YOU WOULDN’T DOWNLOAD A VIRUS… wait, never mind
Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform
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 instability.
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-2 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.




















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