Generation Of Porcine Somatic Cell Lines Carrying Naturally Mutation In MSTN Locus Through CRISPR/Cas9- Mediated Genome Editing

Myostatin,also known as GDF-8 (growth differentiation factor 8),with the typical structure of TGF-P superfamily, is a major negative regulator of skeletal muscle development factor. McPherron (1997) knocked out the MSTN gene in mice for the first time, and found that the MSTN knockout mice showed obvious double muscle trait. It was muscle hyperplasia and hypertrophy that caused a significant increase in muscle in mice. Later, natural mutations of MSTN were found in catties, dogs, sheeps and human. These natural mutations can cause obvious double muscle trait.CRISPR/Cas9 is a newly developed simple and efficient gene editing technology, which is able to identify and cut a specific DNA sequence guided by RNA, forming a double-strand break. Though a non-homologous end joining repair mechanism, it can introduce indels mutations, or through a homologous recombination repair mechanism, it can mediate a single base or long DNA fragments replacement. So far, several groups have successfully knocked out MSTN gene in some large animals by using ZFN, TALEN and CRISPR/Cas9 technologies. However, the double muscle trait produced by these animals were not so strking as those animals produced by the natural mutation of the MSTN gene. Significant increase in the muscles of the forelimb, hind limb and pectoral were not observed. To generate MSTN mutant pigs with apparent double muscle, in this study, through the combination of CRISPR/Cas9 system and ssODNs, we try to create precise mutations similar to that naturally occurred in pMSTN.In order to mimck the natural mutations of MSTN, we selected two MSTN natural mutation sites, which were found in Piedmontese cattle and dogs, respectively. In Piedmontese cattle, a G>A mutation occurs, which encodes tyrosine, in stead of cysteine, disrupting the typical cysteine knot structure of TGF-P superfamily; In dog, a 939-940 dels happens and the number 313 amino acid (cysteine) become stop codon.We first designed gRNA and two single-stranded oligonucleotides containing restriction sites, corresponding to natural mutation of MSTN in Piedmontese cattle and dogs, respectively. Then DNA vectors were electrotransfected into porcine fibroblasts. The cells were treated with G418 for selection of the potential positive clones. The cell conolies were expanded and the mutation pattten was confirmed by PCR and DNA sequence.134 clones with dog's natural mutations were picked.55 of them were tested, 5 homozygous positive clones we achieved (5/55,10%); 70 clones the piedmontese cattle's natural mutations were picked, and 37 colonies were assayed,1 homozygous positive clone was and 2 heterozygous positive clones were identified (3/37,8.1%).Then we conducted somatic cell nuclear transfer by using the positive cells as nuclear donors. When the reconstructed embryso cultured in vitro, the blastocyst rate were 27.3%, appearing normal. The 490 cloned embryos were tentatively transferred into 5 surrogates. One of them was pregnant and developed to term. One cloned pigpet was deliverd and died 5 days after birth. The ear tissues were collected for genotype idetification. PCR test and DNA sequencing showed that that this piget had homozygous mutation of 936G-A found in a natural mutation piedmontese cattle.In summary, for the first time, by combining CRISPR/Cas9 technology with ssODN, we successfully mimick the natural mutations of MSTN in animals, laying foundation for creation of more muscular cloned pigs. In addition, since the mutations in this gene-editing pig have being naturally existed in domestic animals and applied in animal husbandry practice for many years, the genetically modified pork is expected to be accepted by public more easily.