Genes, Vol. 14, Pages 1216: Efficient and Specific Generation of MSTN-Edited Hu Sheep Using C-CRISPR

Genes, Vol. 14, Pages 1216: Efficient and Specific Generation of MSTN-Edited Hu Sheep Using C-CRISPR

Genes doi: 10.3390/genes14061216

Authors: 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

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.