Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase
Targeted epigenome editing relied on zinc fingers or TALEs needing per-target redesign. This paper fused a Dnmt3a-Dnmt3L methyltransferase to dCas9 to target CpG island promoters. The fusion gave 4-5 fold stronger methylation than Dnmt3a alone, repressing target genes.
Key findings
- Developed dCas9-DNMT3A system for targeted DNA methylation. Achieved stable CpG methylation at target loci with heritable silencing. Established targeted epigenetic editing through DNA modification.
Abstract
DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a-Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20-30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/Dnmt3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling.