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

Jiani Wu¹, Jingyuan Yan¹, Changjiang Li¹

¹School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

Analytical Chemistry

|June 29, 2026

Summary

This study introduces a novel, label-free DNA ligase detection system using CRISPR/Cas12a technology. The innovative method offers rapid, highly sensitive detection of DNA ligases, crucial for cancer diagnostics.

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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.