Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltansferase with CRISPR/dCas9

Although associations between DNA methylation and gene expression were established four decades ago, the causal role of DNA methylation in gene expression remains unresolved. Different strategies to address this question were developed; however, all are prone to confounds and may fail to disentangle cause and effect. We developed here a highly effective new method using only deltaCas9(dCas9):gRNA site-specific targeting to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzymatic activity, enabling examination of the role of DNA demethylation per se in living cells. We show that the extensive induction of gene expression achieved by TET/dCas9-based targeting vectors is confounded by DNA methylation-independent activities, inflating the role of DNA methylation in the promoter region. Using this new method, we show that in several inducible promoters, the main effect of DNA methylation is silencing basal promoter activity. We find no credible evidence of off-target DNA demethylation as a consequence of gRNA binding. Thus, the effect of demethylation of the promoter region in these genes is small, while induction of gene expression by different inducers is large and DNA methylation independent. In contrast, targeting demethylation to the pathologically silenced FMR1 gene targets robust induction of gene expression. We also found that standard CRISPR/Cas9 knockout generates a broad unmethylated region around the deletion, which might confound interpretation of CRISPR/Cas9 gene depletion studies. In summary, this new method could be used to reveal the true extent, nature, and diverse contribution to gene regulation of DNA methylation at different regions. Overall design: Whole genome bisulfite sequencing (WGBS) of 5 treatment conditions of NIH-3T3 cells in triplicates, including control untreated NIH-3T3 cells and those expressing (by lentivirus mediated gene-transfer) dCas9 (thereafter removed by lentiviral Cre recombinase) in combination with either a scrambled non-targeting guideRNA (gRNAscr) or a guideRNA targeting the transcription start site of the shorter isoform of IL33 (gRNA3) as well as those expressing dCas9 fused to human TET1 (dCas9-TET) with the same two guideRNAs.