Detection of target DNA with a visual CRISPR-associated hyperbranched rolling circle amplification technique

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• Nucleic acid detection is crucial for diagnostics.
• Existing methods may lack specificity or require complex thermal cycling.
• CRISPR and Hybridization Chain Reaction Amplification (HRCA) offer unique advantages.

Purpose of the Study:

• To develop a novel, highly sensitive, and specific nucleic acid detection technique.
• To integrate CRISPR-Cas9 specificity with the advantages of HRCA.
• To validate the new technique for detecting human papillomavirus (HPV) genes.

Main Methods:

• CRISPR-associated HRCA technique (CART) was developed.
• Target DNA is recognized and cleaved by Cas9/sgRNA.
• Cleaved products are circularized and amplified via isothermal HRCA.

Main Results:

• CART demonstrated high specificity due to Cas9/sgRNA's single-base mismatch recognition.
• HRCA provided isothermal reaction conditions and high amplification efficiency.
• The technique successfully detected various human papillomavirus (HPV) genes with high sensitivity.

Conclusions:

• CART is a novel and effective method for nucleic acid detection.
• The technique combines the strengths of CRISPR and HRCA for improved performance.
• CART shows promise for sensitive and specific molecular diagnostics, including HPV detection.