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Optimization of cabbage (Brassica oleracea var. capitata L.) protoplast transformation for genome editing using CRISPR/Cas9
Genome editing techniques, such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated systems (CRISPR/Cas9) are undoubtedly becoming an indispensable tool for improving food crops and tackling agricultural challenges. In the present study, key factors affecting transformatio...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10577288/ https://www.ncbi.nlm.nih.gov/pubmed/37849838 http://dx.doi.org/10.3389/fpls.2023.1245433 |
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author | Stajič, Ester Kunej, Urban |
author_facet | Stajič, Ester Kunej, Urban |
author_sort | Stajič, Ester |
collection | PubMed |
description | Genome editing techniques, such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated systems (CRISPR/Cas9) are undoubtedly becoming an indispensable tool for improving food crops and tackling agricultural challenges. In the present study, key factors affecting transformation efficiency, such as PEG4000 concentration, incubation time, and plasmid amount were evaluated to achieve efficient delivery of CRISPR/Cas9 vector into cabbage protoplasts. Using amplicon sequencing, we confirmed a significant effect of PEG4000 concentration and incubation time on the induced target mutations. By optimizing the transformation protocol, editing efficiency of 26.4% was achieved with 40 µg of plasmid and 15 minutes incubation with 50% PEG4000. While these factors strongly affected the mutation rate, the viability of the transformed protoplasts remained high. Our findings would be useful for successful genome editing in cabbage and other brassicas, as well as in research areas such as gene function analysis and subcellular localization that rely on transient transformation methods in protoplasts. |
format | Online Article Text |
id | pubmed-10577288 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-105772882023-10-17 Optimization of cabbage (Brassica oleracea var. capitata L.) protoplast transformation for genome editing using CRISPR/Cas9 Stajič, Ester Kunej, Urban Front Plant Sci Plant Science Genome editing techniques, such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated systems (CRISPR/Cas9) are undoubtedly becoming an indispensable tool for improving food crops and tackling agricultural challenges. In the present study, key factors affecting transformation efficiency, such as PEG4000 concentration, incubation time, and plasmid amount were evaluated to achieve efficient delivery of CRISPR/Cas9 vector into cabbage protoplasts. Using amplicon sequencing, we confirmed a significant effect of PEG4000 concentration and incubation time on the induced target mutations. By optimizing the transformation protocol, editing efficiency of 26.4% was achieved with 40 µg of plasmid and 15 minutes incubation with 50% PEG4000. While these factors strongly affected the mutation rate, the viability of the transformed protoplasts remained high. Our findings would be useful for successful genome editing in cabbage and other brassicas, as well as in research areas such as gene function analysis and subcellular localization that rely on transient transformation methods in protoplasts. Frontiers Media S.A. 2023-10-02 /pmc/articles/PMC10577288/ /pubmed/37849838 http://dx.doi.org/10.3389/fpls.2023.1245433 Text en Copyright © 2023 Stajič and Kunej https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Plant Science Stajič, Ester Kunej, Urban Optimization of cabbage (Brassica oleracea var. capitata L.) protoplast transformation for genome editing using CRISPR/Cas9 |
title | Optimization of cabbage (Brassica oleracea var. capitata L.) protoplast transformation for genome editing using CRISPR/Cas9 |
title_full | Optimization of cabbage (Brassica oleracea var. capitata L.) protoplast transformation for genome editing using CRISPR/Cas9 |
title_fullStr | Optimization of cabbage (Brassica oleracea var. capitata L.) protoplast transformation for genome editing using CRISPR/Cas9 |
title_full_unstemmed | Optimization of cabbage (Brassica oleracea var. capitata L.) protoplast transformation for genome editing using CRISPR/Cas9 |
title_short | Optimization of cabbage (Brassica oleracea var. capitata L.) protoplast transformation for genome editing using CRISPR/Cas9 |
title_sort | optimization of cabbage (brassica oleracea var. capitata l.) protoplast transformation for genome editing using crispr/cas9 |
topic | Plant Science |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10577288/ https://www.ncbi.nlm.nih.gov/pubmed/37849838 http://dx.doi.org/10.3389/fpls.2023.1245433 |
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