A Two-tiered functional screen identifies herpesviral transcriptional modifiers and their essential domains

While traditional methods for studying large DNA viruses allow the creation of individual mutants, CRISPR/Cas9 can be used to rapidly create thousands of mutant dsDNA viruses in parallel, enabling the pooled screening of entire viral genomes. Here, we applied this approach to Kaposi's sarcoma-associated herpesvirus (KSHV) by designing a sgRNA library containing all possible ~22,000 guides targeting the 154 kilobase viral genome, corresponding to one cut site approximately every 8 base pairs. We used the library to profile viral sequences involved in transcriptional activation of late genes, whose regulation involves several well characterized features including dependence on viral DNA replication and a known set of viral transcriptional activators. Upon phenotyping all possible Cas9-targeted viruses for transcription of KSHV late genes we recovered these established regulators and identified a new required factor (ORF46), highlighting the utility of the screening pipeline. By performing targeted deep sequencing of the viral genome to distinguish between knock-out and in-frame alleles created by Cas9, we identify the DNA binding but not catalytic domain of ORF46 to be required for viral DNA replication and thus late gene expression. Our pooled Cas9 tiling screen followed by targeted deep viral sequencing represents a two-tiered screening paradigm that may be widely applicable to dsDNA viruses. Author summaryWhile most cancers are caused by mutations to the cell's DNA, others are instead induced by viruses known as oncogenic viruses or oncoviruses. These cancers provide unique challenges but also opportunities for treatment and prevention. Here, we study a human oncogenic virus called Kaposi's sarcoma-associated herpesvirus. It, along with related oncoviruses, relies on a unique form of transcription to express essential viral genes, and we sought to further understand how this class of genes is regulated, potentially leading to therapeutic vulnerabilities. Here we use CRISPR/Cas9 to create and measure the ability of thousands of mutant viruses to express this key class of viral genes. This allowed us to identify a novel viral gene that is required, but this gene is known to have multiple functions. By combining this CRISPR/Cas9 approach with direct viral sequencing, we were able to identify the specific function that is required. While our results contribute specifically to the study of this unique step in the viral life cycle, our methods can be applied to many open questions in the study of these oncoviruses.

Automated summary

This study demonstrates the use of CRISPR/Cas9 to rapidly create and screen thousands of mutant viruses in Kaposi's sarcoma-associated herpesvirus (KSHV). By analyzing the expression of viral genes in these mutant viruses, a novel required gene was identified and its specific function was characterized using targeted deep viral sequencing.