High-efficiency detection of APE1 using a defective PAM-driven CRISPR-Cas12a self-catalytic biosensor

Mar 29, 2025Biosensors & bioelectronics

Highly sensitive detection of APE1 using a self-activating CRISPR-based sensor

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Abstract

DEP-Cas-APE achieved a detection limit as low as 7.66 × 10U μL within 30 minutes.

A novel signal amplification strategy using defective PAM-modified DNA probes was developed for detecting APE1 activity.
The approach combines Cas12a trans-cleavage with a self-catalytic circuit to enhance signal transformation and amplification.
Testing confirmed that DEP-Cas-APE effectively detects APE1 in complex biological samples, including lung cancer patient serum.
The method is capable of distinguishing between cancerous and normal samples.
A point-of-care testing platform was created by integrating DEP-Cas-APE with a colorimetric assay using gold nanoparticles.

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The trans-cleavage activity of the CRISPR-Cas system offers tremendous potential for developing highly sensitive and selective molecular diagnostic tools. However, conventional methods often face challenges such as limited catalytic efficiency of single Cas proteins and the necessity of complex multi-enzyme preamplification steps. To address these limitations, we present a novel defective PAM-mediated CRISPR-Cas12a self-catalytic signal amplification strategy, termed DEP-Cas-APE, for the rapid, sensitive, and specific detection of apurinic/apyrimidinic endonuclease 1 (APE1) activity. This approach integrates defective PAM-modified DNA probes to synergize Cas12a trans-cleavage with self-catalytic circuit, achieving efficient signal transformation and amplification under isothermal, one-step conditions. We systematically investigated the influence of defective PAM sequences containing apurinic/apyrimidinic (AP) sites on Cas12a activation and validated the feasibility of the DEP-Cas-APE strategy in detecting APE1. Under optimized conditions, DEP-Cas-APE achieved a detection limit as low as 7.66 × 10U μLwithin 30 min using a simple isothermal reaction. Additionally, we developed a point-of-care testing (POCT) platform by integrating DEP-Cas-APE with a colorimetric assay based on gold nanoparticles (AuNPs), enabling portable, equipment-free detection. This sensitive and selective strategy successfully detected APE1 in complex biological samples, including serum from lung cancer patients, and demonstrated the ability to distinguish cancerous from normal samples. DEP-Cas-APE represents a robust and versatile platform for advancing CRISPR-Cas12a biosensing technologies, offering new opportunities for molecular diagnostics and clinical research. -8 -1

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High-efficiency detection of APE1 using a defective PAM-driven CRISPR-Cas12a self-catalytic biosensor

Abstract

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