Development and Application of Novel CRISPR-Based Epigenome Editors

Abstract

<p>The eukaryotic epigenome has an instrumental role in determining and maintaining cell identity and function. Epigenetic components such as DNA methylation, histone tail modifications, chromatin accessibility, and DNA architecture are tightly correlated to central cellular processes, while their dysregulation manifests in aberrant gene expression and disease. The ability to specifically edit the epigenome holds the promise of enhancing understanding how epigenetic modifications function and enabling manipulation of cell phenotype for scientific or therapeutic purposes. Genome targeting technologies, such as the CRISPR/Cas9 system, have successfully been harnessed to create epigenome editing tools to alter gene expression. Prominently, two leading CRISPR-based technologies, CRISPRa and CRISPRi, were shown to be highly specific and effective in controlling gene transcription levels. These tools, however, often lead to formation of complexes that affect a multitude of endogenous factors, thus mitigating our ability to elucidate the role of individual epigenetic marks. Moreover, changes in epigenetic marks are associated with numerous health conditions, therefore the development of tools that can modify specific marks may help in creating disease models, or the restoration of a “healthy” epigenome. We first created a suite of CRISPR-based epigenome modifiers (CRISPR-GEMs) that were aimed to catalyze the removal or addition of specific histone tail marks. Next, we tested a few promising CRISPR-GEMs on multiple target genes to characterize their effect on gene expression and chromatin marks. Furthermore, we utilized these tools to deepen our insights into the relationship of individual histone marks and gene expression in different contexts and to better our understanding of the kinetics and dynamics of several of these novel tools alongside existing ones. Additionally, we decided to use the CRISPRa platform to explore senescence, a cellular process that is at the epicenter of aging and has been shown to play a key role in various age-related diseases. Using the CRISPRa platform in an inducible-senescence cell model, we found and validated multiple transcription factors (TFs) that regulate senescence-associated growth arrest (SAGA). Lastly, we characterized genetic pathways that are pivotal to successful inhibition of SAGA, thereby demonstrating a new application of epigenome editing in a senescence model that enhanced our understanding of the pathways that govern SAGA.</p>