Characterization of Trachinotus blochii mstnb gene and construction of cognate gRNA vector.

Skeletal muscle development is negatively regulated by the myostatin b (mstnb) gene in finfishes. CRISPR-Cas9-mediated knockout of mstnb has been used to develop fish strains with increased muscle mass. Trachinotus blochii (Silver Pompano), a high-value, moderately sized (~500 g), and cultivable marine finfish, is a promising candidate for mstnb knockout using CRISPR/Cas9. However, limited studies exist on the characterization of T. blochii mstnb and no CRISPR-based knockout studies have been reported in this species. This study aimed to partially characterize the mstnb gene of T. blochii and develop a CRISPR/Cas9-based guide RNA (gRNA) expression vector for its knockout. The T. blochii mstnb sequence available in NCBI was used as a reference to design exon- and intron-specific primers for PCR amplification and sequencing. The sequence revealed three exons and two introns. A predicted 1131 bp open reading frame encodes a 376 amino acid protein containing conserved domains typical of the TGF-β family which includes an N-terminal signal peptide, a propeptide region, a conserved RARR cleavage motif, and a C-terminal GF domain containing nine conserved cysteine residues. Secondary and 3D structure predictions confirmed the protein's functional integrity. Sequence analysis revealed novel putative polymorphisms, including SNPs and a (CA)n microsatellite. Phylogenetic analysis clustered T. blochii with related Trachinotus and other Carangiformes species. A gRNA targeting exon 1 was designed using CRISPOR and successfully cloned into expression vectors. This novel sequence information can aid population-level studies and genetic marker discovery. The constructed gRNA vectors can facilitate CRISPR/Cas9-mediated mstnb knockout in T. blochii to study gene function and develop a fleshy strain.