2. Academic Validation
  3. Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform

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

  • Nature. 2020 Jun;582(7813):561-565. doi: 10.1038/s41586-020-2294-9.
Tran Thi Nhu Thao  # 1 2 3,  Fabien Labroussaa  # 2 4,  Nadine Ebert  # 1 2,  Philip V'kovski 1 2,  Hanspeter Stalder 1 2,  Jasmine Portmann 1 2,  Jenna Kelly 1 2,  Silvio Steiner 1 2 3,  Melle Holwerda 1 2 3 5,  Annika Kratzel 1 2 3,  Mitra Gultom 1 2 3 5,  Kimberly Schmied 1 2,  Laura Laloli 1 2 3 5,  Linda Hüsser 1 2,  Manon Wider 5,  Stephanie Pfaender 1 2 6,  Dagny Hirt 1 2,  Valentina Cippà 2 4,  Silvia Crespo-Pomar 2 4,  Simon Schröder 7,  Doreen Muth 7 8,  Daniela Niemeyer 7 8,  Victor M Corman 7 8,  Marcel A Müller 7 8 9,  Christian Drosten 7 8,  Ronald Dijkman 1 2 5,  Joerg Jores 10 11,  Volker Thiel 12 13
Affiliations

Affiliations

  • 1 Institute of Virology and Immunology (IVI), Bern, Switzerland.
  • 2 Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
  • 3 Graduate School for Biomedical Science, University of Bern, Bern, Switzerland.
  • 4 Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
  • 5 Insitute for Infectious Diseases, University of Bern, Bern, Switzerland.
  • 6 Department for Molecular and Medical Virology, Ruhr-Universität Bochum, Bochum, Germany.
  • 7 Institute of Virology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.
  • 8 German Centre for Infection Research, associated partner Charité, Berlin, Germany.
  • 9 Martsinovsky Institute of Medical Parasitology, Tropical and Vector Borne Diseases, Sechenov University, Moscow, Russia.
  • 10 Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland. joerg.jores@vetsuisse.unibe.ch.
  • 11 Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland. joerg.jores@vetsuisse.unibe.ch.
  • 12 Institute of Virology and Immunology (IVI), Bern, Switzerland. volker.thiel@vetsuisse.unibe.ch.
  • 13 Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland. volker.thiel@vetsuisse.unibe.ch.
  • # Contributed equally.
PMID: 32365353 DOI: 10.1038/s41586-020-2294-9
Abstract

Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate in Escherichia coli owing to the size and occasional instability of the genome1-3. 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 to genetically reconstruct diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Pneumoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step in Saccharomyces cerevisiae using transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak.

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