利用单碱基编辑系统定点编辑哈萨克羊MSTN基因的研究

doi:10.11843/j.issn.0366-6964.2021.08.009

Abstract

Abstract: Myostatin (MSTN) can negatively regulate the growth and development of skeletal muscle. In this study, the MSTN gene exon of Kazakh sheep was edited to create a stop codon in order to knock out the MSTN gene by using cytidine base editor (CBE). Two sgRNAs targeting to loci across exons of MSTN gene of Kazakh sheep were designed in the study. The sgRNAs were ligated to pGL3-U6-sgRNA-PGK-puromycin plasmid and then co-transfected with pCMV-AncBE4 max-P2A-GFP plasmid into Kazakh sheep fetal fibroblasts. The Cruiser^TM assay, Sanger sequencing and TA clone analysis were performed. The results showed that two sgRNAs could create stop codons in the MSTN gene exons of Kazakh sheep. The editing efficiency of the STOPsg-1 target site was 26.7%, and the editing efficiency of the STOPsg-2 target site was 6.7%. In this study, the CBE (AncBE4 max) technique was successfully used to edit the coding region of MSTN gene in fetal fibroblasts of Kazak sheep in the present study, which lay a technical foundation for breeding Kazakh sheep with precise editing of MSTN gene.

Key words: Kazakh sheep, base editors, MSTN

CLC Number:

S826.2

YAO Xudong, MENG Yaqi, REN Xiumeiao, GUO Yanhua, TANG Hong, ZHANG Yiyuan, WANG Limin, ZHOU Ping. Study of Kazakh Sheep MSTN Gene Site-directed Editing Based on the Base Editing System[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(8): 2162-2170.

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Study of Kazakh Sheep MSTN Gene Site-directed Editing Based on the Base Editing System

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