Solid-Phase Collateral Cleavage System Based on CRISPR/Cas12 and Its Application toward Facile One-Pot Multiplex...

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• The CRISPR/Cas12 system offers potential for advanced nucleic acid detection.
• Cas12's collateral cleavage activity upon dsDNA recognition is key for signal transduction.
• Current methods for genotyping can be complex and time-consuming.

Purpose of the Study:

• To develop a simplified, one-pot multiplex dsDNA detection method using CRISPR/Cas12.
• To apply solid-phase collateral cleavage (SPCC) for enhanced genotyping.
• To create a sensitive and specific dsDNA sensor with minimal operational steps.

Main Methods:

• Developed a sensor utilizing immobilized Cas12-crRNA and ssDNA reporters on a solid phase.
• Implemented a solid-phase collateral cleavage (SPCC) reaction for signal generation.
• Designed a dual-target dsDNA sensor with separate spots for Cas12-crRNA and fluorophore-labeled ssDNA reporters.

Main Results:

• Achieved one-pot multiplex dsDNA detection with SPCC, reducing fluorescence by 42.1-57.3% upon target binding.
• Demonstrated high specificity with two-base resolution, distinguishing between different DNA sequences.
• Established a detectable concentration for target dsDNA as low as 10^-9 M.

Conclusions:

• The SPCC-based CRISPR/Cas12 system enables efficient and specific one-pot multiplex dsDNA detection.
• This approach significantly simplifies operational steps for nucleic acid analysis.
• Future applications include comprehensive genotyping using sensor arrays and automated reagent immobilization.