Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos.
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.
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.
Yuyu
Y
Niu
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.
Bin
B
Shen
State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China.
Yiqiang
Y
Cui
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.
Yongchang
Y
Chen
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.
Jianying
J
Wang
State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China.
Lei
L
Wang
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.
Yu
Y
Kang
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
Xiaoyang
X
Zhao
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.
Wei
W
Si
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
Wei
W
Li
Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Sun Yat-Sen University, Guangzhou 510080, China.
Andy Peng
AP
Xiang
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.
Jiankui
J
Zhou
State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China.
Xuejiang
X
Guo
State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China.
Ye
Y
Bi
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.
Chenyang
C
Si
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.
Bian
B
Hu
State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China.
Guoying
G
Dong
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.
Hong
H
Wang
State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China.
Zuomin
Z
Zhou
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.
Tianqing
T
Li
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.
Tao
T
Tan
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.
Xiuqiong
X
Pu
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.
Fang
F
Wang
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.
Shaohui
S
Ji
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
Qi
Q
Zhou
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.
Xingxu
X
Huang
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.
Weizhi
W
Ji
State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China. Electronic address: shajh@njmu.edu.cn.
Jiahao
J
Sha
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004622:Embryo, Mammalian / Q000378:metabolism; D005260:Female; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D008252:Macaca fascicularis / Q000235:genetics*; D008969:Molecular Sequence Data; D009030:Mosaicism; D016415:Sequence Alignment