A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

  1. Yunkai Zhu
  2. Fei Feng
  3. Gaowei Hu
  4. Yuyan Wang
  5. Yin Yu
  6. Yuanfei Zhu
  7. Wei Xu
  8. Xia Cai
  9. Zhiping Sun
  10. Wendong Han
  11. Rong Ye
  12. Di Qu
  13. Qiang Ding
  14. Xinxin Huang
  15. Hongjun Chen
  16. Wei Xu
  17. Youhua Xie
  18. Qiliang Cai
  19. Zhenghong Yuan
  20. Rong Zhang

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2020.08.25.266775: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: The experiment protocol has been approved by the Animal Ethics Committee of School of Basic Medical Sciences at Fudan University.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableAnimal experiments: Six to ten week-old male hamsters were used in the study in the BSL-3 laboratory of Fudan University.
    Cell Line AuthenticationContamination: All cell lines were tested routinely and free of mycoplasma contamination.

    Table 2: Resources

    Antibodies
    SentencesResources
    Live cells were incubated with the recombinant protein, S1 domain of SARS-CoV-2 spike C-terminally fused with Fc (Sino Biological #40591-V02H, 1μg/ml), or the anti-ACE2 antibody (Sino Biological #10108-RP01, 1 μg/ml) at 4 °C for 30 min.
    anti-ACE2
    suggested: None
    The antibodies used are as follows: rabbit anti-COMMD3 (proteintech #26240-1-AP, 1:800), rabbit anti-VPS35 (proteintech #10236-1-AP,1:500), rabbit anti-CCDC22 (proteintech #16636-1-AP, 1:1000), rabbit anti-NPC1 (proteintech #13926-1-AP, 1:1000), rabbit anti-NPC2 (proteintech #19888-1-AP, 1:800), rabbit anti-CCDC53 (proteintech #24445-1-AP, 1:500), rabbit anti-COMMD1 (proteintech #11938-1-AP, 1:2000), mouse anti-SNX27 (Abcam #ab77799, 1:1000), rabbit anti-SNX17 (proteintech, #10275-1-AP, 1:2000), rabbit anti-LDLR (proteintech, #10785-1-AP, 1:1000), rabbit anti-LRP1 (Abcam #ab92544, 1:5000), rabbit anti-SARS-Cov-2 spike S2 (Sino Biological #40590-T62, 1:1000), rabbit anti-β-actin (proteintech #20536-1-AP, 1:2000).
    anti-COMMD3
    suggested: (Proteintech Cat# 26240-1-AP, RRID:AB_2880440)
    anti-VPS35
    suggested: (Proteintech Cat# 10236-1-AP, RRID:AB_2215216)
    anti-CCDC22
    suggested: (Proteintech Cat# 16636-1-AP, RRID:AB_2072065)
    anti-NPC1
    suggested: (Proteintech Cat# 13926-1-AP, RRID:AB_2152050)
    anti-NPC2
    suggested: (Proteintech Cat# 19888-1-AP, RRID:AB_10639363)
    anti-CCDC53
    suggested: (Proteintech Cat# 24445-1-AP, RRID:AB_2879551)
    anti-COMMD1
    suggested: (Proteintech Cat# 11938-1-AP, RRID:AB_2083542)
    anti-SNX27
    suggested: (Abcam Cat# ab77799, RRID:AB_10673818)
    anti-SNX17
    suggested: (Proteintech Cat# 10275-1-AP, RRID:AB_2192394)
    #10275-1-AP
    suggested: (Proteintech Cat# 10275-1-AP, RRID:AB_2192394)
    anti-LDLR
    suggested: (Proteintech Cat# 10785-1-AP, RRID:AB_2281164)
    #10785-1-AP
    suggested: (Proteintech Cat# 10785-1-AP, RRID:AB_2281164)
    anti-LRP1
    suggested: (Abcam Cat# ab92544, RRID:AB_2234877)
    anti-SARS-Cov-2
    suggested: None
    anti-β-actin
    suggested: None
    The HRP-conjugated secondary antibodies include: Goat anti-mouse (sigma #A4416, 1:5000), goat anti-rabbit (thermo fisher #31460, 1:5000), goat anti-human (sigma #A6029, 1:5000).
    anti-rabbit
    suggested: (Thermo Fisher Scientific Cat# 31460, RRID:AB_228341)
    anti-human (sigma #A6029
    suggested: (Sigma-Aldrich Cat# A6029, RRID:AB_258272)
    For quantification studies, after probing with primary antibodies, membranes were incubated with goat anti-rabbit IRDye 800CW secondary antibody (LI-COR #926-32211, 1:10000), goat anti-rabbit IRDye 680RD secondary antibody (LI-COR #926-68071, 1:10000) or goat anti-mouse IRDye 800CW secondary antibody (LI-COR #926-32210, 1:10000), then developed and analyzed with the Odyssey CLx Imaging System.
    #926-32211
    suggested: (LI-COR Biosciences Cat# 926-32211, RRID:AB_621843)
    #926-68071
    suggested: (LI-COR Biosciences Cat# 926-68071, RRID:AB_10956166)
