CRISPR/Cas9-mediated exploration and disruption of herpes simplex virus type 1

Abstract

Herpesviruses are widespread human pathogens that establish lifelong infections in nearly the entire population. Following primary infection, these viruses can enter a latent state characterized by minimal viral gene expression and evasion of immune surveillance. As a result, the host immune system cannot eradicate latent viruses, and no existing therapeutic interventions can fully eliminate them. In this dissertation, novel strategies to eradicate herpesvirus infections were explored using advanced genome-editing technology. Specifically, the prokaryotic antiviral CRISPR/Cas9 system was adapted to selectively target and inactivate the genomes of human herpesviruses. This approach proved highly effective in eliminating infections caused by several members of the Herpesviridae family, including Epstein–Barr virus (EBV), herpes simplex virus type 1 (HSV-1), and human cytomegalovirus (HCMV). When multiple CRISPR/Cas9 constructs directed against HSV-1 were applied simultaneously, complete suppression of viral replication was achieved. Comparative analyses with conventional antiviral treatments currently used in clinical practice demonstrated that CRISPR/Cas9-mediated targeting was more effective in controlling HSV-1 replication and in reducing the emergence of drug-resistant variants. These findings highlight the potential of CRISPR/Cas9 as the basis for developing curative therapeutic strategies against HSV-1 infection. Given that HSV-1 not only causes recurrent oral lesions but can also lead to chronic keratitis and vision loss, a curative treatment would provide significant medical and societal benefit. Beyond direct genome targeting, this work also employed CRISPR/Cas9 as a powerful discovery tool to identify host factors essential for viral infection. Genome-wide loss-of-function screens in human HAP1 cells revealed multiple host genes exploited by viruses during infection. Similar screening with the Modified Vaccinia Ankara (MVA) smallpox virus identified additional host determinants critical for viral replication. The elucidation of these host–virus interactions offers fundamental insights into viral pathogenesis and reveals promising molecular targets for the development of future antiviral therapeutics.