Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform

  1. Tran Thi Nhu Thao
  2. Fabien Labroussaa
  3. Nadine Ebert
  4. Philip V’kovski
  5. Hanspeter Stalder
  6. Jasmine Portmann
  7. Jenna Kelly
  8. Silvio Steiner
  9. Melle Holwerda
  10. Annika Kratzel
  11. Mitra Gultom
  12. Laura Laloli
  13. Linda Hüsser
  14. Manon Wider
  15. Stephanie Pfaender
  16. Dagny Hirt
  17. Valentina Cippà
  18. Silvia Crespo-Pomar
  19. Simon Schröder
  20. Doreen Muth
  21. Daniela Niemeyer
  22. Marcel A. Müller
  23. Christian Drosten
  24. Ronald Dijkman
  25. Joerg Jores
  26. Volker Thiel

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Abstract

Reverse genetics has been an indispensable tool revolutionising our insights into viral pathogenesis and vaccine development. Large RNA virus genomes, such as from Coronaviruses, are cumbersome to clone and to manipulate in E. coli hosts due to size and occasional instability 1-3 . Therefore, an alternative rapid and robust reverse genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform for the genetic reconstruction of diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Paramyxoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples, or synthetic DNA, and reassembled in one step in Saccharomyces cerevisiae using transformation associated recombination (TAR) cloning to maintain the genome as a yeast artificial chromosome (YAC). T7-RNA polymerase has been used to generate infectious RNA, which was then used to rescue viable virus. Based on this platform we have been able to engineer and resurrect chemically-synthetized clones of the recent epidemic SARS-CoV-2 4 in only a week after receipt of the synthetic DNA fragments. The technical advance we describe here allows to rapidly responding to emerging viruses as it enables the generation and functional characterization of evolving RNA virus variants - in real-time - during an outbreak.

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  1. ScreenIT

    SciScore for 10.1101/2020.02.21.959817: (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.

    Table 2: Resources

    No key resources detected.

    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.

    About SciScore

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  2. Version published to 10.1101/2020.02.21.959817 on bioRxiv