Application of Cas9-mediated Core-genome-wide Targeted Sequencing for pathogen detection

Accurate and rapid detection of pathogens is vital for optimal and timely treatment in clinical practice. Currently available methods based on Next-Generation Sequencing (NGS) to detect pathogens have various drawbacks, including limited sensitivity, high cost, long experiment times and/or low flexibility. In this study, we developed COMETS (Cas9-mediated Core-genome-wide Targeted Sequencing), an accurate, scalable and cost-effective pathogen identification workflow. Taking advantage of the programmability and flexibility of CRISPR-Cas9 system, COMETS could enrich multiple species-specific regions in core-genome wide. Using genomic DNA of 11 common species, we determined the feasibility of COMETS, showing 437.1-fold enrichment on average. Furthermore, we evaluated the specificity and sensitivity of COMETS, which exhibited no cross-reactivity among close-related species and a detection limit ranging from 222.5 to 1308.3 copies/reaction. Finally, COMETS was applied on respiratory samples of patients and successfully identified most of the clinical isolates. These results indicate that COMETS could serve as a promising tool for diagnosis of infections in the future.