New virus-based CRISPR system accelerates heritable genome editing in tomato
image: Overview of the tobacco rattle virus-mediated genome editing system in tobacco plants. A 20-nucleotide target sequence is cloned before the sgRNA scaffold under the PEBV promoter in the pTRV2-PEBV plasmid. Agrobacterium (GV3101) cultures harboring both pTRV1 and the engineered pTRV2-PEBV vectors are co-infiltrated into the cotyledons of Cas9-expressing tobacco plants. After agro-infiltration, their systemic leaves are analyzed for the presence of indel mutations at the targeted site. Eventually, tobacco mutants are isolated by screening the harvested seeds, eliminating the need for additional tissue culture. Image link: https://academic.oup.com/view-large/figure/507805838/uhae364f1.tif
Credit: Horticulture Research
CRISPR-Cas9 has revolutionized plant genome editing, yet its application in tomato remains limited due to the plant’s recalcitrance to transformation and regeneration. Traditional methods rely on lengthy and genotype-dependent tissue culture procedures, restricting their use in many commercial cultivars. Viral delivery systems such as potato virus X and tomato spotted wilt virus have been explored, but often fail to generate heritable mutants because they cannot reach germline cells. TRV provides an alternative vehicle that can infect apical meristems, but earlier TRV-based systems achieved low efficiency in tomato. Due to these challenges, it was necessary to develop a tomato-optimized virus-induced genome editing strategy.
A research team from Sejong University in Seoul, Republic of Korea, has successfully created an efficient, heritable TRV-mediated genome editing system for tomato. The study, published (DOI: 10.1093/hr/uhae364) on April 1, 2025, in Horticulture Research, describes how the scientists constructed a SlUBI10-promoter-driven Cas9 line and combined it with a TRV vector carrying mobile guide RNAs fused to the tomato Single Flower Truss (SFT) sequence. This improved design enabled the production of SlPDS knockout seeds without any tissue culture, demonstrating a major breakthrough in tomato functional genomics.
To overcome poor transgene expression and low sgRNA mobility in previous systems, the researchers replaced the Arabidopsis UBI10 promoter with the tomato SlUBI10 promoter, ensuring high Cas9 expression in germline tissues. They also identified the tomato ortholog of the FT gene, SFT, and extracted a 105-base mobile RNA sequence to construct a tomato-specific sgRNA scaffold. When the SlPDS gene, which controls carotenoid biosynthesis, was targeted using this TRV-SFT system, infected seedlings exhibited photobleaching symptoms—a visual marker of gene disruption. Deep sequencing revealed mutation rates of 20–71% in systemic leaves, while some fruits yielded seeds that germinated entirely as white seedlings, confirming homozygous knockouts. The efficiency of generating heritable mutants ranged from 15% to 100%, depending on the fruit tested. Remarkably, these results were achieved without tissue culture, demonstrating that both Cas9 and sgRNA were successfully delivered to the shoot apical meristem. The system thus provides a powerful framework for producing stable genome edits in tomato and potentially other Solanaceae crops.
“Our virus-induced genome editing platform eliminates one of the biggest barriers in tomato biotechnology—the dependence on tissue culture,” said Professor Kyung-Nam Kim, corresponding author of the study. “By optimizing both the Cas9 expression promoter and the sgRNA mobility element for tomato, we achieved efficient heritable gene editing directly through viral delivery. This approach not only simplifies the workflow but also broadens the accessibility of genome editing for tomato breeders and molecular biologists seeking to study gene function in diverse cultivars.”
The tomato-optimized TRV-VIGE system represents a transformative tool for rapid crop improvement and gene function analysis. Once a Cas9-expressing line is established, researchers can easily introduce new guide RNAs through simple viral infection, accelerating the production of mutant lines for traits such as yield, quality, and stress tolerance. Beyond tomato, this strategy could be adapted to related crops in the Solanaceae family, including potato, pepper, and eggplant. By drastically shortening the time from gene targeting to heritable mutation, the system has the potential to reshape plant breeding and functional genomics pipelines in horticultural research.
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References
DOI
Original Source URL
https://doi.org/10.1093/hr/uhae364
Funding information
This study was supported by the program of NSFC [32060700], the National Guidance Foundation for Local Science and Technology Development of China [[2023] 009] and [2022-1-52], Guiyang Science and Technology Plan Project [Construction Technology Contract [2023] 48–21], the Science and Technology Project of Guizhou Province, China (Qiankehe Foundation-ZK [2022]) (Grant No.702076222101).
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, 2024. 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.