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.
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