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A CRISPR-Cas12a-derived biosensing platform for the highly sensitive detection of diverse small molecules.

ABSTRACT: Besides genome editing, CRISPR-Cas12a has recently been used for DNA detection applications with attomolar sensitivity but, to our knowledge, it has not been used for the detection of small molecules. Bacterial allosteric transcription factors (aTFs) have evolved to sense and respond sensitively to a variety of small molecules to benefit bacterial survival. By combining the single-stranded DNA cleavage ability of CRISPR-Cas12a and the competitive binding activities of aTFs for small molecules and double-stranded DNA, here we develop a simple, supersensitive, fast and high-throughput platform for the detection of small molecules, designated CaT-SMelor (CRISPR-Cas12a- and aTF-mediated small molecule detector). CaT-SMelor is successfully evaluated by detecting nanomolar levels of various small molecules, including uric acid and p-hydroxybenzoic acid among their structurally similar analogues. We also demonstrate that our CaT-SMelor directly measured the uric acid concentration in clinical human blood samples, indicating a great potential of CaT-SMelor in the detection of small molecules.

SUBMITTER: Liang M

PROVIDER: S-EPMC6694116 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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A CRISPR-Cas12a-derived biosensing platform for the highly sensitive detection of diverse small molecules.

Liang Mindong M Li Zilong Z Wang Weishan W Liu Jiakun J Liu Leshi L Zhu Guoliang G Karthik Loganathan L Wang Man M Wang Ke-Feng KF Wang Zhong Z Yu Jing J Shuai Yuting Y Yu Jiaming J Zhang Lu L Yang Zhiheng Z Li Chuan C Zhang Qian Q Shi Tong T Zhou Liming L Xie Feng F Dai Huanqin H Liu Xueting X Zhang Jingyu J Liu Guang G Zhuo Ying Y Zhang Buchang B Liu Chenli C Li Shanshan S Xia Xuekui X Tong Yaojun Y Liu Yanwen Y Alterovitz Gil G Tan Gao-Yi GY Zhang Li-Xin LX

Nature communications 20190814 1

Besides genome editing, CRISPR-Cas12a has recently been used for DNA detection applications with attomolar sensitivity but, to our knowledge, it has not been used for the detection of small molecules. Bacterial allosteric transcription factors (aTFs) have evolved to sense and respond sensitively to a variety of small molecules to benefit bacterial survival. By combining the single-stranded DNA cleavage ability of CRISPR-Cas12a and the competitive binding activities of aTFs for small molecules an ...[more]

PMID: 31413315

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Project description:BackgroundCOVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component.MethodsWe tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19.ResultsThe OR-DETECTR detection process can be completed in one tube, which takes approximately 50 min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/µl input (RNA standard) and 1 copy/µl input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/µl input.ConclusionsThe OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.

Dataset Information

A CRISPR-Cas12a-derived biosensing platform for the highly sensitive detection of diverse small molecules.

Publications

A CRISPR-Cas12a-derived biosensing platform for the highly sensitive detection of diverse small molecules.

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