Targeted Cytosine Methylation using dCas9- methyltransferases

Mammalian genomes exhibit complex patterns of gene expression regulated, in part, by DNA methylation. The advent of engineered DNA methyltransferases (MTases) to target DNA methylation to specific sites in the genome will accelerate many areas of biological research. However, targeted MTases require clear design rules to direct site-specific DNA methylation and minimize the unintended effects of off-target DNA methylation. We report a targeted MTase composed of an artificially split CpG MTase (sMTase) with one fragment fused to a catalytically-inactive Cas9 (dCas9) that directs the functional assembly of sMTase fragments at the targeted CpG site. We precisely map RNA-programmed DNA methylation to targeted CpG sites as a function of distance and orientation from the protospacer adjacent motif (PAM). Expression of the dCas9-sMTase in mammalian cells led to predictable and efficient (up to ~70%) DNA methylation at targeted sites. Multiplexing sgRNAs enabled targeting methylation to multiple sites in a single promoter and to multiple sites in multiple promoters. Targeted methyltransferase results in some methylation at non-target CpG sites. We provide evidence that non-target methylation primarily occurs with dCas9 bound at its target protospacer site, as opposed to dCas9 bound at an unintended site or dCas9 not bound to DNA at all. We show that shortening the linker between the dCas9 and the sMTase increases methylation frequency at target sites located at select distances from the dCas9 binding site, decreases methylation frequency at target sites located at most other distances, and does not reduce methylation at off-target sites. Significant reduction in off-target methylation results from mutations designed to decrease the methyltransferase domain’s affinity for DNA. The mutations also restrict methylation of target sites to a narrow range of distances away from the dCas9 binding site. Our programmable de novo MTase tools might be used for studying mechanisms of initiation, spreading and inheritance of DNA methylation, and for proof of concept of therapeutic gene silencing