The potential for gene-editing to increase muscle growth in pigs: experiences with editing myostatin

Abstract Gene-editing holds promise as a new technique for growth promotion in livestock, especially in the face of increased opposition to traditional methods of growth promotion like feed additives. However, to date, there has been only limited progress toward models of growth promotion through gene-editing. The vast majority of gene-editing projects have focused on a single gene, myostatin (MSTN), with several reports of successful editing events. These attempts have been limited by the low efficiency of successful edits and issues of viability. The use of both microinjection and somatic cell nuclear transfer appear to be susceptible to these viability issues. Herein, we report a successful editing of myostatin in pigs using a zinc-finger nuclease targeted at exon 3 of myostatin. Overall, the successful editing rate was 1% (37 edited cell lines/3616 screened cell lines). Edits included a variety of small indels and larger deletions. One male and one female cell line with a deletion of one thymidine residue (− 1 T) were selected for somatic cell nuclear transfer. Of the ~ 900 embryos transferred, there were 12 live births (1 male, 11 females) but only 5 female pigs survived to sexual maturity. These animals were bred to commercial sires to expand the population and determine potential interactive effects between myostatin mutations and the naturally-occurring g.3072G > A mutation in insulin-like growth factor 2. Even in subsequent generations (F0xWT, F1xF0, F1xF1), viability of piglets continued to be poor and was associated with the progeny’s proportion of the original clone genome, even in pigs lacking MSTN loss-of-function (LOF) mutations. However, viability of pigs with myostatin LOF (MSTN −/−) was especially poor as none of the 37 animals born were viable. Sequencing of cloned pigs indicated that off-target effects did not explain this poor viability. Reducing the percentage of the cloned genome by outcrossing successfully improved viability of MSTN +/+ and MSTN ± pigs, but not of MSTN −/− pigs. Characterization of MSTN ± pigs did reveal an increase in muscle growth and body weight compared with MSTN +/+ pigs. Therefore, in order for the promise of gene-editing of myostatin for growth promotion in livestock to be fulfilled, issues of viability of offspring and efficacy of editing have yet to be overcome. Additionally, gene-editing targets other than myostatin must be explored.