Next-generation CRISPR-based diagnostic tools for human diseases

Accurate diagnosis of human diseases is crucial for effective prevention, clinical treatment, and prognosis. The World Health Organization (WHO) has provided guidelines for molecular diagnostic methods, suggesting that the ideal method should meet the ASSURED criteria: affordable, sensitive, specific, user-friendly, rapid, robust, equipment-free, and deliverable to end-users.However, traditional molecular detection methods have certain limitations, including the need for expensive reagents and equipment, skilled technicians, lengthy processing time, high labor intensity, low sensitivity, and limited applicability in remote areas and real-time scenarios. The rapidly evolving CRISPR/Cas systems have revolutionized molecular diagnostics by providing a low-cost, ver-satile, and powerful toolbox. In this review, we will first introduce the general working principle of the CRISPR toolbox in diagnosis. We will then present various nucleic acid and non-nucleic acid detection methods based on CRISPR/Cas systems for diagnosing both infectious and non-infectious diseases, with a particular focus on the detection of single nucleotide polymorphisms (SNPs). Finally, we discuss the challenges faced by CRISPR mo-lecular diagnostics and expect more groundbreaking applications in disease diagnostics through rational design and multidisciplinary engineering to enable multiplexed, preamplification-free, point-of-care testing (POCT) and continuous monitoring of disease.