Treating Influenza and SARS-CoV-2 via mRNA-encoded Cas13a

  1. Emmeline L. Blanchard
  2. Daryll Vanover
  3. Swapnil Subhash Bawage
  4. Pooja Munnilal Tiwari
  5. Laura Rotolo
  6. Jared Beyersdorf
  7. Hannah E. Peck
  8. Nicholas C Bruno
  9. Robert Hincapie
  10. M.G. Finn
  11. Frank Michel
  12. Eric R. Lafontaine
  13. Robert J. Hogan
  14. Chiara Zurla
  15. Philip J. Santangelo

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Abstract

Here, Cas13a has been used to target and mitigate influenza virus A (IAV) and SARS-CoV-2 using a synthetic mRNA-based platform. CRISPR RNAs (crRNA) against PB1 and highly conserved regions of PB2 were screened in conjunction with mRNA-encoded Cas13a. Screens were designed such that only guides that decreased influenza RNA levels in a Cas13-mediated fashion, were valid. Cas13a mRNA and validated guides, delivered post-infection, simulating treatment, were tested in combination and across multiplicities of infection. Their function was also characterized over time. Similar screens were performed for guides against SARS-CoV-2, yielding multiple guides that significantly impacted cytopathic effect. Last, the approach was utilized in vivo , demonstrating the ability to degrade influenza RNA in a mouse model of infection, using polymer-formulated, nebulizer-based mRNA delivery. Our findings demonstrate the applicability of Cas13a in mitigating respiratory infections both in vitro and in a mouse model, paving the way for future therapeutic use.

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  1. ScreenIT

    SciScore for 10.1101/2020.04.24.060418: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All work with live SARS-CoV-2 was performed inside a certified Class II Biosafety Cabinet in a BSL3 laboratory in compliance with all State and Federal guidelines and with the approval of the UGA Institutional Biosafety Committee (IBC)
    RandomizationAnimals were randomly distributed among experimental groups.
    BlindingResearchers were blinded to animal group allocation during data acquisition.
    Power Analysisnot detected.
    Sex as a biological variableAnimal studies: Six- to 8-week-old female BALB/c mice (Jackson Laboratories) were maintained under pathogen-free conditions in individually ventilated and watered cages kept at negative pressure.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Influenza virus stocks (H1N1 Influenza virus A/WSN/33) were prepared in MDCK cells.
    MDCK
    suggested: CLS Cat# 602280/p823_MDCK_(NBL-2, RRID:CVCL_0422)
    Viral stocks were generated by infecting Vero E6 cells (ATCC, C1008) at ~95% confluency in 150cm2 flasks with SARS-CoV-2 at an MOI of 0.1 PFU/cell.
    Vero E6
    suggested: RRID:CVCL_XD71)
    In vitro anti-viral assay with IAV: A549 cells were seeded overnight at a density of 120,000-130,000 per well in a 24 well plate.
    A549
    suggested: None
    In vitro anti-viral assay with SARS-CoV-2: Vero cells were seeded overnight at ~80% confluency in a 6 well plate.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Experimental Models: Organisms/Strains
    SentencesResources
    Animal studies: Six- to 8-week-old female BALB/c mice (Jackson Laboratories) were maintained under pathogen-free conditions in individually ventilated and watered cages kept at negative pressure.
    BALB/c
    suggested: RRID:IMSR_ORNL:BALB/cRl)
    Software and Algorithms
    SentencesResources
    An ROI of the same size was then used for all images and the sum intensity was calculated by Volocity.
    Volocity
    suggested: (Volocity 3D Image Analysis Software, RRID:SCR_002668)
    Lung luminescence was then quantified using Living Image software (Perkin Elmer).
    Living Image
    suggested: (Living Image software, RRID:SCR_014247)
    Data was analyzed using GraphPad Prism 7.04.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 43. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.04.24.060418v1 on bioRxiv