Abstract
DNA methylation is a stable epigenetic modification, playing a critical role in the regulation of gene expression. Dysregulation of DNA methylation is implicated in the pathogenesis of numerous diseases including cancer. High level of promoter DNA methylation is associated with transcriptional silencing. However, we identified Early B-cell Factor 3 (EBF3) as a potential epigenetic driver of melanoma metastasis displaying high promoter methylation associated with increased mRNA expression. To establish the causal role of DNA methylation in directly altering gene transcription, it is necessary to exclusively edit the methylation status of the target loci.
The emergence of CRISPR/dCas9-based editing systems have shown a more specific and efficient method for targeted manipulation of DNA methylation compared to traditional epigenomic editing technologies. However, despite the recent improvements in CRISPR/dCas9 technology, off-target effects are still a major concern.
Here, I describe the initial construction of plasmid components of CRISPR-dCas9-SunTag demethylation system required for the editing the EBF3 promoter region. Moreover, an evaluation of the system’s potential off-target effects in relation with locus-specific guide- RNA (gRNA) targeting was analysed by the presence of Cas9 activity. Analysis of Cas9 activity on putative off-target locations was performed using targeted Illumina Miseq Sequencing. Limited Cas9 activity have been observed in these off-target sites confirming the specificity of gRNA targeting.
Overall, these work has laid an initial platform for further investigation of the relationship between DNA methylation and transcriptional regulation.