Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses

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1. Version published to 10.1016/j.cell.2020.12.004

   Jan 1, 2021
2. 

   ScreenIT

   Sep 28, 2020

   SciScore for 10.1101/2020.09.24.312298: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	Contamination: Cell lines were tested negative for mycoplasma contamination.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Primary antibodies were detected by incubating membranes with 1:5000 dilution of HRP-conjugated (Southern Biotech) secondary anti-mouse and anti-rabbit antibodies for 1 h at room temperature.	anti-mouse suggested: None anti-rabbit suggested: None
   The following primary antibodies and their dilutions were used in this study: GAPDH (SCBT, sc-32233) at 1:1000, ACE2 (R&D Systems, AF933) at 1:1000, …

   SciScore for 10.1101/2020.09.24.312298: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	Contamination: Cell lines were tested negative for mycoplasma contamination.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Primary antibodies were detected by incubating membranes with 1:5000 dilution of HRP-conjugated (Southern Biotech) secondary anti-mouse and anti-rabbit antibodies for 1 h at room temperature.	anti-mouse suggested: None anti-rabbit suggested: None
   The following primary antibodies and their dilutions were used in this study: GAPDH (SCBT, sc-32233) at 1:1000, ACE2 (R&D Systems, AF933) at 1:1000, TMPRSS2 (Abcam, ab92323) at 1:1000.	GAPDH suggested: (Santa Cruz Biotechnology Cat# sc-32233, RRID:AB_627679) ACE2 suggested: (GenWay Biotech Inc. Cat# 18-661-15169-0.1 mg, RRID:AB_514759) TMPRSS2 suggested: (Abcam Cat# ab92323, RRID:AB_10585592)
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture: Huh7.5.1 (gift from Frank Chisari), HEK293FT (Thermo Scientific), Vero cells (ATCC) and A549-ACE2 cells (gift from Olivier Schwartz) were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Omega Scientific), penicillin/streptomycin (Gibco), non-essential amino acids (Gibco) and L-glutamine (Gibco) at 37C and 5% CO2.	A549-ACE2 suggested: None
   Lentivirus was produced in HEK293FT by co-transfection of cDNA containing lentiviral plasmid together with pCMV-dR8.2 dvpr (Addgene, #8455, gift from Bob Weinberg), pCMV-VSV-G (Addgene, #8454, gift from Bob Weinberg) and pAdVAntage (Promega) using FugeneHD (Promega).	HEK293FT suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
   Viral titers were determined by standard plaque assay using either Huh7.5.1 cells (OC43 and 229E) or Vero cells (SARS-CoV-2).	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
   Genome-wide CRISPR screens: Huh7.5.1-Cas9 cells were generated by lentiviral transduction with lentiCas9-blast (Addgene, #52962, gift from Feng Zhang) and subsequently selected with blasticidin for 7 days.	Huh7.5.1-Cas9 suggested: None
   To generate CRISPR KO libraries, a total of 240 million Huh7.5.1-Cas9-blast or Huh7.5.1-Cas9-blast+ACE2-IRES-TMPRSS2-hygro cells were transduced with lentivirus of the human GeCKO v2 library (Addgene, #1000000049, gift from Feng Zhang) at a moi of 0.4 and subsequently selected using puromycin and expanded for 7 days.	Huh7.5.1-Cas9-blast suggested: None Huh7.5.1-Cas9-blast+ACE2-IRES-TMPRSS2-hygro suggested: None
   Cell viability assay: Huh7.5.1 cells were treated with compounds at the same concentrations and durations as in infection assays.	Huh7.5.1 suggested: RRID:CVCL_E049)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Cell culture: Huh7.5.1 (gift from Frank Chisari), HEK293FT (Thermo Scientific), Vero cells (ATCC) and A549-ACE2 cells (gift from Olivier Schwartz) were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Omega Scientific), penicillin/streptomycin (Gibco), non-essential amino acids (Gibco) and L-glutamine (Gibco) at 37C and 5% CO2.	Huh7.5.1 suggested: RRID:CVCL_E049)
   Software and Algorithms
   Sentences	Resources
   A total of 60 million mutagenized cells for each GeCKO sublibrary (A and B) were collected for genomic DNA extraction to assess the sgRNA representation of the starting population.	GeCKO suggested: (Gecko, RRID:SCR_009001)
   The gene ontology enrichment of the individual screens was run on genes with MaGECK positive score <= 0.005 using the GO Biological Processes of the Molecular Signatures Database (MSigDB).	GO Biological suggested: None
   The resulting network was clustered into subnetworks using the GLay Cytoscape plugin 68.	Cytoscape suggested: (Cytoscape, RRID:SCR_003032)

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   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).

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

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   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.

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   Read the original source
3. 

   Version published to 10.1101/2020.09.24.312298v1 on bioRxiv

   Sep 24, 2020