CRISPR/Cas9-mediated mutation of Mstn confers growth performance in Culter alburnus juveniles

Myostatin is a member of the TGF-β superfamily and functions as a negative regulator for skeletal muscle development and growth. It has become the most targeted gene in aquaculture that used for selective breeding. Previous studies involved in genome editing in several fish species confirmed that CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) system was highly efficient with lower off-target effect, however, no reports were raised in Culter alburnus. In this study, we employed CRISPR/Cas9 gene editing system to successfully disrupt mstn gene by co-injection with Cas9 protein and the targeted sgRNA in C. alburnus. Various Indel mutations were obtained with 82% knockout efficiency in the F0 generation by PCR sequencing. In addition, mutations in mstn that induced by CRISPR/Cas9 were detected in the F1 generation by individually mating the wild-type female with the F0 generation of mstn-KO male at sexual maturity. More importantly, the body weight and length were significantly elevated in mstn ± group when compared to those of the control. As expected in mstn ± group, the expression level of mstn was sharply reduced, whereas a slight increase was observed in two growth-related genes (myod and myog). Moreover, higher numbers of muscle fibers were observed in mstn ± group, meaning that growth performance in mstn ± individuals might be represented by increasing the number of muscle fibers. Taken together, our current study successfully obtained a site-specific modification of mstn using CRISPR/Cas9 technology, and these results provided a new insight for facilitating topmouth culter genetic studies and breeding.