A Single‐Enzyme Activated CRISPR‐Cas12a Nano System via Subtly Balanced dsDNA for Kinetic‐Gated UDG Detection and Spatiotemporal Cellular Imaging

ABSTRACT Uracil‐DNA glycosylase (UDG) is a key enzyme in base excision repair and an important biomarker for genomic stability and disease. In many reported sensing systems, uracil excision is coupled to signal generation through additional downstream BER processing steps, resulting in an indirect readout of UDG activity. Here, we report a single‐enzyme activated CRISPR‐Cas12a nanosystem driven by a subtly balanced double‐stranded DNA (dsDNA) substrate. This dsDNA serves as a kinetic gatekeeper that maintains Cas12a in an inert state until UDG‐mediated uracil excision disrupts the balance, lowering the energy barrier for crRNA invasion and initiating Cas12a trans‐cleavage. This conformationally gated mechanism directly converts a uracil excision event into an amplified CRISPR response without requiring sequential enzymatic processing. The system achieves a 1840‐fold discrimination ratio and an ultralow detection limit of 5 × 10 −7 U/mL. Furthermore, a genetically encoded variant enables nuclear localization of Cas12a and dsDNA sensors for in situ imaging of endogenous UDG. The platform visualizes UDG dynamics across distinct cell cycle phases, realizing spatiotemporal mapping of repair activity in living cells. This work introduces a new activation paradigm for CRISPR‐Cas12a via subtly balanced dsDNA and establishes a generalizable strategy for precise molecular sensing in complex biological environments.