A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing in Cichorium intybus L.
Root chicory (Cichorium intybus L. var. sativum) is used to extract inulin, a fructose polymer used as a natural sweetener and prebiotic. However, bitter tasting sesquiterpene lactones, giving chicory its known flavour, need to be removed during inulin extraction. To avoid this extraction and associ...
| Main Authors: | , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-04-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2023.1111110/full |
| _version_ | 1797847921870241792 |
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| author | Umberto Salvagnin Katharina Unkel Thorben Sprink Paul Bundock Robert Sevenier Milica Bogdanović Slađana Todorović Katarina Cankar Johanna Christina Hakkert Elio Schijlen Ronald Nieuwenhuis Maria Hingsamer Veronika Kulmer Michael Kernitzkyi Dirk Bosch Stefan Martens Mickael Malnoy |
| author_facet | Umberto Salvagnin Katharina Unkel Thorben Sprink Paul Bundock Robert Sevenier Milica Bogdanović Slađana Todorović Katarina Cankar Johanna Christina Hakkert Elio Schijlen Ronald Nieuwenhuis Maria Hingsamer Veronika Kulmer Michael Kernitzkyi Dirk Bosch Stefan Martens Mickael Malnoy |
| author_sort | Umberto Salvagnin |
| collection | DOAJ |
| description | Root chicory (Cichorium intybus L. var. sativum) is used to extract inulin, a fructose polymer used as a natural sweetener and prebiotic. However, bitter tasting sesquiterpene lactones, giving chicory its known flavour, need to be removed during inulin extraction. To avoid this extraction and associated costs, recently chicory variants with a lower sesquiterpene lactone content were created by inactivating the four copies of the germacrene A synthase gene (CiGAS-S1, -S2, -S3, -L) which encode the enzyme initiating bitter sesquiterpene lactone biosynthesis in chicory. In this study, different delivery methods for CRISPR/Cas9 reagents have been compared regarding their efficiency to induce mutations in the CiGAS genes, the frequency of off-target mutations as well as their environmental and economic impacts. CRISPR/Cas9 reagents were delivered by Agrobacterium-mediated stable transformation or transient delivery by plasmid or preassembled ribonucleic complexes (RNPs) using the same sgRNA. All methods used lead to a high number of INDEL mutations within the CiGAS-S1 and CiGAS-S2 genes, which match the used sgRNA perfectly; additionally, the CiGAS-S3 and CiGAS-L genes, which have a single mismatch with the sgRNA, were mutated but with a lower mutation efficiency. While using both RNPs and plasmids delivery resulted in biallelic, heterozygous or homozygous mutations, plasmid delivery resulted in 30% of unwanted integration of plasmid fragments in the genome. Plants transformed via Agrobacteria often showed chimerism and a mixture of CiGAS genotypes. This genetic mosaic becomes more diverse when plants were grown over a prolonged period. While the genotype of the on-targets varied between the transient and stable delivery methods, no off-target activity in six identified potential off-targets with two to four mismatches was found. The environmental impacts (greenhouse gas (GHG) emissions and primary energy demand) of the methods are highly dependent on their individual electricity demand. From an economic view - like for most research and development activities - employment and value-added multiplier effects are high; particularly when compared to industrial or manufacturing processes. Considering all aspects, we conclude that using RNPs is the most suitable method for genome editing in chicory since it led to a high efficiency of editing, no off-target mutations, non-transgenic plants with no risk of unwanted integration of plasmid DNA and without needed segregation of transgenes. |
| first_indexed | 2024-04-09T18:19:05Z |
| format | Article |
| id | doaj.art-a3ea00da587d46a49fd37d39f1b5e97e |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-04-09T18:19:05Z |
| publishDate | 2023-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-a3ea00da587d46a49fd37d39f1b5e97e2023-04-12T11:21:57ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2023-04-011410.3389/fpls.2023.11111101111110A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing in Cichorium intybus L.Umberto Salvagnin0Katharina Unkel1Thorben Sprink2Paul Bundock3Robert Sevenier4Milica Bogdanović5Slađana Todorović6Katarina Cankar7Johanna Christina Hakkert8Elio Schijlen9Ronald Nieuwenhuis10Maria Hingsamer11Veronika Kulmer12Michael Kernitzkyi13Dirk Bosch14Stefan Martens15Mickael Malnoy16Fondazione Edmund Mach (FEM), Centro Ricerca e Innovazione, San Michele all’Adige, TN, ItalyJulius Kuehn-Institute (JKI), Federal Research Centre for Cultivated Plants, Institute for Biosafety in Plant Biotechnology, Quedlinburg, GermanyJulius Kuehn-Institute (JKI), Federal Research Centre for Cultivated Plants, Institute for Biosafety in Plant Biotechnology, Quedlinburg, GermanyKeygene N.V., Agro Business Park 90, Wageningen, NetherlandsKeygene N.V., Agro Business Park 90, Wageningen, NetherlandsDepartment