Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform.
It's quite amazing. For starters, the work was completed in less than 6 weeks, since the first genomic sequence of the SARS-CoV-2 virus was released on Jan 10 and the paper was submitted on Feb 22. Most of that time was preparation of the DNA fragments.
9 DNA fragments
The first step was to PCR amplify DNA segments spanning the genome. This was done by RT-PCR. Usually one verifies the sequence has not been mutated in the PCR reaction (I don't see this in the paper).
Alternatively one can just order synthetic DNA of < 8 kb these days (with a 10 day turnaround)! link NPR story
Cloning
Some DNA fragments are unstable when cloned into E. coli. So they used YACs (yeast artificial chromosomes). An additional advantage is that all 8 fragments can be assembled into the final product in one step! (TAR-cloning)
In vitro transcription
The clones are made with a promoter for phage T7 RNA polymerase upstream and poly A tail downstream followed by a restriction site for Pac I. YAC DNA was prepared, cut with Pac I and transcribed with T7 polymerase.
Transfection
The transcript was then transfected into mammalian cells, together with mRNA for one viral protein. The system is a bit more complicated than that: there are two cell types. But it seems easy enough, and the result is that the transfected cell system produces virus.
The virus can then be cloned (classic terminology, progeny from a single individual produced) and grown in culture. Simply amazing.
Attenuating mutations from viruses for the live-attenuated vaccine for SARS-CoV can be assembled into live SARS-CoV-2 and tested for growth properties and attenuation, and then investigated as vaccines.
