PROJECT SUMMARY/ABSTRACT Herpesviruses such as Herpes simplex virus (HSV) 1 and 2 are ubiquitous DNA viruses that establish lifelong infections. After primary infection, they enter latency and occasionally reactivate, causing recurrent disease. HSV 1 and 2 establish latency in neurons, and reactivation causes lesions of the facial or genital area. HSV-1 and 2 lack vaccines or curative treatments and new therapeutic strategies against chronic HSV diseases are critically needed. During an infection, cells are frequently co-infected by multiple virions, and therapeutic approaches that rely on viral co-infection have great potential. We recently invented a CRISPR-based “viral gene drive” that relies on viral co-infection to replace wild-type viruses with an engineered version. Our strategy was inspired by similar methods developed in insects and uses CRISPR-Cas9 and homologous recombination to efficiently propagate a genetic modification in the viral population, ultimately reducing viral levels. Our discovery uncovered a new way to engineer herpesviruses for therapeutic and research purposes. Our long-term goal is to design gene drives that could be used as curative therapies for HSV infection. Patients suffering from chronic HSV disease could be treated with an engineered virus that recombines with wild-type viruses in the latent reservoir and prevents viral outbreaks. In this innovative application, we propose to determine if a viral gene drive can spread to the latent reservoir and inactivate wild-type viruses in mice latently infected with HSV-1. In specific aim 1, we will use mouse models of HSV-1 infection to test if a gene drive can suppress viral shedding in mice persistently infected with wild-type HSV-1. This will establish the potential of gene drives to cure chronic HSV-1 infection. In specific aim 2, we will use fluorescently-labeled viruses and mathematical modeling to quantify co-infection frequency during gene drive propagation in vivo. The rationale is that gene drives rely on co-infection, and that a better understanding of the biology of co-infection is needed for the development of our innovative therapeutic strategy. We will determine how often a gene drive reaches latently infected neurons and establish if a gene drive can efficiently inactivate the latent reservoir. Altogether, this project will test the therapeutic potential of a breakthrough technology and may lead to novel therapies that significantly improve human health.