Repurposing the CRISPR-Cas9 system for targeted DNA methylation

Epigenetic studies relied so far on correlations be- tween epigenetic marks and gene expression pat- tern. Technologies developed for epigenome edit- ing now enable direct study of functional relevance of precise epigenetic modifications and gene reg- ulation. The reversible nature of epigenetic modi- fications, including DNA methylation, has been al- ready 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 con- sisting of deactivated Cas9 (dCas9) nuclease and cat- alytic 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 tar- geted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methy- lation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression.