TopCas: Topology‐Gated Cas12a via DNA‐RNA Chimeric Circular crRNA for Amplification‐Free Nucleic Acid Detection and Conditional Gene Editing

ABSTRACT Controlled activation of CRISPR‐Cas12a is critical for achieving conditional gene editing and molecular diagnostics. As an indispensable component for forming an active complex, CRISPR RNA (crRNA) represents a key route to regulate Cas12a activity. Here, we establish TopCas (Topology‐gated Cas12a via DNA‐RNA Chimeric Circular crRNA) as a platform for preamplification‐free nucleic acid detection and conditional gene editing. Within TopCas, the circular crRNA sterically constrains Cas12a's nuclease activity until target‐activated complexes trans‐cleave the DNA segment of the chimeric crRNA, converting the circular guide into its linear form and initiating an autocatalytic cascade that culminates in fluorophore release and signal amplification. By the same mechanism, the system conditionally activates Cas12a's gene‐editing function (cis‐cleavage) exclusively in the presence of specific nucleic acid targets (e.g., viral DNA or RNA). We demonstrate that TopCas affords high specificity and sensitivity in nucleic acid detection, supports accurate detection in clinical viral nucleic acid samples, and shows potential for in vivo real‐time molecular imaging, while also demonstrating the feasibility of conditional gene editing. This innovative chimeric circular crRNA‐Cas12a system not only provides a new tool for precise disease diagnostics but also offers a promising strategy for personalized therapeutic intervention.