    After three washes, cells were incubated with the secondary goat anti-mouse antibody conjugated with Alexa Fluor 555 (Thermo #A-21424, 2 μg/ml) for 2 h at room temperature, followed by staining with 4’,6-diamidino-2-phenylindole (DAPI).
    anti-mouse
    suggested: (Molecular Probes Cat# A-21424, RRID:AB_141780)
    Cells were stained with rabbit polyclonal antibody against SARS-CoV nucleocapsid protein (Rockland, 200-401-A50, 0.5μg/ml) overnight at 4°C, incubated with the secondary goat anti-rabbit HRP-conjugated antibody for 2 h at room temperature.
    SARS-CoV nucleocapsid protein
    suggested: (Creative Diagnostics Cat# DMAB8869, RRID:AB_2392503)
    Experimental Models: Cell Lines
    SentencesResources
    Cells: Vero E6 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), HEK 293T (ATCC # CRL-3216), HeLa (ATCC #CCL-2), A549 (kindly provided by M.S. Diamond, Washington University), and Calu-3 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) all were cultured at 37°C in Dulbecco’s Modified Eagle Medium (Hyclone #SH30243.01) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM Sodium pyruvate, 1× non-essential amino acids, and 100 U/ml of Penicillin-Streptomycin.
    HEK 293T
    suggested: None
    HeLa
    suggested: None
    A549
    suggested: None
    The A549-ACE2 and HeLa-ACE2 clonal cell lines were generated by transduction of lentivector expressing the human ACE2 gene as described bellow.
    HeLa-ACE2
    suggested: None
    Similarly, the bulk Vero-TMPRSS2 cells were generated by transduction of lentivector expressing the human TMPRSS2 and selected with puromycin.
    Vero-TMPRSS2
    suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
    This virus stock underwent three rounds of plaque-purification in Vero E6 cells in the presence of trypsin and designated as SH01-Sfull (thereafter as Sfull).
    Vero E6
    suggested: None
    The sgRNA plasmid library was packaged in 293FT cells after co-transfection with psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) at a ratio of 2:2:1 using Fugene®HD (Promega).
    293FT
    suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
    For the CRISPR sgRNA screen, A549-ACE2-Cas9 cells were generated by transduction of A549-ACE2 cell line with a packaged lentivirus expressing the mCherry derived from the lentiCas9-Blast (Addgene #52962) that the blasticidin resistance gene was replaced by mCherry.
    A549-ACE2-Cas9
    suggested: None
    A549-ACE2
    suggested: None
    Cells were inoculated with same MOI of Sfull or Sdel (Vero, Vero-TMPRSS2, MOI 0.01; A549-ACE2, MOI 2; Calu-3, MOI 0.1) for 1 h.
    Vero
    suggested: None
    Biotinylation of plasma membrane proteins: Gene-edited A549-ACE or Calu-3 cells seeded in 6-well plate 24 h prior to experiment were chilled on ice for 10 min, and labeled with 2.5 mg/ml Biotin (Thermo fisher #21331) in PBS for 30 min on ice.
    Calu-3
    suggested: None
    Software and Algorithms
    SentencesResources
    The sgRNA sequences targeting specific genes were extracted using the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) and cutadapt 1.8.1, and further analyzed for sgRNA abundance and gene ranking by a published computational tool (MAGeCK)(Li et al., 2014) (see Supplementary Tables 1 and 2).
    http://hannonlab.cshl.edu/fastx_toolkit/
    suggested: (FASTX-Toolkit, RRID:SCR_005534)
    Images were collected using an Operetta High Content Imaging System (PerkinElmer), and processed using the ImageJ program (http://rsb.info.nih.gov/ij/).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Statistical analysis: Statistical significance was assigned when P values were < 0.05 using Prism Version 8 (GraphPad).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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.

    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.

  2. Version published to 10.1038/s41467-021-21213-4
  3. Version published to 10.1101/2020.08.25.266775v1 on bioRxiv