Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs
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SciScore for 10.1101/2021.05.19.444823: (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 not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication Contamination: All cell lines were regularly screened for the absence of mycoplasma contamination. Table 2: Resources
Antibodies Sentences Resources They were washed three times with PBS and then incubated for 30 minutes with Alexa-488-conjugated donkey anti-mouse IgG and Alexa594-conjugated goat anti-rabbit IgG secondary antibodies (both from Jackson Immunoresearch) in 5% GS in PBS supplemented with 1 μg/ml DAPI (4′,6- diamidino-2-phenylindole). Alexa-488-conjugated donkey anti-mouse IgGsuggested: Noneanti-rabbit IgGsuggested: NoneThe medium was … SciScore for 10.1101/2021.05.19.444823: (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 not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication Contamination: All cell lines were regularly screened for the absence of mycoplasma contamination. Table 2: Resources
Antibodies Sentences Resources They were washed three times with PBS and then incubated for 30 minutes with Alexa-488-conjugated donkey anti-mouse IgG and Alexa594-conjugated goat anti-rabbit IgG secondary antibodies (both from Jackson Immunoresearch) in 5% GS in PBS supplemented with 1 μg/ml DAPI (4′,6- diamidino-2-phenylindole). Alexa-488-conjugated donkey anti-mouse IgGsuggested: Noneanti-rabbit IgGsuggested: NoneThe medium was replaced with 5%FCS-supplemented DMEM complemented with a mouse monoclonal anti-VSV-G antibody (CliniSciences, clone 8G5F11, final concentration 1 μg/mL) to neutralize residual viral input, as described (Condor Capcha et al., 2020). anti-VSV-Gsuggested: (Absolute Antibody Cat# Ab01401-2.0, RRID:AB_2883992)M, deoxycholate 0,1%) supplemented with sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 100 mM DTT, 0.02% bromophenol blue), resolved by SDS-PAGE and analyzed by immunoblotting using primary antibodies against ACE2 (ProteinTech 21115-1-P) and Actin (Sigma-Aldrich A1978), followed by HRP-conjugated anti-rabbit or anti-mouse immunoglobulin antibodies and chemiluminescence Clarity or Clarity max substrate (Bio-Rad). ACE2suggested: NoneActinsuggested: (Sigma-Aldrich Cat# A1978, RRID:AB_476692)anti-rabbitsuggested: Noneanti-mouse immunoglobulinsuggested: NoneExperimental Models: Cell Lines Sentences Resources Cell lines: Human HEK293T, Caco-2, Calu-3, A549, Huh-7, and Huh7.5.1, simian Vero E6 and LLC-MK2, dog MDCK cells were maintained in complete Dulbecco’s modified Eagle medium (DMEM) (Gibco) supplemented with 10% foetal bovine serum and penicillin/streptomycin. A549suggested: NoneHuh-7suggested: NoneMDCKsuggested: NoneFor CRISPR-Cas9-mediated gene disruption, Calu-3, Caco-2 and A549-ACE2, cells stably expressing Cas9 or dCas9-VP64 were first generated by transduction with LX_311-Cas9 or XPR_BRD109, respectively, followed by blasticidin selection at 10 µg/ml. Caco-2suggested: NoneA549-ACE2suggested: NoneLentiviral production and transduction: Lentiviral vector stocks were obtained by polyethylenimine (PEI; for LentiGuide vectors) or Lipofectamine 3000 (Thermo Scientific; for XPR_502 vectors)-mediated multiple transfections of 293T cells in 6-well plates with vectors expressing Gag-Pol, the miniviral genome, the Env glycoprotein at a ratio of 1:1:0.5. 293Tsuggested: NoneCRISPR KO screens: Vero E6, Caco-2-ACE2 and Calu-3 cells were spin infected for 2h at 1000g with LX_311-Cas9 lentiviral vector at a high MOI and in the presence of polybrene (4 µg/mL). Calu-3suggested: None120 million Calu-3-dCas9-VP64 cells were then transduced with the Calabrese library in two biological replicates (for sublibrary A) or in one replicate (for sublibrary B) at a low MOI (∼0.3-0.5). Calu-3-dCas9-VP64suggested: NoneViral supernatants were titrated by plaque assays in Vero E6 cells. Vero E6suggested: RRID:CVCL_XD71)Infections of Calu-3 were performed at MOI 300 for HCoV-229E-Renilla (as measured on Huh7.5.1 cells) and MOI 0.1 for HCoV-NL63 (as measured on LLC-MK2 cells) and infection efficiency was analyzed 3 days later by measuring Renilla activity or 5 days later by RT-qPCR for HCoV-229E-Renilla and HCoV-NL63, respectively. Huh7.5.1suggested: RRID:CVCL_E049)HCoV-229E-Renillasuggested: NoneMERS-CoV production and infection: To produce MERS-CoV, HEK-293T cells were transfected with a bacmid containing a full-length cDNA clone of the MERS-CoV genome (a king gift of Dr Luis Enjuanes; (Almazán et al., 2013) and overlaid six hours later with Huh7 cells. HEK-293Tsuggested: NoneAfter lysis of Huh7 cells, cell supernatants were collected and the virus was further amplified on Huh7 cells. Huh7suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)The pCSI-SpikeRBD expression vector was transfected in HEK293T cells using the PEIpro® transfection reagent. HEK293Tsuggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)Experimental Models: Organisms/Strains Sentences Resources Seasonal coronavirus production and infection: HCoV-229E-Renilla was a gift from Volker Thiel (van den Worm et al., 2012) and was amplified for 5-7 days at 33°C in Huh7.5.1 cells in 5% FCS-containing DMEM. HCoV-229E-Renillasuggested: NoneRecombinant DNA Sentences Resources Plasmids and constructs: The lentiviral vector expressing ACE2 (pRRL.sin.cPPT.SFFV/ACE2, Addgene 145842) has been described (Rebendenne et al., 2021). pRRL.sin.cPPT.SFFV/ACE2suggested: NoneThe pLX_311-Cas9 (Addgene 96924) and pXPR_BRD109, which express Cas9 and dCas9-VP64, respectively, have been described (Sanson et al., 2018). pLX_311-Cas9suggested: RRID:Addgene_96924)pXPR_BRD109suggested: NoneGuide RNA coding oligonucleotides were annealed and ligated into BsmBI-digested LentiGuide-Puro or pXPR_502 vectors, as described (Addgene). pXPR_502suggested: RRID:Addgene_96923)pcDNA3.1_spike_del19 was a gift from Raffaele De Francesco (Addgene 155297). pcDNA3.1_spike_del19suggested: NoneCRISPR KO screens: Vero E6, Caco-2-ACE2 and Calu-3 cells were spin infected for 2h at 1000g with LX_311-Cas9 lentiviral vector at a high MOI and in the presence of polybrene (4 µg/mL). LX_311-Cas9suggested: NoneSpike pseudotype production: 293T cells were seeded in a 6 well plate prealably coated with poly-Lysine (Sigma-Aldrich) and, 1 day later, transfected with 5 μg of an expression plasmid coding either VSV-G (pMD.G) or SARS-CoV-2 Spike del19 (pcDNA3.1_spike_del19) using Lipofectamine 2000 (Thermo Scientific). VSV-Gsuggested: RRID:Addgene_138479)pMD . Gsuggested: NoneTriplicate reactions were run according to the manufacturer’s instructions using a ViiA7 Real Time PCR system (ThermoFisher Scientific). pRdRp and pNL63 (which respectively contains fragments amplified from SARS-CoV-2- and NL63-infected cell RNAs using primers RdRp_for and RdRp_rev, and NL-63F2 and NL-63R2, cloned into pPCR-Blunt II-TOPO) was diluted in 20 ng/ml salmon sperm DNA to generate a standard curve to calculate relative cDNA copy numbers and confirm the assay linearity (detection limit: 10 molecules of RdRp per reaction). pRdRpsuggested: NonepNL63suggested: NonepPCR-Bluntsuggested: NoneThe RBD-mFc fusion sequence was then cloned into a pCSI vector for expression in mammalian cells, as previously described (Giovannini et al., 2013). pCSIsuggested: NoneThe pCSI-SpikeRBD expression vector was transfected in HEK293T cells using the PEIpro® transfection reagent. pCSI-SpikeRBDsuggested: NoneSoftware and Algorithms Sentences Resources The files containing the guide-level and gene-level residual z-scores for each screen are being deposited on Gene Expression Omnibus (GEO) Gene Expression Omnibussuggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)About 10,000 to 20,000 cells were counted per condition in each experiment using homemade macros running in ImageJ. ImageJsuggested: (ImageJ, RRID:SCR_003070)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.
- 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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