Knockout Of MSTN Gene By CRISPR/Cas9 In Ovine Muscle Satellite Cells

CRISPR(clustered regulatory interspaced short palindromic repeat) found in bacteria and archaeagenome which contains multiple short repeats loci, provides acquired immunity against invadingforeign DNA via RNA-guided DNA cleavage. CRISPR/Cas9 system can direct sequence-specificcleavage and generate a double-strand break(DSB) by Cas9 proteins. Based on this, various geneticmethods, including gene targeting, gene addition, gene correction etc., are being designed to manipulatethe genomes of different species at specific loci. Myostatin is a transforming growth factor-β familymember that negatively regulates muscle mass. Naturally occurring MSTN mutations have been foundto result in similar double-muscled phenotypes. The disruption of the MSTN gene also causes apronounced increase in skeletal muscle mass.To knockout the myostatin gene in ovine muscle satellite cells by CRISPR/Cas9 system and obtainMSTN knockout cell lines, four plasmids were designed and constructed to target the MSTN gene, andconfirmed correct by sequencing. Muscle satellite cells are isolated by the method of two-stepenzymatic digestion. And they were identified based on the morphology and biological characters. Thecorrect plasmids were delivered into muscle satellite cells in vitro, and the targeting efficiency wasdetected using Sanger sequence and SURVEYOR assay kit. The stable transfected cell colonies wereobtained via limiting dilution procedure. The mutation types were established by TA-cloning, and thesequences were analysed using bioinformatics program.Following induced several days, different muscle cells fused to form myotubes. And the expressionof MyoG, MyoD, Myf5 genes, which were specific expressions in muscle satellite cells, were examinedby RT-PCR. The correct plasmids were delivered into the muscle satellite cells in vitro, and the resultsfrom sequencing demonstrated that the pX330-target1 and pX330-target4 plasmids can successfullyknockout MSTN. The targeting efficiency was up to 24.20% and 10.18% respectively. TwelveMSTN-knockout cell colonies were obtained via limiting dilution, and one of them is homozygousmutation. Several indel mutations were discovered at specific site, and some of them were frame-shiftmutations. Therefore, we conclude that the CRISPR/Cas9 system we constructed can apply to the MSTNgene editing in sheep efficiently, and the gene knockout cell clones have potential application ingenerating MSTN knockout sheep.