A virally encoded high resolution screen of cytomegalovirus host dependencies

Genetic screens have transformed our ability to interrogate cellular factor requirements in infection, yet current approaches are limited in their sensitivity, biased towards early stages of infection and provide only simplistic phenotypic information which is often based on infected cell survival. Here, by engineering human cytomegalovirus to express sgRNA libraries directly from the viral genome, we developed a sensitive, versatile, viral centric approach that allows profiling of different stages along viral infection in a pooled format. Using this approach, which we termed VECOS (Virus Encoded CRISPR-based direct readOut Screening system), we identified hundreds of novel dependency and restriction factors and we quantified their direct effects on viral genome replication, viral particle secretion and infectiousness of secreted particles, providing a multi-dimensional perspective on viral-host interactions. These high resolution measurements reveal that perturbations that alter late stages in HCMV life cycle mostly regulate HCMV particle quality rather than quantity, defining correct virion assembly as a critical stage that is heavily reliant on viral-host interactions. Overall, VECOS facilitates systematic high resolution dissection of human proteins' role along the infection cycle, providing a roadmap for in-depth dissection of host–herpesvirus interactions. CRISPR/Cas9 based screen perfomed by creating an sgRNA library encoded in Human Cytomegalovirus (HCMV), where each virus expresses one sgRNA targeting one of 2000 genes in the human genome. Cas9-expressing fibroblasts were infected with the library at a low MOI, and sgRNA abundance was determined via deep sequuencing of sgRNA amplicon sequences, amplified from the viral genome. Samples were collected at 10 days post infection, and sgRNA abundance was determined in the infected cells, in the secreted virus, and in the infectious virus population (by sequencing virses that established infection in WT fibroblasts. Each condition was done in triplicates, and the selection in Cas9-cells was performed in 3 sequential rounds. The input library was sequenced in three biological replicates, and the plasmid and BAC cloning stages were also sequenced. To verify the effectivity of knockout by sgRNA expressed from the viral genome, viruses targeting two human genes (RRM2 and TRIP10) were used to infect Cas9 fibroblasts and the cells were harvested at different times post infection (0, 10, 24, 48, 72 hours), and PCR amplicons of the targeted seuqences in the human genome were deep sequenced.