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Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination

The ongoing pandemic coronavirus (CoV) disease 2019 (COVID-19) by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has already caused substantial morbidity, mortality, and economic devastation. Reverse genetic approaches to generate recombinant viruses are a powerful tool to characterize and und...

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Autores principales: Herrmann, Alexandra, Jungnickl, Doris, Cordsmeier, Arne, Peter, Antonia Sophia, Überla, Klaus, Ensser, Armin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8507965/
https://www.ncbi.nlm.nih.gov/pubmed/34638527
http://dx.doi.org/10.3390/ijms221910188
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author Herrmann, Alexandra
Jungnickl, Doris
Cordsmeier, Arne
Peter, Antonia Sophia
Überla, Klaus
Ensser, Armin
author_facet Herrmann, Alexandra
Jungnickl, Doris
Cordsmeier, Arne
Peter, Antonia Sophia
Überla, Klaus
Ensser, Armin
author_sort Herrmann, Alexandra
collection PubMed
description The ongoing pandemic coronavirus (CoV) disease 2019 (COVID-19) by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has already caused substantial morbidity, mortality, and economic devastation. Reverse genetic approaches to generate recombinant viruses are a powerful tool to characterize and understand newly emerging viruses. To contribute to the global efforts for countermeasures to control the spread of SARS-CoV-2, we developed a passage-free SARS-CoV-2 clone based on a bacterial artificial chromosome (BAC). Moreover, using a Lambda-based Red recombination, we successfully generated different reporter and marker viruses, which replicated similar to a clinical isolate in a cell culture. Moreover, we designed a full-length reporter virus encoding an additional artificial open reading frame with wild-type-like replication features. The virus-encoded reporters were successfully applied to ease antiviral testing in cell culture models. Furthermore, we designed a new marker virus encoding 3xFLAG-tagged nucleocapsid that allows the detection of incoming viral particles and, in combination with bio-orthogonal labeling for the visualization of viral RNA synthesis via click chemistry, the spatiotemporal tracking of viral replication on the single-cell level. In summary, by applying BAC-based Red recombination, we developed a powerful, reliable, and convenient platform that will facilitate studies answering numerous questions concerning the biology of SARS-CoV-2.
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spelling pubmed-85079652021-10-13 Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination Herrmann, Alexandra Jungnickl, Doris Cordsmeier, Arne Peter, Antonia Sophia Überla, Klaus Ensser, Armin Int J Mol Sci Article The ongoing pandemic coronavirus (CoV) disease 2019 (COVID-19) by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has already caused substantial morbidity, mortality, and economic devastation. Reverse genetic approaches to generate recombinant viruses are a powerful tool to characterize and understand newly emerging viruses. To contribute to the global efforts for countermeasures to control the spread of SARS-CoV-2, we developed a passage-free SARS-CoV-2 clone based on a bacterial artificial chromosome (BAC). Moreover, using a Lambda-based Red recombination, we successfully generated different reporter and marker viruses, which replicated similar to a clinical isolate in a cell culture. Moreover, we designed a full-length reporter virus encoding an additional artificial open reading frame with wild-type-like replication features. The virus-encoded reporters were successfully applied to ease antiviral testing in cell culture models. Furthermore, we designed a new marker virus encoding 3xFLAG-tagged nucleocapsid that allows the detection of incoming viral particles and, in combination with bio-orthogonal labeling for the visualization of viral RNA synthesis via click chemistry, the spatiotemporal tracking of viral replication on the single-cell level. In summary, by applying BAC-based Red recombination, we developed a powerful, reliable, and convenient platform that will facilitate studies answering numerous questions concerning the biology of SARS-CoV-2. MDPI 2021-09-22 /pmc/articles/PMC8507965/ /pubmed/34638527 http://dx.doi.org/10.3390/ijms221910188 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Herrmann, Alexandra
Jungnickl, Doris
Cordsmeier, Arne
Peter, Antonia Sophia
Überla, Klaus
Ensser, Armin
Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination
title Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination
title_full Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination
title_fullStr Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination
title_full_unstemmed Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination
title_short Cloning of a Passage-Free SARS-CoV-2 Genome and Mutagenesis Using Red Recombination
title_sort cloning of a passage-free sars-cov-2 genome and mutagenesis using red recombination
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8507965/
https://www.ncbi.nlm.nih.gov/pubmed/34638527
http://dx.doi.org/10.3390/ijms221910188
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