RESUMO
Despite the increased interest in epigenetic research, its progress has been hampered by a lack of satisfactory tools to control epigenetic factors in specific genomic regions. Until now, many attempts to manipulate DNA methylation have been made using drugs but these drugs are not target-specific and have global effects on the whole genome. However, due to new genome editing technologies, potential epigenetic factors can now possibly be regulated in a site-specific manner. Here, we demonstrate the utility of CRISPR/Cas9 to modulate methylation at specific CpG sites and to elicit gene expression. We targeted the murine Oct4 gene which is transcriptionally locked due to hypermethylation at the promoter region in NIH3T3 cells. To induce site-specific demethylation at the Oct4 promoter region and its gene expression, we used the CRISPR/Cas9 knock-in and CRISPR/dCas9-Tet1 systems. Using these two approaches, we induced site-specific demethylation at the Oct4 promoter and confirmed the up-regulation of Oct4 expression. Furthermore, we confirmed that the synergistic effect of DNA demethylation and other epigenetic regulations increased the expression of Oct4 significantly. Based on our research, we suggest that our proven epigenetic editing methods can selectively modulate epigenetic factors such as DNA methylation and have promise for various applications in epigenetics.
