一种使用多模式报告基因进行多种读值的理性设计的 CRISPR/Cas12a 检测法。

A Rationally Designed CRISPR/Cas12a Assay Using a Multimodal Reporter for Various Readouts.

机构信息

CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.

University of Chinese Academy of Sciences, 19 Yuquan Road, Shijingshan District, Beijing 100049, China.

出版信息

Anal Chem. 2023 Aug 8;95(31):11741-11750. doi: 10.1021/acs.analchem.3c01876. Epub 2023 Jul 28.

Abstract

The CRISPR/Cas systems offer a programmable platform for nucleic acid detection, and CRISPR/Cas-based diagnostics (CRISPR-Dx) have demonstrated the ability to target nucleic acids with greater accuracy and flexibility. However, due to the configuration of the reporter and the underlying labeling mechanism, almost all reported CRISPR-Dx rely on a single-option readout, resulting in limitations in end-point result readouts. This is also associated with high reagent consumption and delays in diagnostic reports due to protocol differences. Herein, we report for the first time a rationally designed Cas12a-based multimodal universal reporter (CAMURE) with improved sensitivity that harnesses a dual-mode reporting system, facilitating options in end-point readouts. Through systematic configurations and optimizations, our novel universal reporter achieved a 10-fold sensitivity enhancement compared to the DETECTR reporter. Our unique and versatile reporter could be paired with various readouts, conveying the same diagnostic results. We applied our novel reporter for the detection of staphylococcal enterotoxin A due to its high implication in staphylococcal food poisoning. Integrated with loop-mediated isothermal amplification, our multimodal reporter achieved 10 CFU/mL sensitivity and excellent specificity using a real-time fluorimeter, in-tube fluorescence, and lateral flow strip readouts. We also propose, using artificially contaminated milk samples, a fast (2-5 min) Triton X-100 DNA extraction approach with a comparable yield to the commercial extraction kit. Our CAMURE could be leveraged to detect all gene-encoding SEs by simply reprogramming the guide RNA and could also be applied to the detection of other infections and disease biomarkers.

摘要

CRISPR/Cas 系统为核酸检测提供了一个可编程的平台,基于 CRISPR/Cas 的诊断(CRISPR-Dx)已经证明了其能够以更高的准确性和灵活性靶向核酸。然而,由于报告器的配置和基础标记机制,几乎所有报道的 CRISPR-Dx 都依赖于单一选项的读数,从而导致终点结果读数的局限性。这也与试剂消耗量大以及由于协议差异导致诊断报告延迟有关。在这里,我们首次报道了一种基于 Cas12a 的合理设计的多模态通用报告器(CAMURE),该报告器具有更高的灵敏度,利用了双模式报告系统,为终点读数提供了多种选择。通过系统的配置和优化,我们的新型通用报告器与 DETECTR 报告器相比,灵敏度提高了 10 倍。我们独特而通用的报告器可以与各种读数配对,传达相同的诊断结果。我们应用我们的新型报告器来检测葡萄球菌肠毒素 A,因为它在葡萄球菌食物中毒中具有很高的相关性。与环介导等温扩增集成后,我们的多模态报告器在实时荧光计、管内荧光和侧流条读数上实现了 10 CFU/mL 的灵敏度和出色的特异性。我们还提出了一种使用 Triton X-100 快速(2-5 分钟)提取 DNA 的方法,与商用提取试剂盒相比,该方法具有相当的产量,可用于人工污染的牛奶样本。我们的 CAMURE 可以通过简单地重新编程向导 RNA 来检测所有基因编码的 SE,也可以应用于其他感染和疾病生物标志物的检测。

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