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Functional Validation of cas9/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8′OH1 loci
Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in t...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8269138/ https://www.ncbi.nlm.nih.gov/pubmed/34210100 http://dx.doi.org/10.3390/ijms22137038 |
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author | Michalski, Krzysztof Hertig, Christian Mańkowski, Dariusz R. Kumlehn, Jochen Zimny, Janusz Linkiewicz, Anna M. |
author_facet | Michalski, Krzysztof Hertig, Christian Mańkowski, Dariusz R. Kumlehn, Jochen Zimny, Janusz Linkiewicz, Anna M. |
author_sort | Michalski, Krzysztof |
collection | PubMed |
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 cas9/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 cas9/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 ABA 8′-HYDROXYLASE 1 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. |
format | Online Article Text |
id | pubmed-8269138 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-82691382021-07-10 Functional Validation of cas9/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8′OH1 loci Michalski, Krzysztof Hertig, Christian Mańkowski, Dariusz R. Kumlehn, Jochen Zimny, Janusz Linkiewicz, Anna M. Int J Mol Sci Article Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/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 cas9/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 ABA 8′-HYDROXYLASE 1 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. MDPI 2021-06-29 /pmc/articles/PMC8269138/ /pubmed/34210100 http://dx.doi.org/10.3390/ijms22137038 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Michalski, Krzysztof Hertig, Christian Mańkowski, Dariusz R. Kumlehn, Jochen Zimny, Janusz Linkiewicz, Anna M. Functional Validation of cas9/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8′OH1 loci |
title | Functional Validation of cas9/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8′OH1 loci |
title_full | Functional Validation of cas9/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8′OH1 loci |
title_fullStr | Functional Validation of cas9/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8′OH1 loci |
title_full_unstemmed | Functional Validation of cas9/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8′OH1 loci |
title_short | Functional Validation of cas9/GuideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8′OH1 loci |
title_sort | functional validation of cas9/guiderna constructs for site-directed mutagenesis of triticale aba8′oh1 loci |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8269138/ https://www.ncbi.nlm.nih.gov/pubmed/34210100 http://dx.doi.org/10.3390/ijms22137038 |
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