Volume 27, No 7, Jul 2017

ISSN: 1001-0602 
EISSN: 1748-7838 2018 
impact factor 17.848* 
(Clarivate Analytics, 2019)

Volume 27 Issue 7, July 2017: 933-945   |  Open Access

ORIGINAL ARTICLES

One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs

Erwei Zuo^1,*, Yi-Jun Cai^1,*, Kui Li^1,*, Yu Wei^1,2,4,*, Bang-An Wang^3,*, Yidi Sun⁶, Zhen Liu^1,8, Jiwei Liu¹, Xinde Hu¹, Wei Wei^1,5, Xiaona Huo¹, Linyu Shi^1,7, Cheng Tang^1,8, Dan Liang², Yan Wang¹, Yan-Hong Nie¹, Chen-Chen Zhang¹, Xuan Yao^1,8, Xing Wang^1,8, Changyang Zhou^1,8, Wenqin Ying¹, Qifang Wang¹, Ren-Chao Chen¹, Qi Shen¹, Guo-Liang Xu³, Jinsong Li², Qiang Sun¹, Zhi-Qi Xiong¹ and Hui Yang¹

¹Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
²State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
³State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China
⁴Shanghai University, Shanghai 200444, China
⁵College of Animal Science and Technology, Guangxi University, Nanning, Guangxi 530004, China
⁶Key Lab of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
⁷Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China
⁸College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China Correspondence: Hui Yang, E-mail: huiyang@ion.ac.cn; Zhi-Qi Xiong, E-mail: xiongzhiqi@ion.ac.cn; Qiang Sun,(qsun@ion.ac.cn)

The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs (spaced 10-200 bp apart) that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficiencies (100% on Arntl and 91% on Prrt2) in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models.

10.1038/cr.2017.81

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