Advances in the application of CRISPR/Cas9 gene editing technology in woody fruit trees

The CRISPR/Cas9 system is a groundbreaking gene-editing tool, composed of clustered regularly interspaced short palindromic repeats (CRISPR) and the associated protein Cas9. The core mechanism of this system relies on a single-guide RNA (sgRNA) to guide the Cas9 nuclease to precisely locate and cleave the target DNA sequence, thereby inducing double-strand breaks (DSBs). Subsequently, cells initiate two primary DNA repair pathways: Non-Homologous End Joining (NHEJ) and HomologyDirected Repair (HDR). NHEJ typically results in small insertions or deletions, which can lead to gene mutation, whereas HDR enables accurate gene modification by utilizing a repair template. Since its introduction, the CRISPR/Cas9 technology has transformed the field of genome editing, offering unprecedented opportunities for innovation in scientific research, agriculture, and medicine. In the context of woody fruit tree research, CRISPR/Cas9 has been extensively applied and has become a pivotal tool for accelerating genetic improvement. In the development of the CRISPR/Cas9 system, researchers have concentrated on designing and optimizing Cas9 gene expression vectors to enhance editing efficiency and specificity. Research has demonstrated that codon optimization of the Cas9 gene for plant systems can markedly enhance the translation efficiency of Cas9 protein in plant cells. Moreover, integrating the modified Cas9 gene into vectors with various promoters can further improve both the efficiency and specificity of gene editing. For example, the utilization of strong promoters can elevate the expression levels of the Cas9 protein, thereby increasing editing efficiency. In contrast, tissue-specific promoters allow for targeted regulation of Cas9 expression within specific tissues or cell types. Concurrently, the meticulous design of sgRNA expression vectors is essential for achieving successful gene editing outcomes. Small RNA promoters, such as U3 and U6, are extensively employed for sgRNA expression due to their high transcriptional efficiency and stability. These promoters facilitate precise sgRNA transcription, thereby significantly enhancing the overall effectiveness of the gene editing process. In the context of genetic improvement of woody fruit trees, CRISPR/Cas9 technology has exhibited substantial application potential. Through precise editing of genes associated with fruit color, flavor, and nutritional composition, this technology effectively enhances both the visual appeal and sensory qualities of fruits. For example, in the regulation of fruit color, targeted editing of key genes involved in the anthocyanin biosynthesis pathway—such as chalcone synthase (CHS) gene and dihydroflavonol reductase (DFR) gene—can effectively modulate anthocyanin production, thereby altering fruit pigmentation to achieve more vibrant hues or meet specific market preferences. Regarding the modulation of fruit sweetness and acidity, genes associated with sugar and organic acid metabolism play a crucial role. Editing genes such as sucrose phosphate synthase (SPS) and citrate synthase (CS) enables precise regulation of the sweetness-to-acidity ratio, thereby enhancing the overall flavor profile of fruits. Beyond influencing external appearance and taste, CRISPR/Cas9 technology has also proven instrumental in regulating fruit development and morphology. By modifying genes involved in cell division and elongation, such as cyclin-dependent kinase regulatory subunit (CYCD), researchers can control the rate and extent of cellular proliferation, directly influencing fruit size. This level of precision enables the development of fruits in a range of sizes to accommodate consumer preferences and market requirements. Furthermore, editing AP1 gene allows for the optimization of floral organ architecture, which facilitates pollination and fertilization processes. This not only contributes to improved fruit morphology but also significantly enhances fruit set, ultimately leading to increased yield. In the context of pathogen resistance, CRISPR/Cas9 technology offers innovative strategies for improving disease resistance in woody fruit trees. By targeting genes involved in plant immune responses and disease resistance signaling pathways, this approach can enhance the ability of fruit trees to recognize and defend against a broad spectrum of pathogens, including fungi, bacteria, and viruses. For example, targeted modification of the key regulatory factor NPR1 (non- expressor of pathogenesis- related genes) in the systemic acquired resistance (SAR) pathway can activate the expression of downstream disease resistance genes, thereby enhancing the resistance of fruit trees to various pathogens. Additionally, upregulating the expression of pathogenesis-related protein genes (PR genes) can further strengthen the immune response of fruit trees, improving their resilience to infectious agents. However, despite its promising potential in the genetic improvement of woody fruit trees, the practical application of CRISPR/Cas9 technology still encounters several challenges. First, the editing efficiency and specificity of the CRISPR/Cas9 system require further enhancement, particularly in perennial woody species, in which extended growth cycles, complex genomes, and diverse genetic backgrounds pose significant obstacles to successful gene editing. Second, the rapid identification of individuals carrying desired gene mutations remains a critical challenge that needs to be addressed. Conventional screening methods are often labor-intensive and time-consuming, failing to meet the efficiency demands of large- scale agricultural production. Therefore, future research should prioritize the optimization of gene editing tools and systems and researchers aim to develop more efficient and precise CRISPR/Cas9 platforms. Concurrently, integrating advanced biotechnological techniques—such as tissue culture and genetic transformation—can accelerate the breeding process and shorten the development cycle for new fruit tree varieties. Moreover, by advancing studies on fruit yield and quality improvement, as well as deepening the understanding of fruit tree immune systems, innova-tive strategies and methodologies can be developed to support the genetic enhancement and sustainable production of woody fruit trees. This not only facilitates the fulfillment of market demands for highquality fruits but also contributes significantly to enhancing the economic and social value of modern agricultural practices.