadata

Field	Value
Publication number	US-10711280-B2
Application number	US-201816224413-A
Country	US
Kind code	B2
Filing date	Dec 18, 2018
Priority date	Dec 11, 2013
Publication date	Jul 14, 2020
Grant date	Jul 14, 2020

Abstract

Compositions and methods are provided for modifying a genomic locus of interest in a eukaryotic cell, a mammalian cell, a human cell or a non-human mammalian cell using a large targeting vector (LTVEC) comprising various endogenous or exogenous nucleic acid sequences as described herein. Further methods combine the use of the LTVEC with a CRISPR/Cas system. Compositions and methods for generating a genetically modified non-human animal comprising one or more targeted genetic modifications in their germline are also provided.

First claim

We claim:

1. An in vitro method for modifying a genome at a genomic locus of interest in a mouse embryonic stem (ES) cell, comprising:
introducing into the mouse ES cell a Cas9 protein or a nucleic acid encoding the Cas9 protein, a guide RNA or a DNA encoding the guide RNA, wherein the guide RNA comprises a CRISPR RNA that hybridizes to a CRISPR target sequence at the genomic locus of interest and a tracrRNA, and a large targeting vector (LTVEC) that is at least 10 kb in size and comprises an insert nucleic acid flanked by:
(i) a 5′ homology arm that is homologous to a 5′ target sequence at the genomic locus of interest; and
(ii) a 3′ homology arm that is homologous to a 3′ target sequence at the genomic locus of interest,
wherein the guide RNA is designed to avoid recognition of any sequence in the insert nucleic acid, and
wherein following introducing into the mouse ES cell the Cas9 protein or the nucleic acid encoding the Cas9 protein, the guide RNA or the DNA encoding the guide RNA, and the LTVEC, the genome of the mouse ES cell is modified to comprise a targeted genetic modification comprising (i) deletion of a region of the genomic locus of interest wherein the deletion is at least 30 kb and/or (ii) insertion of the insert nucleic acid at the genomic locus of interest wherein the insertion is at least 30 kb.

2. The method of claim 1 , wherein the targeted genetic modification comprises the deletion of the region of the genomic locus of interest, and the guide RNA is designed to create a double strand break within the region of the genomic locus of interest targeted for deletion.

3. The method of claim 2 , wherein the CRISPR target sequence is within the 5′ end of the region of the genomic locus of interest targeted for deletion.

4. The method of claim 3 , wherein the CRISPR target sequence is 50 to 1000 base pairs from the deletion endpoint.

5. The method of claim 2 , wherein the CRISPR target sequence is within the 3′ end of the region of the genomic locus of interest targeted for deletion.

6. The method of claim 5 , wherein the CRISPR target sequence is 50 to 1000 base pairs from the deletion endpoint.

7. The method of claim 1 , wherein the CRISPR target sequence is located in an intron, an exon, a promoter, an enhancer, a regulatory region, or any non-protein coding region.

8. The method of claim 1 , wherein the CRISPR target sequence is located within a coding region of a gene or within a regulatory region that influences expression of the gene.

9. The method of claim 1 , wherein the guide RNA comprises SEQ ID NO: 2, 3, 4, 5, 6, 7, or 8.

10. The method of claim 9 , wherein the guide RNA comprises SEQ ID NO: 3, 4, 5, or 7.

11. The method of claim 1 , wherein the CRISPR RNA and the tracrRNA are introduced as a single nucleic acid molecule comprising the CRISPR RNA and the tracrRNA.

12. The method of claim 11 , wherein the single nucleic acid molecule comprises the CRISPR RNA and the tracrRNA fused together in the form of a single guide RNA (sgRNA).

13. The method of claim 1 , wherein the CRISPR RNA and the tracrRNA are introduced separately.

14. The method of claim 1 , wherein the method comprises introducing into the mouse ES cell:
(a) the guide RNA and the Cas9 protein;
(b) the guide RNA and the nucleic acid encoding the Cas9 protein, wherein the nucleic acid encoding the Cas9 protein is a messenger RNA (mRNA) encoding the Cas9 protein; or
(c) the DNA encoding the guide RNA and the nucleic acid encoding the Cas9 protein, wherein the nucleic acid encoding the Cas9 protein is DNA.