for Plant Physiology, Institute for Biological Research “Siniša Stanković”-National Institute of Republic of Serbia, University of Belgrade, Belgrade, SerbiaDepartment for Plant Physiology, Institute for Biological Research “Siniša Stanković”-National Institute of Republic of Serbia, University of Belgrade, Belgrade, SerbiaWageningen Plant Research, Wageningen University & Research, Wageningen, NetherlandsWageningen Plant Research, Wageningen University & Research, Wageningen, NetherlandsWageningen Plant Research, Wageningen University & Research, Wageningen, NetherlandsWageningen Plant Research, Wageningen University & Research, Wageningen, NetherlandsJoanneum Research Forschungsgesellschaft mbH, Graz, AustriaJoanneum Research Forschungsgesellschaft mbH, Graz, AustriaJoanneum Research Forschungsgesellschaft mbH, Graz, AustriaWageningen Plant Research, Wageningen University & Research, Wageningen, NetherlandsFondazione Edmund Mach (FEM), Centro Ricerca e Innovazione, San Michele all’Adige, TN, ItalyFondazione Edmund Mach (FEM), Centro Ricerca e Innovazione, San Michele all’Adige, TN, ItalyRoot chicory (Cichorium intybus L. var. sativum) is used to extract inulin, a fructose polymer used as a natural sweetener and prebiotic. However, bitter tasting sesquiterpene lactones, giving chicory its known flavour, need to be removed during inulin extraction. To avoid this extraction and associated costs, recently chicory variants with a lower sesquiterpene lactone content were created by inactivating the four copies of the germacrene A synthase gene (CiGAS-S1, -S2, -S3, -L) which encode the enzyme initiating bitter sesquiterpene lactone biosynthesis in chicory. In this study, different delivery methods for CRISPR/Cas9 reagents have been compared regarding their efficiency to induce mutations in the CiGAS genes, the frequency of off-target mutations as well as their environmental and economic impacts. CRISPR/Cas9 reagents were delivered by Agrobacterium-mediated stable transformation or transient delivery by plasmid or preassembled ribonucleic complexes (RNPs) using the same sgRNA. All methods used lead to a high number of INDEL mutations within the CiGAS-S1 and CiGAS-S2 genes, which match the used sgRNA perfectly; additionally, the CiGAS-S3 and CiGAS-L genes, which have a single mismatch with the sgRNA, were mutated but with a lower mutation efficiency. While using both RNPs and plasmids delivery resulted in biallelic, heterozygous or homozygous mutations, plasmid delivery resulted in 30% of unwanted integration of plasmid fragments in the genome. Plants transformed via Agrobacteria often showed chimerism and a mixture of CiGAS genotypes. This genetic mosaic becomes more diverse when plants were grown over a prolonged period. While the genotype of the on-targets varied between the transient and stable delivery methods, no off-target activity in six identified potential off-targets with two to four mismatches was found. The environmental impacts (greenhouse gas (GHG) emissions and primary energy demand) of the methods are highly dependent on their individual electricity demand. From an economic view - like for most research and development activities - employment and value-added multiplier effects are high; particularly when compared to industrial or manufacturing processes. Considering all aspects, we conclude that using RNPs is the most suitable method for genome editing in chicory since it led to a high efficiency of editing, no off-target mutations, non-transgenic plants with no risk of unwanted integration of plasmid DNA and without needed segregation of transgenes.https://www.frontiersin.org/articles/10.3389/fpls.2023.1111110/fullchicorygenome editingCRISPR/Cas9RNPsprotoplastsgermacrene A synthase |
| spellingShingle | Umberto Salvagnin Katharina Unkel Thorben Sprink Paul Bundock Robert Sevenier Milica Bogdanović Slađana Todorović Katarina Cankar Johanna Christina Hakkert Elio Schijlen Ronald Nieuwenhuis Maria Hingsamer Veronika Kulmer Michael Kernitzkyi Dirk Bosch Stefan Martens Mickael Malnoy A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing in Cichorium intybus L. Frontiers in Plant Science chicory genome editing CRISPR/Cas9 RNPs protoplasts germacrene A synthase |
| title | A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing in Cichorium intybus L. |
| title_full | A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing in Cichorium intybus L. |
| title_fullStr | A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing in Cichorium intybus L. |
| title_full_unstemmed | A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing in Cichorium intybus L. |
| title_short | A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing in Cichorium intybus L. |
| title_sort | comparison of three different delivery methods for achieving crispr cas9 mediated genome editing in cichorium intybus l |
| topic | chicory genome editing CRISPR/Cas9 RNPs protoplasts germacrene A synthase |
| url | https://www.frontiersin.org/articles/10.3389/fpls.2023.1111110/full |
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