METHODS AND COMPOSITIONS FOR THE TARGETED MODIFICATION OF A GENOME
First Claim
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.
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Accused Products
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.
105 Citations
27 Claims
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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Specification