Suppression of Epstein-Barr virus infection in nasopharyngeal carcinoma cells through CRISPR/Cas9 targeting of EBNA1, OriP and W repeats

Abstract

Epstein-Barr virus (EBV) infects more than 90 % of the world's adult population. Persistent infection with EBV is strongly associated with nasopharyngeal carcinoma (NPC), the incidence of which is geographically restricted and particularly high in Southern China and Hong Kong. EBV infection favors host cell growth and survival to facilitate NPC development. Eliminating EBV has therapeutic effect but existing methods could only inhibit viral protein or RNA expression incompletely and transiently. CRISPR/Cas9 has recently emerged as a powerful and versatile tool for targeted engineering of cellular genome. We have recently demonstrated the feasibility of CRISPR/Cas9 editing of EBV genome in latently infected cells. This editing results in complete and permanent disruption of EBV genes. No off-target cleavage was noted. The next technical challenge is to harness CRISPR/Cas9 to eliminate EBV latent infection. In this study we explored CRISPR/Cas9 targeting of an EBV essential gene EBNA1 towards elimination of EBV infection in NPC cells. We identified gRNAs that have optimal EBNA1-targeting and EBV-suppressing activity but minimal off-target effects. The gRNAs were introduced into EBV-infected NPC cells together with Cas9 nuclease. Furthermore, gRNAs and Cas9 were delivered to NPC cells via adeno-associated virus vector and a replication-deficient recombinant EBV. The levels of EBV DNA in infected NPC cells were remarkably decreased. Raji cell infection assays indicated a significant reduction of the infectivity of EBV recovered from NPC cells. Our work provides not only a new platform technology for suppressing and eliminating DNA virus infection, but also a new strategy to cure EBV infection using CRISPR/Cas9 targeting of EBNA1.