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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...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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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. |
format | Online Article Text |
id | pubmed-9321979 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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