CRISPR/Cas9-mediated myostatin b knockout enhances growth performance and muscle hypertrophy in grass carp (Ctenopharyngodon idella)

Aquaculture is critical for global food security, yet genetic improvement in farmed fish has progressed more slowly than in terrestrial livestock. Myostatin (MSTN), a conserved negative regulator of skeletal muscle growth in vertebrates, presents a prime target for enhancing aquaculture productivity. In this study, we employed CRISPR/Cas9 ribonucleoprotein (RNP) complexes to disrupt mstnb , the orthologous gene of livestock mstn , in grass carp ( Ctenopharyngodon idella ), the most economically important freshwater aquaculture species in China. Co-injection of five single-guide RNAs (sgRNAs) targeting mstnb exon 1 with Cas9 protein into single-cell embryos achieved 62.0–97.8 % editing efficiency in F 0 mosaic individuals, with an overall population efficiency of 67.1 %. At 7 months post-hatching, mstnb -knockout (KO) fish displayed 17.5 % greater body weight, 8.8 % increased body width, and 7.6 % higher body height compared to control siblings. Histological analysis revealed marked muscle fiber hypertrophy, with cross-sectional area increases of 9.0–26.3 % in cephalosome, trunk, and rump muscles. KO fish also displayed reduced hepatic lipid and glycogen accumulation, indicative of improved metabolic efficiency. While muscle amino acid composition remained largely unchanged, we observed a modest decrease in ω-3 polyunsaturated fatty acids (e.g., EPA and DHA). This study marks the first successful application of CRISPR/Cas9 in grass carp, demonstrating that mstnb disruption enhances growth and muscle development without substantially compromising meat quality. Our results underscore the potential of genome editing for sustainable aquaculture breeding, offering a promising strategy to address rising global protein demands.