A Thermodynamically Asymmetric Molecular Switch for Reversible Blockade of Split crRNA-Mediated CRISPR-Cas12a Enables Logic-Gated Detection of Dual Biomarkers

The identification of split crRNA functionality in Cas12a has prompted the development of various chemically modified systems for regulating its activity. Although these approaches are useful, they are often expensive, technically demanding, and Cas12a function may be compromised after deblocking. In the present study, instead of modifying the split crRNA, we demonstrate that a simple ssDNA suppressor can inactivate the split crRNA-mediated CRISPR–Cas12a system by hybridizing with the tcrRNA scaffold. The influence of suppressor length and hybridization position on suppression efficiency was systematically examined. Inhibition was further designed to be reversible by incorporating an abasic site into the blocker: APE1 cleaves this site, destabilizes the shorter flanking segment, and liberates tcrRNA, thereby reactivating Cas12a. Exploiting this mechanism, we established a logic-gated sensing platform named STC (suppressor-mediated tcrRNA scaffold blocking for CRISPR–Cas12a logic-gated biosensing platform), which supports simultaneous detection of APE1 (a non-nucleic acid analyte) and miRNA (a nucleic acid analyte) under isothermal conditions. A thermodynamically asymmetric molecular switch was employed to reduce background leakage while retaining efficient target-induced activation. The platform exhibits high sensitivity and specificity along with strong programmability. With a reagent cost estimated at only $0.21 per test, this method offers a straightforward and inexpensive means of controlling split crRNA-based Cas12a activity and represents a versatile tool for CRISPR-enabled molecular diagnostics.