An optimized microRNA detection platform based on PAM formation-regulated CRISPR/Cas12a activation.

Area of Science:

• Biotechnology
• Molecular Biology
• Biosensing

Background:

• MicroRNAs (miRNAs) are crucial biomarkers for disease diagnosis and prognosis, particularly in cancer.
• CRISPR/Cas12a systems combined with hybridization chain reaction (HCR) offer a promising avenue for sensitive miRNA detection.

Purpose of the Study:

• To compare two hairpin DNA design strategies for CRISPR/Cas12a-HCR systems to enhance miRNA detection compatibility and sensitivity.
• To develop an optimized miRNA detection platform utilizing the most effective HCR strategy.

Main Methods:

• Comparison of two hairpin DNA designs in HCR for CRISPR/Cas12a compatibility.
• Optimization of probe sequences and reaction conditions for a miRNA detection platform.
• Evaluation of the platform's ability to detect different miRNAs, single-stranded DNA, and base mutations.

Main Results:

• The "PAM Formation" strategy, where PAM sites form after HCR, significantly improved detection sensitivity compared to other designs.
• An optimized miRNA detection platform was developed, capable of identifying various miRNAs by modifying probe sequences.
• The platform demonstrated reliability in detecting target miRNA expression levels across different cell lines and distinguishing DNA mutations.

Conclusions:

• The developed CRISPR/Cas12a-HCR biosensor platform, particularly with the "PAM Formation" strategy, offers high sensitivity and versatility for miRNA detection.
• This platform shows significant potential for early and accurate clinical diagnosis of cancers and other diseases.
• The system's ability to detect mutations and differentiate miRNA expression levels underscores its diagnostic utility.