Abstract
Genome editing of late myogenic regulators provides a way to dissect the mechanisms through which transcriptional programs and growth-related signaling pathways shape muscle gene expression programs in farmed fish. This study disrupted myogenic regulatory factor 4 (MRF4) in Nile tilapia using CRISPR/Cas9 to examine downstream transcriptional changes in fast skeletal muscle across the trunk, belly, and head regions. Adult F0 crispants carried a frameshift mutation that truncated the basic helix–loop–helix domain and showed an approximate 80–85% reduction in MRF4 mRNA across the trunk, belly, and head muscles. The expression of 23 genes representing myogenic regulatory factors, MEF2 paralogs, structural and contractile components, non-myotomal regulators, cell adhesion and fusion-related transcripts, and growth-related genes within the GH–IGF–MSTN axis was quantified and compared between wild-type and MRF4-crispants. Expressions of major structural genes remained unchanged despite MRF4 depletion, whereas MyoG and MyoD were upregulated together with MEF2B and MEF2D, indicating strong transcriptional compensation. Twist1, ID1, PLAU, CDH15, CHRNG, NCAM1, MYMK, GHR, and FGF6 were also significantly elevated, while IGF1 was reduced, and MSTN remained stable. Together, these results show that MRF4 loss is associated with coordinated transcriptional changes in regulatory and growth-related pathways, while major fast-muscle structural and contractile transcript levels remain stable, thereby highlighting candidate transcriptional targets for future studies that will evaluate links to muscle phenotype and growth performance in Nile tilapia.