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Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum)
Cichorium intybus var. foliosum (witloof) is an economically important crop with a high nutritional value thanks to many specialized metabolites, such as polyphenols and terpenoids. However, witloof plants are rich in sesquiterpene lactones (SL) which are important for plant defense but also impart...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8525355/ https://www.ncbi.nlm.nih.gov/pubmed/34713228 http://dx.doi.org/10.3389/fgeed.2020.604876 |
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author | De Bruyn, Charlotte Ruttink, Tom Eeckhaut, Tom Jacobs, Thomas De Keyser, Ellen Goossens, Alain Van Laere, Katrijn |
author_facet | De Bruyn, Charlotte Ruttink, Tom Eeckhaut, Tom Jacobs, Thomas De Keyser, Ellen Goossens, Alain Van Laere, Katrijn |
author_sort | De Bruyn, Charlotte |
collection | PubMed |
description | Cichorium intybus var. foliosum (witloof) is an economically important crop with a high nutritional value thanks to many specialized metabolites, such as polyphenols and terpenoids. However, witloof plants are rich in sesquiterpene lactones (SL) which are important for plant defense but also impart a bitter taste, thus limiting industrial applications. Inactivating specific genes in the SL biosynthesis pathway could lead to changes in the SL metabolite content and result in altered bitterness. In this study, a CRISPR/Cas9 genome editing workflow was implemented for witloof, starting with polyethylene glycol (PEG) mediated protoplast transfection for CRISPR/Cas9 vector delivery, followed by whole plant regeneration and mutation analysis. Protoplast transfection efficiencies ranged from 20 to 26 %. A CRISPR/Cas9 vector targeting the first exon of the phytoene desaturase (CiPDS) gene was transfected into witloof protoplasts and resulted in the knockout of CiPDS, giving rise to an albino phenotype in 23% of the regenerated plants. Further implementing our protocol, the SL biosynthesis pathway genes germacrene A synthase (GAS), germacrene A oxidase (GAO), and costunolide synthase (COS) were targeted in independent experiments. Highly multiplex (HiPlex) amplicon sequencing of the genomic target loci revealed plant mutation frequencies of 27.3, 42.7, and 98.3% in regenerated plants transfected with a CRISPR/Cas9 vector targeting CiGAS, CiGAO, and CiCOS, respectively. We observed different mutation spectra across the loci, ranging from consistently the same +1 nucleotide insertion in CiCOS across independent mutated lines, to a complex set of 20 mutation types in CiGAO across independent mutated lines. These results demonstrate a straightforward workflow for genome editing based on transfection and regeneration of witloof protoplasts and subsequent HiPlex amplicon sequencing. Our CRISPR/Cas9 workflow can enable gene functional research and faster incorporation of novel traits in elite witloof lines in the future, thus facilitating the development of novel industrial applications for witloof. |
format | Online Article Text |
id | pubmed-8525355 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-85253552021-10-27 Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum) De Bruyn, Charlotte Ruttink, Tom Eeckhaut, Tom Jacobs, Thomas De Keyser, Ellen Goossens, Alain Van Laere, Katrijn Front Genome Ed Genome Editing Cichorium intybus var. foliosum (witloof) is an economically important crop with a high nutritional value thanks to many specialized metabolites, such as polyphenols and terpenoids. However, witloof plants are rich in sesquiterpene lactones (SL) which are important for plant defense but also impart a bitter taste, thus limiting industrial applications. Inactivating specific genes in the SL biosynthesis pathway could lead to changes in the SL metabolite content and result in altered bitterness. In this study, a CRISPR/Cas9 genome editing workflow was implemented for witloof, starting with polyethylene glycol (PEG) mediated protoplast transfection for CRISPR/Cas9 vector delivery, followed by whole plant regeneration and mutation analysis. Protoplast transfection efficiencies ranged from 20 to 26 %. A CRISPR/Cas9 vector targeting the first exon of the phytoene desaturase (CiPDS) gene was transfected into witloof protoplasts and resulted in the knockout of CiPDS, giving rise to an albino phenotype in 23% of the regenerated plants. Further implementing our protocol, the SL biosynthesis pathway genes germacrene A synthase (GAS), germacrene A oxidase (GAO), and costunolide synthase (COS) were targeted in independent experiments. Highly multiplex (HiPlex) amplicon sequencing of the genomic target loci revealed plant mutation frequencies of 27.3, 42.7, and 98.3% in regenerated plants transfected with a CRISPR/Cas9 vector targeting CiGAS, CiGAO, and CiCOS, respectively. We observed different mutation spectra across the loci, ranging from consistently the same +1 nucleotide insertion in CiCOS across independent mutated lines, to a complex set of 20 mutation types in CiGAO across independent mutated lines. These results demonstrate a straightforward workflow for genome editing based on transfection and regeneration of witloof protoplasts and subsequent HiPlex amplicon sequencing. Our CRISPR/Cas9 workflow can enable gene functional research and faster incorporation of novel traits in elite witloof lines in the future, thus facilitating the development of novel industrial applications for witloof. Frontiers Media S.A. 2020-12-21 /pmc/articles/PMC8525355/ /pubmed/34713228 http://dx.doi.org/10.3389/fgeed.2020.604876 Text en Copyright © 2020 De Bruyn, Ruttink, Eeckhaut, Jacobs, De Keyser, Goossens and Van Laere. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Genome Editing De Bruyn, Charlotte Ruttink, Tom Eeckhaut, Tom Jacobs, Thomas De Keyser, Ellen Goossens, Alain Van Laere, Katrijn Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum) |
title | Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum) |
title_full | Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum) |
title_fullStr | Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum) |
title_full_unstemmed | Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum) |
title_short | Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum) |
title_sort | establishment of crispr/cas9 genome editing in witloof (cichorium intybus var. foliosum) |
topic | Genome Editing |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8525355/ https://www.ncbi.nlm.nih.gov/pubmed/34713228 http://dx.doi.org/10.3389/fgeed.2020.604876 |
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