Establishment of a Reverse Genetics System for SARS-CoV-2 Using Circular Polymerase Extension Reaction (preprint)

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). While the development of specific treatments and a vaccine is urgently needed, functional analyses of SARS-CoV-2 have been limited by the lack of convenient mutagenesis methods. In this study, we established a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. Notably, the construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (~5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. We hope that our reverse genetics system will contribute to the further understanding of SARS-CoV-2.Funding: This work was supported from the Ministry of Health, Labor and Welfare of Japan and the Japan Agency for Medical Research and Development (AMED; http//www.amed.go.jp/) (JP20wm0225002, JP20he0822006, JP20fk0108264, JP20he0822008, JP20wm0225003, JP20fk0108267, JP19fk0108113, and JP20wm0125010), and the Japan Society for the Promotion of Science KAKENHI (JP19K24679). S. Torii is supported by a JSPS Research Fellowships for young scientists (https//www.jsps.go.jp/english/e-grants/) (19J12641). Conflict of Interest We have no conflicts of interest to declare.Ethical Approval All experiments involving SARS-CoV-2 were performed in biosafety level-3 laboratories, following the standard biosafety protocols approved by the Research institute for Microbial Diseases at Osaka University.