Project description:Fusion of active protein domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been widely used for epigenome editing, but the specificities of these engineered proteins have still not been fully investigated. Targeted methylation of specific gene loci offers a direct approach to perturb DNA methylation-associated biological processes. In this study, we generated and validated the global off-target characteristics of CRISPR-guided DNA methyltransferases (CRISPRme) by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from S. pyogenes. Using targeted quantitative bisulfite pyrosequencing and whole genome bisulfite sequencing (WGBS), we prove that CRISPRme can efficiently methylate the CpG dinucleotides flanking its target sites in genomic loci (uPA and TGFBR3) in human cells (HEK293T) with CpG-methylation levels exceeding 70% for some target sites. Using qPCR, fluorescent reporter cells, and RNA sequencing, we found that CRISPRme can mediate transient inhibition of gene expression which appears to result from Cas9-mediated interference with transcription rather than de novo DNA methylation. Analyses of whole genome methylation did not identify global methylation changes, however a substantial number of CRISPRme off-target differentially methylated regions (DMR, over 6000) were still identified. The majority of these DMRs were hypermethylated both in cells expressing CRISPRme alone and cells expressing CRISPRme together with gRNAs. These off-target hypermethylated sites were enriched in gene bodies, introns, 5’UTR, CGI shores, Alu sequences, open chromatin and PAM rich regions, but not correlated with off-target binding sites predicted by ChIP-seq. Our results prove that CRISPRme allows for efficient RNA-guided methylation of endogenous CpGs, however with high frequencies of off-target methylation indicating that further improvements of the specificity of CRISPR-dCas9 based DNA methylation modifiers are still required.

Project description: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.

Dataset Information

Targeted DNA methylation in vivo using an engineered dCas9-MQ1 fusion protein (RRBS)

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