Editing the cystine knot motif of MSTN enhances muscle development of Liang Guang Small Spotted pigs.

Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) family, and functions as an inhibitor of muscle growth. Disrupting the inhibitory effect of MSTN on growth can provide an effective way to increase the muscle yield of livestock and poultry. The cysteine knot motif of TGF-β can stabilize the structure of MSTN protein and plays an important regulatory role in the biological function of MSTN. Accordingly, in this study, we used the CRISRP/Cas9 to edit the exon 3 of MSTN in the kidney cells of Liang Guang Small Spotted pig (LPKCs), in order to disrupt the cysteine knot motif of MSTN and remove the inhibitory effect of MSTN on its target genes.MSTN-edited LPKCs were obtained through fluorescence-activated cell sorting (FACS) and used as donor cells for somatic cell nuclear transfer (SCNT) to generate cloned embryos, which were then transferred to surrogate sows to finally obtain eight MSTN-edited Liang Guang Small Spotted piglets. Among them, two survived to 10 days old. Genotyping revealed that these two piglets were gene edited heterozygotes with base deletion and substitution occurred within the coding sequence of C106 and C108 at the cystine knot motif of MSTN. These changes resulted in frameshift mutations, and conversion of C106 and C108 to other amino acids. More developments of muscles were observed at the shoulders and hips of the heterozygotes of MSTN-edited Liang Guang Small Spotted pigs. H&E analysis showed that the cross-sectional area (CSA) of myofiber inMSTN-edited pigs was significantly decreased, and the number of myofiber were significantly increased. Western blot analysis showed that the disruption of C106 and C108 did not affect the expression of MSTN protein, but significantly up-regulated the expression of its target genes such as Myf5, MyoD, Myogenin and other myogenic regulatory factors. In summary, the gene-edited pig model obtained in this study did not cause complete loss of MSTN expression, and could retain other biological functions of MSTN, thereby promoting muscle growth while minimizing the potential adverse effects on complete loss of MSTN in the Liang Guang Small Spotted pigs.