title: Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep.
abstract: The CRISPR/Cas9 system provides a flexible approach for genome engineering of genetic loci. Here, we successfully achieved precise gene targeting in sheep by co-injecting one-cell-stage embryos with Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and BCO2). We carefully examined the sgRNAs:Cas9-mediated targeting effects in injected embryos, somatic tissues, as well as gonads via cloning and sequencing. The targeting efficiencies in these three genes were within the range of 27-33% in generated lambs, and that of simultaneously targeting the three genes was 5.6%, which demonstrated that micro-injection of zygotes is an efficient approach for generating gene-modified sheep. Interestingly, we observed that disruption of the MSTN gene resulted in the desired muscle hypertrophy that is characterized by enlarged myofibers, thereby providing the first detailed evidence supporting that gene modifications had occurred at both the genetic and morphological levels. In addition, prescreening for the off-target effect of sgRNAs was performed on fibroblasts before microinjection, to ensure that no detectable off-target mutations from founder animals existed. Our findings suggested that the CRISPR/Cas9 method can be exploited as a powerful tool for livestock improvement by simultaneously targeting multiple genes that are responsible for economically significant traits.
authors: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
authors: Xiaolong
authors: X
authors: Wang
authors: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
authors: Yiyuan
authors: Y
authors: Niu
authors: 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.
authors: Jiankui
authors: J
authors: Zhou
authors: College of Life Science, Yulin University, Yulin 719000, China.
authors: Honghao
authors: H
authors: Yu
authors: Ningxia Tianyuan Sheep Farm, Hongsibu, 751999, China.
authors: Qifang
authors: Q
authors: Kou
authors: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
authors: Anmin
authors: A
authors: Lei
authors: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
authors: Xiaoe
authors: X
authors: Zhao
authors: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.|College of Life Science, Yulin University, Yulin 719000, China.
authors: Hailong
authors: H
authors: Yan
authors: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
authors: Bei
authors: B
authors: Cai
authors: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
authors: Qiaoyan
authors: Q
authors: Shen
authors: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
authors: Shiwei
authors: S
authors: Zhou
authors: College of Life Science, Yulin University, Yulin 719000, China.
authors: Haijing
authors: H
authors: Zhu
authors: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
authors: Guangxian
authors: G
authors: Zhou
authors: Ningxia Tianyuan Sheep Farm, Hongsibu, 751999, China.
authors: Wenzhi
authors: W
authors: Niu
authors: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
authors: Jinlian
authors: J
authors: Hua
authors: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
authors: Yu
authors: Y
authors: Jiang
authors: 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.
authors: Xingxu
authors: X
authors: Huang
authors: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
authors: Baohua
authors: B
authors: Ma
authors: College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
authors: Yulin
authors: Y
authors: Chen
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D006984:Hypertrophy / Q000235:genetics; D008297:Male; D009132:Muscles / Q000378:metabolism* / Q000473:pathology; D009135:Muscular Diseases / Q000235:genetics* / Q000662:veterinary; D055435:Myostatin / Q000235:genetics*; D012756:Sheep; D012757:Sheep Diseases / Q000235:genetics*
chemical_list: D055435:Myostatin