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Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated with protein CAS9) is a genome-editing tool that has been extensively used in the last five years because of its novelty, affordability, and feasibility. This technology has been developed in many plant species...

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Autores principales: Bernard, Guillaume, Gagneul, David, Santos, Harmony Alves Dos, Etienne, Audrey, Hilbert, Jean-Louis, Rambaud, Caroline
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6429391/
https://www.ncbi.nlm.nih.gov/pubmed/30845784
http://dx.doi.org/10.3390/ijms20051155
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author Bernard, Guillaume
Gagneul, David
Santos, Harmony Alves Dos
Etienne, Audrey
Hilbert, Jean-Louis
Rambaud, Caroline
author_facet Bernard, Guillaume
Gagneul, David
Santos, Harmony Alves Dos
Etienne, Audrey
Hilbert, Jean-Louis
Rambaud, Caroline
author_sort Bernard, Guillaume
collection PubMed
description CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated with protein CAS9) is a genome-editing tool that has been extensively used in the last five years because of its novelty, affordability, and feasibility. This technology has been developed in many plant species for gene function analysis and crop improvement but has never been used in chicory (Cichorium intybus L.). In this study, we successfully applied CRISPR/Cas9-mediated targeted mutagenesis to chicory using Agrobacterium rhizogenes-mediated transformation and protoplast transfection methods. A U6 promoter (CiU6-1p) among eight predicted U6 promoters in chicory was selected to drive sgRNA expression. A binary vector designed to induce targeted mutations in the fifth exon of the chicory phytoene desaturase gene (CiPDS) was then constructed and used to transform chicory. The mutation frequency was 4.5% with the protoplast transient expression system and 31.25% with A. rhizogenes-mediated stable transformation. Biallelic mutations were detected in all the mutant plants. The use of A. rhizogenes-mediated transformation seems preferable as the regeneration of plants is faster and the mutation frequency was shown to be higher. With both transformation methods, foreign DNA was integrated in the plant genome. Hence, selection of vector (transgene)-free segregants is required. Our results showed that genome editing with CRISPR/Cas9 system can be efficiently used with chicory, which should facilitate and accelerate genetic improvement and functional biology.
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spelling pubmed-64293912019-04-10 Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory Bernard, Guillaume Gagneul, David Santos, Harmony Alves Dos Etienne, Audrey Hilbert, Jean-Louis Rambaud, Caroline Int J Mol Sci Article CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated with protein CAS9) is a genome-editing tool that has been extensively used in the last five years because of its novelty, affordability, and feasibility. This technology has been developed in many plant species for gene function analysis and crop improvement but has never been used in chicory (Cichorium intybus L.). In this study, we successfully applied CRISPR/Cas9-mediated targeted mutagenesis to chicory using Agrobacterium rhizogenes-mediated transformation and protoplast transfection methods. A U6 promoter (CiU6-1p) among eight predicted U6 promoters in chicory was selected to drive sgRNA expression. A binary vector designed to induce targeted mutations in the fifth exon of the chicory phytoene desaturase gene (CiPDS) was then constructed and used to transform chicory. The mutation frequency was 4.5% with the protoplast transient expression system and 31.25% with A. rhizogenes-mediated stable transformation. Biallelic mutations were detected in all the mutant plants. The use of A. rhizogenes-mediated transformation seems preferable as the regeneration of plants is faster and the mutation frequency was shown to be higher. With both transformation methods, foreign DNA was integrated in the plant genome. Hence, selection of vector (transgene)-free segregants is required. Our results showed that genome editing with CRISPR/Cas9 system can be efficiently used with chicory, which should facilitate and accelerate genetic improvement and functional biology. MDPI 2019-03-06 /pmc/articles/PMC6429391/ /pubmed/30845784 http://dx.doi.org/10.3390/ijms20051155 Text en © 2019 by the authors. 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 (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Bernard, Guillaume
Gagneul, David
Santos, Harmony Alves Dos
Etienne, Audrey
Hilbert, Jean-Louis
Rambaud, Caroline
Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory
title Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory
title_full Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory
title_fullStr Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory
title_full_unstemmed Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory
title_short Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory
title_sort efficient genome editing using crispr/cas9 technology in chicory
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6429391/
https://www.ncbi.nlm.nih.gov/pubmed/30845784
http://dx.doi.org/10.3390/ijms20051155
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