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Evaluation of CRISPR/Cas9 Constructs in Wheat Cell Suspension Cultures
Despite intensive optimization efforts, developing an efficient sequence-specific CRISPR/Cas-mediated genome editing method remains a challenge, especially in polyploid cereal species such as wheat. Validating the efficacy of nuclease constructs prior to using them in planta is, thus, a major step o...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9916915/ https://www.ncbi.nlm.nih.gov/pubmed/36768486 http://dx.doi.org/10.3390/ijms24032162 |
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author | Michalski, Krzysztof Ziąbska, Paulina Sowa, Sławomir Zimny, Janusz Linkiewicz, Anna M. |
author_facet | Michalski, Krzysztof Ziąbska, Paulina Sowa, Sławomir Zimny, Janusz Linkiewicz, Anna M. |
author_sort | Michalski, Krzysztof |
collection | PubMed |
description | Despite intensive optimization efforts, developing an efficient sequence-specific CRISPR/Cas-mediated genome editing method remains a challenge, especially in polyploid cereal species such as wheat. Validating the efficacy of nuclease constructs prior to using them in planta is, thus, a major step of every editing experiment. Several construct evaluation strategies were proposed, with PEG-mediated plasmid transfection of seedling-derived protoplasts becoming the most popular. However, the usefulness of this approach is affected by associated construct copy number bias and chromatin relaxation, both influencing the outcome. Therefore, to achieve a reliable evaluation of CRISPR/Cas9 constructs, we proposed a system based on an Agrobacterium-mediated transformation of established wheat cell suspension cultures. This system was used for the evaluation of a CRISPR/Cas9 construct designed to target the ABA 8′-hydroxylase 1 gene. The efficiency of editing was verified by cost-effective means of Sanger sequencing and bioinformatic analysis. We discuss advantages and potential future developments of this method in contrast to other in vitro approaches. |
format | Online Article Text |
id | pubmed-9916915 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-99169152023-02-11 Evaluation of CRISPR/Cas9 Constructs in Wheat Cell Suspension Cultures Michalski, Krzysztof Ziąbska, Paulina Sowa, Sławomir Zimny, Janusz Linkiewicz, Anna M. Int J Mol Sci Communication Despite intensive optimization efforts, developing an efficient sequence-specific CRISPR/Cas-mediated genome editing method remains a challenge, especially in polyploid cereal species such as wheat. Validating the efficacy of nuclease constructs prior to using them in planta is, thus, a major step of every editing experiment. Several construct evaluation strategies were proposed, with PEG-mediated plasmid transfection of seedling-derived protoplasts becoming the most popular. However, the usefulness of this approach is affected by associated construct copy number bias and chromatin relaxation, both influencing the outcome. Therefore, to achieve a reliable evaluation of CRISPR/Cas9 constructs, we proposed a system based on an Agrobacterium-mediated transformation of established wheat cell suspension cultures. This system was used for the evaluation of a CRISPR/Cas9 construct designed to target the ABA 8′-hydroxylase 1 gene. The efficiency of editing was verified by cost-effective means of Sanger sequencing and bioinformatic analysis. We discuss advantages and potential future developments of this method in contrast to other in vitro approaches. MDPI 2023-01-21 /pmc/articles/PMC9916915/ /pubmed/36768486 http://dx.doi.org/10.3390/ijms24032162 Text en © 2023 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 | Communication Michalski, Krzysztof Ziąbska, Paulina Sowa, Sławomir Zimny, Janusz Linkiewicz, Anna M. Evaluation of CRISPR/Cas9 Constructs in Wheat Cell Suspension Cultures |
title | Evaluation of CRISPR/Cas9 Constructs in Wheat Cell Suspension Cultures |
title_full | Evaluation of CRISPR/Cas9 Constructs in Wheat Cell Suspension Cultures |
title_fullStr | Evaluation of CRISPR/Cas9 Constructs in Wheat Cell Suspension Cultures |
title_full_unstemmed | Evaluation of CRISPR/Cas9 Constructs in Wheat Cell Suspension Cultures |
title_short | Evaluation of CRISPR/Cas9 Constructs in Wheat Cell Suspension Cultures |
title_sort | evaluation of crispr/cas9 constructs in wheat cell suspension cultures |
topic | Communication |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9916915/ https://www.ncbi.nlm.nih.gov/pubmed/36768486 http://dx.doi.org/10.3390/ijms24032162 |
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