Abstract: 【Objective】 Using a single base editor to introduce a stop codon at the exon 2 of myostatin(MSTN) gene in Ningxiang pig,and obtaining kidney fibroblast cells with MSTN gene silencing,which was the foundation for the later breeding of MSTN base-editing pigs.【Method】 In this study,a single guide RNA (single guide RNA,sgRNA) was designed at the exon 2 of MSTN gene,and connected to the pMLM3636-puro plasmid to form a recombinant expression vector pMLM3636-puro-MSTN,co-transfected into kidney fibroblast cells with YE1-BE3-FNLS containing the red fluorescent base editor.Under the conditions of red fluorescence and puromycin,single cell colonies were picked.After confirmed by sequencing,the protein expression of the target mutant cell lines was analyzed.【Result】 Base site directed mutation occurred in the exon 2 of MSTN gene,the amino acid sequence of the target site was changed from tryptophan (TGG) to a stop codon (TAA),and the G→A mutation rate was 5.5%.The results of Western blotting showed that the MSTN protein expression in the experimental group No.10 single clone cells decreased by 60% compared with the wild type.【Conclusion】 In this study,single base editing technology was used to introduce stop codon in the coding region of MSTN gene of Ningxiang pig,so that translation was terminated early,resulting in a significant decrease in protein expression,which laid a foundation for the production of MSTN base mutant pigs.

Key words: single base editing; Ningxiang pig; MSTN gene; stop codon