Molecular Plant Breeding 2025, Vol.16, No.3, 202-210 http://genbreedpublisher.com/index.php/mpb 206 4 Application Strategies of CRISPR/Cas9 in Flavor Improvement of Golden Pitaya 4.1 Gene knockout and multi-gene stacking Gene knockout (knock-out) is the most commonly used strategy of CRISPR/Cas9, which completely loses its function by inducing frameshift mutations or large fragment deletions in the target gene. For negatively regulated flavor genes, knockout often improves flavor (Wang et al., 2021). Tomato SlINVINH1 and SlVPE5 inhibitors can increase sugar content by using CRISPR single gene knockout. In golden pitaya, if the function of a gene is confirmed to inhibit sugar accumulation or promote acid formation, then knockout can produce an improvement effect. For complex flavor traits, multi-gene regulatory networks are often involved, and knocking out a single gene alone has limited effect. In this case, a multi-gene stacking editing strategy can be used to knock out two or more related genes at the same time to achieve a synergistic effect. For example, Wang et al. (2021) hybridized the SlINVINH1 and SlVPE5 knockout lines to obtain a tomato line with further improved sugar content. In terms of improving sweetness, it is possible to consider editing several key sites in the golden pitaya at the same time (such as knocking out a sugar inhibitor and an acidity promoter at the same time) to achieve the dual effects of higher sugar and lower acid. 4.2 Base editing and site-directed mutagenesis Base editing is a CRISPR-derived technology that does not produce DNA double-strand breaks. It catalyzes base changes at the target site through a fused deaminase to achieve precise point mutations (Miao et al., 2024). For example, the key site encoding the enzyme is mutated to a more active allele. Assuming that a certain aroma synthase in the golden pitaya has lower activity due to a difference in one amino acid, a guide RNA can be designed to target the codon and change it to the codon corresponding to the high-activity allele through base editing, thereby improving the aroma synthesis efficiency. In addition to base editing, site-directed mutagenesis/insertion (also known as site-directed modification) can also be completed using traditional CRISPR in combination with homologous recombination templates. For example, in order to enhance the regulation of a certain transcription factor on a flavor gene, several cis-element sequences can be inserted into its promoter to increase expression. This requires providing a repair template so that the cell can integrate the new sequence into the target site through homologous recombination (Miao et al., 2024). However, the efficiency of homologous recombination in plant cells is generally low, especially in new varieties whose regeneration system is not yet sound. Therefore, gene knockout is still the main means of fruit flavor improvement. Base editing is expected to be gradually applied as an emerging technology, while site-specific insertion and more precise regulation and transformation are still in the exploratory stage. 4.3 Gene editing vector and strategy optimization For the application of CRISPR/Cas9 in golden pitaya flavor improvement, it is necessary to comprehensively consider factors such as editing strategy (knockout vs. mutation), vector system (stable genetic transformation vs. transient RNP), and target number (single gene vs. multiple genes). Reasonable experimental design and technical optimization will significantly improve editing efficiency and breeding process. Domestic teams have carried out gene editing practices on fruits such as strawberries, such as using CRISPR to knock out pectinase genes to improve strawberry firmness and storage (López-Casado et al., 2023). These experiences can provide useful reference for the flavor editing of golden pitaya. 5 Research Progress and Case Analysis of Gene Editing Technology to Improve the Flavor of Golden Pitaya 5.1 Successful cases of gene editing of related fruit flavors As a model fruit vegetable, the research on flavor gene editing of tomatoes is the most abundant. For example, in terms of improving sweetness, CDPK27/26 double knockout tomatoes were completed by the Chinese Academy of Agricultural Sciences and the University of Florida, which significantly increased the sugar content of the fruit without compromising yield (Zhang et al., 2024). In terms of acidity regulation, a study in 2022 reported that knocking out SlALMT9 reduced the malic acid content of tomato fruit by about half, and tried to cultivate
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