Generation of pigs with a Belgian Blue mutation in MSTNusing CRISPR/Cpf1-assisted ssODN-mediated homologous recombination

CRISPR/Cpf1 has emerged recently as an effective tool for genome editing in many organisms, but its use in pigs to generate precise genetic modifications has seldom been described. Myostatin (MSTN) is a well-characterized negative regulator of muscle development, and natural mutations in this gene cause a double-muscled phenotype in many species. However, to the best of our knowledge, no naturally occurring mutation in MSTNhas been found in pigs. In addition, no living pig models with sophisticated modifications orthologous to natural mutations in MSTNhave yet been reported. In this study, we exploited the CRISPR/Cpf1 system to introduce a predefined modification orthologous to the natural MSTNmutation found in Belgian Blue cattle (thus known as the Belgian Blue mutation). Our research demonstrated that the cutting efficiency of CRISPR/Cpf1 was 12.3% in mixed porcine fetal fibroblasts in drug free medium, and 41.7% in clonal colonies obtained using G418 selection. Then, the Cpf1-sgRNA vector, ssODN template, and a self-excision cassette were co-transfected into porcine fetal fibroblasts. After G418 selection, 8 clonal colonies were examined and 5 with genetic modification were found. Of these 5, 2 harbored the precise 11-bp deletion. Using 1 heterozygous clonal colony, 2 cloned Duroc piglets were successfully generated, which was heterozygous for the Belgian Blue mutation. In summary, our results demonstrate that CRISPR/Cpf1 system can be used efficiently to generate double-stranded breaks, and also to mediate homologous recombination to introduce precise genomic modifications in pigs.