Targeted Epigenetic Editing Using a dCas9- SunTag Methylation System

Abstract

DNA methylation is a stable and somatically heritable epigenetic process. It is classically understood as a gene silencing mechanism, whereby hypermethylation of gene promoters is largely associated with transcriptional silencing. However, emerging research in the past decade has found a different association, particularly in the context of cancer and metastasis. These studies have demonstrated a seemingly paradoxical correlation between high levels of promoter methylation and subsequent transcriptional activation. As such, we are yet to fully unravel the complex mechanism underlying this relationship. This warrants further investigations to better understand this dynamic molecular mechanism and thus its implications in cancer pathobiology and metastasis.

Previously established methods of manipulating DNA methylation have largely involved the use of chemical drugs, such as decitabine. These drugs act globally on the epigenome and are thus non-specific. Importantly, as they are inhibitors of DNA methylation, they cannot definitively demonstrate that an increase in promoter methylation is directly responsible for an increase in gene expression. Therefore, by using a CRISPR-based system for targeted epigenetic editing, the precise mechanism of paradoxical gene activation by DNA methylation can now be investigated.

Our group has recently demonstrated this paradoxical correlation in a study characterising epigenetic changes in metastatic melanoma. The Early B-cell Factor 3 (EBF3) gene promoter region was found to be substantially hypermethylated in metastatic cell lines compared to paired, primary cell lines. This increase in methylation was also found to be associated with a subsequent increase in gene expression. These findings suggest that EBF3 in human melanoma cell lines is a good candidate gene to investigate this complex relationship.

Here, I aim to establish a methylation editing system and apply it to the EBF3 promoter region. This is a three-component CRISPR system incorporating the SUperNova Tag (SunTag), which allows for the recruitment of multiple effector proteins. In this project, I use the DNA methyltransferase 3A (DNMT3A) effector protein to induce active methylation at a target locus within the EBF3 promoter region in melanoma cell lines.

Methylation changes have been evaluated using methylation-specific Illumina iSeq sequencing. Variable levels of targeted methylation have been observed, of up to 38.0% absolute methylation change between edited and unedited samples. This project represents important progress in investigating the promoter methylation of EBF3 in more detail, as well as laying out an essential platform to further investigate methylation changes and causal mechanisms of gene expression alteration in future studies.