CRISPR-based next-generation molecular diagnostics for bone infection.

Bone and joint infections, notably osteomyelitis and periprosthetic joint infections, present a profound clinical challenge characterized by severe morbidity, prolonged hospitalizations, and substantial healthcare costs. Effective management is persistently hindered by diagnostic uncertainty. Conventional microbiological cultures yield false-negative results in up to 50% of cases, largely due to prior antibiotic exposure, biofilm formation, and fastidious organisms. Concurrently, the escalating prevalence of antimicrobial resistance (AMR) among orthopedic pathogens demands rapid and highly accurate diagnostic alternatives. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) technologies have recently emerged as robust nucleic acid detection platforms. These systems offer attomolar sensitivity and single-nucleotide specificity while remaining compatible with portable, instrument-free readout formats. In this review, we critically evaluate the current diagnostic landscape of bone infections, highlighting the limitations of conventional and early molecular methods. We specifically examine how the distinct mechanistic properties of varied Cas effectors (Cas12, Cas13, Cas14, and CasΦ) can transform clinical diagnostics through single-nucleotide polymorphism (SNP) discrimination, RNA-based viability assessment, and PAM-independent detection. Furthermore, we explore the application of these features in stratifying pathogens within culture-negative and polymicrobial infections, directly identifying AMR genes for targeted therapy, and facilitating point-of-care testing in intraoperative environments. Finally, we assess the primary barriers to clinical translation, namely, complex sample preprocessing in bone matrices, the lack of standardized diagnostic thresholds, and the need for prospective studies demonstrating improved patient outcomes, ultimately outlining essential research priorities to transition CRISPR diagnostics from laboratory proof-of-concept to routine orthopedic practice.