Functional Validation of <i>cas9</i>/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale <i>ABA8′OH1 loci</i>
Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although <i>cas9</i>/guide (g)RNA constructs are straightforward to assemble and can be customized to target virt...
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| Format: | Article |
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MDPI AG
2021-06-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/13/7038 |
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| author | Krzysztof Michalski Christian Hertig Dariusz R. Mańkowski Jochen Kumlehn Janusz Zimny Anna M. Linkiewicz |
| author_facet | Krzysztof Michalski Christian Hertig Dariusz R. Mańkowski Jochen Kumlehn Janusz Zimny Anna M. Linkiewicz |
| author_sort | Krzysztof Michalski |
| collection | DOAJ |
| description | Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although <i>cas9</i>/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated <i>cas9</i>/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale <i>ABA 8′-HYDROXYLASE 1</i> gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes. |
| first_indexed | 2024-03-10T09:56:42Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T09:56:42Z |
| publishDate | 2021-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-5fa8b0a4796345afb616ed024bb6fd582023-11-22T02:18:21ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-06-012213703810.3390/ijms22137038Functional Validation of <i>cas9</i>/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale <i>ABA8′OH1 loci</i>Krzysztof Michalski0Christian Hertig1Dariusz R. Mańkowski2Jochen Kumlehn3Janusz Zimny4Anna M. Linkiewicz5GMO Controlling Laboratory, Plant Biotechnology and Cytogenetics Department, Plant Breeding and Acclimatization Institute—National Research Institute, Radzików, 05-870 Błonie, PolandPlant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), 06466 Seeland, GermanyLaboratory of Seed Production and Plant Breeding Economics, Department of Seed Science and Technology, Plant Breeding and Acclimatization Institute—National Research Institute, Radzików, 05-870 Błonie, PolandPlant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), 06466 Seeland, GermanyGMO Controlling Laboratory, Plant Biotechnology and Cytogenetics Department, Plant Breeding and Acclimatization Institute—National Research Institute, Radzików, 05-870 Błonie, PolandGMO Controlling Laboratory, Plant Biotechnology and Cytogenetics Department, Plant Breeding and Acclimatization Institute—National Research Institute, Radzików, 05-870 Błonie, PolandCas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although <i>cas9</i>/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated <i>cas9</i>/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale <i>ABA 8′-HYDROXYLASE 1</i> gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.https://www.mdpi.com/1422-0067/22/13/7038genome editingCRISPRprotoplaststargeted mutagenesisTREX2construct validation |
| spellingShingle | Krzysztof Michalski Christian Hertig Dariusz R. Mańkowski Jochen Kumlehn Janusz Zimny Anna M. Linkiewicz Functional Validation of <i>cas9</i>/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale <i>ABA8′OH1 loci</i> International Journal of Molecular Sciences genome editing CRISPR protoplasts targeted mutagenesis TREX2 construct validation |
| title | Functional Validation of <i>cas9</i>/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale <i>ABA8′OH1 loci</i> |
| title_full | Functional Validation of <i>cas9</i>/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale <i>ABA8′OH1 loci</i> |
| title_fullStr | Functional Validation of <i>cas9</i>/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale <i>ABA8′OH1 loci</i> |
| title_full_unstemmed | Functional Validation of <i>cas9</i>/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale <i>ABA8′OH1 loci</i> |
| title_short | Functional Validation of <i>cas9</i>/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale <i>ABA8′OH1 loci</i> |
| title_sort | functional validation of i cas9 i guiderna constructs for site directed mutagenesis of triticale i aba8 oh1 loci i |
| topic | genome editing CRISPR protoplasts targeted mutagenesis TREX2 construct validation |
| url | https://www.mdpi.com/1422-0067/22/13/7038 |
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