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

  1. Katherine Chan
  2. Adrian Granda Farias
  3. Hunsang Lee
  4. Furkan Guvenc
  5. Patricia Mero
  6. Kamaldeep Aulakh
  7. Kevin R. Brown
  8. Shahan Haider
  9. Edyta Marcon
  10. Ulrich Braunschweig
  11. Amy Hin Yan Tong
  12. Shuye Pu
  13. Andrea Habsid
  14. Natasha Christie-Holmes
  15. Patrick Budylowski
  16. Audrey Astori
  17. Ayoob Ghalami
  18. Samira Mubareka
  19. Arinjay Banerjee
  20. Karen L. Mossman
  21. Jack Greenblatt
  22. Scott D. Gray-Owen
  23. Brian Raught
  24. Benjamin J. Blencowe
  25. Mikko Taipale
  26. Jason Moffat

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Abstract

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.

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

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    1. ScreenIT

      SciScore for 10.1101/2021.06.28.450244: (What is this?)

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

      Table 1: Rigor

      Ethicsnot detected.
      Sex as a biological variablegenome-scale CRISPR library (Addgene #90294) was used to perform pooled CRISPR knockout screens in Vero E6, Caco-2, Huh-7, Calu-3 and HEK293+A+T cells(Hart et al., 2017)
      Randomizationnot detected.
      Blindingnot detected.
      Power Analysisnot detected.
      Cell Line AuthenticationContamination: Cells were regularly monitored for mycoplasma contamination.

