Targeted DNA methylation in human cells using engineered dCas9-methyltransferases.

Sci Rep
Authors
Keywords
Abstract

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. Here 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. 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.

Year of Publication
2017
Journal
Sci Rep
Volume
7
Issue
1
Pages
6732
Date Published
2017 07 27
ISSN
2045-2322
DOI
10.1038/s41598-017-06757-0
PubMed ID
28751638
PubMed Central ID
PMC5532369
Links
Grant list
DP1 DK105602 / DK / NIDDK NIH HHS / United States