A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs

Abstract

Detection of DNA methylation in the genome has been possible for decades; however, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded our understanding of the direct effect of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this. Recent adaptations of genome editing technologies, including fusion of the DNMT3A DNA methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to alter DNA methylation at desired loci. Here, we show that these tools exhibit consistent off-target DNA methylation deposition in the genome, limiting their capabilities to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA methylation. dC9Sun-D3A is tunable, specific, and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target methylation by dC9-D3A. Furthermore, we used dC9Sun-D3A to demonstrate the binding sensitivity to DNA methylation for CTCF and NRF1 in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest off-target DNA methylation levels reported to date, allowing accurate functional determination of the role of DNA methylation at single loci.

Engineering of Effector Domains for Targeted DNA Methylation with Reduced Off-Target Effects

“…It resulted in increased DNA methylation activity and was applied in several studies for gene silencing [17][18][19][20]. Another advancement was the introduction of dCas9 as a programmable DNA binding domain, which was used in several projects aiming for targeted DNA methylation [19][20][21][22][23][24][25][26][27].…”

“…This system consists of dCas9 fused to an array of peptide sequences, which are derived from the GCN4 protein and recognized by a single-chain antibody (scFv-GCN4). Co-expression of dCas9-SunTag and scFv-GCN4 fused to chromatin-modifying enzymes results in the recruitment of multiple effector domains to the target locus and may potentially increase on-target editing efficiency, which was demonstrated with the TET1 catalytic domain used for DNA demethylation [29,30] as well as the DNMT3A catalytic domain used for DNA methylation [24,26].…”

Functions and mechanisms of epigenetic inheritance in animals

“…The recent developments in CRISPR-Cas9driven targeted epigenome remodel ling applied to easily tractable model organisms such as zebrafish could tackle these challenges. For example, www.nature.com/nrm the fusion of the Cas9 protein to catalytic domains of DNMTs [179][180][181] or of histone modifying enzymes 182 could enable targeted epigenetic modification of genomes that could then be monitored through generations. This tar geted modification will facilitate precise functional stud ies that do not depend on often lethal gene knockouts, which can hamper the interrogation of heritable effects, and will also allow the study of the potential depend ence of MEI phenomena on the DNA sequence.…”

Gene editing and CRISPR in the clinic: current and future perspectives

“…The dCas9-DNMT3A fusions can accurately methylate DNA across a region of ∼35-320 bp from the DNA-bound fusion protein and can be multiplexed with several sgRNAs to span a larger target region of the genome [270][271][272]. Alternatively, methylation across a larger genomic region can be edited using the dCas9-SunTag-DNMT3A system, that can recruit multiple DNMT3As to a target site and hypermethylated a region of ∼4.5 kb [274,275]. Multimerization can also improve methylation and using dCas9-DNMT3a/DNMT3L could lead to more potent and widespread DNA methylation of CpG islands, and additionally caused DNA methylation spreading from a target site [276].…”