One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering

被引:2994
作者
Wang, Haoyi [1 ]
Yang, Hui [1 ]
Shivalila, Chikdu S. [1 ,2 ]
Dawlaty, Meelad M. [1 ]
Cheng, Albert W. [1 ,3 ]
Zhang, Feng [4 ,5 ]
Jaenisch, Rudolf [1 ,3 ]
机构
[1] Whitehead Inst Biomed Res, Cambridge, MA 02142 USA
[2] MIT, Dept Biol, Cambridge, MA 02139 USA
[3] MIT, Computat & Syst Biol Program, Cambridge, MA 02139 USA
[4] MIT, McGovern Inst Brain Res, Dept Brain & Cognit Sci, Dept Biol Engn, Cambridge, MA 02139 USA
[5] Broad Inst MIT & Harvard, Cambridge, MA 02142 USA
关键词
ZINC-FINGER NUCLEASES; EMBRYO MICROINJECTION; POSTNATAL-DEVELOPMENT; KNOCKOUT RATS; BACTERIA; SYSTEMS; CELLS; CAS9; TET2; ENDONUCLEASE;
D O I
10.1016/j.cell.2013.04.025
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming inter-crossing 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.
引用
收藏
页码:910 / 918
页数:9
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