Site-Specific DNA Methylation in Human Cells Using Engineered Cas9-Methyltransferases

Mammalian genomes exhibit complex patterns of gene expression, which are regulated, in part, by levels of DNA methylation. High levels of promoter methylation are correlated with repressed gene expression, and the ability to harness site-specific DNA methylation in vivowould accelerate many areas of biological research and could affect novel epigenetic therapies. Existing approaches attempt to bias methylation to specific sites using active DNA methyltransferases fused to DNA binding domains. However, intact DNA methyltransferases retain their inherent DNA binding and methylation activity resulting in significant off-target effects that limits their use as a research tool and would be a serious obstacle for any therapeutic application.

To circumvent this limitation, we designed a tool for highly specific DNA methylation in human cells by splitting a bacterial CpG DNA methyltransferase (M.SssI) into two inactive fragments. We fused this split methyltransferase (sMTase) to a catalytically inactive (endonuclease dead) CRISPR/Cas9 (dCas9). The sMTase are localized to targeted sites on promoters using RNA guide strands, which leads to site-specific DNA methylation in promoter regions. Targeted sites are easily changed within the promoter (and to other promoter regions) by changing guide RNA sequences. Additionally, multiplexing RNA guide strands enables targeting several sites in a promoter region for gene repression in human cells. Our site-specific de novo DNA methylation tool will allow analysis of (1) initiation, spreading and inheritance of DNA methylation patterns, (2) the downstream effects of DNA methylation on transcriptional activation, and (3) nucleation of higher order chromatin structures. Moreover, because a growing number of diseases have an epigenetic etiology, site-specific DNA methylation of disease-promoting genes holds enormous untapped therapeutic potential.