Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

Abstract

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 confounded and 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 methylation 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 our new method, we show that in several inducible promoters, the main effect of DNA methylation is silencing basal promoter activity. 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.

Intrauterine Hypoxia and Epigenetic Programming in Lung Development and Disease

“…In addition, evolving gene editing approaches, including the use of nucleases such as TALENs (transcription activator-like effector nucleases), zinc-finger nucleases, and CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPRassociated 9) to instigate a sequence-specific change in the DNA [132][133][134], encouraged the development of gene expression regulation, epigenetic modification, functional gene screening, gene diagnosis, and therapeutic drug discovery [135][136][137][138][139][140]. For example, dCas9 can be fused with DNMTs or TETs to regulate DNA methylation, thereby regulating specific gene expression [141][142][143]. Similarly, dCas9 can be fused with HDACs or HATs to regulate chromatin structure, thereby regulating gene expression [144][145][146].…”

Enhancer-associated H3K4 methylation safeguards in vitro germline competence

“…Congruently, we found that H3K4me1/2 deficient cells displayed increased mCpG levels within PGCLC enhancers. Therefore, although the importance of mCpG for enhancer activity and gene expression are not fully established [78][79][80] , it is tempting to speculate that the lack of DNA methylation in flies render them less sensitive to H3K4me1/2 loss. Nevertheless, Trr catalytic mutant fly embryos displayed aberrant phenotypes under stress conditions, suggesting that H3K4me1/2 might help to fine-tune enhancer activity under suboptimal conditions.…”