Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs

Abstract

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

Kinase-independent activity of DYRK1A promotes viral entry of highly pathogenic human coronaviruses

“…We previously identified DYRK1A as a pro-viral gene for both SARS-CoVs and MERS-CoV in Vero-E6 cells and Calu-3 cells 22,23 . Additional recent independent screens in Vero-E6 and Calu-3 cells have confirmed our initial finding of DYRK1A as a host dependency factor for SARS-CoV-2 33,34 .…”

“…1A ). In another screen, DYRK1A was the third most strongly enriched hit after ACE2 33 . We generated single-cell knockout (KO) cells of DYRK1A in Vero-E6 cells using CRISPR/Cas9 to validate screening results and clarify the pro-viral mechanism underlying DYRK1A activity in coronavirus infection ( Fig.…”

“…During the COVID-19 pandemic, a number of genome-wide CRISPR/Cas9 screens have been performed to unveil host factors that regulate SARS-CoV-2 infection 22,23,[27][28][29][30][31][32][33]91,92 . We previously identified DYRK1A as a pro-viral gene for both SARS-CoVs and MERS-CoV in Vero-E6 cells and Calu-3 cells 22,23 .…”

“…Our group and others recently identified DYRK1A as a critical host factor for SARS-CoV-2, MERS-CoV, and chimeric HKU5 (bat coronavirus) expressing the SARS-CoV-1 spike (HKU5-SARS-CoV-1-S) in a genome-wide CRISPR/Cas9 inactivation screen in Vero-E6 cells 22,33 . In two additional independent genome-wide CRISPR screens in Calu-3 human immortalized lung cancer cells, DYRK1A was also identified as a top pro-viral gene for SARS-CoV-2 23,33 . By comparing the top 10,000 enriched genes ranked by z-score for SARS-CoV-2 across Vero-E6 and Calu-3 cells, we identified DYRK1A as second most strongly enriched gene after only ACE2 22,23 (Fig.…”

“…While reproducibility between screens was excellent when performed on the same cell type, there was otherwise limited overlap across hits in these loss of function screens [27][28][29][30][31][32] . One notable exception was DYRK1A, which was a top enriched hit in both Vero-E6 and Calu-3 cells 22,23,33,34 . Interestingly, DYRK1A was not a hit in cell lines exogenously overexpressing ACE2, such as A549, Huh7.5, or HeLa cells [27][28][29][30][31][32] .…”