Patent ID: 9546384
Date: 2017-01-17
CPC Classifications: A01K,C12N

Claim:
1. A method for producing an F0 generation mouse that comprises a targeted genetic modification at a genomic locus of interest, the method comprising: (a) contacting a mouse ES cell in vitro with a Cas9protein, a CRISPR RNA that hybridizes to a CRISPR target sequence at the genomic locus of interest, 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 following contacting the mouse ES cell with the Cas9 protein, the CRISPR RNA, and the tracrRNA in the presence of the LTVEC, the genome of the mouse ES cell is modified to comprise a targeted genetic modification comprising deletion of a region of the genomic locus of interest wherein the deletion is at least 30 kb and/or insertion of the insert nucleic acid at the genomic locus of interest wherein the insertion is at least 30 kb; (b) identifying the modified mouse ES cell comprising the targeted genetic modification at the genomic locus of interest; (c) introducing the modified mouse ES cell into a mouse host embryo; and (d) gestating the mouse host embryo in a surrogate mother, wherein the surrogate mother produces the F0 generation mouse comprising the targeted genetic modification at the genomic locus of interest.