Flexible regulation of CRISPR/Cas12a activity by spatial confinement effect

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostic Assays

Background:

• CRISPR/Cas12a trans-cleavage activity is crucial for molecular diagnostics.
• Current methods using freely diffusing CRISPR/Cas12a components require complex designs and modifications.
• There is a need for simpler, more broadly applicable CRISPR/Cas12a systems.

Purpose of the Study:

• To demonstrate a novel method for modulating CRISPR/Cas12a activity using spatial confinement on fluid membranes (CAS-FLIER).
• To explore the impact of crRNA length and reporter accessibility on Cas12a trans-cleavage.
• To develop a sensitive and point-of-care diagnostic assay for HIV RNA detection.

Main Methods:

• Utilized spatially confined Cas12a on fluid membranes (CAS-FLIER).
• Adjusted crRNA length and duplex-strand reporter characteristics to modulate Cas12a activity.
• Incorporated a DNA reverse-transcriptor for one-pot HIV RNA detection.
• Adapted the assay to a lateral-flow format for visual readout.

Main Results:

• Demonstrated precise, scalable control over Cas12a trans-cleavage activity by tuning membrane confinement and reporter accessibility.
• Achieved co-activation of confined Cas12a by both single-stranded DNA (ssDNA) and RNA inputs.
• Developed a highly sensitive, one-pot assay for HIV RNA detection using the CAS-FLIER system.
• Validated the assay's compatibility with a lateral-flow format for visual, point-of-care use.

Conclusions:

• Spatial confinement of Cas12a on fluid membranes offers a simple yet effective strategy for modulating its trans-cleavage activity.
• The CAS-FLIER system overcomes limitations of freely diffusing systems, enabling novel activation mechanisms.
• The developed HIV RNA detection assay shows significant potential as a point-of-care diagnostic tool.