Repurposing the CRISPR-Cas9 system for targeted DNA methylation

TLDR

Epigenetic studies have largely been correlative, but recent epigenome‑editing technologies enable direct functional interrogation of precise modifications, and although reversible DNA methylation has been used non‑selectively in cancer therapy, a selective, locus‑specific approach is still lacking. The study aims to develop a locus‑specific epigenetic editing strategy that can selectively and heritably alter gene expression. The authors engineered a CRISPR‑Cas9 system comprising dCas9 fused to the catalytic domain of DNMT3A, guided by a 20‑bp RNA adjacent to an NGG site, to direct DNA methylation to chosen loci. They showed that the dCas9‑DNMT3A fusion can induce specific, heritable CpG methylation over a ~35‑bp window, that multiple guides can extend methylation across larger promoter regions, and that methylation of IL6ST and BACH2 promoters reduced their expression.

Abstract

Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co–expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression.