Molecular Characterization, Expression Analysis, and CRISPR/Cas9 Mediated Gene Disruption of Myogenic Regulatory...

Myogenic regulator factors (MRFs) are essential for skeletal muscle development in vertebrates, including fish. This study aimed to characterize the role of myogenic regulatory factor 4 (MRF4) in muscle development in Nile tilapia by cloning NT-MRF4 from muscle tissues. To explore the function of NT-MRF4, CRISPR/Cas9 gene editing was employed. The NT-MRF4 cDNA was 1146 bp long and had encoded 225 amino acids, featuring a myogenic basic domain, a helix-loop-helix domain, and a nuclear localization signal. NT-MRF4 mRNA was exclusively expressed in adult muscle tissues, with expression also observed during embryonic and larval stages. Food-deprived Nile tilapia exhibited significantly lower NT-MRF4 mRNA levels than the controls while re-feeding markedly increased expression. The CRISPR/Cas9 gene editing of NT-MRF4 successfully generated two types of gene disruption, leading to a frame-shift mutation in the NT-MRF4 protein. Expression analysis of MRF and MEF2 genes in gene-edited (GE) Nile tilapia revealed that MyoG expressions nearly doubled compared to wild-type (WT) fish, suggesting that MyoG compensates for the loss of MRF4 function. Additionally, MEF2b, MEF2d, and MEF2a expressions significantly increased in GE Nile tilapia, supporting continued muscle development. Overall, these findings suggest that NT-MRF4 regulates muscle development, while MyoG may compensate for its inactivation to sustain normal muscle growth.