Allosteric ribozyme-driven crRNA switch for the amplification-free detection of biomolecules.

Currently, CRISPR-mediated biosensors are concentrating on the design of the crRNA or the activator strand to regulate the trans-cleavage activity of Cas12a. Herein, we report an allosteric ribozyme-driven crRNA switch-regulated CRISPR/Cas12a sensor for amplification-free detection of biomolecules. An allosteric ribozyme is meticulously engineered to connect the target recognition sequence with the 5' binding arm of the hammerhead ribozyme, resulting in the formation of a hairpin structure through complementary hybridization. The presence of target induces the conformational change in the allosteric module and disrupts the hairpin structure, restoring multiple-turnover cleavage RNA activity of ribozyme. Then, the activated ribozyme specifically cuts the cleavage site of the substrate-locked crRNA and releases the native crRNA to initiate CRISPR/Cas12a functions for signal reporting. The reported biosensor exhibited high sensitivity and excellent specificity for miR-155 and adenosine triphosphate (ATP) detection, giving the detection limits of 256 fM and 160 nM, respectively. For clinical validation, our proposed strategy can quantify miR-155 expression levels in cells and serum of cancer patients. Furthermore, we also demonstrate that the allosteric ribozyme-driven crRNA switch can be easily compatible with lateral flow assays, realizing visualization and the portable monitoring of target. Hence, the biosensor not only has outstanding potential in point-of-care testing, but also enables the detection of various biomolecules by flexibly substituting target recognition sequences for molecular diagnosis in the clinic.