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Systematic Genome-Scale Identification of Host Factors for SARS-Cov-2 Infection Across Models Yields a Core Single Gene Dependency; ACE2

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Systematic Genome-Scale Identification of Host Factors for SARS-Cov-2 Infection Across Models Yields a Core Single Gene Dependency; ACE2 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.28.450244; this version posted June 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. TITLE Systematic genome-scale identification of host factors for SARS-CoV-2 infection across models yields a core single gene dependency; ACE2. AUTHORS Katherine Chan1, Adrian Granda Farias1,2,*, Hunsang Lee1,*, Furkan Guvenc2,*, Patricia Mero1, Kamaldeep Aulakh1, Kevin R. Brown1, Shahan Haider1, Edyta Marcon1, Ulrich Braunschweig1, Amy Hin Yan Tong1, Shuye Pu1, Andrea Habsid1, Natasha Christie-Holmes4, Patrick Budylowski2, Audrey Astori5, Ayoob Ghalami6, Samira Mubareka7,8, Arinjay Banerjee9,10, Karen L. Mossman11, Jack Greenblatt1,2, Scott D. Gray-Owen2, Brian Raught5, Benjamin J. Blencowe1,2, Mikko Taipale1,2, Jason Moffat1,2,3 1Donnelly Center, 160 College Street, University of Toronto, Toronto, Ontario, Canada, M5S3E1 2Department of Molecular Genetics, 1 King’s College Circle, University of Toronto, Toronto, Ontario, Canada, M5S1A8 3Institute for Biomedical Engineering, Rosebrugh Building, 164 College Street, Room 407, University of Toronto, Toronto, Ontario, Canada, M5S3G9 4Combined Containment Level 3 Unit, Temerty Faculty of Medicine, University of Toronto Toronto, Ontario, Canada, M5S3E1 5Princess Margaret Cancer Center, Toronto, Ontario, Canada 6Office of Environmental Health & Safety, University of Toronto, Toronto, Ontario, Canada 7Sunnybrook Research Institute, Toronto, Ontario, Canada, M5S3E1 8Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada 9Vaccine and Infectious Disease Organization, Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada 10Department of Biology, University of Waterloo, Waterloo, Ontario, Canada 11Department of Medicine, McMaster University, Hamilton, Ontario, Canada * de-notes equal contributions Correspondence: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.28.450244; this version posted June 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. IN BRIEF SARS-CoV-2, depends on host cell components for infection and replication. Genome-wide CRISPR screens were performed in multiple human cell lines to elucidate common host dependencies required for SARS-CoV-2 infection. Only ACE2, the cognate SARS-CoV-2 entry receptor, was common amongst cell lines, while all other host genes identified were cell line specific, several of which converged on pathways involved in cell signalling, lipid metabolism, immune pathways, and chromatin modulation. Overall, a network of host factors was identified that will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate productive targeting of host genes and pathways. HIGHLIGHTS - Genome-wide CRISPR screens for SARS-CoV-2 in multiple human cell lines - Identification of wide-ranging cell-type dependent genetic dependencies for SARS-CoV-2 infection - ACE2 is the only common host factor identified across different cell types 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.28.450244; this version posted June 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. SUMMARY SARS-CoV-2, depends on host cell components for replication, therefore the identification of virus-host dependencies offers an effective way to elucidate mechanisms involved in viral infection. Such host factors may be necessary for infection and replication of SARS-CoV-2 and, if druggable, presents an attractive strategy for anti-viral therapy. We performed genome wide CRISPR knockout screens in Vero E6 cells and 4 human cell lines including Calu-3, Caco-2, Hek293 and Huh7 to identify genetic regulators of SARS-CoV-2 infection. Our findings identified only ACE2, the cognate SARS-CoV-2 entry receptor, as a common host dependency factor across all cell lines, while all other host genes identified were cell line specific including known factors TMPRSS2 and CTSL. Several of the discovered host-dependency factors converged on pathways involved in cell signalling, lipid metabolism, immune pathways and chromatin modulation. Notably, chromatin modulator genes KMT2C and KDM6A in Calu-3 cells had the strongest impact in preventing SARS-CoV-2 infection when perturbed. Overall, the network of host factors that have been identified will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate the development of host-directed therapies. 