Nuclei were stained with Hoechst 33342 (ThermoFisher Scientific, cat. a member of the of viruses that can cause respiratory infections of varying severity. The cellular host factors and pathways co-opted during SARS-CoV-2 and related coronavirus life cycles remain ill defined. To address this space, we performed genome-scale CRISPR knockout screens during contamination by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E). These screens uncovered host factors and pathways with pan-coronavirus and virus-specific functional functions, including major dependency on glycosaminoglycan biosynthesis, sterol regulatory element-binding protein (SREBP) signaling, bone morphogenetic protein (BMP) signaling, and glycosylphosphatidylinositol biosynthesis, as well as a requirement for several poorly characterized proteins. We identified an absolute requirement for the VMP1, TMEM41, and TMEM64 (VTT) domain-containing protein transmembrane protein 41B (TMEM41B) for contamination by SARS-CoV-2 and three seasonal coronaviruses. This human coronavirus host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus pandemics. family, which is composed of enveloped positive-sense RNA viruses with large (>30 kb) genomes that can infect a variety of vertebrate hosts (Cui et?al., 2019). Seasonal human SKLB610 CoVs (HCoVs), such as the beta-CoV OC43, as well as the alpha-CoVs NL63 and 229E can cause moderate to moderate upper respiratory tract infections with cold-like symptoms in humans (Cui et?al., 2019). In stark contrast, highly pathogenic beta-CoVs have been responsible for multiple fatal?outbreaks in the 21st century, including SARS-CoV (2003), Middle East respiratory syndrome coronavirus (MERS-CoV; 2012), and SARS-CoV-2 (2019) (Cui et?al., 2019). The spread of SARS-CoV and MERS-CoV was contained in part because of?their comparatively low transmissibility (Cui et?al., 2019). However, SARS-CoV-2 spreads more readily and remains largely?uncontrolled across the globe, presenting an urgent health crisis. A complete understanding of the host factors and pathways co-opted by SARS-CoV-2 and other CoVs for execution of their life cycles could contribute to development of therapies to treat COVID-19 and increase preparedness for potential future outbreaks. Large-scale forward genetic approaches based on RNA interference, insertional mutagenesis, and CRISPR have proven to be powerful for identifying host factors required for contamination by different viruses (examined in Puschnik et?al., 2017). Here we performed parallel genome-scale CRISPR-Cas9 knockout screens to generate an extensive functional catalog of host factors required for contamination by SARS-CoV-2 and three seasonal CoVs (HCoV-OC43, HCoV-NL63, and HCoV-229E). We recognized multiple genes and pathways with pan-CoV and virus-specific functional functions, including factors involved in glycosaminoglycan (GAG) biosynthesis, sterol regulatory element-binding protein (SREBP) signaling, bone morphogenetic protein (BMP) signaling, and glycosylphosphatidylinositol (GPI) biosynthesis, as well as several poorly characterized proteins, such as transmembrane protein 41B (TMEM41B). We show that this VMP1, TMEM41, and TMEM64 (VTT) domain-containing protein TMEM41B is usually a critical host factor required for contamination by SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E as well as several flaviviruses of high interest to public health (Hoffmann et?al., 2020a [this issue of scores from (B) and (C) for SARS-CoV-2 screens at 37C and 33C, respectively. (E) Subset of significantly enriched genes from SARS-CoV-2 screens at 37C and 33C. We performed a series of parallel genetic screens by transducing Huh-7. 5-Cas9 cells with the Brunello library followed by antibiotic selection and growth SKLB610 for 7? days to ensure CRISPR-based knockout of host factor genes prior to CoV contamination. In this context, cells expressing sgRNAs targeting genes required for computer virus contamination or virus-induced death should survive, whereas those expressing neutral sgRNAs or sgRNAs targeting genes irrelevant to contamination are expected to deplete. Similarly, cells expressing sgRNAs targeting essential genes with no roles in computer virus contamination or virus-induced death are expected to deplete under mock-infected (uninfected) and virus-infected conditions. SARS-CoV-2 screens were performed in triplicate at two physiologically relevant temperatures, 33C and 37C, to mimic the temperatures of the upper and lower airways, respectively (Vkovski et?al., 2020). Surviving cells were harvested 5?days post-infection TNFRSF4 and subjected to genomic DNA extraction and screen deconvolution using high-throughput sequencing. Several quality control (QC) metrics exhibited excellent technical overall performance across all SKLB610 screens and biological replicates (Physique?S1 ). First, we confirmed that 76,160 of 76,441 of sgRNAs (99.6%) from your Brunello library SKLB610 were recovered from your plasmid preparation and that all screen libraries were sequenced to saturation (Physique?S1A). Second, pairwise correlation analyses exhibited that biological replicates from each genetic screen clustered together with high correlation coefficients (Physique?S1B). Third, receiver operating characteristic (ROC) curves generated based on the fitness effects of disruption of previously defined neutral and essential genes from your Brunello library.