Engineering CRISPR/Cas9-Loaded Nanocarriers for Targeted Editing of Biofilm-Regulatory Genes in Resistant Food Industry Strains with Advanced Quantitative RNA-Seq Transcriptomic Analysis

چکیده

This comprehensive study elucidates the de novo synthesis and physicochemical fine-tuning of hierarchically engineered multilaminar nanocarrier assemblies encapsulating CRISPR/Cas9 ribonucleoprotein (RNP) complexes, devised for ultra-precise, locus-specific genomic perturbation of essential biofilm-regulatory operons within recalcitrant bacterial biofilm consortia prevalent in agro-industrial food processing ecosystems. Fabrication employed iterative layer-by-layer (LbL) polyelectrolyte self-assembly integrated with surface stealthing via polyethylene glycol (PEG) conjugation and targeting ligand functionalization to optimize endocytic uptake kinetics, circumvent intracellular endonuclease-mediated degradation, and mediate temporospatial endosomal escape culminating in efficacious cytoplasmic RNP bioavailability. Highly specific, computationally optimized single-guide RNAs (sgRNAs) were strategically designed to target conserved genetic loci responsible for exopolysaccharide (EPS) biosynthesis pathways, quorum sensing hierarchical networks, c-di-GMP signaling cascades, and multidrug resistance (MDR) efflux systems, all critically orchestrating biofilm maturation, extracellular polymeric substance (EPS) matrix robustness, and phenotypic antibiotic tolerance. Post delivery, extensive transcriptomic rewiring was quantitatively profiled via ultra-deep, strand-specific RNA sequencing (RNA-Seq), employing stringent normalization and differential expression models complemented by integrative systems biology platforms including gene set enrichment analysis (GSEA), weighted gene co-expression network analysis (WGCNA), and regulatory motif discovery to elucidate disruption of transcriptional modules underpinning biofilm resilience and antimicrobial defense. Functional validation encompassing confocal laser scanning microscopy (CLSM) and high-throughput crystal violet biofilm biomass quantification assays confirmed significant attenuation of biofilm architectural integrity, EPS matrix deposition, and MDR phenotype suppression, thereby substantiating the genomic editing efficacy. The integrative theranostic nano-genomic platform described herein exemplifies a revolutionary modality for precision microbial biofilm modulation, enabling programmable, scalable, and biosafe biocontrol interventions in industrial food production lines, advancing microbial ecology engineering frontiers, enhancing product sterility, and mitigating entrenched biofilm-driven contamination risks with far-reaching implications for next-generation antimicrobial therapeutics and food safety technologies.