Efficient and Specific Generation of MSTN-Edited Hu Sheep Using C-CRISPR

Rihong Guo; Huili Wang; Chunhua Meng; Hongbing Gui; Yinxia Li; Fang Chen; Chenjian Zhang; Han Zhang; Qiang Ding; Jianli Zhang; Jun Zhang; Yong Qian; Jifeng Zhong; Shaoxian Cao

doi:10.3390/genes14061216

Efficient and Specific Generation of MSTN-Edited Hu Sheep Using C-CRISPR

Genes (Basel). 2023 Jun 2;14(6):1216. doi: 10.3390/genes14061216.

Authors

Rihong Guo^{1

2}, Huili Wang^{1

2}, Chunhua Meng^{1

2}, Hongbing Gui¹, Yinxia Li^{1

2}, Fang Chen^{1

2}, Chenjian Zhang¹, Han Zhang¹, Qiang Ding^{1

2}, Jianli Zhang^{1

2}, Jun Zhang^{1

2}, Yong Qian^{1

2}, Jifeng Zhong^{1

2}, Shaoxian Cao^{1

2

3}

Affiliations

¹ Jiangsu Provincial Engineering Research Center for Precision Animal Breeding, Nanjing 210014, China.
² Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.
³ Key Laboratory of Crop and Animal Integrated Farming, Ministry of Agriculture and Rural Affairs, Nanjing 210014, China.

Abstract

Hu sheep, an indigenous breed in China known for its high fecundity, are being studied to improve their growth and carcass traits. MSTN is a negative regulator of muscle development, and its inactivation results in muscularity. The C-CRISPR system, utilizing multiple neighboring sgRNAs targeting a key exon, has been successfully used to generate genes for complete knockout (KO) monkeys and mice in one step. In this study, the C-CRISPR system was used to generate MSTN-edited Hu sheep; 70 embryos injected with Cas9 mRNA and four sgRNAs targeting exon 3 of sheep MSTN were transferred to 13 recipients. Out of 10 lambs born from five recipients after full-term pregnancies, nine had complete MSTN KO with various mutations. No off-target effects were found. These MSTN-KO Hu sheep showed a double-muscled (DM) phenotype, characterized by a higher body weight at 3 and 4 months old, prominent muscular protrusion, clearly visible intermuscular groves, and muscle hypertrophy. The molecular analysis indicated enhanced AKT and suppressed ERK1/2 signaling in the gluteus muscle of the edited Hu sheep. In conclusion, MSTN complete KO Hu sheep with a DM phenotype were efficiently and specifically generated using C-CRISPR, and the C-CRISPR method is a promising tool for farm animal breeding.

Keywords: CRISPR/Cas9; Hu sheep; MSTN; double muscling phenotype; off-target effects.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Animals, Genetically Modified
CRISPR-Cas Systems*
Female
Mice
Muscle, Skeletal / metabolism
Mutation
Myostatin* / genetics
Myostatin* / metabolism
Pregnancy
Sheep / genetics

Substances

Myostatin

Grants and funding

This study was supported by the Jiangsu Provincial Key R&D Program–Modern Agriculture (grant no. BE2019373) and the Jiangsu Province Agricultural Science and Technology Independent Innovation Fund Project (grant no. CX(21)3128).