CRISPR/Cas13a-triggered Cas12a biosensing method for ultrasensitive and specific miRNA detection

Area of Science:

• Biotechnology
• Molecular Biology
• Biosensing

Background:

• Ultrasensitive detection of trace targets is crucial in molecular diagnostics.
• CRISPR/Cas systems offer powerful tools for nucleic acid detection.
• Existing methods may lack the required sensitivity for certain clinical applications.

Purpose of the Study:

• To develop a dual-amplified biosensing strategy using CRISPR/Cas13a-triggered Cas12a (Cas13a-12a amplification).
• To demonstrate the strategy's efficacy for the ultrasensitive detection of miRNA-155.
• To evaluate the platform's performance in quantifying miRNA levels in clinical samples.

Main Methods:

• A novel Cas13a-12a amplification strategy was designed.
• Target binding activated Cas13a to cleave a blocker strand, releasing a primer strand.
• The released primer strand activated Cas12a for signal amplification via reporter probe cleavage.

Main Results:

• The Cas13a-12a amplification strategy achieved a detection limit as low as 0.35 fM for synthetic miRNA-155.
• This sensitivity was significantly higher than that of a Cas13a-only assay.
• The method accurately quantified miRNA-155 expression levels in cancer patient samples.

Conclusions:

• The developed Cas13a-12a amplification offers a supersensitive and highly specific platform for miRNA detection.
• This strategy holds significant potential for clinical applications in diagnostics.
• The dual-amplification approach enhances CRISPR-based biosensing capabilities.