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.· Frontiers in Plant Science· 0 citations
Cotton leaf curl disease (CLCuD), caused by begomoviruses and their betasatellites, is a major threat to cotton production, especially in South Asia, where periodic viral outbreaks continue to affect cotton yields, quality, and livelihoods. The advent of CRISPR/Cas9 gene-editing technology has transformed plant biotechnology, offering efficient, accurate, and programmable methods for combating viral pathogens at the genetic level. Here, the antiviral efficacy of two most widely used CRISPR/Cas9 binary plant expression vectors, pKSE401 and pHSE401, was tested in Nicotiana benthamiana against Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Multan betasatellite (CLCuMuB). The guide-RNAs (gRNAs) were designed to target viral genes that play significant roles in pathogenicity and replication, with pHSE401 encoding a single gRNA and pKSE401 a multiplex of two gRNAs. Agrobacterium-mediated transient transformation and viral inoculation experiments revealed that both CRISPR/Cas9 vectors effectively delayed symptom onset and reduced virus titers relative to infected controls. Remarkably, the multiplex pKSE401 system was more effective at suppressing viral infection, achieving about a 90% reduction in viral accumulation compared with a 75% reduction by the single gRNA pHSE401 construct. pKSE401-treated plants showed delayed symptom development, reduced severity, and partial recovery, demonstrating the improved efficiency of multiplex genome editing. 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. This research not only contributes to our understanding of CRISPR-based antiviral response mechanisms but also provides a promising avenue for designing broad-spectrum, sustainable resistance against viral epidemics in cotton and other commercially valuable crops.
Farwa Yaqub, Sidra Ashraf, Ahmed Al‐Harrasi et al.· Plant Protection· 0 citations