Project Summary Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus that infects epithelial and endothelial cells, fibroblasts, and monocytes before establishing lifelong latency in hematopoietic stem cells (HSCs). An estimated 50-95% of adults worldwide are infected. Periodic reactivation of latent virus is stimulated by HSC differentiation and does not cause symptoms in healthy, immunocompetent people. However, HCMV reactivation and replication causes serious complications in transplant patients, with lung transplant recipients and allogeneic hematopoietic stem cell transplant (HSCT) patients at the highest risk for experiencing CMV disease. Additionally, HCMV primary infection or reactivation during pregnancy can result in vertical transfer of the virus across the placenta to harm the developing fetus, making HCMV the leading cause of congenital birth defects in the US. Despite this, there is no FDA-approved HCMV vaccine. The complexity of HCMV infection in the host is highlighted by three things: 1) the ability to oscillate between latent and lytic infection cycles, 2) its broad cell tropism in vivo, and 3) the large number of ORFs (>150) in the viral genome. Current techniques to mutate HCMV rely on using fibroblasts, effectively limiting the scope of studies concerning viral tropism and latency factors. I propose a new method for creating recombinant HCMV—edit HCMV DNA using a Sendai virus (SeV) vector to deliver Cas9 and gRNAs targeting specific HCMV genes. SeV, a mouse paramyxovirus, has broad cell tropism and can efficiently deliver transgenes to CD14+ monocytes and CD34+ HSCs. In preliminary experiments, I cloned and rescued SeV-Cas9 viruses with gRNAs targeting components of the HCMV pentamer complex used for viral entry into epithelial cells. These SeV-Cas9 vectors targeted replicating HCMV with great efficiency, edited the desired ORF in 85-95% of virions, and blocked the virus from entering epithelial cells. This project aims to create a comprehensive map of which HCMV ORFs are required for viral entry, spread, and latency in all relevant cell types. I will create SeV-Cas9 vectors to target each HCMV ORF and edit HCMV in fibroblasts, epithelial cells, endothelial cells, and monocytes and analyze how disrupting the same gene in different cell types alters HCMV infection outcomes. Additionally, I will use SeV-Cas9 to target the HCMV latent genome in CD14+ and CD34+ HSC in vitro latency models to identify viral factors regulating latency establishment, maintenance, and reactivation. These project findings will identify which HCMV ORFs are required for HCMV spread among distinct cell populations and which ORFs are critical during latency, two things which have been impossible with current techniques. Thus, reprogramming SeV-Cas9 to target HCMV will revolutionize our ability to study HCMV in clinically relevant cell populations.