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...
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doaj-500d7bbe82eb4b15bda361735addd7b02021-04-02T18:42:26ZengFrontiers Media S.A.Frontiers in Genome Editing2673-34392020-12-01210.3389/fgeed.2020.604876604876Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum)Charlotte De Bruyn0Charlotte De Bruyn1Charlotte De Bruyn2Tom Ruttink3Tom Eeckhaut4Thomas Jacobs5Thomas Jacobs6Ellen De Keyser7Alain Goossens8Alain Goossens9Katrijn Van Laere10Plant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Melle, BelgiumDepartment of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, BelgiumCenter for Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Ghent, BelgiumPlant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Melle, BelgiumPlant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Melle, BelgiumDepartment of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, BelgiumCenter for Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Ghent, BelgiumPlant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Melle, BelgiumDepartment of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, BelgiumCenter for Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Ghent, BelgiumPlant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Melle, BelgiumCichorium 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.https://www.frontiersin.org/articles/10.3389/fgeed.2020.604876/fullgene editingprotoplast transfectionCichorium intybussesquiterpene lactonesphytoene desaturaseHiPlex amplicon sequencing |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Charlotte De Bruyn Charlotte De Bruyn Charlotte De Bruyn Tom Ruttink Tom Eeckhaut Thomas Jacobs Thomas Jacobs Ellen De Keyser Alain Goossens Alain Goossens Katrijn Van Laere |
spellingShingle |
Charlotte De Bruyn Charlotte De Bruyn Charlotte De Bruyn Tom Ruttink Tom Eeckhaut Thomas Jacobs Thomas Jacobs Ellen De Keyser Alain Goossens Alain Goossens Katrijn Van Laere Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum) Frontiers in Genome Editing gene editing protoplast transfection Cichorium intybus sesquiterpene lactones phytoene desaturase HiPlex amplicon sequencing |
author_facet |
Charlotte De Bruyn Charlotte De Bruyn Charlotte De Bruyn Tom Ruttink Tom Eeckhaut Thomas Jacobs Thomas Jacobs Ellen De Keyser Alain Goossens Alain Goossens Katrijn Van Laere |
author_sort |
Charlotte De Bruyn |
title |
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_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_sort |
establishment of crispr/cas9 genome editing in witloof (cichorium intybus var. foliosum) |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Genome Editing |
issn |
2673-3439 |
publishDate |
2020-12-01 |
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. |
topic |
gene editing protoplast transfection Cichorium intybus sesquiterpene lactones phytoene desaturase HiPlex amplicon sequencing |
url |
https://www.frontiersin.org/articles/10.3389/fgeed.2020.604876/full |
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