CRISPR/Cas12a Collateral Cleavage Activity for Sensitive 3'-5' Exonuclease Assay

Area of Science:

• Molecular Biology
• Biochemistry
• Enzyme kinetics

Background:

• 3 -5 exonuclease (Exo III) enzymes are vital for cellular processes and linked to longevity.
• Dysregulation of Exo III is implicated in diseases like cancer, especially under stress.

Purpose of the Study:

• To develop a sensitive and specific method for detecting 3 -5 exonuclease (Exo III) activity.
• To utilize CRISPR/Cas12a technology for enzyme activity detection and inhibitor screening.

Main Methods:

• A CRISPR/Cas12a system was engineered with a hairpin probe (HP) containing an activator sequence.
• Exo III activity was detected by its hydrolysis of the HP's 3 -end, exposing the activator.
• Exposed activator triggered Cas12a collateral cleavage of a DNA signal probe, generating a fluorescent signal.

Main Results:

• The method achieved sensitive detection of Exo III activity down to 0.0073 U/mL.
• The technique demonstrated high specificity, leveraging Exo III's preference for dsDNA 3 -ends.
• The developed assay was successfully applied to screen for Exo III inhibitors.

Conclusions:

• The CRISPR/Cas12a-based approach provides a sensitive and specific platform for Exo III activity detection.
• This method is valuable for biochemical assays and screening potential therapeutic compounds targeting Exo III.