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Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction

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Title: Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction
Authors: Torii, Shiho Browse this author
Ono, Chikako Browse this author
Suzuki, Rigel Browse this author
Morioka, Yuhei Browse this author
Anzai, Itsuki Browse this author
Fauzyah, Yuzy Browse this author
Maeda, Yusuke Browse this author
Kamitani, Wataru Browse this author
Fukuhara, Takasuke Browse this author
Matsuura, Yoshiharu Browse this author
Issue Date: 20-Apr-2021
Publisher: Cell Press
Journal Title: Cell Reports
Volume: 35
Issue: 3
Start Page: 109014
Publisher DOI: 10.1016/j.celrep.2021.109014
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). Although multiple mutations have been observed in SARS-CoV-2, functional analysis of each mutation of SARS-CoV-2 has been limited by the lack of convenient mutagenesis methods. In this study, we establish 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. 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 (similar to 5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. This reverse genetics system may potentially advance further understanding of SARS-CoV-2.
Type: article
Appears in Collections:医学院・医学研究院 (Graduate School of Medicine / Faculty of Medicine) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

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