Novel CRISPR-Cas strategy increases gene editing efficiency in tomatoes by over 9-Fold
image: The impacts of KUDN and BRCA1 on the intramolecular MMEJ patterns at the SlHKT1;2 and SlEPSPS1 sites.
Credit: Horticulture Research
Gene editing in plants remains challenging, with the traditional non-homologous end-joining (cNHEJ) repair pathway often hindering precision. In this study, researchers advanced CRISPR-Cas-based gene targeting (GT) in tomatoes by incorporating a dominant-negative ku80 (KUDN) mutant. This approach shifts the DNA repair mechanism towards homologous recombination (HR), resulting in a significant increase in GT efficiency. The team demonstrated a 1.71 to 9.84-fold improvement in gene targeting at various loci, offering a promising method for precise and efficient plant breeding.
Gene targeting (GT) allows for the precise modification of plant genomes, an essential tool for plant breeding. However, the process is often inefficient due to the dominance of the non-homologous end-joining (cNHEJ) repair pathway, which competes withhomologous recombination (HR) for double-strand break (DSB) repair. In plants, overcoming this challenge is crucial for the practical application of GT. Researchers have explored multiple methods to improve GT efficiency, with some success, but further innovation is needed. Based on these challenges, enhancing HR by disrupting cNHEJ through dominant-negative ku80 (KUDN) could provide a solution.
In a recent study published (DOI: 10.1093/hr/uhae294) in Horticulture Research on October 23, 2024, a team from Gyeongsang National University applied KUDN to CRISPR-Cas-based gene targeting in tomatoes. This novel strategy demonstrated up to a 9.84-fold increase in GT efficiency at key loci. The study underscores the potential for KUDN to enhance gene editing in plants, moving closer to practical applications in agriculture.
By introducing the KUDN mutant into the CRISPR-Cas system, the researchers successfully redirected DNA repair towards HR, bypassing the cNHEJ pathway. At the callus stage, they observed a 1.71 to 3.55-fold improvement in GT efficiency at the SlHKT1;2 and SlEPSPS1 loci. At the plant stage, the GT efficiency increased by up to 9.84-fold, proving the efficacy of the KUDN-based approach. The method enabled the addition of a 9-bp sequence at the SlCAB13 locus, marking a significant advancement in tomato gene editing. This development opens up possibilities for more efficient plant breeding and the creation of genetically modified crops with desirable traits.
Professor Jae-Yean Kim, one of the study's authors, stated, “Our findings represent a major step towards improving CRISPR-Cas-based gene editing in plants. By optimizing the repair pathway choice, we have significantly enhanced GT efficiency in tomatoes, offering new opportunities for precision breeding in agriculture.”
The enhanced gene targeting efficiency achieved by incorporating KUDN into the CRISPR-Cas system could revolutionize plant breeding. This method can be applied to a variety of crops, potentially improving traits such as disease resistance, yield, and nutritional content. The KUDN-based approach could also pave the way for more precise and scalable genetic modifications, advancing the development of genetically modified crops for sustainable agriculture.
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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.