Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep.

Xiaolong Wang,Qifang Kou,Jinlian Hua,Anmin Lei,Yiyuan Niu,Yulin Chen,Wenzhi Niu,Honghao Yu,Guangxian Zhou,Hailong Yan,Xiaoe Zhao,Qiaoyan Shen,Bei Cai,Yu Jiang,Xingxu Huang,Baohua Ma,Shiwei Zhou,Haijing Zhu,Jiankui Zhou

doi:10.1038/srep32271

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.

Highlights

Sheep are an economically important livestock that serve as a resource for various products, as well as a significant disease model in biomedical research, including bone healing[1,2], cardiology[3], and reproduction[4]
These results demonstrate the efficient multiple gene targeting by clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas[9], and provide the first detailed evidence of economic trait improvement by gene targeting in sheep
Genotyping using T7 endonuclease I (T7EI) showed that PCR fragments from genome targeting by sgRNAs were cut into expected bands (Supplementary Fig. S1a,b,d and f), indicating that the CRISPR/ Cas[9] system can mediate efficient genome editing in sheep fibroblasts

Summary

Introduction

Sheep are an economically important livestock that serve as a resource for various products (e.g., meat, wool, and milk), as well as a significant disease model in biomedical research, including bone healing[1,2], cardiology[3], and reproduction[4]. Efficiently targeting multiple loci simultaneously, and the expression of desired traits in sheep remains to be established. To this end, in the present study, we targeted three functional genes, including the myostatin (MSTN), agouti signaling protein (ASIP), and beta-carotene oxygenase 2 (BCO2), by Cas9/sgRNA, and carefully analyzed the phenotype resulting from the disruption of the MSTN gene. The carcass with yellow fat ( known as yellow fat disease or panniculitis), occasionally observed in sheep leads to metabolic diseases and may sometimes be lethal[28] These results demonstrate the efficient multiple gene targeting by CRISPR/Cas[9], and provide the first detailed evidence of economic trait improvement by gene targeting in sheep

Methods

Results

Conclusion