Improving targeted gene editing in Arabidopsis thaliana

Kamoen, L. (2025)

Doctoral Thesis

Understanding DNA repair mechanisms is essential for advancing plant biotechnology and crop improvement. This study investigates how double-strand breaks (DSBs), induced by CRISPR/Cas systems, are repaired in the model plant Arabidopsis thaliana, and how these processes influence Agrobacterium tumefaciens T-DNA integration. DSBs are primarily repaired via classical nonhomologous end joining (cNHEJ) and polymerase theta-mediated end joining (TMEJ), each leading to distinct mutational outcomes. T-DNA integration at induced DSB sites was found to be frequent, with TMEJ playing a key role in mediating attachment of the T-DNA ends. By combining CRISPR-induced DSBs with Agrobacterium-mediated transformation, this research demonstrates a powerful strategy for targeted gene insertion. Finally, a novel method for error-free gene editing is developed, bypassing the low efficiency of homologous recombination. This work enhances our understanding of CRISPR-mediated genome editing and supports the development of precise, efficient strategies for plant genetic engineering.

• T-DNA
• DNA repair
• CRISPR/Cas
• Gene editing
• Non-homologous end-joining (NHEJ)
• Polymerase theta-mediated end-joining (TMEJ)
• Double-strand break repair
• Microhomology

• All authors: Kamoen, L.
• Supervisor: Tijsterman, M.
• Co-supervisor: Pater, B.S. de
• Committee: Meijer, A.H.; Offringa, R.; Attikum, H. van; Puchta, H; Kregten, M van
• Qualification: Doctor (dr.)
• Awarding Institution: Institute of Biology (IBL), Faculty of Science, Leiden University
• Date: 2025-06-10