METHODS AND COMPOSITIONS FOR THE TARGETED MODIFICATION OF A GENOME

摘要

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.

主权项

1. A method for modifying a genome in a mouse cell or a human cell at a genomic locus of interest, comprising contacting the genome with a Cas protein, a CRISPR RNA that hybridizes to a CRISPR target sequence at the genomic locus of interest, and a tracrRNA,
wherein the genome is contacted in the presence of a large targeting vector (LTVEC) that is at least 10 kb and comprises an insert nucleic acid flanked with:
(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 insert nucleic acid is at least 30 kb and/or the 5′ target sequence and the 3′ target sequence are separated by at least 30 kb; wherein following contacting with the Cas protein, CRISPR RNA, and tracrRNA in the presence of the LTVEC, the genome comprises a targeted genetic modification at the genomic locus of interest, wherein the modified genomic locus of interest comprises the insert nucleic acid.