Advances in CRISPR-based molecular biosensing: from pathogen detection to early tumor screening

The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas) system, with its programmable nucleic acid recognition and trans-cleavage-mediated signal amplification capabilities, has evolved from a gene-editing tool into a core technology for next-generation molecular biosensing. This review systematically elucidates the molecular mechanisms of this technology, focusing on the trans-cleavage activity of key proteins such as Cas12, Cas13, and Cas14, in conjunction with the functional characteristics of tool enzymes like Cas9, and the fluorescence, electrochemical, and colorimetric signal readout strategies they employ. Furthermore, this article provides a detailed summary of the application advancements of CRISPR biosensors in the rapid test of pathogens, including viruses, bacteria, and parasites, and early tumor screening, which encompasses the highly sensitive detection of various nucleic acid biomarkers, including gene mutation, methylation, fusion gene, and microRNA. Furthermore, this review summarizes the technological evolution of device integration, from fully automated, closed microfluidics to portable systems, to achieve point-of-care testing. Finally, the article proposes future development directions, including the integration of this technology with nanomaterials and artificial intelligence. This review aims to provide a systematic reference for the further development and application transformation of CRISPR molecular biosensing technology.