A universal two-step strategy for multiple DNA MTase activity: enhancing sensitivity through CRISPR/Cas12a-assisted...

Area of Science:

• Biochemistry
• Molecular Biology
• Enzyme Assays

Background:

• DNA methyltransferase (DNA MTase) activity is crucial for genetic diseases, impacting drug screening, diagnosis, and treatment.
• Accurate detection of DNA MTase activity is essential for advancing research and clinical applications.

Purpose of the Study:

• To develop a universal, efficient, and sensitive detection strategy for DNA MTase activity.
• To establish a novel assay for drug screening, clinical diagnosis, and disease treatment related to DNA methylation.

Main Methods:

• A two-step CRISPR/Cas12a-assisted hyperbranched rolling circle amplification (CA-HRCA) strategy was developed.
• Simultaneous DNA methylation, restriction enzyme digestion, and HRCA amplification were performed in the first step.
• CRISPR/Cas12a collateral cleavage induced by dsDNA products from HRCA was utilized for signal amplification in the second step.

Main Results:

• The CA-HRCA strategy successfully detected Dam MTase and M.SssI MTase with a total detection time of 155 minutes.
• Achieved low limits of detection (LODs) of 7.6 × 10^-4 U mL^-1 for Dam MTase and 1.8 × 10^-4 U mL^-1 for M.SssI MTase.
• Demonstrated excellent specificity, reproducibility, and practical applicability through serum spiked tests and inhibitor evaluation.

Conclusions:

• The proposed CA-HRCA assay offers a simplified and rapid method for DNA MTase activity detection.
• This assay holds significant potential for drug development and clinical diagnostics related to DNA methylation.
• The use of dsDNA activators enhances CRISPR/Cas12a collateral cleavage efficiency for improved sensing performance.