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Journal of Integrative Agriculture  2020, Vol. 19 Issue (4): 1065-1073    DOI: 10.1016/S2095-3119(19)62853-4
Special Issue: Animal Science
Animal Science · Veterinary Medicine Advanced Online Publication | Current Issue | Archive | Adv Search |
The CRISPR/Cas9 induces large genomic fragment deletions of MSTN and phenotypic changes in sheep
DING Yi1*, ZHOU Shi-wei1*, 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
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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.
 
Keywords:  CRISPR/Cas9        sheep        MSTN        large deletion        genetic engineering  
Received: 19 March 2019   Accepted: 04 March 2020
Fund: 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).
Corresponding Authors:  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.

Cite this article: 

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. 2020. The CRISPR/Cas9 induces large genomic fragment deletions of MSTN and phenotypic changes in sheep. Journal of Integrative Agriculture, 19(4): 1065-1073.

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