Tree Genetics and Molecular Breeding 2024, Vol.14, No.6, 269-276 http://genbreedpublisher.com/index.php/tgmb 271 3 Application of CRISPR/Cas9 Technology in Kiwifruit Improvement 3.1 Overview of CRISPR/Cas9 principles and applications CRISPR/Cas9 technology is a revolutionary tool for genome editing that allows for precise modifications at specific genomic loci. It involves the use of a guide RNA (gRNA) to direct the Cas9 protein to a specific DNA sequence, where it introduces double-strand breaks. This system has been widely adopted in various crops, including kiwifruit, due to its simplicity, efficiency, and versatility (Varkonyi-Gasic et al., 2018). The technology has been applied to improve agronomic traits, enhance nutritional value, and develop disease-resistant varieties in fruit crops. 3.2 Functional validation and editing of key trait-related genes using CRISPR In kiwifruit, CRISPR/Cas9 has been used to target and edit genes associated with important traits. For instance, the AcPDS gene, involved in carotenoid biosynthesis, was successfully edited using a paired-sgRNA/Cas9 system, resulting in a high mutagenesis frequency and the induction of an albino phenotype in kiwifruit plantlets (Keul et al., 2022). Additionally, the AcBFT2 gene, which regulates plant dormancy, was targeted to produce an evergrowing phenotype without affecting flowering, demonstrating the potential for climate adaptation (Herath et al., 2022). 3.3 Case studies: improving fruit quality in kiwifruit through CRISPR technology CRISPR/Cas9 has been instrumental in enhancing fruit quality traits in kiwifruit. By targeting genes involved in fruit ripening and bioactive compound synthesis, researchers have been able to modify fruit texture, color, and nutritional content. For example, the manipulation of genes like Lycopene desaturase (PDS) and Pectate lyases (PL) has shown promise in improving fruit quality and extending shelf life (Zhou et al., 2020). These advancements highlight the potential of CRISPR technology to meet consumer demands for high-quality fruit. 3.4 Gene editing research on disease resistance and stress tolerance traits Research on CRISPR/Cas9 in kiwifruit has also focused on enhancing disease resistance and stress tolerance. By editing genes associated with these traits, such as those involved in plant architecture and flowering time, kiwifruit can be made more resilient to environmental stresses and pathogens. This approach not only improves crop yield and quality but also reduces the need for chemical inputs, contributing to sustainable agriculture (Wang et al., 2018). 4 Technical Optimization and Application Challenges 4.1 Key technical challenges in applying CRISPR to kiwifruit Applying CRISPR/Cas9 technology to kiwifruit presents several technical challenges. One major issue is the optimization of the CRISPR system for high efficiency in this specific species. The editing capability can vary significantly depending on the combination of synthetic guide RNA (sgRNA) and Cas9 protein expression devices used. For instance, the development of a paired-sgRNA/Cas9 system has shown promise in increasing mutagenesis frequency, but it requires careful optimization to achieve desired results in kiwifruit (Liu et al., 2021). Additionally, the delivery of CRISPR constructs into kiwifruit cells and ensuring stable integration and expression remain significant hurdles (Wang et al., 2024). 4.2 Off-target effects and issues with gene editing efficiency Off-target effects are a critical concern in CRISPR/Cas9 applications, as unintended edits can lead to undesirable traits or affect plant health. In kiwifruit, the specificity of the CRISPR system must be finely tuned to minimize these effects. The efficiency of gene editing is also variable, with some systems like the polycistronic tRNA-sgRNA cassette (PTG) showing higher efficiency compared to traditional CRISPR/Cas9 systems (Xu et al., 2020). However, achieving consistent and high editing efficiency across different genetic backgrounds and target sites remains a challenge. 4.3 Genetic stability and heritability after kiwifruit gene editing Ensuring genetic stability and heritability of edited traits in kiwifruit is crucial for the practical application of CRISPR technology. Edited traits must be stably inherited across generations to be useful in breeding programs.
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