      Table 2: Resources

      Antibodies
      SentencesResources
      The next day, cells were washed with PBS and incubated with secondary antibody, anti-human FITC-IgG (Fc specific) (ThermoFisher Scientific) at a dilution of 1:400 and for 45 minutes at room temperature.
      anti-human FITC-IgG
      suggested: None
      Percentage of cells infected were determined using HarmonyTM high content imaging and analysis software by comparing cells stained positive with anti-N antibody versus Hoechst actin-stained cells.
      anti-N
      suggested: None
      Subsequently proteins were detected using anti-ACE2 (R&D AF933, RRID:AB_439702), anti-TMPRSS2 (Millipore, MABF2158), anti-CTSL (SantaCruz SC-32320 RRID:AB_626811), ß-Tublin
      anti-CTSL
      detected: (Santa Cruz Biotechnology Cat# sc-32320, RRID:AB_626811)
      (DSHB E7, RRID:AB_528499), anti-HSP90 (SantaCruz, SC-13119, RRID:AB_675659) or anti-FLAG (Sigma, A8592, RRID:AB_439702) antibodies with an appropriate HRP conjugated secondary antibody, anti-mouse-IgG (Cell Signaling Technologies 7076, RRID:AB_330924)
      detected: (DSHB Cat# E7, RRID:AB_528499)
      anti-HSP90
      detected: (Santa Cruz Biotechnology Cat# sc-13119, RRID:AB_675659)
      anti-FLAG
      detected: (Sigma-Aldrich Cat# A8592, RRID:AB_439702)
      anti-mouse-IgG
      suggested: (Proteintech Cat# 10283-1-AP, RRID:AB_2877728)
      anti-mouse-IgG
      detected: (Cell Signaling Technology Cat# 7076, RRID:AB_330924)
      , anti-rabbit-IgG (Cell Signaling Technologies 7074, RRID:AB_2099233) or anti-goat IgG (Jackson ImmunoResearch 805035180, RRID: AB_2340874).
      anti-rabbit-IgG
      detected: (Cell Signaling Technology Cat# 7074, RRID:AB_2099233)
      anti-goat IgG
      detected: (Jackson ImmunoResearch Labs Cat# 805-035-180, RRID:AB_2340874)
      ACE2 flow cytometry analysis: For ACE2 labeling, cells were harvested with 5mM EDTA, washed once with FACS buffer (1%BSA in PBS and 5mM EDTA) and incubated for 40 minutes in FACS buffer containing 0.75ug of human ACE2-488 conjugated antibody (R&D Systems FAB9332) and 1:1000 7-AAD (ThermoFisher Scientific)
      FAB9332
      suggested: None
      Experimental Models: Cell Lines
      SentencesResources
      Calu-3, and UM-UC-4 cells were cultured in EMEM (Wisent) with 10% FBS and 1% penicillin-streptomycin.
      Calu-3
      suggested: None
      UM-UC-4
      suggested: ECACC Cat# 08090501, RRID:CVCL_2749)
      Virus stocks: SARS-CoV-2/SB3 isolate (Banerjee et al., 2020) used here was propagated in Vero E6 cells at 37°C to generate P4 viral stock.
      Vero E6
      suggested: None
      genome-scale CRISPR library (Addgene #90294) was used to perform pooled CRISPR knockout screens in Vero E6, Caco-2, Huh-7, Calu-3 and HEK293+A+T cells(Hart et al., 2017)
      Caco-2
      suggested: None
      Huh-7
      suggested: None
      Lentiviruses were produced by transfecting HEK-293T cells with pPAX2, pVSVG and pLenti6.2 generated above using Lipofectamine 2000 (Invitrogen).
      HEK-293T
      suggested: None
      The conditioned media was collected from transfected cultures, filtered through 0.22um filters and applied to Vero E6, Calu-3 and Huh7 cells at 1:10 ratio of conditioned media : fresh media, in the presence of 8ug/ml polybrene (Sigma)
      Huh7
      suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
      Recombinant DNA
      SentencesResources
      To generate protease overexpression lines, Tmprss2-FLAG, FLAG-Tmprss2 or Cathepsin L-FLAG constructs were cloned into pLenti6.2 plasmid with blasticidin resistance marker using gateway cloning system (Invitrogen).
      pLenti6.2
      suggested: RRID:Addgene_113724)
      Lentiviruses were produced by transfecting HEK-293T cells with pPAX2, pVSVG and pLenti6.2 generated above using Lipofectamine 2000 (Invitrogen).
      pPAX2
      suggested: None
      pVSVG
      suggested: RRID:Addgene_85140)
      Software and Algorithms
      SentencesResources
      Flow cytometry was performed using the LSR Fortessa X20 (BD Biosciences) and analyzed with FlowJo v10 software.
      FlowJo
      suggested: (FlowJo, RRID:SCR_008520)
      Pre-processed paired-end reads were aligned to TKOv3 reference library sequences using Bowtie (v0.12.8) allowing up to two mismatches and one exact alignment (specific parameters: -v2 -m1 -p4 --sam-nohead).
      Bowtie
      suggested: (Bowtie, RRID:SCR_005476)
      Gene expression changes were analyzed by pseudo-aligning pre-trimmed reads to GENCODE v29 transcripts using Salmon v0.14.1 #(Patro R et al., Nat Methods 2017)# and aggregated per gene using the R package tximport #(Soneson C et al., F1000Research 2015)#.
      Salmon
      suggested: (Salmon, RRID:SCR_017036)
      Alternative splicing was analyzed using Vast-tools v2.2.2 (Tapial et al., 2017) in combination with the VastDB Hs2 library released on Dec. 20, 2019 (https://github.com/vastgroup/vast-tools).
      Vast-tools
      suggested: None
      QAPA builds the reference library of 3′ UTRs from all annotated protein-coding genes using GENCODE basic gene annotation (v19) for humans (hg19), supplemented by experimentally defined polyA sites archived in the PolyAsite database (Gruber et al., 2016).
      GENCODE
      suggested: (GENCODE, RRID:SCR_014966)
      PolyAsite
      suggested: None
      Gene set enrichment and network analysis: Analysis of enriched gene functions was performed as ordered queries with g:Profiler ###Ref: https://doi.org/10.1093/nar/gkz369 ### using the TKOv3 library as a custom background.
      g:Profiler
      suggested: (G:Profiler, RRID:SCR_006809)
      All data were recorded with Xcalibur software (ThermoFisher Scientific).
      Xcalibur
      suggested: (Thermo Xcalibur, RRID:SCR_014593)
      Human protein reference sequences from the UniProt Swiss-Prot database were downloaded on 18-06-2020.
      UniProt
      suggested: (UniProtKB, RRID:SCR_004426)

      Results from OddPub: Thank you for sharing your code.

      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.
      • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
      • No protocol registration statement was detected.

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

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    2. Version published to 10.1101/2021.06.28.450244v1 on bioRxiv