ARS-CoV-2 is the etiologic agent of the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV-2 is the third highly pathogenic coronavirus to cross the species barrier in the 21st century after SARS-CoV-1 in 2002-2003 (refs. 1-3 ) and MERS-CoV in 2012 (ref. 4 ). Four additional HCoVs (HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1) are known to circulate seasonally in humans, contributing to approximately one-third of common cold infections 5 . Like SARS-CoV-1 and HCoV-NL63, SARS-CoV-2 entry into target cells is mediated by the angiotensin-converting enzyme 2 (ACE2) receptor [6][7][8][9][10] . The cellular serine protease transmembrane protease serine 2 (TMPRSS2) is used by both SARS-CoV-1 and SARS-CoV-2 for Spike protein priming at the plasma membrane 6,11 . Cathepsins are also involved in SARS-CoV spike protein cleavage and fusion peptide exposure upon entry via an endocytic route, in the absence of TMPRSS2 (refs. [12][13][14][15] ).Several whole-genome KO CRISPR screens for the identification of coronavirus regulators have been reported [16][17][18][19][20][21] . These screens used naturally permissive simian Vero E6 cells of kidney origin 20 ; human Huh7 cells (or derivatives) of liver origin (ectopically expressing ACE2 and TMPRSS2, or not) 16,18,19 ; and A549 cells of lung origin, ectopically expressing ACE2 17,21 . Here, we conducted genome-wide, loss-of-function CRISPR KO screens and gain-of-function CRISPRa screens in several cell lines, including physiologically relevant human Calu-3 cells and Caco-2 cells, of lung and colorectal adenocarcinoma origin, respectively, followed by secondary screens in these cell lines and in Huh7.5.1 and A549 cells. Well-known SARS-CoV-2 host-dependency factors were identified among top hits, such as ACE2 and either TMPRSS2 or cathepsin L (depending on the cell type). We characterized the mechanism of action of the top hits and assessed their effect on other coronaviruses and influenza A orthomyxovirus. Altogether, this study provides insights into the coronavirus life cycle by identifying host factors that modulate replication and might lead to pan-coronavirus strategies for host-directed therapies. ResultsMeta-analysis of CRISPR KO screens highlights the importance of multiple models. Vero E6 cells present high levels of cytopathic effects (CPEs) upon SARS-CoV-2 replication, making them ideal to perform whole-genome CRISPR screens for host factor identification. A Chlorocebus sabaeus single-guide RNA (sgRNA) library was previously successfully used to identify host factors regulating SARS-CoV-2 (isolate USA-WA1/2020) replication 20 . Therefore, we initially repeated whole-genome CRISPR KO screens in Vero E6 cells using the SARS-CoV-2 isolate BetaCoV/France/ IDF0372/2020 (Fig. 1a). Importantly, ACE2 was a top hit (Fig. 1b

Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. We performed genome-wide knockout and activation CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. In addition to identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Moreover, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins, IL6R, and CD44 were identified. We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A orthomyxovirus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.

Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. We performed genome-wide knockout and activation CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. In addition to identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Moreover, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins, IL6R, and CD44 were identified. We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A orthomyxovirus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.