GSE107607
A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs
DNA methylation is a covalent modification of the genome that plays important roles in genome regulation and vertebrate development. Although detection of this modification in the genome has been possible for several decades, the ability to deliberately and specifically manipulate local DNA methylation states  in the genome has been extremely limited. Consequently, this has impeded the direct determination of the consequence 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 outstanding question. Recent adaptations of genome editing technologies, such as the fusion of the DNMT3A methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to provide new tools for altering DNA methylation at desired loci. Here, we performed a deeper analysis of the performance of the tools at a genome wide level which revealed consistent off-target binding events and DNA methylation deposition throughout the genome, limiting the capacity of these tools to generate unambiguous determination of the functional consequences of DNA methylation. To address this issue, 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 binding and methylation by the dC9-D3A direct fusion construct. Furthermore, we used dC9Sun-D3A to test the impact of DNA methylation upon the DNA binding of CTCF and NRF1 upon targeted methylation of their core binding sites, demonstrating the binding sensitivity of these proteins to DNA methylation in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the least amount of off-target DNA methylation reported to date, allowing accurate functional determination of the role of DNA methylation at single loci. Overall design: A direct fusion dCas9-DNMT3A construct was compared to a modular, highly specific dCas9-SunTag + aGCN4-DNMT3A catalytic domain (dC9Sun-D3A) system, where on- and off-target DNA methylation deposition was measured by bsPCR-seq at select loci. dC9Sun-D3A was found to have less off-target effects compared to direct fusion dCas9-DNMT3A while not compromising its high on-target performance. Consequently, the response of targeted DNA methylation at CTCF and NRF1 core binding sites in HeLa cells by dC9Sun-D3A was measured by bsPCR-seq, ChIP-seq and ChIP-BS-seq to assess CTCF and NRF1 DNA methylation tolerance. dC9Sun-D3A was introduced by “hit-and-run epigenetic editing”, using transient transfection, and expression was selected for by either 48h puromycin treatment or cell sorting. DNA or chromatin was extracted from the cells after treatment and selection with controls (DNMT3AMut or mCherry aGCN4 coupled effectors) or dC9Sun-D3A for on-target DNA methylation deposition. WGBS, targeted solution capture bisulfite sequencing (TSC-bs-seq), ChIP-seq and ChIP-BS-seq libraries were prepared for sequencing on the Illumina HiSeq1500. bsPCR-seq libraries were sequenced on Illumina MiSeq.