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Статья
Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform
2021
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 coli owing to the size and occasional instability of the genome1–3. 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, Flaviviridae and Pneumoviridae families. 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 cerevisiae using 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. © 2020, The Author(s), under exclusive licence to Springer Nature Limited.
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Версии
- 1. Version of Record от 2021-04-27
Метаданные
Название журнала
- Nature
Том
- 582
Выпуск
- 7813
Страницы
- 561-565
Ключевые слова
- DNA fragment; RNA polymerase; virus DNA; bacteriophage T7 RNA polymerase; DNA directed RNA polymerase; viral protein; clone; disease treatment; drug development; genetic engineering; genomics; recombination; vaccine; viral disease; yeast; animal cell; Article; controlled study; Coronaviridae; coronavirus disease 2019; Flaviviridae; gene synthesis; genetic engineering; genomic fragment; human; human cell; human tissue; mouse; nonhuman; pandemic; Pneumovirinae; priority journal; Saccharomyces cerevisiae; Severe acute respiratory syndrome coronavirus 2; viral genetics; virus isolation; yeast artificial chromosome; animal; Betacoronavirus; China; Chlorocebus aethiops; Coronavirus infection; genetics; genomics; metabolism; molecular cloning; molecular evolution; mutation; Pneumovirus; procedures; reverse genetics; synthetic biology; Vero cell line; virology; virus genome; virus pneumonia; Zika virus; Coronaviridae; Coronavirus; Escherichia coli; Flaviviridae; RNA viruses; Saccharomyces cerevisiae; SARS coronavirus; Animals; Betacoronavirus; China; Chlorocebus aethiops; Chromosomes, Artificial, Yeast; Cloning, Molecular; Coronavirus Infections; DNA-Directed RNA Polymerases; Evolution, Molecular; Genome, Viral; Genomics; Humans; Mutation; Pandemics; Pneumonia, Viral; Respiratory Syncytial Viruses; Reverse Genetics; Saccharomyces cerevisiae; Synthetic Biology; Vero Cells; Viral Proteins; Zika Virus
Издатель
- Nature Research
Тип документа
- journal article
Источник
- scopus
