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Cell. 2014 Feb 13;156(4):836-43. doi: 10.1016/j.cell.2014.01.027. Epub 2014 Jan 30.

Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos.

Author information

1
Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China; Kunming Biomed International and National Engineering Research Center of Biomedicine and Animal Science, Kunming 650500, China.
2
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.
3
State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China.
4
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
5
Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Sun Yat-Sen University, Guangzhou 510080, China.
6
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.
7
Yunnan Key Laboratory of Primate Biomedical Research, Kunming 650500, China; Kunming Biomed International and National Engineering Research Center of Biomedicine and Animal Science, Kunming 650500, China. Electronic address: wji@kbimed.com.
8
State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China. Electronic address: shajh@njmu.edu.cn.

Abstract

Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of monkeys in biomedical researches has been significantly hindered by the difficulties in producing animals genetically modified at the desired target sites. Here, we first applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, we successfully achieve precise gene targeting in cynomolgus monkeys. We also show that this system enables simultaneous disruption of two target genes (Ppar-γ and Rag1) in one step, and no off-target mutagenesis was detected by comprehensive analysis. Thus, coinjection of one-cell-stage embryos with Cas9 mRNA and sgRNAs is an efficient and reliable approach for gene-modified cynomolgus monkey generation.

PMID:
24486104
DOI:
10.1016/j.cell.2014.01.027
[Indexed for MEDLINE]
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