Amplification-Free CRISPR Diagnostics for Point-of-Care Testing

CRISPR-based diagnostics integrated with nucleic acid pre-amplification have demonstrated profound potential for single-molecule detection. However, the pervasive risk of aerosol contamination during amplification significantly hinders their translation to point-of-care testing (POCT). Although amplification-free CRISPR diagnostics promise a streamlined “sample-to-answer” workflow, their development remains in the nascent stages due to the sluggish cleavage kinetics of natural Cas enzymes and the diffusion limitations of trace targets in homogeneous systems. This review systematically summarizes recent core technological advancements, including molecular engineering of CRISPR/Cas systems, novel signal transduction enhancement mechanisms, and digital detection methodologies based on spatial confinement effects. Furthermore, addressing the “matrix effect” that often compromises analytical sensitivity in clinical scenarios, we highlight advanced pre-treatment strategies for complex biological samples. Finally, we propose that the future of POCT relies on the synergy of multiplexed detection, AI-assisted analysis, and microfluidic integration to ultimately bridge the gap between laboratory innovation and clinical application.