Tomato gene targeting reimagined: A smart detour around DNA repair
image: Impacts of the factors that affect DSB repair pathway choice on the editing efficiency in tomato. A, Plasmid map of the GT tool used in the study. The GT construct was cloned into a geminiviral replicon vector with the boundaries of two long intergenic sequences (LIRs) and one short intergenic sequence (SIR). The replicon is autonomously replicated with the support of the Rep protein/expression cassette. B–C, The impacts of KUDN and BRCA1 on GT (B) and indel mutation (C) efficiency at the callus stage. The efficiencies were assessed by targeted NGS using 21-dpt cotyledon/callus samples. D, The protein expression levels of ttLbCas12a and its KUDN and BRCA1 fusions. Western blot membranes showing bands of the Cas proteins with expected sizes and expression cassettes. Actin levels were used as loading controls. P-values of the t-test for pairwise comparison between the relative protein levels of ttLbCas12a and the fusions are indicated on the top of the bars. All data points are shown as dots in the plots in (B–D). The expression cassettes and Cas configurations are at the bottom of the bars.
Credit: Horticulture Research
Plant genome editing through CRISPR-Cas holds transformative potential, yet its precision remains hampered by low gene targeting (GT) efficiency. This study presents a strategy to enhance CRISPR-based GT in tomato by using a dominant-negative version of the ku80 protein (KUDN), which interferes with the canonical non-homologous end-joining (cNHEJ) pathway. By shifting the DNA repair preference toward homologous recombination (HR), the team achieved 1.7- to 9.8-fold improvements in GT efficiency at specific tomato loci. These results demonstrate that modulating DNA repair dynamics can significantly improve precise and heritable edits, offering a new tool for advanced plant breeding.
Gene targeting (GT) via homologous recombination (HR) is a powerful tool for precise genome editing, but its application in plants is limited due to the dominance of canonical non-homologous end-joining (cNHEJ) in double-strand break (DSB) repair. CRISPR-based editing often fails to achieve high-fidelity integration of DNA templates, limiting the scalability of complex edits in agriculture. Strategies to boost GT efficiency have focused on increasing donor DNA delivery or promoting DSB formation, but few effectively redirect repair toward HR. Recent insights into DNA repair modulation offer new possibilities for fine-tuning editing outcomes in plants. Due to these limitations, there is a need to explore strategies that bias DSB repair toward HR to enable efficient GT.
Researchers from Gyeongsang National University have developed an enhanced GT tool for tomatoes by fusing a dominant-negative fragment of ku80 (KUDN) with the ttLbCas12a CRISPR enzyme. The study (DOI: 10.1093/hr/uhae294), published on October 23, 2024, in Horticulture Research, demonstrates that this strategy significantly boosts GT efficiency by suppressing the cNHEJ repair pathway. The results show improved performance at both callus and plant stages, achieving up to a 9.84-fold increase in editing precision, and mark a promising step toward more precise plant genome engineering.
The research focused on improving HR-based GT by disrupting the cNHEJ repair mechanism using a KUDN protein fragment. The team fused KUDN to the ttLbCas12a enzyme, creating NKUDN, which showed notable enhancement in GT efficiency—3.55-fold at the SlEPSPS1 locus and 1.71-fold at the SlHKT1;2 locus during the callus stage. In regenerated plants, NKUDN further improved GT rates by up to 9.84-fold. Importantly, the study confirmed that these improvements were not due to increased Cas protein expression but rather the redirection of DSB repair from cNHEJ to HR and microhomology-mediated end joining (MMEJ). Additional experiments demonstrated stable inheritance of the GT alleles across generations and the feasibility of obtaining transgene-free edited plants. Furthermore, the NKUDN construct exhibited high specificity with no off-target effects detected. When applied to an additional locus, SlCAB13, the tool successfully introduced a precise 9-bp insertion, further proving its versatility. These findings suggest that KUDN effectively shifts the repair landscape in favor of precision editing.
“Our results highlight a powerful approach to improving precision genome editing in plants,” said Dr. Jae-Yean Kim, corresponding author of the study. “By interfering with the cNHEJ repair pathway through a KUDN fragment, we created a more favorable environment for HR. This not only boosts GT efficiency but also ensures heritability and specificity—key factors for real-world application in crop breeding. It’s a significant step toward practical, high-precision plant genome engineering.”
This enhanced GT system offers a viable solution for overcoming the low efficiency of CRISPR-mediated HR in plant breeding, particularly in species like tomato where allele selection markers are limited. By increasing the likelihood of precise edits without introducing transgenes, it aligns with regulatory expectations and public acceptance of genome-edited crops. The successful insertion of functional alleles and their stable inheritance across generations also opens doors for multi-trait stacking and complex genome designs. While current efficiency still varies across genomic loci, the modularity of this approach makes it adaptable for other plant species and traits, contributing to the advancement of sustainable and precise crop improvement strategies.
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References
DOI
Original Source URL
https://doi.org/10.1093/hr/uhae294
Funding information
This work was supported by the National Research Foundation of Korea (Program 2020M3A9I4038352, 2020R1A6A1A03044344, 2021R1A5A8029490, 2022R1A2C3010331) and the Program for New Plant Breeding Techniques (NBT, Grant PJ01686702), Rural Development Administration (RDA), Korea.
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.