tetano
Editor, Senior Moderator
Cell Rep
. 2021 Apr 1;109014.
doi: 10.1016/j.celrep.2021.109014. Online ahead of print.
Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction
Shiho Torii[SUP] 1 [/SUP], Chikako Ono[SUP] 1 [/SUP], Rigel Suzuki[SUP] 2 [/SUP], Yuhei Morioka[SUP] 3 [/SUP], Itsuki Anzai[SUP] 3 [/SUP], Yuzy Fauzyah[SUP] 3 [/SUP], Yusuke Maeda[SUP] 3 [/SUP], Wataru Kamitani[SUP] 4 [/SUP], Takasuke Fukuhara[SUP] 5 [/SUP], Yoshiharu Matsuura[SUP] 6 [/SUP]
Affiliations
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 (?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.
Keywords: CPER; SARS-CoV-2; infectious clone; mutagenesis; reverse genetics.
. 2021 Apr 1;109014.
doi: 10.1016/j.celrep.2021.109014. Online ahead of print.
Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction
Shiho Torii[SUP] 1 [/SUP], Chikako Ono[SUP] 1 [/SUP], Rigel Suzuki[SUP] 2 [/SUP], Yuhei Morioka[SUP] 3 [/SUP], Itsuki Anzai[SUP] 3 [/SUP], Yuzy Fauzyah[SUP] 3 [/SUP], Yusuke Maeda[SUP] 3 [/SUP], Wataru Kamitani[SUP] 4 [/SUP], Takasuke Fukuhara[SUP] 5 [/SUP], Yoshiharu Matsuura[SUP] 6 [/SUP]
Affiliations
- PMID: 33838744
- 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 (?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.
Keywords: CPER; SARS-CoV-2; infectious clone; mutagenesis; reverse genetics.