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

ABSTRACT

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.