Efficient epigenetic editing of the zebrafish slc45a2 gene using the CRISPR/dCas9 system and tandem guide RNAs

The CRISPR-dCas9 (deactivated Cas9) system is a programmable DNA-targeting tool engineered by inactivating the nuclease activity of the Cas9 protein. This modification enables precise genomic localization without inducing double-strand breaks (DSBs), making it a versatile platform for diverse applications in molecular biology and therapeutics. However, there are relatively few research reports on the application of CRISPR/dCas9 technology in living organisms, particularly in fish. Therefore, this study aims to check whether CRISPR/dCas9 system was able to induce gene silencing via epigenetic gene editing in fish breeding. To visually demonstrate the gene silencing efficiency of CRISPR/dCas9, we specifically selected the key melanogenesis gene solute carrier family 45 member 2 (Slc45a2) as the target and further examined the impact of CRISPR/dCas9 technology on the changes of body color, gene expression and DNA methylation level of the slc45a2 gene. We found that microinjection of CRISPR/dCas9 with gRNAs targeting the CpG island around the transcription initiation site led to a significant reduction in melanin synthesis. This was particularly evidenced by a marked decrease in melanocyte numbers at 7, 45, and 80 days post fertilization (dpf). Moreover, injection of CRISPR/dCas9-gRNA elevated the methylation level of CpG island and inhibited the slc45a2 gene expression at transcription level. Furthermore, the elevation of methylation level of slc45a2 gene resulted in a notable decline of the gene expression of melanin biosynthesis pathway genes of pyrophosphatase (Inorganic) 4A ( pnp4a ), colony stimulating factor 1 receptor a/b ( csf1ra / b ) and tyrosinase ( tyr ) genes. The effects of gene editing were able to sustain to 80 dpf. The findings in fish indicated that epigenetic gene editing technology is efficient for gene silencing, which will lay the foundation for the application and promotion of this technology in aquaculture fish species.