This article is more of a “new methods & technology” topic than following the usual gene-environment interactions theme. However, this technique offers an exciting breakthrough in efficiently synthesizing and making available the 30,000-base genome of the SARS-CoV-2 (+)-single-stranded RNA [(+)-ssRNA] virus. Because SARS-CoV-2 outbreak virus isolates are not available to health authorities and the scientific community — the RNA genome is urgently needed to build up diagnostics, develop and assess anti-virals and (perhaps) development of a successful vaccine, and establish appropriate in vivo models. Generation of the SARS-CoV-2 (+)-ssRNA virus from chemically-synthesized DNA could bypass the limited availability of virus isolates and would furthermore allow genetic modifications and functional characterization.
The bacterium Escherichia coli is generally used for cloning many viral genomes; however, it has drawbacks for a number of emerging RNA viruses — including coronaviruses — regarding assembly and stablye maintenance of full-length clones. Synthetic genomics is a relatively recent field, fueled by efforts to create a bacterial cell, controlled by a synthetic genome. Genome-wide re-assembly of the ~1.1Mb (million-base) Mycoplasma genome was first attempted using E. coli as an intermediate host, but maintenance of 100-kbp (100 thousand-base-pair) DNA fragments appeared to be very difficult in this host. Therefore, baker’s yeast (Saccharomyces cerevisiae) was chosen to clone, assemble, and mutagenize entire Mycoplasma genomes. The rationale for using a yeast cloning system is the ability of yeast to recombine overlapping DNA fragments inside the cell, which led to development of a technique called “transformation-associated recombination” (TAR) cloning.
Authors [see attached article] show the full functionality of a yeast-based synthetic genomics platform to genetically
reconstruct 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 re-assembled in one step in Saccharomyces cerevisiae, using TAR cloning to maintain the genome as a yeast artificial
T7-RNA polymerase has been used to generate infectious RNA for rescuing viable virus. Based on this platform, authors were able to engineer and resurrect chemically-synthetized clones of the current pandemic SARS-CoV-2 (+)-ssRNA virus in only a week after receipt of the synthetic DNA fragments…!! This fantastic technical advance, described herein, allows a rapid response to emerging viruses — because it enables creation and functional characterization of evolving RNA virus variants — in real-time — during any rapid serious outbreak. 😊
Nature [accelerated publication] 4 May 2020; https://doi.org/10.1038/s41586-020-2294-9