Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction
| Autor: | Itsuki Anzai, Yuzy Fauzyah, Yoshiharu Matsuura, Shiho Torii, Chikako Ono, Yusuke Maeda, Wataru Kamitani, Yuhei Morioka, Rigel Suzuki, Takasuke Fukuhara |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Base pair QH301-705.5 viruses Mutant Mutagenesis (molecular biology technique) medicine.disease_cause Genome General Biochemistry Genetics and Molecular Biology law.invention 03 medical and health sciences Biosafety reverse genetics 0302 clinical medicine CPER law Cricetinae Complementary DNA Report medicine Animals Humans Biology (General) skin and connective tissue diseases Polymerase Mutation Reporter gene biology SARS-CoV-2 fungi infectious clone COVID-19 virus diseases Virology Reverse genetics body regions HEK293 Cells 030104 developmental biology biology.protein Recombinant DNA 030217 neurology & neurosurgery mutagenesis |
| Zdroj: | Cell Reports, Vol 35, Iss 3, Pp 109014-(2021) Cell Reports |
| ISSN: | 2211-1247 |
| Popis: | Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). While 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. 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. This reverse genetics system may potentially advance further understanding of SARS-CoV-2. Graphical Abstract Torii et al. establish a novel PCR-based, bacterium-free reverse genetics system for SARS-CoV-2 by using the CPER method. Recombinant SARS-CoV-2 can be produced with high titers around 2 weeks after amplification of SARS-CoV-2 gene fragments. The method can be applied to generate recombinant SARS-CoV-2 carrying reporter genes or mutations. |
| Databáze: | OpenAIRE |
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