alexa One-step generation of mice carrying mutations in multiple genes by CRISPR Cas-mediated genome engineering.
Genetics & Molecular Biology

Genetics & Molecular Biology

Cloning & Transgenesis

Author(s): Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW,

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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.
This article was published in Cell and referenced in Cloning & Transgenesis

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