Translating CRISPR-based Diagnostics to the Clinical Use: Strategies for Practical, Amplification-free Platform

Recent advances in molecular diagnostics enable precise and objective identification of pathogens at the molecular level.Among these, the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system, in particular, is rapidly emerging as a next-generation molecular diagnostic platform, due to its inherent specificity and programmability.However, constraints in guide RNA (guide ribonucleic acid, gRNA) design, including PAM (protospacer adjacent motif) site requirements and target secondary structure considerations, and insufficient sensitivity continue to hinder its clinical implementation.This review provides a comprehensive overview of CRISPR-based diagnostics for both DNA and RNA targets, with a focus on strategies to overcome PAM constraints, improve target accessibility, and reduce the dependence on pre-amplification.Furthermore, we propose prospective avenues for enhancement, including the integration of sample pretreatment, deep learning-guided gRNA design, and the advancement of multiplexing systems.These advances are expected to accelerate the application of CRISPR diagnostic systems from laboratory research tools into practical clinical platforms, facilitating rapid and effective responses to emerging infectious diseases.