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.28.450244; this version posted June 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. INTRODUCTION Since its identification in late 2019, the highly infectious severe acute respiratory syndrome coronavirus (SARS-CoV-2) has rapidly spread across the world, resulting in over 160 million infections and greater than 3.7 million deaths as of June 9, 2021 (https://www.worldometers.info/coronavirus/). SARS-CoV-2 is closely related to SARS-CoV-1, which caused the 2002-2004 SARS epidemic, as well as MERS-CoV, the virus responsible for respiratory disease outbreaks in 27 countries since 2012 (da Costa et al., 2020). Infections can range from asymptomatic or mild cases to severe respiratory disease, that can lead to acute respiratory distress syndrome, septic shock and multi-organ failure (Vos et al., 2021). SARS-CoV- 2, after SARS-CoV and MERS-CoV, is the third zoonotic coronavirus in the last two decades to rise to epidemic, and now, pandemic proportions. Each of these viral outbreaks can be traced to animal hosts, with zoonosis accounting for human CoV infections. Increasingly close contact with diverse animal species will continue to drive zoonotic transmission of potentially deadly coronaviruses, which underscores the importance of clearly delineating the molecular mechanisms of human CoV viral infection (Banerjee et al., 2021a; Banerjee et al., 2021c). Like all viruses, SARS-CoV-2 is an obligate intracellular pathogen that depends on host cell components for replication. The viral envelope is studded with several proteins, including the Spike (S) glycoprotein that mediates direct virus-host cell interactions (Hartenian et al., 2020; V'Kovski et al., 2021). The SARS-CoV-2 S-protein is a heavily glycosylated homotrimer composed of two subunits, S1 (receptor binding domain - RBD) and S2 (helical domain that mediates membrane fusion), which act in a coordinate manner to attach to the host cell surface and initiate viral entry (Letko et al., 2020). S-protein RBD binds to the host receptor angiotensin-converting enzyme 2 (ACE2), which also serves as the entry receptor for SARS-CoV (Hoffmann et al., 2020). High variability in the RBD was the major determinant of cross species transmission and evolution of SARS-CoV-2 (Starr et al., 2020). Furthermore, mutations in RBD have appeared among pandemic variants, increasing virus transmissibility and potentially compromising efficacy of treatments and vaccines in development (Linsky et al., 2020). ACE2 is highly expressed in the lung, heart, arteries, kidney and intestines, an expression pattern that may in part explain the constellation of symptoms associated with SARS infection (Crackower et al., 2002; Danilczyk et al., 2006; Hashimoto et al., 2012; Imai et al., 2005; Zhang et al., 2020). In addition to ACE2, transmembrane protease serine 2 (TMPRSS2) has been identified as a cofactor that aids viral entry by priming S-protein through cleavage of the S1/S2 and S2’ cleavage sites (Zhu et al., 2021). Similarly, cathepsin L (CTSL) has also been shown to mediate S priming and facilitate intracellular viral release (Zhao et al., 2021). Recent studies show that SARS-CoV-2 has an insertion of a polybasic (RXYR) furin cleavage site at the S1/S2 boundary that is absent from SARS-CoV and other 2b betaCoVs. This could be an additional factor contributing to the increased virulence of SARS-CoV-2, as furin proteases are ubiquitously expressed in humans, providing expanded tissue tropism and pathogenesis (Wang et al., 2020). The SARS-CoV-2 life cycle appears to require a mixed array of host factors. A deeper understanding of the host-pathogen interactions of SARS-CoV-2 across a range of permissive models will help shed light on viral pathogenesis and host defense mechanisms. In fact, 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.28.450244; this version posted June 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. therapeutic strategies targeting host factors are still important to consider as virus-targeted therapies will likely drive viral escape mechanisms and the emergence
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