CRISPR/Cas9-mediated myostatin disruption enhances muscle mass in the olive flounder Paralichthys olivaceus

Myostatin (MSTN), a negative regulator of skeletal muscle development, is the major gene of interest for improving animal breeding productivity, including for aquaculture. We introduced a CRISPR/Cas9 system to disrupt Paralichthys olivaceus MSTN (PoMSTN) using microinjection. Cas9 mRNA and sgRNAs targeting the first exon of the PoMSTN gene were co-injected into embryos of P. olivaceus. On-target mutations on the PoMSTN locus were generated in somatic tissues with 75.6% efficiency, and founders harboring germline mutations were produced in the F0 generation. In the F1 generation, restriction fragment length polymorphism analysis was applied to select biallelic mutants, and heterozygous biallelic mutants with PoMSTN disruption were obtained. PoMSTN-disrupted P. olivaceus exhibited greater body thickness and increased condition factor, indicating the enhancement of muscle mass with muscle hyperplasia. Expression of PoMSTN mRNA and protein was significantly reduced in the muscle of PoMSTN-disrupted P. olivaceus, and the mRNA expression of major myogenic regulatory factors (myogenin, MyoD, and Myf5) was differentially affected in PoMSTN-disrupted mutants. These results suggest that the establishment of a CRISPR/Cas9 system in P. olivaceus provides powerful tools for related genetic studies and breeding.