ABSTRACT
Background Understanding the cellular host factors that promote and inhibit viral entry is important for identifying viral countermeasures. CRISPR whole genome screens can be used to rapidly discover host factors that contribute to or impair viral entry. However, when using the live viruses and cellular lethality for selection, these screens identify large numbers of genes without any specificity for the stage of the viral infection cycle. New screening methods are needed to identify host machinery contributing to specific steps of viral infection. Here, we developed a CRISPR whole genome screen and counter screen strategy based on a pseudoviral platform that allowed identification of genes specific to SARS-CoV-2 spike and vesicular stomatitis virus glycoprotein VSV-G mediated entry.Methods To focus the screen onto the entry step, we used non-lytic fluorescent reporters in combination with a comparative counter screen strategy to distinguish host genes affecting the pseudoviral reporter from those unique to envelope-mediated entry. Screening of SARS-CoV-2 spike and VSV-G on the same lentiviral pseudovirus allowed identification of entry-specific genes relative to genes associated with retro-transcription, integration, and reporter expression from the lentiviral pseudovirus. Second, a Cre-Gag fusion protein in the pseudovirus was used to bypassed retro-transcription and integration by directly activating a floxed GFP reporter upon entry to reduce the number of gene hits and increase specificity for viral entry.Results Our approach correctly identified SARS-CoV-2 and VSV-G receptors ACE2 and LDLR, respectively and distinguished genes associated with retroviral reporter expression from envelope-mediated entry. Moreover, the CRE-Gag fusion/flox reporter increased the screen specificity for viral entry associated genes.Conclusion Overall, this approach provides a new strategy for identifying host genes influencing viral entry without the confounding complexity of live-viral screens which produce long gene lists associated with all aspects of viral pathogenesis and replication. This approach provides a pathway for increasing the specificity of CRISPR whole genome screens for identifying host genes contributing to specific steps in viral infection.