CRISPR/Cas12a Collateral Cleavage Activity for Sensitive 3′–5′ Exonuclease Assay

Author:

Jeung Jae Hoon12ORCID,Han Hyogu13ORCID,Lee Chang Yeol4ORCID,Ahn Jun Ki1ORCID

Affiliation:

1. Material & Component Convergence R&D Department, Korea Institute of Industrial Technology (KITECH), Ansan 15588, Republic of Korea

2. Department of Biological Engineering, College of Engineering, Konkuk University, Seoul 05029, Republic of Korea

3. Department of Chemistry, Gangneung–Wonju National University, Gangneung 25457, Republic of Korea

4. Bionanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak–ro, Yuseong–gu, Daejeon 34141, Republic of Korea

Abstract

This study presents a technique for detecting 3′–5′ exonuclease activity through the use of CRISPR/Cas12a. These enzymes, including 3′–5′ exonuclease (Exo III), perform crucial roles in various cellular processes and are associated with life expectancy. However, imbalances in their expression can increase susceptibility to diseases such as cancer, particularly under prolonged stress. In this study, an activator sequence of CRISPR/Cas12a was constructed on the 5′–end of a hairpin probe (HP), forming a blunt end. When the 3′–end of the HP was hydrolyzed with Exo III activity, the activator sequence of Cas12a was exposed, which led to collateral cleavage of the DNA signal probe and generated a fluorescent signal, allowing sensitive and highly specific Exo III detection. This detection principle relied on the fact that Exo III exclusively cleaves the 3′–end mononucleotide of dsDNA and does not affect ssDNA. Based on this strategy, Exo III activity was successfully assayed at 0.0073 U/mL, demonstrating high sensitivity. In addition, this technique was used to screen candidate inhibitors of Exo III activity.

Funder

Korea Institute of Industrial Technology

Ministry of Agriculture, Food and Rural Affairs

Publisher

MDPI AG

Subject

Clinical Biochemistry,General Medicine,Analytical Chemistry,Biotechnology,Instrumentation,Biomedical Engineering,Engineering (miscellaneous)

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