Proximity-activated guide RNA of CRISPR–Cas12a for programmable diagnostic detection and gene regulation

Abstract The flexibility and programmability of CRISPR–Cas technology have made it one of the most popular tools for biomarker diagnostics and gene regulation. Especially, the CRISPR–Cas12 system has shown exceptional clinical diagnosis and gene editing capabilities. Here, we discovered that although the top loop of the 5′ handle of guide RNA can undergo central splitting, deactivating CRISPR–Cas12a, the segments can dramatically restore CRISPR function through nucleic acid self-assembly or interactions with small molecules and aptamers. This discovery forms the basis of an engineered Cas12a system with a programmable proximity-activated guide RNA (PARC–Cas12a) that links targets of interest to dsDNA. Leveraging the efficient trans- and cis-cleavage of Cas12, our findings further inspired a detection platform design for RNAs or non-nucleic acid biomarkers, enabling highly sensitive and multiplexed analysis. We further demonstrated the feasibility of RNA-controllable gene knockout/knockdown in Escherichia coli. Notably, we successfully validated the gene regulatory capabilities of the PARC–Cas12a system within mammalian cell systems by utilizing the classical theophylline molecule–aptamer system. Our results introduce a programmable toolbox for precise diagnostics and cell regulation, allowing the development of versatile diagnostic tools, complex synthetic biological circuits, and cellular biosensors.