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

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.

CRISPR Tackles Emerging Viral Pathogens

“…As example, Calu-3 and A549 cells, both of which are of lung epithelial origin, revealed ACE2 as the top hit; however, the AP-1 complex subunit gamma-1 (AP1G1), a clatherinadaptor protein expressed as part of the trans-golgi network, is significantly enriched in Calu-3s but not A549 cells. While AP1G1 is potentially mediating the role of either virus endocytosis or egress in Calu-3s, it is possible that this is not the case in A549s, which may use other host factors for these processes [91].…”

“…CRISPRa genome-wide screens have also revealed enrichment for sgRNAs representing the known anti-viral Mucin family members, MUC1, -4, -13 and -21 [85,91]. Mucins are a family of O-glycosylated glycoproteins that are either membrane bound or secreted to form mucosa membranes.…”

Genome-wide CRISPR screens identify GATA6 as a proviral host factor for SARS-CoV-2 via modulation of ACE2

“…Taken together, the data therefore suggest that some of the host entry factors necessary for SARS-CoV-2 infection exhibit cell-line specificity, illustrating the importance of using relevant cell models to maximize the understanding of SARS-CoV-2 entry. Finally, an inspection of data from two recently documented Calu-3 infection screens 21 , 62 revealed that multiple top proviral candidates identified in our screens were enriched at a commensurate extent also in these independent screens (Fig. 1h ).…”

“…IRF6 is below threshold in A549 and Vero-E6 cells. h Comparative analysis of previously reported SARS-CoV-2 knockout screens performed in the Calu-3 cell line 21 , 62 . Top proviral candidates identified in our screens are displayed.…”

Systematic genome-scale identification of host factors for SARS-CoV-2 infection across models yields a core single gene dependency; ACE2

“…This motif is also common in multiple viruses, including SARS-CoV-2, allowing viruses to exploit AP2M1 for internalization from the plasma membrane to intracellular sites (Minakshi and Padhan, 2014; Yuan et al, 2020). Calu-3 knockout screens reported by others (Biering et al, 2021; Rebendenne et al, 2021), also identified members of the clathrin adaptor complex; however, the genes were pro-viral and associated with the AP1 complex, which is found on the trans-Golgi-network and endosomes and is responsible for trafficking proteins between the Golgi and/or endosomes to lysosome (Biering et al ., 2021; Touz et al, 2004). Many viruses are known to hijack endocytic mechanisms for entry to the cell (Robinson et al, 2018).…”