Epigenetic editing of marine medaka ( Oryzias melastigma) fgf2 using CRISPR/dCas9-Tet1CD.

CRISPR/dCas9-mediated epigenetic editing offers a versatile approach for transcriptional regulation without introducing DNA strand breaks. Although this strategy has been explored in a limited number of species, its application in aquatic vertebrates remains largely uncharacterized. In this study, ten-eleven translocation methylcytosine dioxygenase 1 ( tet1) was cloned and molecularly characterized in marine medaka ( Oryzias melastigma). Decitabine treatment identified fibroblast growth factor 2 ( fgf2) as a methylation-sensitive gene, with a regulatory CpG island located within its promoter region. Subsequently, a CRISPR/dCas9-Tet1CD activation system was constructed by fusing the catalytic domain of Tet1 (Tet1CD, Ala1352-Thr2034) to dCas9, enabling locus-specific DNA demethylation. Targeting fgf2, this CRISPR/dCas9-Tet1CD system induced efficient and selective demethylation of the CpG island, resulting in a maximal 2.41-fold increase in fgf2 transcript levels. Whole-genome bisulfite sequencing and transcriptomic analysis confirmed high on-target precision with minimal off-target effects. Epigenetic activation of fgf2 further modulated downstream gene networks associated with growth, promoting durable transcriptional enhancement and increased cellular proliferation. Collectively, these results establish a robust and highly specific epigenetic editing platform in marine medaka, providing a powerful tool for functional genomics studies and regulatory element analysis in aquatic models.