Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase

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.

Designing Epigenome Editors: Considerations of Biochemical and Locus Specificities

“…To test if the multimerization of DNMT3A has a role in targeted DNA methylation, they used DNMT3A mutants with a catalytic core mutation, a polymerization interface mutation, or both. While the mutants with the catalytic core mutation did not methylate the target loci at all, the mutant with polymerization interface mutation showed decreased DNA methylation activity by ~2-fold in vitro which is in agreement with the role of multimerization for DNA methylation activity [41]. …”

“…For example, using ZFs or dCas9 to recruit a stable DNMT3A/DNMT3L single-chain fusion protein versus an individual DNMT3A catalytic domain to an endogenous promoter results in two to four fold higher methylation [36, 41]. This boost in chromatin modifying activity may arise from synergism between the domains rather than simply doubling the number of domains, as DNMT3L is known to stimulate the DNA methylation activity of DNMT3A [42].…”

Epigenetic editing: How cutting-edge targeted epigenetic modification might provide novel avenues for autoimmune disease therapy

“…This was shown to increase specificity up to 1500-fold in traditional dCas9 genetic editing [62]. This approach has demonstrated efficacy in epigenetic editing as well [22, 63], as presented most recently in an article by Stepper et al from 2017, in which a dimeric dCas9-Dnmt3a-Dnmt3l DNA methyltransferase was generated and targeted to three gene promoters. The group noted substantial increases in DNA methylation at targeted regions with only mild off-target methylation in two different genomic regions [63].…”

“…This approach has demonstrated efficacy in epigenetic editing as well [22, 63], as presented most recently in an article by Stepper et al from 2017, in which a dimeric dCas9-Dnmt3a-Dnmt3l DNA methyltransferase was generated and targeted to three gene promoters. The group noted substantial increases in DNA methylation at targeted regions with only mild off-target methylation in two different genomic regions [63]. Moving forward, specificity concerns must be taken into account when designing and implementing epigenetic editing strategies, particularly as we move towards clinical applications of these techniques.…”

CRISPR-based reagents to study the influence of the epigenome on gene expression

“…2b). Similarly, fusion of histone or DNA methyl transferases to Cas9 has been used to prove the influence of specific DNA methylation events 61, 62, 63, 64, 65, 66.…”