Methods and compositions for the targeted modification of a genome
AssigneeRegeneron Pharmaceuticals, Inc.
InventorsDavid Frendewey, Wojtek Auerbach, Ka-Man Venus Lai, David M. Valenzuela, George D. Yancopoulos
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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.
Claims24
An in vitro method for modifying a genome at a genomic locus of interest in a mouse ES cell, comprising:
- contacting the mouse ES cell with a Cas9 protein, 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.
Dependencies are inferred from the claim text.