Epigenetic Reprogramming for Cancer Therapy

Background

Epigenetics refer to heritable changes in gene expression that are not associated with changes in the DNA sequence. DNA methylation is a key epigenetic mark responsible for regulating gene expression. When present in high levels, functions to repress gene expression. The aberrant distribution of DNA methylation is associated with the development of many forms of cancer. Thus the ability to modulate oncogenic epigenetic marks would prove advantageous in developing novel prevention and treatment options.

Current clinical epigenetic therapies have many side effects as they function by globally altering the epigenome and causing a total reduction in DNA methylation. In an effort to target specific epigenetic regions with minimal off-target effects, we utilized CRISPR-Cas technology to achieve site-specific DNA methylation. CRISPRs function through the Cas9 protein that associates with a guide RNA (gRNA) to bind a particular DNA region. While Cas9 typically acts as an endonuclease, we utilized a catalytically-dead Cas9 mutant (dCas9) that binds but does not cleave DNA. A gRNA-associated dCas9 protein, when fused with an enzymatic catalytic domain, exerts localized catalytic action on its bound DNA region.

Project Goal

In our study, we fused the catalytic domain of DNMT3a to dCas9. As a proof of concept, we targeted the dCas9-DNMT3a complex to the p21 tumor suppressor gene. Since p21 is a major negative regulator in the cell cycle and in cell growth, we hypothesized that dCas9-DNMT3a would induce de novo methylation on p21. This would result in an increased p21 methylation profile and increased cell growth rate.