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 coliowing 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, Flaviviridaeand Pneumoviridaefamilies. 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 cerevisiaeusing 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.

Within the past decade, we have seen outbreaks of numerous viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV) 5, ZIKA virus 6, Ebola virus 7 and, at the end of 2019, SARS-CoV-2—which was first detected in Wuhan, Hubei province, China 4, but rapidly developed into a pandemic. During the early phase of the SARS-CoV-2 outbreak, virus isolates were not available to health authorities and the scientific community, even though these isolates are urgently needed to generate diagnostic tools, to develop and assess antivirals and vaccines, and to establish appropriate in vivo models. The generation of the SARS-CoV-2 from chemically synthesized DNA could bypass the limited availability of virus isolates and would furthermore enable genetic modifications and functional characterization. However, although E. coliproved very useful for the cloning of many viral genomes, it has a number of disadvantages when used for the assembly and stable maintenance of full-length molecular clones of emerging RNA viruses, including coronaviruses.