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

Abstract

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.

Inducible CRISPR activation screen for interferon-stimulated genes identifies OAS1 as a SARS-CoV-2 restriction factor

“…Given the apparently potent effects of OAS1 in our experiments, we were surprised that it had not been detected in other recently published genome-wide and ISG-focused screens 29,43,44,85,86 . This discrepancy could be due to the distinct features of the experimental systems used, including different cell types, different infection time points, and low M.O.I.…”

“…Cathepsins are cellular proteases that have been implicated in the entry processes of multiple viruses by activating viral glycoproteins to trigger viral fusion at the endosomal membrane 69 . Our screens found that expression of Cathepsin L (CTSL), an entry factor for coronaviruses 60 , sensitizes cells to SARS-CoV- Given the apparently potent effects of OAS1 in our experiments, we were surprised that it had not been detected in other recently published genome-wide and ISG-focused screens 29,43,44,85,86 . This discrepancy could be due to the distinct features of the experimental systems used, including different cell types, different infection time points, and low M.O.I.…”

“…Using a similar genome-wide gRNA library in a different cell line, Dukhovny et al detected antiviral effectors with activity against Zika Virus 42 . Recent preprints also reported genome-wide CRISPRa screens for SARS-CoV-2 43,44 . While these examples demonstrate the utility of pooled CRISPRa screens for identifying viral restriction factors genome-wide, we aimed to establish a CRISPRa system optimized for efficiently evaluating ISG activities against respiratory viruses.…”

“…infections of IFN-competent cell lines, in which potential paracrine signaling may obscure certain ISG effects. This possibility is generally supported by the relatively few ISGs detected in pooled activation screens [41][42][43][44] , which would otherwise be predicted to be enriched due to their direct antiviral effects. Moreover, as OAS1 has been characterized as proapoptotic 92 , our inducible system and multifactorial analysis strategy may have been uniquely capable to robustly detect its effects.…”

“…Studies in which genome-wide libraries of activating gRNAs uncovered important host factors in viral infection systems provide an important proof of concept to the characterization of ISGs using CRISPRa 41,42 . Importantly, two recent preprints report genome-wide CRISPRa screens for genes with antiviral potential against SARS-CoV-2 43,44 .…”