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Cell. 2013 May 9;153(4):910-8. doi: 10.1016/j.cell.2013.04.025. Epub 2013 May 2.

One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.

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  • 1Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.

Abstract

Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty--8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.

Copyright © 2013 Elsevier Inc. All rights reserved.

PMID:
23643243
[PubMed - indexed for MEDLINE]
PMCID:
PMC3969854
Free PMC Article
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