Using CRISPR/Cas9 approaches to interrogate and delineate viral gene expression programmes

Abstract

Natural Killer/T cell Lymphoma (NKTL) is a highly aggressive form of nasopharyngeal cancer highly correlated with Epstein-Barr Virus (EBV) infection with poor treatment outcomes. EBV plays a major role in determining cancer progression through different epigenetic modifications mainly by causing the overexpression of DNA methyltransferases (DNMTs) and ubiquitin-like PHD and ring-finger domain-containing proteins (UHRFs). The hypermethylation of viral genes by DNMTs, guided by UHRFs, is presumed to be linked to the ability of EBV to avoid immune surveillance. To understand more about how DNMTs and UHRFs affect viral protein expression, we used CRISPR-based approaches, including the conventional gene-disrupting Cas9 and the gene-activating dCas9-VP64 system. We aimed to knockout key DNMT and UHRF genes to explore their roles in regulating the expression of lytic and latent viral genes, the latter including latent membrane protein 1 (LMP1). LMP1 mRNA levels have been found to be highly correlated with patient survival. The impact of these gene knockouts on lytic protein expression such as BZLF1 and BRRF2 that are linked to increased cancer progression is also investigated. Our findings demonstrate that there was a drastic increase in BRRF2 mRNA transcript levels with UHRF1 depletion. DNMT1 and UHRF1 reduction both show a slight decrease in LMP1 levels. However, DNMT3A and UHRF2 knockdowns showed an increase in LMP1 levels and hence, are potential targets for immunotherapy as their disruption may increase the immunogenicity of NKTL.