Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction
Summary: 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 co...
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| Format: | Article |
| Language: | English |
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Elsevier
2021-04-01
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| Series: | Cell Reports |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2211124721003284 |
| _version_ | 1818941731078406144 |
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| author | Shiho Torii Chikako Ono Rigel Suzuki Yuhei Morioka Itsuki Anzai Yuzy Fauzyah Yusuke Maeda Wataru Kamitani Takasuke Fukuhara Yoshiharu Matsuura |
| author_facet | Shiho Torii Chikako Ono Rigel Suzuki Yuhei Morioka Itsuki Anzai Yuzy Fauzyah Yusuke Maeda Wataru Kamitani Takasuke Fukuhara Yoshiharu Matsuura |
| author_sort | Shiho Torii |
| collection | DOAJ |
| description | Summary: 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 (∼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. |
| first_indexed | 2024-12-20T07:00:11Z |
| format | Article |
| id | doaj.art-2b5589cbe431454eb85367643a1a2e11 |
| institution | Directory Open Access Journal |
| issn | 2211-1247 |
| language | English |
| last_indexed | 2024-12-20T07:00:11Z |
| publishDate | 2021-04-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Cell Reports |
| spelling | doaj.art-2b5589cbe431454eb85367643a1a2e112022-12-21T19:49:14ZengElsevierCell Reports2211-12472021-04-01353109014Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reactionShiho Torii0Chikako Ono1Rigel Suzuki2Yuhei Morioka3Itsuki Anzai4Yuzy Fauzyah5Yusuke Maeda6Wataru Kamitani7Takasuke Fukuhara8Yoshiharu Matsuura9Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan; Center for Infectious Diseases Education and Research, Osaka University, Suita, Osaka 565-0871, JapanDepartment of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan; Center for Infectious Diseases Education and Research, Osaka University, Suita, Osaka 565-0871, JapanDepartment of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Sapporo, Hokkaido 060-8638, JapanDepartment of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, JapanDepartment of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, JapanDepartment of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, JapanDepartment of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, JapanDepartment of Infectious Diseases and Host Defense, Graduate School of Medicine, Gunma University, Maebashi, Gunma 371-8511, JapanDepartment of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Sapporo, Hokkaido 060-8638, Japan; Corresponding authorDepartment of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan; Center for Infectious Diseases Education and Research, Osaka University, Suita, Osaka 565-0871, Japan; Corresponding authorSummary: 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 (∼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.http://www.sciencedirect.com/science/article/pii/S2211124721003284SARS-CoV-2reverse geneticsinfectious cloneCPERmutagenesis |
| spellingShingle | Shiho Torii Chikako Ono Rigel Suzuki Yuhei Morioka Itsuki Anzai Yuzy Fauzyah Yusuke Maeda Wataru Kamitani Takasuke Fukuhara Yoshiharu Matsuura Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction Cell Reports SARS-CoV-2 reverse genetics infectious clone CPER mutagenesis |
| title | Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction |
| title_full | Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction |
| title_fullStr | Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction |
| title_full_unstemmed | Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction |
| title_short | Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction |
| title_sort | establishment of a reverse genetics system for sars cov 2 using circular polymerase extension reaction |
| topic | SARS-CoV-2 reverse genetics infectious clone CPER mutagenesis |
| url | http://www.sciencedirect.com/science/article/pii/S2211124721003284 |
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