Methods and compositions for the targeted modification of a genome

Patent metadata

Field	Value
Publication number	US-11820997-B2
Application number	US-202016891978-A
Country	US
Kind code	B2
Filing date	Jun 3, 2020
Priority date	Dec 11, 2013
Publication date	Nov 21, 2023
Grant date	Nov 21, 2023

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 contacting the genome with:
(a) a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated 9 (Cas9) endonuclease; (b) a CRISPR RNA (crRNA) that hybridizes to a CRISPR target sequence at the genomic locus of interest; (c) a trans-activating CRISPR RNA (tracrRNA); and (d) a large targeting vector (LTVEC) comprising an insert nucleic acid that is at least 20 kb in size 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 5′ homology arm is at least 5 kb and/or the 3′ homology arm is at least 5 kb, and

wherein following the contacting, the genome of the mouse ES cell is modified to comprise a targeted genetic modification comprising insertion of the insert nucleic acid at the genomic locus of interest, wherein the insertion is at least 20 kb.

2. The method of claim 1 , wherein the method comprises introducing into the mouse ES cell:
(a) the Cas9 endonuclease, wherein the Cas9 endonuclease is introduced in the form of a protein, a messenger RNA (mRNA) encoding the Cas9 endonuclease, or a DNA encoding the Cas9 endonuclease;
(b) the crRNA, wherein the crRNA is introduced in the form of an RNA or a DNA encoding the crRNA;
(c) the tracrRNA, wherein the tracrRNA is introduced in the form of an RNA or a DNA encoding the tracrRNA; and
(d) the LTVEC.

3. The method of claim 2 , wherein the crRNA and the tracrRNA are introduced as a single nucleic acid molecule comprising the crRNA and the tracrRNA.

4. The method of claim 2 , wherein the method comprises introducing into the mouse ES cell:
(a) the crRNA, the tracrRNA, and the Cas9 endonuclease;
(b) the crRNA, the tracrRNA, and the mRNA encoding the Cas9 endonuclease; or
(c) the DNA encoding the crRNA, the DNA encoding the tracrRNA, and the DNA encoding the Cas9 endonuclease.

5. The method of claim 4 , wherein the Cas9 endonuclease, the crRNA, and the tracrRNA are introduced as a protein-RNA complex.

6. The method of claim 4 , wherein the method comprises introducing into the mouse ES cell the DNA encoding the crRNA, the DNA encoding the tracrRNA, and the DNA encoding the Cas9 endonuclease, and wherein:
(I) (a) the DNA encoding the Cas9 endonuclease is in a first expression construct comprising a first promoter operably linked to a first nucleic acid encoding the Cas9 endonuclease;
(b) the DNA encoding the crRNA is in a second expression construct comprising a second promoter operably linked to a second nucleic acid encoding the crRNA; and
(c) the DNA encoding the tracrRNA is in 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 endonuclease is in a first expression construct comprising a first promoter operably linked to a first nucleic acid encoding the Cas9 endonuclease; and
(b) the DNA encoding the crRNA and the DNA encoding the tracrRNA are in a second expression construct comprising a second promoter operably linked to a second nucleic acid encoding the crRNA 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.

7. The method of claim 1 , wherein the insert nucleic acid is at least 50 kb in size.

8. The method of claim 7 , wherein the insert nucleic acid is at least 100 kb in size.

9. The method of claim 1 , wherein the insertion is at least 50 kb in size.

10. The method of claim 9 , wherein the insertion is at least 100 kb in size.

11. The method of claim 1 , wherein the crRNA and the tracrRNA are fused together in the form of a single guide RNA (sgRNA).

12. The method of claim 1 , wherein the crRNA and the tracrRNA are separate molecules.

13. 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.

14. The method of claim 13 , wherein the deletion is at least 20 kb, and the insert nucleic acid is at least 20 kb, at least 50 kb, or at least 100 kb.

15. The method of claim 13 , wherein the deleted endogenous nucleic acid sequence is from 30 kb to 110 kb, and the insert nucleic acid is from 40 kb to 140 kb.

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

17. The method of claim 16 , 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.

18. The method of claim 17 , 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.

19. The method of claim 18 , 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.

20. The method of claim 1 , wherein the CRISPR target sequence is immediately flanked by a Protospacer Adjacent Motif (PAM) sequence.

21. The method of claim 1 , wherein the targeted genetic modification comprises:
(I) replacement of an endogenous nucleic acid sequence with a homologous nucleic acid sequence or an orthologous nucleic acid sequence;
(II) deletion of an endogenous nucleic acid sequence;
(III) insertion of an exogenous nucleic acid sequence;
(IV) insertion of an exogenous nucleic acid sequence comprising a homologous nucleic acid sequence or an orthologous nucleic acid sequence;
(V) insertion of a selectable marker; or
(VI) a combination thereof.

22. The method of claim 21 , wherein the targeted genetic modification comprises insertion of one or more human antibody heavy chain variable domains, one or more human kappa light chain variable domains, or one or more human lambda light chain variable domains.

23. The method of claim 1 , wherein the crRNA is designed to avoid recognition of any sequence in the insert nucleic acid.

24. The method of claim 1 , wherein the targeted genetic modification comprises a deletion of a region of the genomic locus of interest, and the CRISPR target sequence is within the region of the genomic locus of interest targeted for deletion.

25. The method of claim 24 , wherein the CRISPR target sequence is within the 5′ end of the region of the genomic locus of interest targeted for deletion or the 3′ end of the region of the genomic

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

• C12N15/8509 Primary

  for producing genetically modified animals, e.g. transgenic · CPC title

• A01K67/0276

  Knock-out vertebrates · CPC title

• A01K67/0278

  Knock-in vertebrates, e.g. humanised vertebrates · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title