Blocker-Strand Continuity Regulates CRISPR/Cas12a Activity for Dual-Enzyme-Responsive Fluorescence Sensing

Abstract CRISPR/Cas12a is widely used for nucleic acid analysis because it is programmable and provides trans-cleavage-based signal amplification. However, precise control of Cas12a activity remains important for reliable sensing. Here, we examined how blocker continuity affects Cas12a regulation by comparing continuous and split blockers with the same total number of complementary bases. A continuous ssDNA blocker spanning both the spacer and repeat-derived handle regions stabilized the caged crRNA state more effectively. The split design was less inhibitory, consistent with the heterogeneous complexes and reduced stability predicted by thermodynamic calculations. We introduced uracil sites into the continuous blocker so that sequential processing by two base excision repair (BER) enzymes could disrupt its continuity. This design yielded a dual-enzyme-responsive platform, termed DualEnz-Cas12a. Sequential processing by UDG and APE1 restored Cas12a activity and enabled sensitive detection of either enzyme when the other was supplied in excess. The platform was also applied to proof-of-concept inhibitor testing and analysis of coupled UDG/APE1 activity in cell lysates. These results identify blocker continuity as a useful design parameter for low-leakage, input-responsive Cas12a biosensors. They also provide a general strategy for controlling CRISPR-based diagnostic systems.