Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction.
CPER
SARS-CoV-2
infectious clone
mutagenesis
reverse genetics
Journal
Cell reports
ISSN: 2211-1247
Titre abrégé: Cell Rep
Pays: United States
ID NLM: 101573691
Informations de publication
Date de publication:
20 04 2021
20 04 2021
Historique:
received:
24
09
2020
revised:
03
03
2021
accepted:
26
03
2021
pubmed:
12
4
2021
medline:
4
5
2021
entrez:
11
4
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 33838744
pii: S2211-1247(21)00328-4
doi: 10.1016/j.celrep.2021.109014
pmc: PMC8015404
pii:
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
109014Informations de copyright
Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of interests The authors declare no competing interests.
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