The CRISPR/Cas9 induces large genomic fragment deletions of MSTN and phenotypic changes in sheep

doi:10.1016/S2095-3119(19)62853-4

Journal of Integrative Agriculture ›› 2020, Vol. 19 ›› Issue (4): 1065-1073.DOI: 10.1016/S2095-3119(19)62853-4

收稿日期:2019-03-19 出版日期:2020-04-01 发布日期:2020-03-04

The CRISPR/Cas9 induces large genomic fragment deletions of MSTN and phenotypic changes in sheep

DING Yi^1*, ZHOU Shi-wei^1*, DING Qiang¹, CAI Bei¹, ZHAO Xiao-e², ZHONG Shu¹, JIN Miao-han¹, WANG Xiao-long¹, MA Bao-hua², CHEN Yu-lin¹

¹ College of Animal Science and Technology, Northwest A&F University, Yangling 712100, P.R.China
² College of Veterinary Medicine, Northwest A&F University, Yangling 712100, P.R.China

Received:2019-03-19 Online:2020-04-01 Published:2020-03-04
Contact: Correspondence CHEN Yu-lin, E-mail: chenyulin@nwafu.edu.cn; MA Bao-hua, E-mail: mabh@nwafu.edu.cn
About author:DING Yi, E-mail: 1132600258@qq.com; * These authors contributed equally to this study.
Supported by:
This work was supported by the grants from the National Natural Science Foundation of China (31572369, 31772571, 31872332), the earmarked fund for China Agriculture Research System (CARS-39-12) and the Tan Sheep Breeding Project of Ningxia, China (NXTS201601).

摘要/Abstract

Abstract:

The CRISPR/Cas9 system has been extensively used to engineer genetic loci for the generation of knockouts, insertions, and point mutations in animal models. However, many mutations that have been reported in animals are small insertions or deletions. This study used the CRISPR/Cas9 system to induce large DNA fragment deletions in MSTN via three guide RNAs in sheep. This successfully achieved the precise gene editing of the ovine MSTN gene by injecting both Cas9 mRNA and sgRNAs into embryos at the one-cell stage. Of 10 edited animals, 3 animals (30%) exhibited large genomic fragment deletions (~5 kb). Furthermore, the body weights of these 3 animals were significantly different (P₀<0.0001, P₁₅=0.001, P₃₀=0.005, P₆₀=0.027) between lambs with large deletions and wildtype lambs. In addition, the edited lambs were also significantly different (P₀<0.0001, P₁₅<0.0001, P₃₀=0.002, P₆₀=0.011) compared with wildtype. These results suggest that the generated MSTN knockout sheep is a reliable and effective animal model for further study. Furthermore, this method is time- and labor-saving, and efficient for the creation of animal models for agriculture, biology, and medicine.

Key words: CRISPR/Cas9 , sheep , MSTN , large deletion , genetic engineering

. [J]. Journal of Integrative Agriculture, 2020, 19(4): 1065-1073.

DING Yi, ZHOU Shi-wei, DING Qiang, CAI Bei, ZHAO Xiao-e, ZHONG Shu, JIN Miao-han, WANG Xiao-long, MA Bao-hua, CHEN Yu-lin. The CRISPR/Cas9 induces large genomic fragment deletions of MSTN and phenotypic changes in sheep[J]. Journal of Integrative Agriculture, 2020, 19(4): 1065-1073.

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The CRISPR/Cas9 induces large genomic fragment deletions of MSTN and phenotypic changes in sheep

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