Developing a highly sensitive CRISPR based platform for virus and host functional genomics

Human cytomegalovirus (HCMV) is a highly prevalent pathogen causing severe disease in newborns and immunocompromised adults. It has an exceptionally large and complex genome, densely packed with hundreds of translated open reading frames, many of which are of unknown function. With the aim of systematically defining the functional components of the HCMV genome, we developed a highly sensitive, scalable screening platform based on CRISPR/Cas9 technology that we named VEKOS (Virus Encoded KO System). Previous efforts to perform functional screens for genes essential for virus propagation were limited by the use of indirect readouts, such as host cell viability or fluorescent reporters. Here, by inserting the sgRNA into the viral genome instead of the host cell genome we create a direct readout for viral propagation. Since the sgRNA is encoded within the viral genome, tagging it, any change in abundance of the sgRNA following selection is a direct and sensitive readout for the effect of the sgRNA on viral propagation. Using VEKOS with sgRNAs targeting essential and non-essential viral genes, we could detect strong differential effects on viral propagation. We next applied VEKOS in combination with a high-resolution sgRNA library densely targeting the HCMV genome. We will report on our efforts to create a detailed map of genomic elements important for viral propagation. The results of this screen will shed light on the genome of this complex virus, and promote our understanding of the principles underlying complex genomes.