Variable alterations in frequencies of CRISPR/Cas9-induced nucleotide deletion and insertion in transgenic maize callus tissues exposed to chromatin modifiers.
5 affiliations
- 1Agribiotechnology and Precision Breeding for Food Security National Laboratory, Institute of Plant Biology, HUN-REN Biological Research Centre, Szeged, Hungary.
- 2Functional Cell Biology and Immunology Advanced Core Facility (FCBI-ACF), Hungarian Centre of Excellence for Molecular Medicine (HCEMM), Szeged, Hungary.
- 3Faculty of Medicine, Albert Szent-Györgyi Health Centre, Interdisciplinary R&D and Innovation Centre of Excellence, University of Szeged, Szeged, Hungary.
- 4Doctoral School of Biology, University of Szeged, Szeged, Hungary.
- 5Cereal Research Non-profit Ltd., Szeged, Hungary.
Abstract
Given the growing emphasis on the efficient application of CRISPR/Cas9 in plant genome editing, there is a need for methodologies that enhance targeted mutagenesis efficiency. In the present study, we utilized stable transgenic maize callus tissues harboring active CRISPR/Cas9 vector systems targeting either the abnormal cytokinin response 1 (ARE1) gene or the potassium channel gene (KAT2). The presented DNA sequencing data indicate that the KAT2-targeting vector, which utilizes a 3'-end truncated guide RNA, retained mutagenic activity, albeit at a notably low frequency. We quantified mutagenic events following exposure to elevated temperatures (37 °C for 48 h) and various chromatin-modifying compounds, including sodium butyrate (NaBt), nicotinamide (NA), and 5-azacytidine (AZA). High-temperature treatment alone increased the frequency of nucleotide deletions (5.8-fold relative to the untreated samples) in the ARE1 target gene. Conversely, in the KAT2 gene, both deletions and insertions were stimulated (6.4 and 6.2-fold, respectively). As a novel approach, we evaluated the mutagenic efficacy of chromatin-modifying compounds administered in conjunction with heat treatment. The combined application of NaBt and AZA with elevated temperature was particularly effective in increasing insertion frequencies; in the ARE1 (with NaBt + heat: 17.3-fold) and KAT2 (with AZA + heat: 21.4-fold) genes. Cell cycle analysis of suspension-cultured cells revealed that chromatin-modifying treatments altered cell cycle progression, evidenced by an accumulation of cells in the G2 phase and a significant reduction in the number of mitotic cells. Subsequent DNA sequencing demonstrated a differential increase in the induction of deletions and insertions, alongside an expansion of the deleted regions.
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BibTeX
@article{nagy2026variable,
title = {{Variable alterations in frequencies of CRISPR/Cas9-induced nucleotide deletion and insertion in transgenic maize callus tissues exposed to chromatin modifiers.}},
author = {Bettina Nagy and Aysha Jameel and Gábor V Horváth and Katalin Török and Ferhan Ayaydin and Nikolett László and Divya Teja Dondapati and Sándor Mórocz and Zoltán Zombori and Györgyi Ferenc and Dénes Dudits},
journal = {Frontiers in genome editing},
year = {2026},
month = oct,
doi = {10.3389/fgeed.2026.1924094},
url = {https://europepmc.org/article/MED/42909021},
note = {CC BY},
}
RIS
TY - JOUR
TI - Variable alterations in frequencies of CRISPR/Cas9-induced nucleotide deletion and insertion in transgenic maize callus tissues exposed to chromatin modifiers.
AU - Bettina Nagy
AU - Aysha Jameel
AU - Gábor V Horváth
AU - Katalin Török
AU - Ferhan Ayaydin
AU - Nikolett László
AU - Divya Teja Dondapati
AU - Sándor Mórocz
AU - Zoltán Zombori
AU - Györgyi Ferenc
AU - Dénes Dudits
PY - 2026
DA - 2026/10/07
JO - Frontiers in genome editing
DO - 10.3389/fgeed.2026.1924094
UR - https://europepmc.org/article/MED/42909021
AB - Given the growing emphasis on the efficient application of CRISPR/Cas9 in plant genome editing, there is a need for methodologies that enhance targeted mutagenesis efficiency. In the present study, we utilized stable transgenic maize callus tissues harboring active CRISPR/Cas9 vector systems targeting either the abnormal cytokinin response 1 (ARE1) gene or the potassium channel gene (KAT2). The presented DNA sequencing data indicate that the KAT2-targeting vector, which utilizes a 3'-end truncated guide RNA, retained mutagenic activity, albeit at a notably low frequency. We quantified mutagenic events following exposure to elevated temperatures (37 °C for 48 h) and various chromatin-modifying compounds, including sodium butyrate (NaBt), nicotinamide (NA), and 5-azacytidine (AZA). High-temperature treatment alone increased the frequency of nucleotide deletions (5.8-fold relative to the untreated samples) in the ARE1 target gene. Conversely, in the KAT2 gene, both deletions and insertions were stimulated (6.4 and 6.2-fold, respectively). As a novel approach, we evaluated the mutagenic efficacy of chromatin-modifying compounds administered in conjunction with heat treatment. The combined application of NaBt and AZA with elevated temperature was particularly effective in increasing insertion frequencies; in the ARE1 (with NaBt + heat: 17.3-fold) and KAT2 (with AZA + heat: 21.4-fold) genes. Cell cycle analysis of suspension-cultured cells revealed that chromatin-modifying treatments altered cell cycle progression, evidenced by an accumulation of cells in the G2 phase and a significant reduction in the number of mitotic cells. Subsequent DNA sequencing demonstrated a differential increase in the induction of deletions and insertions, alongside an expansion of the deleted regions.
SN - 2673-3439
ER -