Molecular Characterization, Expression Analysis, and CRISPR/Cas9 Mediated Gene Disruption of Myogenic Regulatory Factor 4 (MRF4) in Nile Tilapia.

Myogenic regulator factors (MRFs) are essential for skeletal muscle development in vertebrates, including fish. This study aimed to characterize the role of () in muscle development in Nile tilapia by cloning from muscle tissues. To explore the function of , CRISPR/Cas9 gene editing was employed. The 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. -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 mRNA levels than the controls while re-feeding markedly increased expression. The CRISPR/Cas9 gene editing of successfully generated two types of gene disruption, leading to a frame-shift mutation in the NT-MRF4 protein. Expression analysis of and genes in gene-edited (GE) Nile tilapia revealed that expressions nearly doubled compared to wild-type (WT) fish, suggesting that compensates for the loss of MRF4 function. Additionally, , , and expressions significantly increased in GE Nile tilapia, supporting continued muscle development. Overall, these findings suggest that regulates muscle development, while may compensate for its inactivation to sustain normal muscle growth.