CRISPR screens for host factors critical for infection by SARS-CoV-2 variants of concern identify GATA6 as a central modulator of ACE2

  1. Ma’ayan Israeli
  2. Yaara Finkel
  3. Yfat Yahalom-Ronen
  4. Nir Paran
  5. Theodor Chitlaru
  6. Ofir Israeli
  7. Inbar Cohen-Gihon
  8. Moshe Aftalion
  9. Reut Falach
  10. Uri Elia
  11. Ital Nemet
  12. Limor Kliker
  13. Michal Mandelboim
  14. Adi Beth-Din
  15. Tomer Israely
  16. Ofer Cohen
  17. Noam Stern-Ginossar
  18. Adi Bercovich-Kinori

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Abstract

The global spread of SARS-CoV-2 led to the most challenging pandemic in this century, posing major economic and health challenges worldwide. Revealing host genes essential for infection by multiple variants of SASR-CoV-2 can provide insights into the virus pathogenesis, and facilitates the development of novel broad-spectrum host-directed therapeutics. Here, employing genome-scale CRISPR screens, we provide a comprehensive data-set of cellular factors that are exploited by WT-SARS-CoV-2 as well as two additional recently emerged variants of concerns (VOCs), Alpha and Beta. These screens identified known and novel host factors critical for SARS-CoV-2 infection, including various components belonging to the Clathrin-dependent transport pathway, ubiquitination and Heparan sulfate biogenesis. In addition, the host phosphatidylglycerol biosynthesis processes appeared to have major anti-viral functions. Comparative analysis of the different VOCs revealed the host factors KREMEN2 and SETDB1 as potential unique candidates required only to the Alpha variant, providing a possible explanation for the increased infectivity of this variant. Furthermore, the analysis identified GATA6, a zinc finger transcription factor, as an essential pro-viral gene for all variants inspected. We revealed that GATA6 directly regulates ACE2 transcription and accordingly, is critical for SARS-CoV-2 cell entry. Analysis of clinical samples collected from SARS-CoV-2 infected individuals showed an elevated level of GATA6, indicating the important role GATA6 may be playing in COVID-19 pathogenesis. Finally, pharmacological inhibition of GATA6 resulted in down-modulation of ACE2 and consequently to inhibition of the viral infectivity. Overall, we show GATA6 represents a target for the development of anti-SARS-CoV-2 therapeutic strategies and reaffirm the value of the CRISPR loss-of-function screens in providing a list of potential new targets for therapeutic interventions.

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  1. Version published to 10.1101/2021.07.19.452809v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.07.19.452809: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: All cell lines tested negative for mycoplasma.

    Table 2: Resources

    Antibodies
    SentencesResources
    Primary antibodies used in this study: Rabbit anti-GATA6 (Abcam ab175349), hyperimmune Rabbit serum from intervenous (i.v) SARS-CoV-2 infected Rabbits.
    anti-GATA6
    suggested: None
    Secondary antibody used was IRDye® 800CW conjugated Goat anti-Rabbit (Licor).
    anti-Rabbit
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: HEK293T (ATCC-CRL-3216), Vero-E6 (ATCC-CRL-1586) and Calu-3 cells (ATCC-HTB-55) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS), 10 mM non-essential amino acids (NEAA), 2 mM L-Glutamine, 1 mM Sodium pyruvate and 1% Penicillin/Streptomycin (all from Biological Industries, Israel) at 37°C, 5% CO2.
    HEK293T
    suggested: None
    Genome-wide CRISPR screens: To generate CRISPR KO libraries, a total of 4*108 Calu-3 cells were transduced with lentivirus of human Brunello Human CRISPR library (Addgene #73179, gift from David Root and John Doench) in the presence of 0.5 mg/ml polybrene (TR-1003, Sigma), at a MOI of 0.3.
    Calu-3
    suggested: None
    Viral titration: Vero-E6 cells were seeded in 12-well plates (5 × 105 cells/well) and grown overnight in growth medium.
    Vero-E6
    suggested: None
    Analysis of GATA6 expression in different cell lines: Published mRNA-seq data used for A549, Vero-E6 and Calu-3 cells are from Kinori et al. 2016, Finkel et al. 2021a and Finkel et al. 2021b., respectively.
    A549
    suggested: None
    Recombinant DNA
    SentencesResources
    Generation of Calu-3 KO cell lines: DNA oligos (Integrated DNA Technologies, Inc.) containing sgRNA sequences (see supplementary Table S1) were annealed and ligated into lentiCRISPRv2 (Addgene, #52961, gift from Feng Zhang).
    lentiCRISPRv2
    suggested: RRID:Addgene_98292)
    Lentivirus was packaged by co-transfection of constructs with the 2nd generation packaging plasmids pMD2.G and PsPax using jetPEI (Polyplus-transfection) into 6-well plates with HEK293T cells according to protocol.
    pMD2.G
    suggested: RRID:Addgene_12259)
    For GATA6 complementation experiment, cells were complemented by transfection of 2.5 μg pBabe 3XFLAG-wt GATA6-3XAU1 puro vector (Addgene #72607) using Lipofectamine 3000 (L3000015 ThermoFisher) according to manufacturer’s instructions.
    pBabe 3XFLAG-wt GATA6-3XAU1 puro
    suggested: None
    HEK293T cells were transfected with 3 μg pBabe 3XFLAG-wt-GATA6-3AU1 puro plasmid (Addgene #76207) using Lipofectamine 3000 (L3000015 ThermoFisher) according to manufacturer’s instructions.
    pBabe 3XFLAG-wt-GATA6-3AU1 puro
    suggested: None
    Software and Algorithms
    SentencesResources
    Twenty-four hours prior to infection with SARS-CoV-2, 1.5*107 Calu-3 library-cells were seeded in 75-cm2 flasks.
    SARS-CoV-2
    suggested: (BioLegend Cat# 944703, RRID:AB_2890874)
    Analysis of CRISPR-Cas9 genetic screen data: MAGeCK v0.5.6 [73] was used to count sgRNA from FASTQ files and to analyze the selection effect of genes based on the change in sgRNA distribution, using the robust rank aggregation (RRA) algorithm with normalization to total reads.
    MAGeCK
    suggested: None
    Pathway and network analysis: Directed scores were used as input for gene set enrichment analysis (GSEA version 4.1) with GO biological process (c5.bp) from MSigDB (version 7.4) [74, 75].
    GSEA
    suggested: (SeqGSEA, RRID:SCR_005724)
    The STRING network was imported into Cytoscape [76].
    STRING
    suggested: (STRING, RRID:SCR_005223)
    Cytoscape
    suggested: (Cytoscape, RRID:SCR_003032)
    Genes were highlighted based on association with selected REACTOME pathways.
    REACTOME
    suggested: (Reactome, RRID:SCR_003485)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2021.07.19.452809v1 on bioRxiv