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

• Published in: Nature (London) Vol. 582; no. 7813; pp. 561 - 565
• Main Authors: Thi Nhu Thao, Tran, Labroussaa, Fabien, Ebert, Nadine, V’kovski, Philip, Stalder, Hanspeter, Portmann, Jasmine, Kelly, Jenna, Steiner, Silvio, Holwerda, Melle, Kratzel, Annika, Gultom, Mitra, Schmied, Kimberly, Laloli, Laura, Hüsser, Linda, Wider, Manon, Pfaender, Stephanie, Hirt, Dagny, Cippà, Valentina, Crespo-Pomar, Silvia, Schröder, Simon, Muth, Doreen, Niemeyer, Daniela, Corman, Victor M., Müller, Marcel A., Drosten, Christian, Dijkman, Ronald, Jores, Joerg, Thiel, Volker
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 25.06.2020; Nature Publishing Group
• Subjects: 42/70; 45/91; 631/326/596; 631/326/596/2078; 631/326/596/4130; Analysis; Animals; Artificial chromosomes; Betacoronavirus - genetics; China - epidemiology; Chlorocebus aethiops; Chromosomes; Chromosomes, Artificial, Yeast - metabolism; Cloning; Cloning, Molecular - methods; Coronaviridae; Coronavirus Infections - epidemiology; Coronavirus Infections - virology; Coronaviruses; COVID-19; Deoxyribonucleic acid; DNA; DNA-directed RNA polymerase; DNA-Directed RNA Polymerases - metabolism; E coli; Epidemics; Evolution, Molecular; Flaviviridae; Fragments; Genetic aspects; Genetic engineering; Genetically modified organisms; Genetics; Genome, Viral - genetics; Genomes; Genomic analysis; Genomics; Genomics - methods; Humanities and Social Sciences; Humans; Methods; Middle East respiratory syndrome; multidisciplinary; Mutation; Pandemics; Pandemics - statistics & numerical data; Pathogenesis; Pneumonia, Viral - epidemiology; Pneumonia, Viral - virology; Pneumoviridae; Recombination; Respiratory diseases; Respiratory Syncytial Viruses - genetics; Reverse engineering; Reverse Genetics - methods; Ribonucleic acid; RNA; RNA polymerase; RNA viruses; Saccharomyces cerevisiae - genetics; SARS-CoV-2; Science; Science (multidisciplinary); Severe acute respiratory syndrome; Severe acute respiratory syndrome coronavirus 2; Synthetic Biology - methods; Vaccine development; Vaccines; Vero Cells; Viral diseases; Viral Proteins - metabolism; Viruses; Yeast; Yeasts; Zika virus; Zika Virus - genetics; China; Switzerland
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/s41586-020-2294-9

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. A yeast-based synthetic genomics platform is used to reconstruct and characterize large RNA viruses from synthetic DNA fragments; this technique will facilitate the rapid analysis of RNA viruses, such as SARS-CoV-2, during an outbreak.