INTRODUCTION
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, has claimed over 6 million deaths worldwide and has disrupted the global economy (COVID-19 Dashboard by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University, accessed July 21, 2023). Mutations in the spike envelope protein of SARS-CoV-2 continue to accumulate contributing to new variants that may have altered infectivity. Additionally, new viruses will emerge with envelopes that influence their transmissibility and pathogenesis as evidenced by the emergence of new SARS-CoV-2 variants and the need for revised vaccines (Harvey et al., 2021; Mannar et al., 2022; Volz et al., 2021; L. Zhang et al., 2020). Thus, developing countermeasures requires new methods that can rapidly identify host factors affecting the viral infection.
CRIPSR/Cas9 has emerged as a powerful tool for identifying host factors affecting the viral life cycle. CRISPR screens against SARS-CoV-2, provide insight into these factors, however, nearly all screens run to date rely on cellular lethality leading to the depletion of host cells bearing all kinds of mutations that decrease viability during infection. These screens are therefore complicated by large numbers of host factors affecting all steps of the viral life cycle including entry, replication, pathogenesis and egress as well as host viability factors influencing energy metabolism, macromolecular synthesis and cell cycle regulation (Baggen et al., 2021; Daniloski et al., 2021; Schneider et al., 2021; Wang et al., 2021; Wei et al., 2021). The lack of specificity in current screening strategies leads to a biased selection of target genes for validation, based on investigator preference, or costly and time-consuming rational validation screens (Baggen et al., 2021; Daniloski et al., 2021; Han et al., 2017; Schneider et al., 2021; Wang et al., 2021; Wei et al., 2021). Replication incompetent pseudoviral vectors, can deliver reporters without triggering cell death and therefore provide a strategy for isolating genes associated with viral entry. Indeed, lentivirus pseudotyped with SARS-CoV-2 spike protein have demonstrated great utility in the pandemic, serving as the basis for viral neutralization assays (Neises et al., 2021). Also, replication competent vesicular stomatitis virus (VSV) expressing SARS-CoV-2 Spike has been used in previously published CRISPR screens (Wei et al., 2021). Yet, improved strategies for identifying host factors affecting viral entry are needed.
Here we describe proof-of-concept findings for a method for identifying genes affecting viral entry of SARS-CoV-2 and VSV-G into target cells. Using the Brunello library, we mutagenized 293T-Ace2 cells and then screened them for entry of SARS-CoV-2 spike and VSV-G pseudoviruses with a GFP reporter (Doench et al., 2016). Comparative analysis of these screens demonstrated the feasibility of focusing CRISPR whole-genome screens down to genes influencing the specific step of envelope mediated entry and allowed identification of genes that were responsible for retro-transcription of reporter genes. Moreover, inclusion of a recently developed Gag-Cre auto-cleaving fusion into the VSV-G pseudoviruses and screening in 293T cells bearing a floxed fluorescent protein reporter increased the specificity for the genes identified to viral entry (Esposito et al., 2016; P. E. Mangeot et al., 2019). This method identified the known ACE2 receptor as a top hit gene in our SARS-CoV-2 spike screen and LDL receptor as one of the top hits in our VSV-G-GagCre screens as well as other putative genes. Overall, this strategy provides a new approach for identifying host genes specific to envelope mediated entry and will be of use for new studies targeting viral entry.