CRISPR/Cas9 system-based myostatin-targeted disruption promotes somatic growth and adipogenesis in loach, Misgurnus anguillicaudatus

Abstract

The mud loach (Misgurnus anguillicaudatus) is one of the most important aquaculture fishes in Asia. The application of the CRISPR/Cas9 system in the cultivation of loach strains with valuable economic traits presents great potential. Myostatin is a candidate factor that can be used for the cultivation of fast-growing loach strains. In this study, the appropriate microinjection dose of the Cas9 protein (600 pg) for genome editing was determined considering both the mutation rate and survival rate of loach embryos. With this microinjection dose of the Cas9 protein, both the exogenous GFP and endogenous myostatin genes were efficiently edited using the CRISPR/Cas9 system. Compared with that in wild-type siblings, the average body weight of the fish in 1-, 2- and 3-month-old myostatin mutant chimeric loaches was significantly increased by 34.9% (P < 0.001), 15.5% (P = 0.024) and 27.8% (P = 0.012), respectively. Mutation of mstn significantly increased the number of muscle fibers and total area of muscle fibers. Also, lipid accumulation was significantly higher in chimeric mutant loach. Additionally, the mRNA levels of myogenesis (myod and myog) and lipogenesis-related genes (scd, fabp1, fabp2 and dgat) were significantly upregulated in chimeric mutants. In particular, the mRNA level of the stearoyl-CoA desaturase gene, whose product catalyses the formation of monounsaturated fatty acids from saturated fatty acids, was increased by 23-fold (P = 0.034) and 736-fold (P < 0.05) in 3 dpf and 1-month-old chimeric loaches, respectively. The gene expression results indicated that the targeted disruption of myostatin activated myogenesis and adipogenesis in loach. Our study is highly valuable for cultivating fast-growing loach strains and understanding the specific role of myostatin in lipogenesis.