Flexible regulation of CRISPR/Cas12a activity by spatial confinement effect

Precise regulation of the trans-cleavage activity of CRISPR/Cas12a has substantially expanded its utility in molecular diagnostics. However, existing strategies rely predominantly on systems with freely diffusing components, necessitating intricate CRISPR RNA (crRNA) designs or specialized chemical modifications, which hinder their simplicity and broader applicability. Here, we demonstrate that the activity of spatially confined Cas12a on fluid membranes (CAS-FLIER) can be facilely modulated by simply adjusting the length of crRNA and the duplex-strand reporters. We reveal that fine-tuning the movement range of membrane-Cas12a and the accessibility of the reporter to Cas12a enables precise, scalable control over trans-cleavage activity. As a proof of concept, we show that the activity of confined Cas12a can be co-activated by single-stranded DNA (ssDNA) and RNA inputs, a capability that remains unattainable in conventional freely diffusing systems. Furthermore, by incorporating a DNA reverse-transcriptor into the CAS-FLIER system, we achieve one-pot, highly sensitive detection of HIV RNA, supporting accurate diagnosis of HIV infection. Notably, this assay is compatible with a lateral-flow format for direct visual readout, highlighting its potential as a point-of-care diagnostic tool for HIV. Collectively, our findings shed new light on modulating Cas12a activity, advancing its applications in molecular diagnostics.