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Author

Aftab Ahmad

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#protein folding Open access Sep 2026

De novo design of modular insecticidal scaffold enhances cotton resistance to pink bollworm and whitefly

A variety of insect pests pose a serious threat to global cotton production. Among these, pink bollworm (PBW) and whiteflies are the most devastating, resulting in substantial yield losses and increased pesticide use. Conventional insect-resistance strategies, which primarily rely on single Bacillus thuringiensis (Bt) genes, often face limitations due to the development of resistance in target pests. To overcome these challenges, the fusion of multiple insecticidal proteins, including Bt toxins and plant-derived lectins, offers a synergistic approach to broaden the spectrum and durability of resistance. The modular insecticidal scaffold exhibited a distinct promoter-dependent expression pattern, highlighting its potential as a customisable platform for targeted cotton protection. Molecular docking revealed favorable interactions of Cry1Ac, Cry1Ab, Vip3A with the pink bollworm cadherin receptor, whereas ASAL showed favorable binding to mannose residues, representing its recognition of mannose-containing glycoconjugates on midgut cells, supporting the structural compatibility of the de novo assembled scaffold. The scaffold carrying these domains was synthesised and cloned into the pCAMBIA1302 vector under constitutive and fibre-specific promoters, and subsequently transformed into a local cotton variety. Transgenic lines harbouring the fibre-specific promoter (GhSCFP) displayed significantly elevated expression in bolls, reaching over a 100-fold increase compared with leaves, whereas the constitutive promoter (CaMV35S) drove higher expression in foliage, consistent with its broader activity profile. Promoter-guided expression patterns strongly influenced insecticidal outcomes: fibre-specific lines showed substantial mortality in PBW (up to 90–95%), whereas constitutive promoter-driven lines exhibited greater suppression of whiteflies (approximately 60–70%). The observed correlation between spatial expression and pest-specific lethality indicates that the scaffold functions as a cohesive insecticidal unit, effectively integrating multiple toxin domains while maintaining spatial regulation. This dual strategy of boll-targeted PBW protection and whole-plant whitefly resistance represents a significant advancement over single-gene Bt approaches, providing a more resilient and comprehensive resistance framework for cotton improvement. Our findings indicate that promoter selection is a crucial factor influencing tissue-specific expression and insecticidal efficacy. The synergistic combination of fibre-specific and constitutive promoters establishes a dual framework for targeted boll protection and broader pest management. This modular, promoter-driven approach holds considerable promise for the development of next-generation, sustainable pest-resistant cotton suitable for integrated pest management strategies.

Saira Azam, Aftab Ahmad, Ayesha Latif et al. · 0 citations
Review Open access Jul 2026

Beyond GMOs: transgene-free gene-edited crops for global food security

Transgene-free gene-editing has transformed the genomic landscape of crops by enabling targeted, precise, and predictable genetic outcomes without integrating any foreign DNA into the host genome. It has significantly reduced production time and costs, and the regulatory burden for transgene-free gene-edited crops, while improving social acceptance compared with classical transgenic crops. This review compares the transgene-free gene-edited, transgenic, and cisgenic crops. We also focus on core methods for developing transgene-free gene-edited crops, particularly ribonucleoprotein (RNP), transient expression, the transgene killer method, and HI-edit technology. We highlight the practical examples summarizing CRISPR applications for transgene-free gene-edited crops, including cereals, legumes, and oilseeds, and horticultural crops. We also analyze the rapidly evolving global regulatory landscape of transgene-free gene-edited crops, including the recent European Union movement towards differentiated oversight for certain “new genomic techniques (NGTs)” that do not introduce foreign DNA, while maintaining the strict risk assessment for complex modifications. We also summarize the social, ethical, and public perception aspects of transgene-free gene-edited crops compared with traditional GMOs. Finally, we highlight the emerging role of AI in developing precise transgene-free gene-edited crops and the contributions these crops make to global food security. Collectively, this evidence supports the growing role of transgene-free gene-edited crops in scientific developments and real-world agricultural deployment, with remaining bottlenecks in delivery for recalcitrant crops, scalable and universal regulation, detection and traceability of the Cas footprints, and equitable access.

Aftab Ahmad, Muhammad Faheem, Annena Ijaz et al. · 0 citations
#gene editing Open access Aug 2026

Comparative evaluation of pKSE401 and pHSE401 gRNA constructs for CRISPR/Cas9-Mediated resistance to cotton leaf curl virus

The results demonstrate the cutting-edge potential of CRISPR/Cas9 multiplex approaches as next-generation methods for designing sustainable resistance to multifaceted plant virus diseases and contribute to understanding of CRISPR-based antiviral response mechanisms.

Farwa Yaqub, Sidra Ashraf, Ahmed al-Harrasi et al. · 0 citations

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