Direct microRNA detection via topologically engineered CRISPR/Cas12a cascade amplification assay

Area of Science:

• Molecular Biology
• Biotechnology
• Biosensing

Background:

• CRISPR/Cas12a is a potent biosensing tool, but direct RNA detection is challenging due to its DNA-targeting nature.
• Existing methods often require RNA amplification steps like reverse transcription.

Purpose of the Study:

• To develop a one-pot CRISPR/Cas12a assay for direct microRNA detection without amplification.
• To enable sensitive and specific quantification of microRNA-21.

Main Methods:

• Utilized a molecular switch probe (MSP) to recognize miRNA-21 and activate Cas12a.
• Incorporated an amplifier probe (AMP) for a self-driven cascade signal amplification.
• Developed a one-pot assay for direct RNA detection.

Main Results:

• Achieved a low detection limit of 2.88 pM for miRNA-21.
• Demonstrated high accuracy (95.5%-108.6% recovery) and precision (2.35%-7.18% RSD).
• Showcased excellent specificity against homologous miRNAs and successfully quantified elevated miRNA-21 in lung cancer cells.

Conclusions:

• The developed CRISPR/Cas12a biosensor offers a simple, reliable method for direct RNA detection.
• This approach bypasses the need for target amplification, streamlining the detection process.
• The assay shows potential for clinical applications, such as cancer diagnostics.