Two CRISPR/Cas12a-based methods for fast and accurate detection of single-base mutations

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• Current single-base mutation detection methods are inefficient, requiring significant time, labor, and cost.
• There is a critical need for rapid, accurate, and cost-effective technologies for detecting single-base alterations.

Purpose of the Study:

• To develop and validate two distinct CRISPR/Cas12a-based approaches for highly specific single-base mutation detection.
• To achieve 100% differentiation between mutant and wild-type strains using fluorescence-based detection.

Main Methods:

• Method 1: Combined Polymerase Chain Reaction (PCR) and CRISPR/Cas12a cleavage, utilizing 3' end primer mismatches to block wild-type amplification.
• Method 2: Isothermal Recombinase Polymerase Amplification (RPA) coupled with CRISPR/Cas12a, employing an introduced mismatched allele-specific (IMAS-RPA) strategy to prevent wild-type product cleavage.

Main Results:

• Method 1 demonstrated high sensitivity (one copy, approx. 6 copies/μL) with easily adjustable parameters.
• Method 2 provided a rapid (within 1 hour) and user-friendly alternative for mutation detection.
• Both methods successfully differentiated single-base mutations from wild-type strains via fluorescence signals.

Conclusions:

• Two distinct, highly specific, and efficient CRISPR/Cas12a-based methods for single-base mutation detection have been established.
• These methods offer flexibility, allowing selection based on laboratory environment and specific needs.
• The developed approaches hold promise for future applications in clinical diagnostics.