Genome-wide, bidirectional CRISPR screens identify mucins as critical host factors modulating SARS-CoV-2 infection

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1. 

   ScreenIT

   Apr 25, 2021

   SciScore for 10.1101/2021.04.22.440848: (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	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Lentiviral production: Low-passage HEK 293FT cells were grown in DMEM supplemented with 10% FBS (D10 media) and passaged using TrypLE (Gibco).	HEK 293FT suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
   150,000 cells/cm2 of 293FT cells in suspension were mixed with the PEI-DNA DMEM mixture and allowed to incubate for 5 minutes at room temperature and then plated in D10 for 48 hours.	293FT suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
   The original stock from BEI was …

   SciScore for 10.1101/2021.04.22.440848: (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	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Lentiviral production: Low-passage HEK 293FT cells were grown in DMEM supplemented with 10% FBS (D10 media) and passaged using TrypLE (Gibco).	HEK 293FT suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
   150,000 cells/cm2 of 293FT cells in suspension were mixed with the PEI-DNA DMEM mixture and allowed to incubate for 5 minutes at room temperature and then plated in D10 for 48 hours.	293FT suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
   The original stock from BEI was passaged through a 0.45 μM syringe filter and then 5 μl was inoculated onto an 80% confluent T175 flasks (Nunc) of Vero-E6 cells to produce our p1 stock.	Vero-E6 suggested: None
   After 48 hours, viral supernatant was harvested, centrifuged at 800xg for 5 minutes to remove cell debris, and used to transduce 800,000 Cas9-expressing Calu-3 cells.	Calu-3 suggested: KCLB Cat# 30055, RRID:CVCL_0609)
   The virus was rescued and passaged in Huh7.5.1 cells multiple times until widespread GFP fluorescence and cytopathic effects were observed.	Huh7.5.1 suggested: RRID:CVCL_E049)
   To rescue the VSVdG-RABV-G, 293FT cells (Thermo Fisher Scientific) were co-cultured with VeroE6 cells in a 6-well plate.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   To produce p2 working SARS-CoV-2 stocks, 5 μl of the p1 stock was inoculated onto 80% confluent T175 flasks of Vero-E6 cells as described above.	p1 suggested: None
   Recombinant DNA
   Sentences	Resources
   PEI (0.71ug PEI/cm2 cell culture area) was mixed with 1 mL of DMEM. pMD2.G, PAX and library plasmid (0.178ug/cm2, at a 0.25 (pMD2.G): 0.5 (psPAX): 1(Library plasmid) ratio were thoroughly mixed with 1 mL DMEM.	pMD2.G suggested: RRID:Addgene_12259) pMD2 suggested: None
   In short, VSVdG-eGFP (Addgene, Plasmid #31842) was modified to insert in frame with the deleted VSV-G a codon optimized SARS-CoV-2-S based on the Wuhan-Hu-1 isolate (GenBank:MN908947.3), which was mutated to remove a putative ER retention domain (K1269A and H1271A).	VSVdG-eGFP suggested: None
   This gene was assembled into VSV-eGFP-dG (Addgene, Plasmid #31842) in frame with the G coding sequence between MluI and NotI to generate VSV-eGPF-RABV-G.	VSV-eGFP-dG suggested: None VSV-eGPF-RABV-G suggested: None
   Cells were transfected with pCAGEN-VSV-N (300ng), pCAGEN-VSV-P (500ng), pCAGEN-VSV-L (200ng), pCAGEN-VSV-G (800ng), pCAGGS-T7 (200ng), and VSV-eGFP-dG-RABV-G (500ng) using JetPrime (Polyplus).	pCAGEN-VSV-N suggested: None pCAGEN-VSV-P suggested: None pCAGEN-VSV-L suggested: None pCAGEN-VSV-G suggested: None pCAGGS-T7 suggested: None
   Software and Algorithms
   Sentences	Resources
   Any genes that exhibited depletion or enrichment in the top 500 hits between the plasmid library and the uninfected control were removed from STRING analysis as they are likely confounded by affecting cellular growth or survival unrelated to viral infection.	STRING suggested: (STRING, RRID:SCR_005223)

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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
2. 

   Version published to 10.1101/2021.04.22.440848v1 on bioRxiv

   Apr 23, 2021