Programming CRISPR Compartmentalization on DNA Origami for Multiplexed Biosensing

CRISPR-Cas12a has emerged as a transformative tool for nucleic acid detection and molecular diagnostics. However, achieving one-pot, orthogonal, multiplexed sensing remains challenging due to the nonspecific trans-cleavage activity of Cas12a. Here, we present a modular DNA origami-based platform that spatially confines and independently controls Cas12a activity, enabling simultaneous detection of multiple DNA targets within a single reaction. This spatial programming approach enables orthogonal one-pot detection of three DNA targets (EGFR L858R/Del19 mutations and HPV16) with minimal cross-talk, demonstrating 60% cleavage efficiency within 120 min at 100 nM. Through integrated molecular dynamics simulations and experimental validation, we identify an optimal ∼6 nm spatial window that maximizes cleavage efficiency, while the platform maintains robust performance in complex biological environments with a 10 nM detection limit in 20% serum. This compartmentalization strategy establishes a framework for spatially programming multi-enzyme systems on DNA nanostructures for synthetic biological circuits.