Mini crRNA-mediated CRISPR/Cas12a system (MCM-CRISPR/Cas12a) and its application in RNA detection

Area of Science:

• Biotechnology
• Molecular Biology
• Biosensing

Background:

• Non-coding RNAs play crucial roles in disease development.
• Accurate detection of non-coding RNAs is essential for disease diagnostics.
• Traditional CRISPR/Cas12a systems require DNA targets, limiting direct RNA detection.

Purpose of the Study:

• To develop a CRISPR/Cas12a-based system for direct non-coding RNA detection.
• To enhance the sensitivity and applicability of non-coding RNA detection methods.
• To overcome the limitations of standard CRISPR/Cas12a systems in RNA analysis.

Main Methods:

• Proposed a mini crRNA-mediated CRISPR/Cas12a system (MCM-CRISPR/Cas12a) by modifying crRNA structure.
• Integrated MCM-CRISPR/Cas12a with hybridization chain reaction (HCR) and cascade signal amplification (CHA).
• Validated the system's specificity and sensitivity using microRNA (miRNA) and long non-coding RNA (lncRNA) targets.

Main Results:

• MCM-CRISPR/Cas12a detected non-coding RNA at picomolar (pM) levels.
• The combined HCR and CHA amplification achieved femtomolar (fM) detection limits for miRNAs and lncRNAs.
• High spiked recovery rates (104.7–109.4%) in human serum demonstrated excellent applicability.

Conclusions:

• The MCM-CRISPR/Cas12a system effectively overcomes the limitations of typical CRISPR/Cas12a for direct non-coding RNA detection.
• The integration with HCR and CHA provides a highly sensitive and specific platform for non-coding RNA analysis.
• This approach holds significant potential for sensitive biomarker detection in disease diagnostics.