Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection

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1. 

   ScreenIT

   Jun 18, 2020

   SciScore for 10.1101/2020.06.16.155101: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After an incubation period of 1 hour at 37°C, the inoculum was removed and cells were washed with PBS before media supplemented with anti-VSV-G clone I4 was added in order to neutralize residual input virus (no antibody was added to cells expressing VSV-G).	anti-VSV-G suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Generation of SARS-CoV-2 stocks: To generate SARS-CoV-2 viral stocks, Huh7.5 cells were inoculated with SARS-CoV-2 isolate USA-WA1/2020 (BEI Resources #NR-52281) at an MOI of approximately 0.01 for three days to generate a P1 stock.	Huh7.5 suggested: RRID:CVCL_7927)
   Virus titer …

   SciScore for 10.1101/2020.06.16.155101: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After an incubation period of 1 hour at 37°C, the inoculum was removed and cells were washed with PBS before media supplemented with anti-VSV-G clone I4 was added in order to neutralize residual input virus (no antibody was added to cells expressing VSV-G).	anti-VSV-G suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Generation of SARS-CoV-2 stocks: To generate SARS-CoV-2 viral stocks, Huh7.5 cells were inoculated with SARS-CoV-2 isolate USA-WA1/2020 (BEI Resources #NR-52281) at an MOI of approximately 0.01 for three days to generate a P1 stock.	Huh7.5 suggested: RRID:CVCL_7927)
   Virus titer was determined by plaque assay using Vero-E6 cells.	Vero-E6 suggested: None
   Cas9 activity was assessed by transducing parental Vero-E6 or Vero-E6-Cas9 cells with pXPR_047 (Addgene 107645), which expresses eGFP and an sgRNA targeting eGFP (72).	Vero-E6-Cas9 suggested: None
   Briefly, 293T cells were transfected with pCAGGS vector expressing the SARS-CoV-2 spike glycoprotein and then inoculated with a replication-deficient VSV vector that contains expression cassettes for Renilla luciferase instead of the VSV-G open reading frame.	293T suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
   Software and Algorithms
   Sentences	Resources
   Screen analysis: Guide sequences were extracted from the sequencing reads with PoolQ version 3.2.9 (Broad Institute, https://portals.broadinstitute.org/gpp/public/software/poolq), using a “CACCG” search prefix, and a counts matrix was generated.	PoolQ suggested: None
   Gene set enrichment and network analysis: We used the STRING enrichment detection tool to identify significantly enriched gene sets, using African green monkey gene symbols, but testing for enrichment across human gene sets.	STRING suggested: (STRING, RRID:SCR_005223)
   We analyzed sets from all available sources provided by that tool, including sets of clusters of proteinprotein interactors in STRING, and excluding the PubMed gene sets.	PubMed suggested: (PubMed, RRID:SCR_004846)
   Total cell numbers were quantified by Gen5 software of brightfield images.	Gen5 suggested: (Gen5, RRID:SCR_017317)

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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: We detected the following sentences addressing limitations in the study:

   Importantly, this study is not without limitations as we used Vero-E6 cells, a type I interferon-deficient non-human primate cell line, rather than primary human cells (48–50). However, Vero-E6 cells are a model cell line for isolating viruses and were selected based on their susceptibility to SARS-CoV-2-induced death. Further, African green monkeys are susceptible to SARS-CoV-2 infection (19–21). Interestingly, we did not detect several genes previously implicated in SARS-CoV-2 infection including TMPRSS2, PIKFyve, and TPC2 suggesting that these genes are either not essential in a Vero cell-intrinsic manner or that functional redundancies exist with other genes (15, 45). Future work is necessary to evaluate the genes identified here in human cells and animal models. The abundance of genes identified here with functional roles in chromatin regulation and histone modification were surprising, as SARS-CoV-2 is an RNA virus that replicates in the cytoplasm. However, these genes highlight the potential importance of epigenetic regulation of SARS-CoV-2 infection and pathogenesis. Epigenetic processes have previously been implicated in regulating antigen presentation and interferon-stimulated gene induction after MERS-CoV and SARS-CoV infection (51–53); however, given that Vero-E6 cells are type I interferon-deficient, distinct mechanism(s) may be at play. Here we identify a novel role for HMGB1 in SARS-CoV-2 susceptibility. HMGB1 is a predominantly nuclear yet pleiotropic protein....

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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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2. 

   Version published to 10.1101/2020.06.16.155101v1 on bioRxiv

   Jun 17, 2020