CRISPR/Cas12a Collateral Cleavage-Driven Transcription Amplification for Direct Nucleic Acid Detection

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• CRISPR/Cas systems offer precise nucleic acid detection.
• Current CRISPR/Cas diagnostics often require preamplification, limiting sensitivity and introducing complexities.
• There is a need for direct, highly sensitive nucleic acid detection methods.

Purpose of the Study:

• To develop a preamplification-free method for Hepatitis B virus (HBV) DNA detection.
• To enhance the sensitivity of CRISPR/Cas12a detection using a light-up RNA aptamer system.
• To establish a universal sensing platform for ultrasensitive nucleic acid detection.

Main Methods:

• Combined CRISPR/Cas12a with a light-up RNA aptamer transcription for signal amplification.
• Designed DNA templates for Broccoli RNA aptamer and kleptamer (Kb) RNA transcription.
• Utilized HBV target recognition to trigger Cas12a cleavage and subsequent aptamer amplification.

Main Results:

• Achieved a detection limit of 22.4 fM for HBV dsDNA.
• Demonstrated excellent sensitivity, specificity, accuracy, and stability in human serum samples.
• The method successfully detected target HBV dsDNA without nucleic acid preamplification.

Conclusions:

• The developed strategy enables direct and ultrasensitive nucleic acid detection.
• The light-up RNA aptamer transcription significantly enhances signal output.
• This approach serves as a versatile platform for advanced nucleic acid-based diagnostics.