1. Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform.
- Author
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Thi Nhu Thao T, Labroussaa F, Ebert N, V'kovski P, Stalder H, Portmann J, Kelly J, Steiner S, Holwerda M, Kratzel A, Gultom M, Schmied K, Laloli L, Hüsser L, Wider M, Pfaender S, Hirt D, Cippà V, Crespo-Pomar S, Schröder S, Muth D, Niemeyer D, Corman VM, Müller MA, Drosten C, Dijkman R, Jores J, and Thiel V
- Subjects
- Animals, COVID-19, China epidemiology, Chlorocebus aethiops, Chromosomes, Artificial, Yeast metabolism, Coronavirus Infections epidemiology, DNA-Directed RNA Polymerases metabolism, Evolution, Molecular, Humans, Mutation, Pandemics statistics & numerical data, Pneumonia, Viral epidemiology, Respiratory Syncytial Viruses genetics, SARS-CoV-2, Saccharomyces cerevisiae genetics, Vero Cells, Viral Proteins metabolism, Zika Virus genetics, Betacoronavirus genetics, Cloning, Molecular methods, Coronavirus Infections virology, Genome, Viral genetics, Genomics methods, Pneumonia, Viral virology, Reverse Genetics methods, Synthetic Biology methods
- 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 genome
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 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.- Published
- 2020
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