Induction of Complex Disease Resistance in Cucumber and Melon by Prime Editing Efficiency Exceeding 80%
Background
CRISPR gene scissors technology corrects genes by randomly cutting and relying on cellular repair mechanisms, which may lead to off-target mutations. In contrast, prime editing (Prime Editing, PE) technology is gaining attention as a next-generation tool capable of precise genome modifications such as insertions, deletions, and substitutions without double-strand breaks.
However, prime editing has faced limitations in dicot crops such as Cucurbitaceae and Solanaceae, where its efficiency is significantly lower. While it shows high activity in monocots like rice and wheat, major dicot crops such as cucumber, melon, and potato exhibit low editing rates, posing a barrier to practical application. To overcome these challenges, improving gene delivery systems and controlling protein expression have become urgent priorities. In particular, incomplete processing of prime editing guide RNA (pegRNA) has been identified as a primary cause of inefficiency. As the development of disease-resistant crops becomes increasingly urgent due to climate change, continuous efforts have been made to overcome the genome editing efficiency barriers in Cucurbitaceae crops.
Key Findings
To surpass the limitations of existing plant prime editing, the research team introduced three key optimization steps.
First, spectinomycin was introduced as a selection marker, significantly improving transformation efficiency in Cucurbitaceae tissue culture.
Second, the tomato elongation factor 1-alpha (SlEF1Ξ±) promoter was incorporated to maximize the expression of editing proteins.
Third, a Csy4 ribonuclease (Csy4)-based self-cleavage method was integrated to ensure that pegRNAs are accurately processed according to design specifications.
The resulting composite genome editing system, particularly the Csy4-PE6d model, achieved an average editing efficiency of 80.83% at target sites in cucumber. In certain gene regions, the editing success rate reached 100%. Among the transgenic lines, 36.43% exhibited homozygous editing, where both parental alleles were corrected.
To confirm the scalability of this platform, the research team conducted the same experiments on melon, pumpkin, and potato cells. The results showed significant editing activity in these crops, demonstrating the potential of this system as a universal precision breeding tool.
Furthermore, the team applied this technology to edit the CsSGR gene in cucumber, which is involved in aging and pathogen susceptibility. As a result, they successfully produced cucumber lines with strong resistance to bacterial angular leaf spot and downy mildew. The edited traits were stably inherited by subsequent generations.
Significance and Outlook
This study is significant in that it has enhanced the genome editing efficiency of Cucurbitaceae crops, which were previously considered highly challenging, and has directly led to the development of disease-resistant crops. By securing cucumber lines resistant to complex diseases, the path is now open to reduce the use of chemical pesticides and improve agricultural productivity.
The combination of the tomato promoter and Csy4 is expected to expand into breeding strategies for various dicot crops in the future. This technology has the potential to become a platform for rapidly responding to pathogen mutations.
Nevertheless, several tasks must be addressed before commercialization. It must be verified whether the gene scissors tool remains in the cell as an external genetic material and is completely removed by the final crop stage. A biosafety evaluation is also required to assess the impact of modified genes on the ecosystem. Additionally, harmonizing the diverse regulatory standards for genome-edited crops across countries will be a key factor in determining the pace of adoption in agriculture.
The prime editing (PE) system is a precise genome editing technology that works efficiently in monocots; however, its application is limited by low editing efficiency in dicots, particularly Cucurbitaceae and Solanaceae plants. Here, we first significantly improved the transformation efficiency by introducing spectinomycin in cucurbits, then used the tomato elongation factor 1-alpha (SlEF1Ξ±) promoter to enhance PE protein expression, and incorporated the Csy4 ribonuclease to process pegRNAs, collectively addressing multiple constraints limiting PE efficiency in cucurbits. The optimized PE systems, particularly Csy4-PE6d, achieved an average desired editing frequency of 80.83% at targeted loci in cucumber via stable genetic transformation, with frequencies reaching up to 100% at certain sites. Moreover, Csy4-PE6d generated homozygous edits in 36.43% of transgenic lines and demonstrated robust editing activity in melon, pumpkin, and potato. Using the Csy4-PE6d tool, we generated heritable edited cucumber lines with dual resistance to bacterial angular leaf spot and downy mildew by targeting the CsSGR gene. Collectively, this optimized system substantially enhances PE efficiency in Cucurbit crops, providing an effective solution to common challenges such as low editing efficiency and limited heritability in these species.
The optimized prime editing platform proposed in this study has the potential to revolutionize the Cucurbitaceae crop breeding industry. Traditional breeding methods require multiple generations of crossbreeding and selection to obtain desired traits, often taking at least five to ten years.
In contrast, the application of high-efficiency prime editing technology allows for the fixation of desired resistance traits through precise base editing in a single generation, significantly shortening the variety development cycle to within one to two years. In particular, seed companies are expected to be able to respond rapidly at the molecular level to new viruses and fungal diseases that are spreading quickly due to climate change.
For example, the simultaneous occurrence of downy mildew and bacterial angular leaf spot due to rising temperatures can be resolved through the development of resistant lines. This will directly translate into concrete benefits for agriculture, including enhanced harvest stability and reduced economic burdens on farmers by lowering the costs of purchasing and applying chemical pesticides.