CRISPR/Cas9-Mediated Editing of Terpene Synthase Gene Reduces Volatile Emission and Attraction of Hypothenemus hampei to Coffea arabica Fruits

Abstract

The coffee berry borer (CBB), Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytinae), is one of the most destructive pests of coffee, causing significant losses in yield, bean quality, and overall crop profitability. Current integrated pest management strategies reduce damage but cannot prevent insects from locating and infesting coffee fruit. Because host selection by CBB relies on olfactory cues, modifying the emission of key volatile compounds represents a potential strategy to reduce pest attraction. This study aimed to decrease CBB attraction by editing genes involved in the biosynthesis of the monoterpenes limonene and pinene, which participate in host recognition. Agrobacterium-mediated transformation was performed in Coffea arabica seedlings, and gene editing used a binary vector carrying sgRNAs targeting terpene synthase genes, the cas9 gene, and the bar selection marker. Nine cas9-positive plants were obtained, including six of them also positive for bar, with seven plants exhibiting non-synonymous mutations at the target site for limonene synthase. Quantitative PCR (qPCR) analysis revealed a significant reduction in transcript levels in edited fruit, ranging from 95.5% to 99.2% compared to wild-type controls. Furthermore, GC-MS analysis confirmed a reduction of approximately 55% in limonene emission in variants exhibiting low-frequency mutations. Olfactometry bioassays revealed significantly reduced attraction, with only 17–36% of H. hampei females orienting toward edited fruit compared with 64–83% for wild-type controls (p < 0.05). These results suggest that targeted editing of limonene synthase can modulate volatile emissions and alter host-seeking behavior in H. hampei, providing a proof-of-concept for engineering crop plants with reduced pest attraction and offering a complementary, sustainable strategy for pest management in coffee production systems.