(569g) RNA-Programmed DNA Methylation

Mammalian genomes exhibit complex patterns of gene expression regulated, in part, by DNA methylation. The study of the mechanisms of initiation, spreading and inheritance of DNA methylation is hindered without tools to efficiently and reproducibly direct methylation patterns to precise sites in model systems. Fusion of DNA methyltransferases (MTases) to DNA binding domains can bias methylation to CpG sites near the DNA binding domain’s target sequence. However, since the MTase activity of the fusion does not require binding of the DNA binding domain to the chromosome, off-target methylation results. The most effective targeted MTases will require clear design rules to direct site-specific DNA methylation and minimize the unintended off-target DNA methylation. Here we report a targeted MTase composed of an artificially split CpG MTase (sMTase) with one fragment fused to a catalytically-inactive CRISPR/Cas9 protein (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 DNA methylation at targeted sites as directed by guide RNAs. Multiplexing guide RNAs enabled targeting methylation to multiple sites in a single promoter and to multiple sites in multiple promoters. This programmable de novo MTase tool might be used for studying mechanisms of initiation, spreading and inheritance of DNA methylation, and for therapeutic gene silencing.