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 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. Ausführliche Beschreibung

Gespeichert in:

Autor*in:	Charlotte De Bruyn [verfasserIn] Tom Ruttink [verfasserIn] Tom Eeckhaut [verfasserIn] Thomas Jacobs [verfasserIn] Ellen De Keyser [verfasserIn] Alain Goossens [verfasserIn] Katrijn Van Laere [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2020

Schlagwörter:	gene editing protoplast transfection Cichorium intybus sesquiterpene lactones phytoene desaturase HiPlex amplicon sequencing

Übergeordnetes Werk:	In: Frontiers in Genome Editing - Frontiers Media S.A., 2020, 2(2020)
Übergeordnetes Werk:	volume:2 ; year:2020

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.3389/fgeed.2020.604876

Katalog-ID:	DOAJ059258144

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author_variant	c d b cdb c d b cdb c d b cdb t r tr t e te t j tj t j tj e d k edk a g ag a g ag k v l kvl
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allfields	10.3389/fgeed.2020.604876 doi (DE-627)DOAJ059258144 (DE-599)DOAJ500d7bbe82eb4b15bda361735addd7b0 DE-627 ger DE-627 rakwb eng TP248.13-248.65 QH426-470 Charlotte De Bruyn verfasserin aut Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum) 2020 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier 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. gene editing protoplast transfection Cichorium intybus sesquiterpene lactones phytoene desaturase HiPlex amplicon sequencing Biotechnology Genetics Charlotte De Bruyn verfasserin aut Charlotte De Bruyn verfasserin aut Tom Ruttink verfasserin aut Tom Eeckhaut verfasserin aut Thomas Jacobs verfasserin aut Thomas Jacobs verfasserin aut Ellen De Keyser verfasserin aut Alain Goossens verfasserin aut Alain Goossens verfasserin aut Katrijn Van Laere verfasserin aut In Frontiers in Genome Editing Frontiers Media S.A., 2020 2(2020) (DE-627)1695605101 (DE-600)3017800-9 26733439 nnns volume:2 year:2020 https://doi.org/10.3389/fgeed.2020.604876 kostenfrei https://doaj.org/article/500d7bbe82eb4b15bda361735addd7b0 kostenfrei https://www.frontiersin.org/articles/10.3389/fgeed.2020.604876/full kostenfrei https://doaj.org/toc/2673-3439 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 2 2020
spelling	10.3389/fgeed.2020.604876 doi (DE-627)DOAJ059258144 (DE-599)DOAJ500d7bbe82eb4b15bda361735addd7b0 DE-627 ger DE-627 rakwb eng TP248.13-248.65 QH426-470 Charlotte De Bruyn verfasserin aut Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum) 2020 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier 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. gene editing protoplast transfection Cichorium intybus sesquiterpene lactones phytoene desaturase HiPlex amplicon sequencing Biotechnology Genetics Charlotte De Bruyn verfasserin aut Charlotte De Bruyn verfasserin aut Tom Ruttink verfasserin aut Tom Eeckhaut verfasserin aut Thomas Jacobs verfasserin aut Thomas Jacobs verfasserin aut Ellen De Keyser verfasserin aut Alain Goossens verfasserin aut Alain Goossens verfasserin aut Katrijn Van Laere verfasserin aut In Frontiers in Genome Editing Frontiers Media S.A., 2020 2(2020) (DE-627)1695605101 (DE-600)3017800-9 26733439 nnns volume:2 year:2020 https://doi.org/10.3389/fgeed.2020.604876 kostenfrei https://doaj.org/article/500d7bbe82eb4b15bda361735addd7b0 kostenfrei https://www.frontiersin.org/articles/10.3389/fgeed.2020.604876/full kostenfrei https://doaj.org/toc/2673-3439 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 2 2020
