Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here, we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, MERS-CoV, bat coronavirus HKU5 expressing the SARS-CoV-1 spike, and VSV expressing the SARS-CoV-2 spike. We identify known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways including HMGB1 and the SWI/SNF chromatin remodeling complex that are SARS-lineage and pan-coronavirus specific, respectively. We show HMGB1 regulates ACE2 expression and is critical for viral entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. Together this identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS-lineage specific and pan-coronavirus host factors that regulate susceptibility to highly pathogenic coronaviruses.

Highlights d ChIRP-MS of SARS-CoV-2 RNA identifies viral RNA-host protein interaction networks d Comparative analysis identifies SARS-specific and multiviral RNA-protein complexes d SARS-CoV-2 interactome-focused CRISPR screens reveal a broad antiviral response d Host mitochondria serve as a general organelle platform for anti-SARS-CoV-2 immunity

Base editor screens link sequence variation and gene function.

Cas12a RNA-guided endonucleases are promising tools for multiplexed genetic perturbations because they can process multiple guide RNAs expressed as a single transcript and subsequently cleave target DNA. However, their widespread adoption has lagged behind Cas9-based strategies due to low activity and the lack of a well-validated pooled screening toolkit. Here we describe the optimization of enhanced AsCas12a (enAsCas12a) for pooled, combinatorial genetic screens in human cells. By assaying the activity of thousands of guides, we refine on-target design rules and develop a comprehensive set of off-target rules to predict and exclude promiscuous guides. We also identify 38 direct repeat variants that can substitute for the wild-type sequence. We validate our optimized AsCas12a toolkit by screening for synthetic lethalities in OVCAR8 and A375 cancer cells, discovering an interaction between MARCH5 and WSB2 . Finally, we show that enAsCas12a delivers similar performance to Cas9 in genome-wide dropout screens but at greatly reduced library size, which will facilitate screens in challenging models.

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-β signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis. Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 , represents the greatest public health threat in a century. More than 7,500,000 people have been infected with more than 420,000 deaths globally (1). Novel therapeutics and vaccines are desperately needed. Coronaviruses are enveloped, positive-sense RNA viruses with genomes of approximately 30 kb that exhibit broad host-range among birds and mammals and are typically transmitted via the respiratory route (2, 3). There are four circulating seasonal coronaviruses in humans (NL63, OC43, 229E, and HKU1) and three highly pathogenic zoonotic coronaviruses (SARS-CoV, MERS, and SARS-CoV-2), none of which have effective antiviral drugs or vaccines (4-7).Viral entry, the first stage of the SARS-CoV-2 life cycle, is mediated by the viral spike protein. The receptor binding domain of spike binds to the cell surface receptor angiotensinconverting enzyme 2 (ACE2), a major determinant of host range and cell tropism (8,9). The coronavirus spike protein requires two proteolytic processing steps prior to entry. The first cleavage event occurs at the interface of the S1 and S2 domains of the spike protein (10, 11). This can occur in the producer cell, the extracellular environment, or in the endosome and can be mediated by several proteases including furin and the plasma membrane protease TMPRSS2 (12)(13)(14). A second proteolytic event is required within S2 to expose the viral fusion peptide and enable membrane fusion. This second cleavage event can occur at the target cell plasma membrane by TMPRSS2 or in the endosome by Cathepsin L (14,15). Upon viral membrane fusion, the viral RNA is released into the cytoplasm where it is translated and establishes viral replication and transcription complexes before assembling and budding (16)(17)(18). The host genes that mediate these processes largely remain elusive.Identification of host factors essential for infection is critical to inform mechanisms of COVID-19 pathogenesis, reveal variation in host susceptibility, and identify novel host-directed therapies, which may have efficacy against current and future pand...