On May 4, local time, the international top academic journal “Nature” published an online study in the form of “Accelerated Article Preview” (Accelerated Article Preview) from a number of scientific research institutions in Switzerland, Germany, and Russia. Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform “. Based on the known gene sequence of the new coronavirus, the team quickly constructed a live new coronavirus in yeast by reverse genetics in the experiment for the first time.
The corresponding author is Volker Thiel of the Institute of Virology and Immunology, University of Bern, Switzerland Joerg Jores of the Department of Infectious Diseases and Pathological Biology, School of Veterinary Medicine. The research work was published online on the preprint website BioRxiv as early as February 21, local time, without peer review.
It is worth noting that this is a basic research work on virus reconstruction based on the known virus genome. It is irrelevant to the “synthetic”. The new coronavirus constructed in the laboratory in this study is a virus reconstruction study based on the published viral genome sequence after the outbreak.
The research team pointed out that the chemical synthesis of new coronavirus, especially before the new outbreak virus has been successfully isolated, can help scientists to provide infectious viruses to health departments and laboratories Strains can also be genetically modified and functionally characterized for individual genes, so as to gain time to respond quickly to outbreaks.
The paper mentioned that reverse genetics is considered an indispensable tool, which has completely changed our understanding of viral pathogenesis and vaccine development. Large RNA virus genomes, such as the coronavirus genome, are difficult to clone and manipulate in E. coli hosts due to their large and unstable genomes. The research team reported a yeast-based synthetic genomics platform this time,It is used for gene reconstruction of various RNA viruses, including members of Coronaviridae, Flaviviridae and Paramyxoviridae.
The research team first tested yeast-based in other RNA viruses (such as murine hepatitis virus MHV) The accuracy of the synthetic genomics platform of the team tested the gene cloning ability of the murine hepatitis virus A59 strain containing green fluorescent protein (MHV GFP). The results showed that YAC accounted for 90% of the correctly assembled MHV genome in the tested clones, which shows The assembly efficiency of the virus in yeast is very high.
It is also using this platform that the researchers have engineered and resurrected the new coronavirus within one week after receiving the synthetic DNA fragments.
Specifically, the research team divided the viral gene components into 12 fragments with a size of 0.5kbp- 3.4kbp. At the same time, in order to facilitate serological diagnosis and tracking in cell culture, the research team designed the synthetic new coronavirus to express GFP (green fluorescent protein). Therefore, the research team divided Fragment 11 into three subfragments containing the GFP sequence. The GFP sequence was inserted into ORF7a (Open Reading Frame, ORF), resulting in a total of 14 fragments.
The research team asked the reagent company to chemically synthesize the above 14 DNA fragments, placed an order on January 14, and got 12 of them on February 4. The cloning of fragments 5 and 7 in E. coli had some problems and could not be completed. However, the research team happened to obtain a new coronavirus sample (SARS-CoV-2 / München1.1 / 2020/929), they decided to obtain fragments 5 and 7 by RT-PCR amplification.
Using TAR cloning, for all six new coronavirus constructs, the researchers obtained molecular clones that were correctly assembled. Subsequently, using transformation-coupled recombination technology (TAR), the homologous recombination system of yeast was used to piece together these DNA sequences according to the sequences repeated at the ends. After obtaining the complete viral sequence, this DNA sequence was transcribed into viral RNA using T7 RNA polymerase, and the RNA was introduced into VeroE6 (monkey kidney cells) using electroporation technology, so that the cells were infected, and the supernatant of these cells was cultured The liquid (containing the released virus particles) is injected into other culture media, and can infect other cells.
The research team further stated, “We can engineer the chemically synthesized clones of the newly popular new coronavirus within one week after obtaining the synthetic DNA fragments of the virus And resurrection. “The recombination of the virus has high efficiency and accuracy. Normally, more than 90% of the clones are correct.
It is worth noting that in addition to the new coronavirus, the research team also reported the synthesis Construction of MHV (murine hepatitis virus, a coronavirus) and MERS-Cov et al. Construction of HCov-229E and Zika virus is still in progress.