A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern

  1. Long T. Nguyen
  2. Nicolas C. Macaluso
  3. Brianna L.M. Pizzano
  4. Melanie N. Cash
  5. Jan Spacek
  6. Jan Karasek
  7. Megan R. Miller
  8. John A. Lednicky
  9. Rhoel R. Dinglasan
  10. Marco Salemi
  11. Piyush K. Jain

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Abstract

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  1. Version published to 10.1016/j.ebiom.2022.103926
  2. ScreenIT

    SciScore for 10.1101/2021.10.15.21265066: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: The sample collection followed the guidelines approved by UF Institutional Review Board (IRB202000781) and through Evaluating the Molecular Epidemiology of Coronavirus (COVID-19) in Florida (IRB202000633).
    IRB: The sample collection followed the guidelines approved by UF Institutional Review Board (IRB202000781) and through Evaluating the Molecular Epidemiology of Coronavirus (COVID-19) in Florida (IRB202000633).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Recombinant DNA
    SentencesResources
    The gene sequence was then codon-optimized using the GenSmart™ Codon Optimization tool (Genscript) for bacterial protein expression, synthesized by Twist Bioscience, and cloned into PET28a+ vector.
    PET28a+
    suggested: None
    Software and Algorithms
    SentencesResources
    The reaction mixture was then transferred to the qPCR StepOne Plus system (Thermofisher), and the binary complex temperature melting profile was recorded over a temperature range of 25-99°C with a ramp rate of 1%/second.
    Thermofisher
    suggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)
    Illumina’s DRAGEN pipeline was used to derive sample consensus sequences, which were filtered based on a minimum of 70% coverage of the genome.
    DRAGEN
    suggested: None
    One-pot BrCas12b detection assay: LAMP primers targeting wild-type SARS-CoV-2 and variants of concern were designed using NEB® LAMP Primer Design Tool (New England Biolabs) and PrimerExplorer v5 at https://primerexplorer.jp/e/14.
    LAMP
    suggested: (LAMP, RRID:SCR_001740)

    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: We did not find any issues relating to colormaps.

    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.

    Results from scite Reference Check: We found no unreliable references.

    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.

  3. Version published to 10.1101/2021.10.15.21265066 on medRxiv