Allosteric Activation of Cas12a via PAM Topological Engineering for Direct and Rapid Detection of Nucleases

Abstract A contemporary question in the intensely active field of CRISPR‐Cas12a‐based molecular diagnostics is how to simplify the multistep conversion process for detecting nonnucleic acid targets. Herein we describe an allosteric Y ‐shaped DNA structure for C as12a activati o n via p rotospacer‐adjacent motif (PAM) topological e ngineering (Y‐COPE) to achieve straightforward and diverse nuclease monitoring. The newly designed topological structure of the Y‐COPE is characterized by a split PAM embedded at the three‐way junction and protospacers flanking both sides. This unique spatial configuration of the PAM effectively prevents Cas12a activation. Upon target cleavage, the released truncated fragments can dynamically correct the PAM, which promptly restores the dsDNA conformation for Cas12a activation and accomplishes signal output. Theoretical calculation results revealed that, compared with the canonical dsDNA activator, in the Y‐COPE, there was a 1.8 Å increase in the center distance between Lys595 of Cas12a and the PAM, which led to a 24.2 kcal mol −1 increase in binding free energy. This clearly revealed the underlying inhibition mechanism of the topological configuration of the PAM for Cas12a activation. This study advances the understanding of the dynamic response of Cas12a to topological PAM conformations and introduces the universal concept of CRISPR‐based nonnucleic acid detection to benefit the next‐generation molecular diagnostics.