Functional single-cell genomics of human cytomegalovirus infection

The complex life cycle of herpesviruses is orchestrated by the interplay of host factors and hundreds of viral genes. Understanding how they work together and how perturbations of viral and host factors impact infection represents both a fundamental problem in virology and the basis for designing antiviral interventions. Here, we use CRISPR screening to study infection of human cytomegalovirus (HCMV) in primary cells, comprehensively defining the functional contribution of each viral and host factor. We then record the transcriptomes of tens of thousands of single cells, and monitor how genetic perturbation of critical host and viral factors alters the timing, course, and progression of infection. We find that normally, the large majority of cells follow a stereotypical transcriptional trajectory. Perturbing critical host factors does not change this trajectory per se, but can either stall, delay or accelerate progression along the trajectory, allowing us to pinpoint systematically the stage of infection at which each host factor acts. Conversely, perturbation of viral factors can create distinct abortive trajectories. Our results reveal a dichotomy between the roles of host and viral factors and more generally provide a road map for functional dissection of host-pathogen interactions. Overall design: This repository includes six individual experiments, all conducted in human foreskin fibroblasts (HFFs) infected with human cytomegalovirus (HCMV). (1) A pooled CRISPR nuclease (CRISPRn) screen with a library tiling the entirety of the HCMV genome, identifying viral factors that affect survival of infected cells. (2) A pooled CRISPR interference (CRISPRi) screen with a human genome-wide library, identifying host factors that affect survival of infected cells. (3) A pooled CRISPR nuclease (CRISPRn) screen with a human genome-wide library, identifying host factors that affect survival of infected cells. (4) A time course of HCMV infection in genetically unperturbed fibroblasts, recorded by single-cell RNA sequencing. (5) A pooled single-cell CRISPRi experiment (CRISPRi Perturb-seq) targeting host factors, recorded by single-cell RNA sequencing. (6) A pooled single-cell CRISPRn experiment (CRISPRn Perturb-seq) targeting both viral and host factors, recorded by single-cell RNA sequencing.