Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system.
Recent advances in the study of the CRISPR/Cas9 system have provided a precise and versatile approach for genome editing in various species. However, the applicability and efficiency of this method in large animal models, such as the goat, have not been extensively studied. Here, by co-injection of one-cell stage embryos with Cas9 mRNA and sgRNAs targeting two functional genes (MSTN and FGF5), we successfully produced gene-modified goats with either one or both genes disrupted. The targeting efficiency of MSTN and FGF5 in cultured primary fibroblasts was as high as 60%, while the efficiency of disrupting MSTN and FGF5 in 98 tested animals was 15% and 21% respectively, and 10% for double gene modifications. The on- and off-target mutations of the target genes in fibroblasts, as well as in somatic tissues and testis of founder and dead animals, were carefully analyzed. The results showed that simultaneous editing of several sites was achieved in large animals, demonstrating that the CRISPR/Cas9 system has the potential to become a robust and efficient gene engineering tool in farm animals, and therefore will be critically important and applicable for breeding.
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Xiaolong
X
Wang
Shaanxi Provincial Engineering and Technology Research Center of Cashmere Goats, Yulin University, Yulin 719000, China.|Life Science Research Center, Yulin University, Yulin 719000, China.
Honghao
H
Yu
College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
Anmin
A
Lei
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.|School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
Jiankui
J
Zhou
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Wenxian
W
Zeng
Shaanxi Provincial Engineering and Technology Research Center of Cashmere Goats, Yulin University, Yulin 719000, China.|Life Science Research Center, Yulin University, Yulin 719000, China.
Haijing
H
Zhu
School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
Zhiming
Z
Dong
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Yiyuan
Y
Niu
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Bingbo
B
Shi
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Bei
B
Cai
Shaanxi Provincial Engineering and Technology Research Center of Cashmere Goats, Yulin University, Yulin 719000, China.|Life Science Research Center, Yulin University, Yulin 719000, China.
Jinwang
J
Liu
Shaanxi Provincial Engineering and Technology Research Center of Cashmere Goats, Yulin University, Yulin 719000, China.|Life Science Research Center, Yulin University, Yulin 719000, China.
Shuai
S
Huang
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.|Shaanxi Provincial Engineering and Technology Research Center of Cashmere Goats, Yulin University, Yulin 719000, China.|Life Science Research Center, Yulin University, Yulin 719000, China.
Hailong
H
Yan
College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
Xiaoe
X
Zhao
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Guangxian
G
Zhou
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Xiaoling
X
He
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Xiaoxu
X
Chen
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Yuxin
Y
Yang
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Yu
Y
Jiang
Shaanxi Provincial Engineering and Technology Research Center of Cashmere Goats, Yulin University, Yulin 719000, China.|Life Science Research Center, Yulin University, Yulin 719000, China.
Lei
L
Shi
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Xiue
X
Tian
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Yongjun
Y
Wang
College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
Baohua
B
Ma
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.|School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
Xingxu
X
Huang
Shaanxi Provincial Engineering and Technology Research Center of Cashmere Goats, Yulin University, Yulin 719000, China.|Life Science Research Center, Yulin University, Yulin 719000, China.
Lei
L
Qu
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Yulin
Y
Chen
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D051521:Fibroblast Growth Factor 5 / Q000737:chemistry / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism; D018390:Gene Targeting; D006041:Goats / Q000235:genetics*; D008845:Microinjections; D008969:Molecular Sequence Data; D055435:Myostatin / Q000737:chemistry / Q000235:genetics*; D010641:Phenotype; D017393:RNA Editing; D000094704:RNA, Guide, CRISPR-Cas Systems; D016415:Sequence Alignment; D015053:Zygote / Q000378:metabolism*
D055435:Myostatin; D000094704:RNA, Guide, CRISPR-Cas Systems; D051521:Fibroblast Growth Factor 5