Epigenetic editing of Indian Medaka fgf2 using CRISPR/dCas9-tet1CD

CRISPR/dCas9-based epigenetic editing circumvents the issue of DNA damage and provide another more flexible and diverse means of epigenetic regulation. Currently, the application of epigenetic editing technologies is limited to a few species, and there is a lack of comprehensive assessment. In this study, we first conducted molecular cloning and characterization of the ten-eleven translocation methylcytosine dioxygenase 1 (tet1) gene in Indian medaka (Oryzias melastigma). Through decitabine treatment, we identified fibroblast growth factor 2 (fgf2) as a target gene of DNA methylation, specifically pinpointing a CpG island within the fgf2 promoter region. Subsequently, we constructed a CRISPR/dCas9-tet1CD activation system by fusing tet1 Catalytic Domain (tet1CD, Ala1352–Tyr2034) to the dCas9 protein. Using fgf2 as the target gene, we demonstrated the efficiency, specificity, and duration of this CRISPR/dCas9-based activation system. The system achieved efficient DNA demethylation of the CpG island in fgf2 promoter region, resulting in approximately 2-fold increase in gene expression levels. Whole-genome bisulfite sequencing (WGBS) and transcriptome analysis also revealed the minimal off-target effects. The epigenetic activation of fgf2 induced changes in the expression of growth-related genes, resulting in a durable increase in gene expression and cell growth. Collectively, our findings establish an efficient and precise epigenetic editing system for Indian medaka. This highlights the potential of CRISPR/dCas9-based epigenetic editing as a powerful tool for functional genomics studies in aquatic species. Keywords: Epigenetic editing; CRISPR/dCas9; tet1; DNA demethylation; fgf2; Oryzias melastigma