Bioscience Methods 2026, Vol.17, No.1, 9-22 http://bioscipublisher.com/index.php/bm 16 One way is to remove negative regulators so the plant’s defense stays more active. In grapevine, scientists deleted VvNPR3, a repressor in the salicylic acid (SA) pathway, and the plants became more resistant to mildew (Moffa et al., 2025). Pineapple also has NPR1, NPR3, and NPR4. If AcNPR3 is edited, SA defenses could be stronger, leading to more PR proteins and more antimicrobial compounds. MicroRNAs also control resistance genes. In tomato, miR482 family members lower the activity of NBS-LRR genes. Removing miR482b and miR482c by CRISPR increased resistance to Phytophthora infestans (Zhu et al., 2022; Li, 2024). Pineapple likely has similar miRNAs. Editing them, or their binding sites on target genes, could release defense genes from suppression. By using CRISPR/Cas9 to mutate the miRNA genes or their binding sites on target genes, pineapple’s immune regulators could escape post-transcriptional suppression. For example, if pineapple has a miRNA that downregulates a key defense gene under stress, disrupting that miRNA (or its target site on the gene’s mRNA) would result in higher expression of the defense gene and a more robust response to infection. Regulatory network editing can therefore achieve polygenic resistance effects: altering one regulator can upregulate a battery of defense genes. However, care is needed to avoid autoimmune or growth penalty phenotypes that sometimes result from constitutive defense activation. Fine-tuning is possible – for instance, editing a promoter to slightly reduce a repressor’s expression rather than a full knockout might yield a balanced outcome. Nonetheless, the precision of CRISPR allows testing of various allele modifications. Other possible targets in pineapple include AcJAZ genes (repressors in the JA pathway), WRKY repressors, or MAPK phosphatases that turn off defense signals. Editing these could make the immune response last longer or react faster. Instead of adding new resistance genes, this strategy maximizes use of pineapple’s existing defense arsenal by lifting internal brakes. Successful demonstrations in other crops provide a roadmap – e.g. the miRNA editing in tomato and EDR1 knockout in wheat (Zhang et al., 2017) – that can be followed in pineapple once comparable regulatory elements are identified. 4.4 Multiplex gene editing for pyramiding resistance traits The wide range of pathogens makes single-gene resistance ineffective for protecting against all diseases. CRISPR/Cas9 technology enables multiplexed editing which allows scientists to modify multiple gene targets within one organism at the same time thus making it possible to stack resistance traits through pyramiding without needing extensive breeding programs. The approach shows great promise for pineapple because it enables the simultaneous disruption of multiple susceptibility genes and defense regulators which results in broad-spectrum disease resistance against major pathogens. Multiplex editing can be done by giving the plant multiple sgRNAs with Cas9, or by using Cas12a (Cpf1), which can process many guide RNAs together (Schepler-Luu et al., 2023; Li et al., 2025). In rice, Oliva et al. (2019) edited three susceptibility gene promoters (OsSWEET11, OsSWEET13, OsSWEET14) at the same time. They made small changes in each promoter’s effector-binding site using one construct. The new rice plants resisted all tested strains of Xanthomonas. This result came in one generation, saving years compared with traditional breeding. A similar method could work for pineapple. One CRISPR/Cas9 plasmid could target AcMLO, AcEDR1, and AcNPR3. A plant edited in all three genes could gain fungal resistance frommlo, stronger basic immunity from edr1, and more SA signaling fromnpr3. Another set of edits could aim at an S gene for heart rot and another for black rot, giving resistance to both at once. The slow growth rate of pineapple makes simultaneous editing of multiple genes a highly advantageous approach. The single-step process produces results more quickly than the multi-year process of modifying individual genes through successive alterations. Other fruit crops prove it can be done. The researchers used CRISPR to disable
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