Input-Triggered Allosteric Activation of CRISPR–Cas12a by Modular Loop-Engineered Hairpins

Achieving precise control over CRISPR-Cas12a activity remains a fundamental challenge in the development of versatile sensing platforms, particularly for applications in diagnostics. Herein, we report an allosteric strategy that employs modular loop-engineered hairpin (MLEH) to precisely control Cas12a activation, thereby establishing a plug-and-play sensing framework with enhanced versatility. The MLEH comprises both the loop‑embedded ssDNA activator sequence and its complementary blocking domain. The input-triggered linearization of the MLEH via toehold-mediated strand displacement governs the accessibility of the activator, thereby controlling the binding and activation of Cas12a. A distinct advantage of this strategy is its elimination of the reliance on protospacer adjacent motif (PAM) sequences, customized CRISPR RNA (crRNA), or external activators. By merely substituting the responsive region of the MLEH, the system can be flexibly reconfigured to detect a wide range of targets, encompassing nucleic acids, proteins, and small molecules. MLEH-Cas12a broadens the scope of CRISPR technologies, offering a robust tool for diverse diagnostic applications. Collectively, our work delineates a novel allosteric regulatory mechanism for Cas12a activation and highlights the potential of engineered hairpins as key components in next-generation molecular sensing platforms, overcoming current limitations in CRISPR-based diagnostics and enabling broader target detection.