allfields_unstemmed	10.3389/fgeed.2020.604876 doi (DE-627)DOAJ059258144 (DE-599)DOAJ500d7bbe82eb4b15bda361735addd7b0 DE-627 ger DE-627 rakwb eng TP248.13-248.65 QH426-470 Charlotte De Bruyn verfasserin aut Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum) 2020 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier 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. gene editing protoplast transfection Cichorium intybus sesquiterpene lactones phytoene desaturase HiPlex amplicon sequencing Biotechnology Genetics Charlotte De Bruyn verfasserin aut Charlotte De Bruyn verfasserin aut Tom Ruttink verfasserin aut Tom Eeckhaut verfasserin aut Thomas Jacobs verfasserin aut Thomas Jacobs verfasserin aut Ellen De Keyser verfasserin aut Alain Goossens verfasserin aut Alain Goossens verfasserin aut Katrijn Van Laere verfasserin aut In Frontiers in Genome Editing Frontiers Media S.A., 2020 2(2020) (DE-627)1695605101 (DE-600)3017800-9 26733439 nnns volume:2 year:2020 https://doi.org/10.3389/fgeed.2020.604876 kostenfrei https://doaj.org/article/500d7bbe82eb4b15bda361735addd7b0 kostenfrei https://www.frontiersin.org/articles/10.3389/fgeed.2020.604876/full kostenfrei https://doaj.org/toc/2673-3439 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 2 2020
allfieldsGer	10.3389/fgeed.2020.604876 doi (DE-627)DOAJ059258144 (DE-599)DOAJ500d7bbe82eb4b15bda361735addd7b0 DE-627 ger DE-627 rakwb eng TP248.13-248.65 QH426-470 Charlotte De Bruyn verfasserin aut Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum) 2020 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier 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. gene editing protoplast transfection Cichorium intybus sesquiterpene lactones phytoene desaturase HiPlex amplicon sequencing Biotechnology Genetics Charlotte De Bruyn verfasserin aut Charlotte De Bruyn verfasserin aut Tom Ruttink verfasserin aut Tom Eeckhaut verfasserin aut Thomas Jacobs verfasserin aut Thomas Jacobs verfasserin aut Ellen De Keyser verfasserin aut Alain Goossens verfasserin aut Alain Goossens verfasserin aut Katrijn Van Laere verfasserin aut In Frontiers in Genome Editing Frontiers Media S.A., 2020 2(2020) (DE-627)1695605101 (DE-600)3017800-9 26733439 nnns volume:2 year:2020 https://doi.org/10.3389/fgeed.2020.604876 kostenfrei https://doaj.org/article/500d7bbe82eb4b15bda361735addd7b0 kostenfrei https://www.frontiersin.org/articles/10.3389/fgeed.2020.604876/full kostenfrei https://doaj.org/toc/2673-3439 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 2 2020
allfieldsSound	10.3389/fgeed.2020.604876 doi (DE-627)DOAJ059258144 (DE-599)DOAJ500d7bbe82eb4b15bda361735addd7b0 DE-627 ger DE-627 rakwb eng TP248.13-248.65 QH426-470 Charlotte De Bruyn verfasserin aut Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum) 2020 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier 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. gene editing protoplast transfection Cichorium intybus sesquiterpene lactones phytoene desaturase HiPlex amplicon sequencing Biotechnology Genetics Charlotte De Bruyn verfasserin aut Charlotte De Bruyn verfasserin aut Tom Ruttink verfasserin aut Tom Eeckhaut verfasserin aut Thomas Jacobs verfasserin aut Thomas Jacobs verfasserin aut Ellen De Keyser verfasserin aut Alain Goossens verfasserin aut Alain Goossens verfasserin aut Katrijn Van Laere verfasserin aut In Frontiers in Genome Editing Frontiers Media S.A., 2020 2(2020) (DE-627)1695605101 (DE-600)3017800-9 26733439 nnns volume:2 year:2020 https://doi.org/10.3389/fgeed.2020.604876 kostenfrei https://doaj.org/article/500d7bbe82eb4b15bda361735addd7b0 kostenfrei https://www.frontiersin.org/articles/10.3389/fgeed.2020.604876/full kostenfrei https://doaj.org/toc/2673-3439 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 2 2020
language	English
source	In Frontiers in Genome Editing 2(2020) volume:2 year:2020
sourceStr	In Frontiers in Genome Editing 2(2020) volume:2 year:2020
format_phy_str_mv	Article
institution	findex.gbv.de
topic_facet	gene editing protoplast transfection Cichorium intybus sesquiterpene lactones phytoene desaturase HiPlex amplicon sequencing Biotechnology Genetics
isfreeaccess_bool	true
container_title	Frontiers in Genome Editing
