Generation of fast-growth grass carp by mutation of mstnb via CRISPR/Cas9 system

Myostatin ( mstn ) is a highly conserved negative regulator of myogenesis and myofiber growth in vertebrates. In recent years, the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) system has been widely used for genetic breeding in aquaculture. Targeted disruption of mstn in various teleost species has led to enhanced growth traits. In this study, two mstn paralogs, designated mstna and mstnb , were identified in grass carp ( Ctenopharyngodon idella ). Tissue distribution analysis using RT-PCR revealed that mstnb , but not mstna , was expressed in muscle tissue, suggesting a potential role for the mstnb gene in grass carp muscle growth. To investigate the role of mstnb in skeletal muscle growth, we adopted CRISPR/Cas9 to disrupt the mstnb gene by co-injecting Cas9 protein and sgRNA into one-cell stage embryos. Approximately 85 % of injected F0 individuals carried mutations at the mstnb target site, including various frameshift indel mutations. Compared to their wild-type counterparts, the mstnb F0 mutants showed significantly increased body weight and length. Quantitative real-time PCR analysis confirmed a significant reduction in mstnb expression in the F0 mutants. Additionally, two growth-related genes, myod1 and vegfba , exhibited significant upregulation in mutant muscle tissue. Histological examination revealed no significant difference in the thickness of individual muscle fibers between WT siblings and the F0 mutants. However, a significant increase in the total number of muscle fibers was observed in the mstnb F0 mutants. We successfully generated mstnb mutants with enhanced muscle growth, which may promote grass carp production in aquaculture practice. Overall, our study provided a valuable resource for the genetic breeding of grass carp.