CRISPR–Nanoparticle Delivery Systems for Precision Gene Editing in Plant Pathogens

Abstract

Plant pathogen species pose a perpetual challenge to food production worldwide and require innovative and targeted approaches for disease control. CRISPR-Cas genome editing is a highly versatile tool with great potential for targeted disruption of virulence genes in pathogen species. Nevertheless, widespread use of CRISPR-Cas in plant pathologies remains a challenge because of inefficient and toxic delivery of CRISPR-Cas elements into pathogen cells. In this study, a CRISPR-Cas delivery system assisted with nanoparticles was established and optimized for precision editing of fungal pathogen Fusarium oxysporum. CRISPR elements were incorporated into polymeric nanoparticles that prevent degradation and ensure controlled release in pathogen cells. Physicochemical analysis revealed the uniform nanoscale size, colloid stability, and efficient loading capacity of CRISPR, and the pH-dependent release property effectively triggered rapid activation of the fungal cells. The nanoparticle-based delivery system remarkably enhanced the uptake and nuclear entry of the CRISPR components compared to the free system and effectively triggered specific gene deletion in a virulence-related gene. The functional studies clearly indicated the induction of significant reductions in fungal growth, virulence factors, and disease-causing efficiency in the host-pathogen interaction models. Crucially, the off-target experiment confirmed the high specificity of the edited sites and the lack of insertions or deletions in the host genome, indicating the biosafety of the proposed approach. Taken together, these results show that the application of CRISPR nanoparticle delivery systems effectively overcomes critical biological barriers in plant pathogens and provides a targeted and environmentally safe approach to traditional chemical-based fungicides. The study opens up the next frontiers of precision gene-editing-based sustainable control of plant diseases using CRISPR-based nanoparticle technologies in modern agriculture.