Proximity-Inducible CRISPR/Cas12a Activity by Scaffold RNA Assembly for Sensing Applications

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostic Assays

Background:

• The CRISPR/Cas12a system is a programmable tool for diagnostics.
• Current engineered crRNA strategies face limitations in target range, specificity, and complexity.

Purpose of the Study:

• To develop an advanced crRNA strategy overcoming limitations of existing CRISPR/Cas12a diagnostic systems.
• To create a versatile platform for detecting diverse molecular targets with high specificity.

Main Methods:

• Developed a proximity-assembly and activate (PAA) strategy using split dumbbell activators.
• Engineered terminally modified target-binding modules that reassemble on scaffold RNA.
• Activated Cas12a trans-cleavage activity through reconstructed functional crRNA.

Main Results:

• Achieved universal detection of both nucleic acid and non-nucleic acid targets.
• Demonstrated reduced background signal and suppressed nonspecific leakage due to strict target dependency.
• Successfully detected miRNA-21, ATP, and anti-Dig antibody with single-base discrimination.
• Detected endogenous miRNA-21 in breast cancer patient samples, distinguishing them from healthy controls.

Conclusions:

• The PAA strategy presents a modular, plug-and-play, and versatile platform for molecular diagnostics.
• This approach significantly enhances specificity and operational simplicity for CRISPR/Cas12a-based assays.
• The platform holds substantial potential for advancing clinical diagnostics and precision medicine.