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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...

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Autores principales: Michalski, Krzysztof, Hertig, Christian, Mańkowski, Dariusz R., Kumlehn, Jochen, Zimny, Janusz, Linkiewicz, Anna M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
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.
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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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