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Int J Biochem Cell Biol. 2014 Jan;46:49-55. doi: 10.1016/j.biocel.2013.10.010. Epub 2013 Nov 20.

One-step generation of different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering.

Author information

1
MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, National Resource Center for Mutant Mice, Nanjing 210061, China.
2
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
3
Key Laboratory of Molecular Virology and Immunology, Institute Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 225 South Chongqing Road, Shanghai, China.
4
Key Laboratory of Molecular Virology and Immunology, Institute Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 225 South Chongqing Road, Shanghai, China. Electronic address: qbleng@sibs.sc.cn.
5
MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, National Resource Center for Mutant Mice, Nanjing 210061, China. Electronic address: xingxuhuang@mail.nju.edu.cn.

Abstract

Taking advantage of the multiplexable genome engineering feature of the CRISPR/Cas9 system, we sought to generate different kinds of immunodeficient mouse strains by embryo co-microinjection of Cas9 mRNA and multiple sgRNAs targeting mouse B2m, Il2rg, Prf1, Prkdc, and Rag1. We successfully achieved multiple gene modifications, fragment deletion, double knockout of genes localizing on the same chromosome, and got different kinds of immunodeficient mouse models with different heritable genetic modifications at once, providing a one-step strategy for generating different immunodeficient mice which represents significant time-, labor-, and money-saving advantages over traditional approaches. Meanwhile, we improved the technology by optimizing the concentration of Cas9 and sgRNAs and designing two adjacent sgRNAs targeting one exon for each gene, which greatly increased the targeting efficiency and bi-allelic mutations.

KEYWORDS:

Cas9; Gene targeting; Immunodeficient; Mouse; Multiple

PMID:
24269190
DOI:
10.1016/j.biocel.2013.10.010
[Indexed for MEDLINE]

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