Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza

  1. Timothy R. Abbott
  2. Girija Dhamdhere
  3. Yanxia Liu
  4. Xueqiu Lin
  5. Laine Goudy
  6. Leiping Zeng
  7. Augustine Chemparathy
  8. Stephen Chmura
  9. Nicholas S. Heaton
  10. Robert Debs
  11. Tara Pande
  12. Drew Endy
  13. Marie La Russa
  14. David B. Lewis
  15. Lei S. Qi

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Abstract

The outbreak of the coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), has infected more than 100,000 people worldwide with over 3,000 deaths since December 2019. There is no cure for COVID-19 and the vaccine development is estimated to require 12-18 months. Here we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN ( P rophylactic A ntiviral C RISPR in huMAN cells), for viral inhibition that can effectively degrade SARS-CoV-2 sequences and live influenza A virus (IAV) genome in human lung epithelial cells. We designed and screened a group of CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs for cleaving SARS-CoV-2. The approach is effective in reducing respiratory cell viral replication for H1N1 IAV. Our bioinformatic analysis showed a group of only six crRNAs can target more than 90% of all coronaviruses. The PAC-MAN approach is potentially a rapidly implementable pan-coronavirus strategy to deal with emerging pandemic strains.

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  1. ScreenIT

    SciScore for 10.1101/2020.03.13.991307: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    The transfection complex solution was distributed evenly to HEK293T cultures dropwise.
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    The following day, Lenti-X 293T cells were transfected with 27.18 μg of SARS-CoV-2 reporter, 23.76 μg of dR8.91 and 2.97 μg of pMD2.
    293T
    suggested: None
    Cas13d A549 cells were plated in two 100 mm dishes.
    A549
    suggested: None
    Software and Algorithms
    SentencesResources
    We used Bowtie 1.2.2 to align crRNAs to the human transcriptome (HG38; including non-coding RNA) and removed crRNAs that mapped to the human transcriptome with fewer than 2 mismatches.
    Bowtie
    suggested: (Bowtie, RRID:SCR_005476)
    Conservation Plot Creation: All the complete SARS-CoV-2 genome sequences were aligned with the SARS-CoV and MERS-CoV NCBI Refseq sequences by MAFFT using the --auto flag.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    The alignment was visualized using the Geneious Prime software (Geneious 2020.0.4; https://www.geneious.com).
    Geneious
    suggested: (Geneious, RRID:SCR_010519)
    Statistical analyses were done using a two-sided t-test with unequal variance in Excel to calculate p values.
    Excel
    suggested: None
    Raw data were processed with Cytobank (BD) software.
    Cytobank
    suggested: (Cytobank, RRID:SCR_014043)

    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04280705CompletedAdaptive COVID-19 Treatment Trial (ACTT)

    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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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.

    About SciScore

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  2. Version published to 10.1101/2020.03.13.991307v1 on bioRxiv