CrispHunter-enabled systematic crRNA design and structure-optimized hairpin probes for enhanced CRISPR-Cas12a...

Effective control of genetically diverse pathogens necessitates rapid and accurate diagnostics, as their genomic variability undermines assay reliability and complicates public health interventions. Herein, we introduce a CRISPR-Cas12a-based molecular diagnostic platform integrating robust in silico crRNA design via our CrispHunter pipeline with a high-performance detection module featuring structure-optimized hairpin probes. We validated this approach using Burkholderia pseudomallei, whose high pathogenicity, genetic diversity, and diagnostic evasion exemplify the challenges of detecting heterogeneous pathogens. Applying CrispHunter to 3245 publicly available B. pseudomallei genomes, we systematically identified 12 highly conserved crRNA candidates and optimized a penta-crRNA strategy that reduced the limit of detection (LOD) from the previously reported 100 pM to 7.5 pM, thereby minimizing the risk of false negatives due to pathogen genomic diversity. Engineered hairpin-structured molecular beacons with iteratively optimized loop and stem lengths further enhanced assay performance, lowering the LOD to 2.1 pM without pre-amplification while improving signal-to-noise ratio by 42-fold, response sensitivity by 47-fold, and reducing time-to-positive from 56 to 3 min with excellent specificity. Employing recombinase polymerase amplification further enhanced sensitivity, lowering the LOD to 14.82 copies/μL, which is significantly below the concentrations typically found in clinical specimens. The modular architecture of CrispHunter, together with the optimized CRISPR-Cas12a detection platform, provides a versatile framework for rapid, sensitive, and specific molecular diagnostics. This strategy enhances detection of B. pseudomallei and can be readily adapted for the diagnosis of other genetically diverse pathogens, supporting broader applications in infectious disease management and public health.