15. The method of claim 14 , wherein the Cas9 protein and the guide RNA are introduced as a protein-RNA complex.

16. The method of claim 14 , wherein the method comprises introducing in to the mouse ES cell the DNA encoding the guide RNA and the nucleic acid encoding the Cas9 protein, wherein the nucleic acid encoding the Cas9 protein is DNA, and wherein:
(I) (a) the DNA encoding the Cas9 protein is in the form of a first expression construct comprising a first promoter operably linked to a first nucleic acid encoding the Cas9 protein;
 (b) the DNA encoding the CRISPR RNA of the guide RNA is in the form of a second expression construct comprising a second promoter operably linked to a second nucleic acid encoding the CRISPR RNA; and
 (c) the DNA encoding the tracrRNA of the guide RNA is in the form of a third expression construct comprising a third promoter operably linked to a third nucleic acid encoding the tracrRNA;
 wherein the first, second, and third promoters are active in the mouse ES cell, and
 wherein the first, second, and third expression constructs are on a single nucleic acid molecule or on multiple nucleic acid molecules; or
(II) (a) the DNA encoding the Cas9 protein is in the form of a first expression construct comprising a first promoter operably linked to a first nucleic acid encoding the Cas9 protein; and
 (b) the DNA encoding the CRISPR RNA of the guide RNA and the DNA encoding the tracrRNA of the guide RNA are in the form of a second expression construct comprising a second promoter operably linked to a second nucleic acid encoding a gRNA comprising the CRISPR RNA and the tracrRNA;
 wherein the first and second promoters are active in the mouse ES cell, and
 wherein the first and second expression constructs are on a single nucleic acid molecule or on separate nucleic acid molecules.

17. The method of claim 1 , wherein the targeted genetic modification comprises simultaneous deletion of an endogenous nucleic acid sequence at the genomic locus of interest and insertion of the insert nucleic acid at the genomic locus of interest.

18. The method of claim 17 , wherein the deleted endogenous nucleic acid sequence is from 30 kb to about 110 kb, and the insert nucleic acid is from about 40 kb to about 140 kb.

19. The method of claim 1 , wherein the targeted genetic modification is a biallelic genetic modification.

20. The method of claim 19 , wherein:
(a) the biallelic genetic modification comprises deletion of an endogenous nucleic acid sequence and insertion of the insert nucleic acid at the genomic locus of interest in two homologous chromosomes; or
(b) the modified mouse ES cell is compound heterozygous or hemizygous at the genomic locus of interest.

21. The method of claim 20 , wherein the targeted genetic modification at the genomic locus of interest in one chromosome comprises deletion of an endogenous nucleic acid sequence and insertion of the insert nucleic acid.

22. The method of claim 21 , wherein the targeted genetic modification comprises: (1) deletion of an endogenous nucleic acid sequence at the genomic locus of interest in first and second homologous chromosomes; and (2) insertion of the insert nucleic acid into the genomic locus of interest in the first homologous chromosome and disruption of the genomic locus of interest in the second homologous chromosome.

23. The method of claim 1 , wherein the LTVEC is at least 40 kb; or
wherein the targeted genetic modification comprises deletion of a region of the genomic locus of interest wherein the deletion is at least 30 kb, and the LTVEC is at least 15 kb.

24. The method of claim 1 , wherein the targeted genetic modification comprises insertion of the insert nucleic acid at the genomic locus of interest, wherein the insert nucleic acid is at least 40 kb; or
wherein the targeted genetic modification comprises deletion of a region of the genomic locus of int

Assignees

• Regeneron Pharma

Inventors

• Frendewey David
• Auerbach Wojtek
• Lai Ka-Man Venus
• Valenzuela David M
• Yancopoulos George D

Classifications

• C12N15/907 Primary

  in mammalian cells · CPC title

• A01K2217/072

  maintaining or altering function, i.e. knock in · CPC title

• C12N15/85

  for animal cells · CPC title

• A01K2227/105

  Murine · CPC title

• A01K2267/0362

  Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes · CPC title