A ligation-driven CRISPR-Cas biosensing platform for non-nucleic acid target detections

Area of Science:

• Biotechnology
• Molecular Biology
• Biosensing

Background:

• CRISPR-Cas systems are primarily known for nucleic acid detection.
• Expanding CRISPR applications to non-nucleic acid targets remains a challenge.
• Sensitive detection of biomolecules like NAD+, ATP, and PNK is crucial in various biological studies.

Purpose of the Study:

• To develop a CRISPR-Cas12a sensing platform for sensitive detection of non-nucleic acid targets.
• To utilize a DNA ligation reaction as a trigger for Cas12a activation.
• To demonstrate the platform's applicability for detecting NAD+, ATP, and polynucleotide kinase (PNK).

Main Methods:

• A CRISPR-Cas12a system was engineered to be activated by DNA ligation.
• Biomolecular targets (NAD+, ATP, PNK) were used to trigger specific DNA ligation reactions.
• The generated DNA duplexes were shown to activate the collateral nuclease activity of Cas12a.
• Amplified fluorescence signals were measured for sensitive detection.

Main Results:

• The developed platform successfully detected non-nucleic acid targets including NAD+, ATP, and PNK.
• The DNA ligation-triggered Cas12a activation resulted in amplified fluorescence signals.
• The system demonstrated high sensitivity for the target biomolecules.
• This approach expands the utility of CRISPR-Cas systems beyond nucleic acid detection.

Conclusions:

• A novel CRISPR-Cas12a sensing platform activated by DNA ligation has been successfully developed.
• This platform offers a sensitive and versatile method for detecting non-nucleic acid biomolecules.
• The findings present an alternative strategy to broaden the application scope of CRISPR-Cas systems in biological detection.