Abstract
In mammalian cells, DNA methylation critically regulates gene expression and thus has pivotal roles in myriad of physiological and pathological processes. Here we report a novel method for targeted DNA demethylation using the widely used clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. Initially, modified single guide RNAs (sgRNAs) (sgRNA2.0) were constructed by inserting two copies of bacteriophage MS2 RNA elements into the conventional sgRNAs, which would facilitate the tethering of the Tet1 catalytic domain (Tet-CD), in fusion with dCas9 or MS2 coat proteins, to the targeted gene loci. Subsequently, such system was shown to significantly upregulate transcription of the target genes, including RANKL, MAGEB2 or MMP2, which was in close correlation to DNA demethylation of their neighboring CpGs in the promoters. In addition, the dCas9/sgRNA2.0-directed demethylation system appeared to afford efficient demethylation of the target genes with tenuous off-target effects. Applications of this system would not only help us understand mechanistically how DNA methylation might regulate gene expression in specific contexts, but also enable control of gene expression and functionality with potential clinical benefits.
Highlights
DNA methylation, an epigenetic process by addition of a methyl group to DNA, mainly occurs at the fifth carbon of cytosine base within CpG dinucleotide
To construct the dCas9-mediated demethylation system, the D10A and H840A substitutions were introduced into Cas9 to form dCas9, which was subsequently fused to Tet1-CD using a flexible linker (Figure 1a and Supplementary Figure S1) to create dCas9-Tet1-CD
Guided by sgRNA2.0 targeting a specific sequence of the genome, both dCas9-Tet1-CD and MS2-Tet1-CD could be simultaneously recruited to the target gene locus
Summary
DNA methylation, an epigenetic process by addition of a methyl group to DNA, mainly occurs at the fifth carbon of cytosine base within CpG dinucleotide. DNA methylation regulates gene expression and has critical roles in myriad of physiological and pathological processes, which include, but are not limited to, cell development and differentiation, genome imprinting and tumorigenesis [1]. DNA hypermethylation in genes of tumor suppressors would silence their expression and contribute to multiple types of human cancers. Many enzymes have been found to catalyze active DNA demethylation with distinct mechanisms [3, 4]. Tet (ten eleven translocation) dioxygenase-catalyzed 5-methylcytosine oxidation was reported to promote DNA demethylation with Tet catalytic domain (Tet-CD) as the smallest functional module [5, 6]. Attempts in targeted DNA demethylation using transcription activator-like effector -fused TET1-CD have been tried to activate target genes [7]. Its broader use has been restricted as the transcription activator-like effectorbased strategies necessitate cumbersome design and assembly, being unsuitable for high-throughput applications
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