1. Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform
- Author
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Simon Schröder, Melle Holwerda, Silvio Steiner, Philip V'kovski, Tran Thi Nhu Thao, Fabien Labroussaa, Jenna N. Kelly, Marcel A. Müller, Annika Kratzel, Silvia Crespo-Pomar, Nadine Ebert, Laura Laloli, Daniela Niemeyer, Joerg Jores, Hanspeter Stalder, Jasmine Portmann, Doreen Muth, Christian Drosten, Dagny Hirt, Manon Wider, Volker Thiel, Stephanie Pfaender, Valentina Cippà, Linda Hüsser, Ronald Dijkman, and Mitra Gultom
- Subjects
0303 health sciences ,030306 microbiology ,Viral pathogenesis ,viruses ,RNA ,RNA virus ,Computational biology ,Biology ,biology.organism_classification ,Reverse genetics ,Synthetic genomics ,03 medical and health sciences ,biology.protein ,Coronaviridae ,Polymerase ,030304 developmental biology ,Subgenomic mRNA - 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 instability1-3. 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-24 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.
- Published
- 2020
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