CRISPR/Cas12a-Enhanced Cascade Amplification for Ultra-sensitive DNA Ligase Detection

Area of Science:

• Biochemistry
• Molecular Biology
• Genomics

Background:

• DNA ligases are vital for genomic stability and serve as cancer biomarkers.
• Existing detection methods for DNA ligases are often complex, costly, and lack sensitivity.

Purpose of the Study:

• To develop an integrated, label-free detection system for DNA ligases.
• To leverage CRISPR/Cas12a activity initiated by DNA ligase for enhanced detection.

Main Methods:

• A novel system where DNA ligase acts as a molecular gatekeeper for CRISPR/Cas12a activation.
• Utilizing the difference in affinity between nicked and intact activators for Cas12a-crRNA.
• Employing a circular DNA-mediated autocatalytic cascade for signal amplification.

Main Results:

• Achieved an ultimate limit of detection (LOD) of 2.59 × 10^-6 U/mL.
• Reached analytical sensitivity comparable to established methods in 30 minutes (LOD of 6.12 × 10^-5 U/mL).
• Demonstrated high selectivity and validated in quantifying endogenous DNA ligase in tumor cell extracts.

Conclusions:

• The ligase-gated CRISPR cascade offers a sensitive, rapid, and label-free platform for DNA ligase detection.
• This system provides a robust framework for clinical diagnostics and enzymology research.
• The technology significantly compresses diagnostic timeframes and enhances sensitivity for low-abundance targets.