Systematic genome-scale identification of host factors for SARS-CoV-2 infection across models yields a core single gene dependency; ACE2
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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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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
Ethics not detected. Sex as a biological variable 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) Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication Contamination: Cells were regularly monitored for mycoplasma contamination. Table 2: Resources
Antibodies Sentences Resources 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-IgGsuggested: NonePercentage of cells infected were … 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
Ethics not detected. Sex as a biological variable 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) Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication Contamination: Cells were regularly monitored for mycoplasma contamination. Table 2: Resources
Antibodies Sentences Resources 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-IgGsuggested: NonePercentage 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-Nsuggested: NoneSubsequently 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-CTSLdetected: (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-HSP90detected: (Santa Cruz Biotechnology Cat# sc-13119, RRID:AB_675659)anti-FLAGdetected: (Sigma-Aldrich Cat# A8592, RRID:AB_439702)anti-mouse-IgGsuggested: (Proteintech Cat# 10283-1-AP, RRID:AB_2877728)anti-mouse-IgGdetected: (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-IgGdetected: (Cell Signaling Technology Cat# 7074, RRID:AB_2099233)anti-goat IgGdetected: (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) FAB9332suggested: NoneExperimental Models: Cell Lines Sentences Resources Calu-3, and UM-UC-4 cells were cultured in EMEM (Wisent) with 10% FBS and 1% penicillin-streptomycin. Calu-3suggested: NoneUM-UC-4suggested: 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 E6suggested: Nonegenome-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-2suggested: NoneHuh-7suggested: NoneLentiviruses were produced by transfecting HEK-293T cells with pPAX2, pVSVG and pLenti6.2 generated above using Lipofectamine 2000 (Invitrogen). HEK-293Tsuggested: NoneThe 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) Huh7suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)Recombinant DNA Sentences Resources 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). pLenti6.2suggested: RRID:Addgene_113724)Lentiviruses were produced by transfecting HEK-293T cells with pPAX2, pVSVG and pLenti6.2 generated above using Lipofectamine 2000 (Invitrogen). pPAX2suggested: NonepVSVGsuggested: RRID:Addgene_85140)Software and Algorithms Sentences Resources Flow cytometry was performed using the LSR Fortessa X20 (BD Biosciences) and analyzed with FlowJo v10 software. FlowJosuggested: (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). Bowtiesuggested: (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)#. Salmonsuggested: (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-toolssuggested: NoneQAPA 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). GENCODEsuggested: (GENCODE, RRID:SCR_014966)PolyAsitesuggested: NoneGene 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:Profilersuggested: (G:Profiler, RRID:SCR_006809)All data were recorded with Xcalibur software (ThermoFisher Scientific). Xcalibursuggested: (Thermo Xcalibur, RRID:SCR_014593)Human protein reference sequences from the UniProt Swiss-Prot database were downloaded on 18-06-2020. UniProtsuggested: (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.
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- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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