Mar 30, 2020

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

BioRxiv : the Preprint Server for Biology
D. M. Sapozhnikov, Moshe Szyf


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 robus...Continue Reading

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Mentioned in this Paper

Observational Study
Transcriptional Regulation
Laboratory Procedures
Transcription, Genetic
Gene Expression

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