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Efficient Method for Generating Point Mutations in the Vaccinia Virus Genome Using CRISPR/Cas9

The vaccinia virus (VACV) was previously used as a vaccine for smallpox eradication. Nowadays, recombinant VACVs are developed as vaccine platforms for infectious disease prevention and cancer treatment. The conventional method for genome editing of the VACV is based on homologous recombination, whi...

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Autores principales: Boutin, Laetitia, Mosca, Estelle, Iseni, Frédéric
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9321979/
https://www.ncbi.nlm.nih.gov/pubmed/35891539
http://dx.doi.org/10.3390/v14071559
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author Boutin, Laetitia
Mosca, Estelle
Iseni, Frédéric
author_facet Boutin, Laetitia
Mosca, Estelle
Iseni, Frédéric
author_sort Boutin, Laetitia
collection PubMed
description The vaccinia virus (VACV) was previously used as a vaccine for smallpox eradication. Nowadays, recombinant VACVs are developed as vaccine platforms for infectious disease prevention and cancer treatment. The conventional method for genome editing of the VACV is based on homologous recombination, which is poorly efficient. Recently, the use of CRISPR/Cas9 technology was shown to greatly improve the speed and efficiency of the production of recombinant VACV expressing a heterologous gene. However, the ability to rapidly recover viruses bearing single nucleotide substitutions is still challenging. Notwithstanding, ongoing studies on the VACV and its interaction with the host cell could benefit from viral gene targeted mutagenesis. Here, we present a modified version of the CRISPR/Cas9 system for the rapid selection of mutant VACV carrying point mutations. For this purpose, we introduced a silent mutation into the donor gene (which will replace the wildtype gene) that serves a double function: it is located in the PAM (NGG) sequence, which is essential for Cas9 cleavage, and it alters a restriction site. This silent mutation, once introduced into the VACV genome, allows for rapid selection and screening of mutant viruses carrying a mutation of interest in the targeted gene. As a proof of concept, we produced several recombinant VACVs, with mutations in the E9L gene, upon which, phenotypic analysis was performed.
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spelling pubmed-93219792022-07-27 Efficient Method for Generating Point Mutations in the Vaccinia Virus Genome Using CRISPR/Cas9 Boutin, Laetitia Mosca, Estelle Iseni, Frédéric Viruses Article The vaccinia virus (VACV) was previously used as a vaccine for smallpox eradication. Nowadays, recombinant VACVs are developed as vaccine platforms for infectious disease prevention and cancer treatment. The conventional method for genome editing of the VACV is based on homologous recombination, which is poorly efficient. Recently, the use of CRISPR/Cas9 technology was shown to greatly improve the speed and efficiency of the production of recombinant VACV expressing a heterologous gene. However, the ability to rapidly recover viruses bearing single nucleotide substitutions is still challenging. Notwithstanding, ongoing studies on the VACV and its interaction with the host cell could benefit from viral gene targeted mutagenesis. Here, we present a modified version of the CRISPR/Cas9 system for the rapid selection of mutant VACV carrying point mutations. For this purpose, we introduced a silent mutation into the donor gene (which will replace the wildtype gene) that serves a double function: it is located in the PAM (NGG) sequence, which is essential for Cas9 cleavage, and it alters a restriction site. This silent mutation, once introduced into the VACV genome, allows for rapid selection and screening of mutant viruses carrying a mutation of interest in the targeted gene. As a proof of concept, we produced several recombinant VACVs, with mutations in the E9L gene, upon which, phenotypic analysis was performed. MDPI 2022-07-18 /pmc/articles/PMC9321979/ /pubmed/35891539 http://dx.doi.org/10.3390/v14071559 Text en © 2022 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
Boutin, Laetitia
Mosca, Estelle
Iseni, Frédéric
Efficient Method for Generating Point Mutations in the Vaccinia Virus Genome Using CRISPR/Cas9
title Efficient Method for Generating Point Mutations in the Vaccinia Virus Genome Using CRISPR/Cas9
title_full Efficient Method for Generating Point Mutations in the Vaccinia Virus Genome Using CRISPR/Cas9
title_fullStr Efficient Method for Generating Point Mutations in the Vaccinia Virus Genome Using CRISPR/Cas9
title_full_unstemmed Efficient Method for Generating Point Mutations in the Vaccinia Virus Genome Using CRISPR/Cas9
title_short Efficient Method for Generating Point Mutations in the Vaccinia Virus Genome Using CRISPR/Cas9
title_sort efficient method for generating point mutations in the vaccinia virus genome using crispr/cas9
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9321979/
https://www.ncbi.nlm.nih.gov/pubmed/35891539
http://dx.doi.org/10.3390/v14071559
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