Integrating CRISPR-Cas12a with a crRNA-Mediated Catalytic Network for the Development of a Modular and Sensitive Aptasensor

Aug 10, 2022ACS synthetic biology

A sensitive and modular sensor using CRISPR-Cas12a and RNA-guided reactions

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Abstract

This method achieved a limit of detection value of 0.16 μM for adenosine triphosphate (ATP).

The Cas12a-CMCAN aptasensor utilizes a crRNA-mediated catalytic nucleic acid network to enhance sensitivity.
This approach enables multiturnover cleavage activity and an inherent recycling amplification mechanism.
A linear detection range was established from 0.30 to 175 μM ATP.
The sensor successfully detected ATP levels in diluted human serum samples.
The system's design allows for easy modification to detect various targets by changing aptamer sequences.

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Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a, which exhibits excellent target DNA-activated-cleavage activity under the guidance of a programmable CRISPR RNA (crRNA), has shown great promise in next-generation biosensing technology. However, current CRISPR-Cas12a-based biosensors usually improve sensitivity by the initial nucleic acid amplification, while the distinct programmability and predictability of the crRNA-guided target binding process has not been fully exploited. Herein, we, for the first time, propose a modular and sensitive CRISPR-Cas12a fluorometric aptasensor by integrating an enzyme-free and robust crRNA-mediated catalytic nucleic acid network, namely, Cas12a-CMCAN, in which crRNA acts as an initiator to actuate cascade toehold-mediated strand displacement reactions (TM-SDRs). As a proof of concept, adenosine triphosphate (ATP) was selected as a model target. Owing to the multiturnover of CRISPR-Cas12a-cleavage and the inherent recycling amplification network, this method achieved a limit of detection value of 0.16 μM (20-fold lower than direct Cas12a-based ATP detection) with a linear range from 0.30 to 175 μM. In addition, Cas12a-CMCAN can be successfully employed to detect ATP levels in diluted human serum samples. Considering the simplicity, sensitivity, and easy to tune many targets by changing aptamer sequences, the Cas12a-CMCAN sensing method is expected to offer a heuristic idea for the development of CRISPR-Cas12a-based biosensors and unlock its potential for general and convenient molecule diagnostics. trans trans

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Integrating CRISPR-Cas12a with a crRNA-Mediated Catalytic Network for the Development of a Modular and Sensitive Aptasensor

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