Muscle growth in targeted knockout common carp (Cyprinus carpio) mstn gene with low off-target effects

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The gene mstn is a negative regulator of skeletal muscle growth and development in vertebrates, including teleosts. Common carp (Cyprinus carpio) is an economically important food fish, and an increase in its skeletal muscle mass, and improvement in growth without introducing exogenous DNA is highly desirable. In our study, sgRNA/Cas9 ribonucleoprotein (RNP) (62 ng/μL Cas9 and 186 ng/μL cc sgRNA2) was used to knockout (KO) the mstn gene of common carp, and subsequent hatchability, survivability, body deformity and growth performance of the mstn+/− F0 common carp was monitored till 90 days post-hatching (dph). Insertion-deletion (indel) mutation of 1–11 bp was observed in the first exon of the mstn gene of KO common carp, and approximately, 88–95% mutation was observed in the target protein-coding site of this gene. Off-target effects, including skeletal deformity, and other phenotypic abnormality were not observed in mstn+/− F0 common carp. Total body length, body weight, width of the body, and the body depth of mstn+/− F0 common carp were significantly (p < 0.001) higher than the wild-type (WT) phenotype. Double muscle phenotype was observed in mstn+/− F0 common carp from 30 dph onwards. Skeletal muscle fiber density in mstn+/− F0 common carp was 51.4% (p < 0.001) higher than the WT phenotype. Increase of muscle mass in mstn+/− F0 common carp was attributed to hyperplasia (49.5% increases) and hypertrophy (11% increase), both. Expression of mstn transcripts in skeletal muscle of mstn+/− F0 common carp was lower than that of WT phenotype. The quantitative mRNA expression level of myogenic regulatory factors (MRFs), myogenic differentiation (myod), myogenic factor 5 (myf5), and myogenin (myog) was found up-regulated from 36 dph onwards in mstn+/− F0 common carp, in comparison to WT phenotypes. Our study shows that, CRISPR/Cas9 based RNPs can be effectively used to target mstn gene in common carp for an increase in its skeletal muscle mass.