One-pot CRISPR diagnostics for the rapid detection of Candida species and antimicrobial resistance biomarkers

Antimicrobial resistance (AMR) is a growing public health threat, where drug-resistant Candida infections are associated with increased hospital costs, lengths of stay, and high mortality rates. Current diagnostic methods are slow and labor-intensive, causing delays in diagnosis and patient treatment. In this research, we propose a rapid CRISPR-based diagnostic for identifying Candida pathogens and antifungal resistance biomarkers in clinical applications. One-pot CRISPR assay designs are optimized for three clinically relevant Candida species, achieving attomolar sensitivity with synthetic DNA and validating performance on cultured isolates. Strategies are developed to engineer CRISPR gRNA to control one-pot reaction kinetics, enhancing discrimination of single nucleotide polymorphism (SNP) resistance mutations associated with azole and echinocandin antifungals. This research expands our understanding of the design of one-pot CRISPR reactions for sensitive detection of DNA, while establishing a foundation for faster, more precise AMR diagnostics to improve surveillance and outbreak management of fungal infections.