Advancing CRISPR-based biosensors for enhanced surveillance of emerging pathogens

Abstract

Sensitive and specific on-site detection of emerging pathogens is pivotal for improving surveillance of contaminants across agricultural, food, and clinical settings. CRISPR technology offers promising molecular detection in combination with isothermal nucleic acid amplification and novel transduction strategies for facile signal recognition, promising programmable and scalable assays for the rapid and on-site identification of emerging pathogens of concern. This dissertation advances CRISPR-based biosensing technologies for rapidly detecting antimicrobial resistance, bacteria, and viruses in complex matrices. Three unique CRISPR-based assays were developed by combining different CRISPR systems with nucleic acid amplification and innovative nanomaterials for visual detection. First, a CRISPR-Cas12a gold nanoparticle (AuNP) biosensor was developed for the rapid and multiplexed detection of three antimicrobial resistance genes. Second, a graphene-oxide CRISPR-Cas12a system was engineered for the fluorescent detection of bacterial infections for sepsis prevention. Third, a CRISPR-AsCas12f split-G-quadruplex (split-G4) sensor was developed for the rapid and user-friendly on-site detection of norovirus in leafy greens. Lastly, a microfluidic chip is presented for quantitative pathogenic RNA detection utilizing loop-mediated isothermal amplification (LAMP) and passive digitization in a portable device. Collectively, the presented developments highlight the versatility of CRISPR sensing platforms for the next frontier of molecular diagnostics. The technologies established in this thesis demonstrate strong analytical sensitivity, molecular design flexibility, and broad applicability across clinical, agricultural, and food surveillance.