Lanthanide Metal-Organic Framework-Integrated CRISPR-Cas Technology for Amplification-free Gene Mutation Assay

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Materials Science

Background:

• Nucleic acid amplification is a significant challenge in CRISPR-based diagnostics, affecting speed, simplicity, and accuracy.
• Existing methods often require complex amplification steps, hindering rapid point-of-care applications.

Purpose of the Study:

• To develop an amplification-free CRISPR-Cas12a diagnostic platform for sensitive and specific gene mutation detection.
• To integrate lanthanide metal-organic frameworks (Ln-MOFs) for a novel fluorescence-based readout mechanism.

Main Methods:

• A CRISPR-Cas12a system was designed to trigger alkaline phosphatase (ALP) release upon target recognition.
• Released ALP hydrolyzed p-nitrophenyl phosphate (pNPP), generating phosphate ions.
• Phosphate ions modulated the fluorescence of a Eu-TPTC (lanthanide metal-organic framework) sensor, enabling ratiometric detection.

Main Results:

• The platform achieved amplification-free detection of the BRAF V600E mutation with a sensitivity of 0.1 pM.
• Demonstrated high specificity and anti-interference capabilities.
• Clinical sample analysis showed complete agreement with quantitative PCR results.

Conclusions:

• The Ln-MOF-integrated CRISPR-Cas12a platform offers a sensitive, specific, and rapid method for gene mutation analysis.
• Eliminating amplification streamlines the workflow, making it suitable for clinical diagnostics.
• This approach represents a significant advancement in molecular diagnostic technologies.