RNA reporter based CRISPR/Cas12a biosensing platform for sensitive detection of circulating tumor DNA

Deng, Fei, Zhang, Chengchen, Hall, Tim and Goldys, Ewa (2023) RNA reporter based CRISPR/Cas12a biosensing platform for sensitive detection of circulating tumor DNA. In 2023 45th Annual International Conference of the IEEE Engineering in Medicine and Biology Conference, EMBC 2023 - Proceedings. IEEE.. (doi:10.1109/EMBC40787.2023.10340759).

Abstract

CRISPR/Cas biotechnology provides an exceptional platform for biosensor development. To date, the reported CRISPR/Cas biosensing systems have shown extraordinary performance for nucleic acids, small molecules, small proteins and microorganism detection. The CRISPR/Cas12a biosensing system, as a typical example, has been well established and applied for both nucleic acids and non-nucleic acids target detection. However, all established CRISPR/Cas12a biosensing systems are based on DNA reporters, which potentially limits further application.In this study, we established an RNA reporter based CRISPR/Cas12a biosensing system. A basic biosensing system was evaluated, and the limit of detection was found to be 1 nM. Afterwards, we optimized this biosensing system using both temperature and chemical enhancers. The final optimal biosensing system (with DTT & 37°C) shows fluorescence signal increased by a factor of ~10 compared with the basic system. The optimal biosensing system was further applied for the detection of circulating tumor DNA (ctDNA), which shows over 4 orders of magnitude detection range from 1pM to 25 nM, with the limit of detection of 1pM. This RNA reporter based CRISPR/Cas12a biosensing system provides an effective platform for nucleic acids quantification.Clinical Relevance-This research provides a novel approach for ctDNA diagnostics, which is an attractive biomarker for noninvasive monitoring of tumor growth, response, and spread.

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Contributors

Author: Chengchen Zhang

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