A proximity-allosteric Adapter unlocks Cas12a exponential amplification for versatile homogeneous assay

The integration of proximity-triggered homogeneous assays with CRISPR/Cas12a offers a promising route to "mix-and-read" bioanalysis, yet early efforts have faced substantial limitations. Proximity-based recognition enabled high specificity, but often introduced steric hindrance that severely suppressed Cas12a activation. Meanwhile, conventional Cas12a systems relied on linear trans-cleavage, which struggled to achieve the aM-level sensitivity required for early disease detection. It is clear that a CRISPR/Cas12a-based assay is needed that overcomes steric hindrance, achieves exponential signal amplification, and maintains the simplicity of homogeneous detection.

Herein, we report a dual-functional "Smart Adapter" that seamlessly couples proximity recognition with Cas12a exponential amplification. The Smart Adapter operates as a kinetically trapped molecular cage that effectively sequesters the Cas12a activator to ensure minimal background. Target binding induces a proximity-driven allostery that unlocks this cage, liberating the activator from steric constraints to initiate Cas12a activity. This primary activation serves as a catalytic seed, triggering a cascading destabilization of the remaining Adapters. By acting as both an allosteric activator and a signal propagator, the Adapter ensures high-specificity transduction and ultrasensitive amplification. The resulting Smart Proximity-Activated Cas12a Exponential Amplification (SPACE) platform achieves a detection limit (LOD) as low as 500 aM for the BCR/ABL p210 gene and enables precise discrimination of p210 minor subtypes in clinical samples. Furthermore, the platform attains a detection sensitivity of 100 fg/mL for p-Tau217 protein.

This versatile strategy provides a robust framework for precise low-abundance biomarker detection, resolving CRISPR/Cas12a bottlenecks and offering a reliable mix-and-read platform for early diagnosis.