SHERLOCK: nucleic acid detection with CRISPR nucleases.,Nature Protocols

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SHERLOCK: nucleic acid detection with CRISPR nucleases.
Nature Protocols ( IF 13.1 ) Pub Date : 2019-09-23 , DOI: 10.1038/s41596-019-0210-2
Max J Kellner ₁ , Jeremy G Koob ₁ , Jonathan S Gootenberg _{1,

2,

3} , Omar O Abudayyeh _{1,

2,

3} , Feng Zhang _{1,

2,

3,

4}

Affiliation

Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.

中文翻译：

SHERLOCK：使用 CRISPR 核酸酶进行核酸检测。

核酸的快速检测是临床诊断和生物技术应用中不可或缺的一部分。我们最近建立了一个基于 CRISPR 的诊断平台，将核酸预扩增与 CRISPR-Cas 酶学相结合，用于特异性识别所需的 DNA 或 RNA 序列。该平台被称为特异性高灵敏度酶报告解锁 (SHERLOCK)，可对临床相关样本中的 RNA 或 DNA 进行多重、便携式和超灵敏检测。在这里，我们提供了设置 SHERLOCK 测定的分步说明，其中包括重组酶介导的 DNA 或 RNA 聚合酶预扩增，以及随后的 Cas13 或 Cas12 介导的检测，通过荧光和比色读数在 1 小时内提供结果。设置时间少于 15 分钟。我们还提供了设计高效 CRISPR RNA (crRNA) 和等温扩增引物的指南，并讨论了多重和定量 SHERLOCK 检测分析的重要注意事项。

更新日期：2019-09-23

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