Scaffold RNA Reengineering for a Leakage-Suppressed CRISPR/Cas12a Cascade System Toward Precise Nucleic Acid Diagnostics.

Amplification-free CRISPR detection has attracted significant attention for its capacity to circumvent the intricacies of nucleic acid amplification. Yet, the persistent challenge of high background signals remains a critical bottleneck, motivating intensive efforts toward background suppression strategies. Here, we present a 3'-end DNA extension of scaffold RNA (3'D-SR) conformational switch to drive the cascade-activatable and leakage-suppressed Cas12a system, a mechanism we denote as CALCas. By engineering the crRNA of Cas12a system and introducing steric hindrance as an activation barrier, we reshape crRNA into a "threshold-controlled" smart molecular switch, enabling Cas12a to shift from "inhibition" to "precise activation". Unlike other Cas cascade reaction generally suffer from internal self-activation that leads to uncontrolled background increase, this newly developed framework, leveraging a DNA-mediated allosteric regulation mechanism and a leakage-suppressed design based on inherent steric hindrance, effectively solves the dilemma between signal enlargement and minor background variation. Through rational design, this cascaded CRISPR system achieves femtomolar sensitivity (over 10,000-fold higher than single Cas12a system), enables rapid HPV16/18 detection, and reduces assay time to under 30 min. This engineered crRNA approach unlocks a new pathway to reprogram Cas12a's enzymatic activities with a stabilized benchmark signal that can be critical in precise quantitative nucleic acid detection.