authorswithroles_txt_mv	Charlotte De Bruyn @@aut@@ Tom Ruttink @@aut@@ Tom Eeckhaut @@aut@@ Thomas Jacobs @@aut@@ Ellen De Keyser @@aut@@ Alain Goossens @@aut@@ Katrijn Van Laere @@aut@@
publishDateDaySort_date	2020-01-01T00:00:00Z
hierarchy_top_id	1695605101
id	DOAJ059258144
language_de	englisch
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callnumber-first	T - Technology
author	Charlotte De Bruyn
spellingShingle	Charlotte De Bruyn misc TP248.13-248.65 misc QH426-470 misc gene editing misc protoplast transfection misc Cichorium intybus misc sesquiterpene lactones misc phytoene desaturase misc HiPlex amplicon sequencing misc Biotechnology misc Genetics Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum)
authorStr	Charlotte De Bruyn
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topic_title	TP248.13-248.65 QH426-470 Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum) gene editing protoplast transfection Cichorium intybus sesquiterpene lactones phytoene desaturase HiPlex amplicon sequencing
topic	misc TP248.13-248.65 misc QH426-470 misc gene editing misc protoplast transfection misc Cichorium intybus misc sesquiterpene lactones misc phytoene desaturase misc HiPlex amplicon sequencing misc Biotechnology misc Genetics
topic_unstemmed	misc TP248.13-248.65 misc QH426-470 misc gene editing misc protoplast transfection misc Cichorium intybus misc sesquiterpene lactones misc phytoene desaturase misc HiPlex amplicon sequencing misc Biotechnology misc Genetics
topic_browse	misc TP248.13-248.65 misc QH426-470 misc gene editing misc protoplast transfection misc Cichorium intybus misc sesquiterpene lactones misc phytoene desaturase misc HiPlex amplicon sequencing misc Biotechnology misc Genetics
format_facet	Elektronische Aufsätze Aufsätze Elektronische Ressource
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hierarchy_parent_title	Frontiers in Genome Editing
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title	Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum)
ctrlnum	(DE-627)DOAJ059258144 (DE-599)DOAJ500d7bbe82eb4b15bda361735addd7b0
title_full	Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum)
author_sort	Charlotte De Bruyn
journal	Frontiers in Genome Editing
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author_browse	Charlotte De Bruyn Tom Ruttink Tom Eeckhaut Thomas Jacobs Ellen De Keyser Alain Goossens Katrijn Van Laere
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class	TP248.13-248.65 QH426-470
format_se	Elektronische Aufsätze
author-letter	Charlotte De Bruyn
doi_str_mv	10.3389/fgeed.2020.604876
author2-role	verfasserin
title_sort	establishment of crispr/cas9 genome editing in witloof (cichorium intybus var. foliosum)
callnumber	TP248.13-248.65
title_auth	Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum)
abstract	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.
abstractGer	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.
abstract_unstemmed	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.
collection_details	GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ SSG-OLC-PHA GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700
title_short	Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum)
url	https://doi.org/10.3389/fgeed.2020.604876 https://doaj.org/article/500d7bbe82eb4b15bda361735addd7b0 https://www.frontiersin.org/articles/10.3389/fgeed.2020.604876/full https://doaj.org/toc/2673-3439
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author2	Charlotte De Bruyn Tom Ruttink Tom Eeckhaut Thomas Jacobs Ellen De Keyser Alain Goossens Katrijn Van Laere
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up_date	2024-07-03T22:35:58.374Z
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Establishment of CRISPR/Cas9 Genome Editing in Witloof (Cichorium intybus var. foliosum)

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