CRISPR/Cas9-mediated epigenetic editing tool: An optimized strategy for targeting de novo DNA methylation with stable status via homology directed repair pathway.

With the development of epigenetic engineering tools for manipulating DNA methylation at the desired locus, the stable maintenance of targeted methylation status in edited cells after division poses a major obstacle. Transient methylation levels, along with the reversible inhibition of the desired genes, by some epigenetic editing techniques, limit their further application in functional and therapeutic studies of critical regulators in the cancer epigenome. In this study, we utilized CRISPR/Cas9-mediated the homology-directed repair (HDR) pathway following double-strand breaks (DSBs) information to target de novo methylation of the vital epigenetic biomarker, O-methylguanine-DNA methyltransferase (MGMT). Based on synthetic single and double stranded methylated repair templates, DNA methylation patterns can be incorporated into the endogenous MGMT gene. In addition, we observed upregulation of DNA methyltransferases (DNMTs) in edited HeLa cells. In particular, we employed an optimized method of using the DNA ligase IV inhibitor SCR7 to markedly enhance the insertion efficiency induced by the long methylated repair template, contributing to the induction of stable methylation alterations through epigenetic recombination after cell division. Additionally, our study confirmed that CRISPR/Cas9-mediated target methylation in a site-specific manner enabled the maintenance of gene silencing in vitro and in vivo. Collectively, we show that a combination of CRISPR/Cas9 components, SCR7 treatment, and the long methylated HDR template can enhance CRISPR/Cas9-directed epigenomic editing efficiency and further induce stable effects on methylation modifications and transcriptional suppression.