Accelerated RNA detection using tandem CRISPR nucleases,Nature Chemical Biology

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Accelerated RNA detection using tandem CRISPR nucleases
Nature Chemical Biology ( IF 12.9 ) Pub Date : 2021-08-05 , DOI: 10.1038/s41589-021-00842-2
Tina Y Liu _{1,

2} , Gavin J Knott _{1,

2,

3} , Dylan C J Smock _{1,

2} , John J Desmarais ₁ , Sungmin Son ₄ , Abdul Bhuiya _{4,

5} , Shrutee Jakhanwal _{1,

2} , Noam Prywes ₂ , Shreeya Agrawal _{2,

4} , María Díaz de León Derby _{4,

5} , Neil A Switz ₆ , Maxim Armstrong ₇ , Andrew R Harris ₄ , Emeric J Charles ₁ , Brittney W Thornton ₁ , Parinaz Fozouni _{8,

9,

10,

11} , Jeffrey Shu _{8,

11} , Stephanie I Stephens _{8,

11} , G Renuka Kumar _{8,

11} , Chunyu Zhao _{8,

12} , Amanda Mok ₁₃ , Anthony T Iavarone ₁₄ , Arturo M Escajeda ₁₅ , Roger McIntosh ₁₅ , Shineui Kim _{1,

2} , Eli J Dugan _{1,

2} , , Katherine S Pollard _{8,

12,

16} , Ming X Tan ₁₅ , Melanie Ott _{8,

11} , Daniel A Fletcher _{4,

5,

12,

17} , Liana F Lareau _{2,

4} , Patrick D Hsu _{2,

4,

13,

17,

18} , David F Savage _{1,

2} , Jennifer A Doudna _{1,

2,

7,

17,

19,

20,

21}

Affiliation

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
Monash Biomedicine Discovery Institute, Department of Biochemistry & Molecular Biology, Monash University, Victoria, Australia.
Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
UC Berkeley, UC San Francisco Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
Department of Physics and Astronomy, San José State University, San José, CA, USA.
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, USA.
Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA.
Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
Chan-Zuckerberg Biohub, San Francisco, CA, USA.
Center for Computational Biology, University of California, Berkeley, Berkeley, CA, USA.
QB3/Chemistry Mass Spectrometry Facility, University of California, Berkeley, Berkeley, CA, USA.
Wainamics Inc., Pleasanton, CA, USA.
Department of Epidemiology & Biostatistics, University of California, San Francisco, San Francisco, CA, USA.
California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA.
Berkeley Stem Cell Center, University of California, Berkeley, Berkeley, CA, USA.
Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, USA.
Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA.
Gladstone Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA, USA.

Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR–Cas nucleases offer programmable RNA-guided RNA recognition that triggers cleavage and release of a fluorescent reporter molecule, but long reaction times hamper their detection sensitivity and speed. Here, we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 molecules per µl of RNA in 20 min. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA extracted from respiratory swab samples with quantitative reverse transcriptase PCR (qRT–PCR)-derived cycle threshold (C_t) values up to 33, using a compact detector. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables sensitive, direct RNA detection in a format that is amenable to point-of-care infection diagnosis as well as to a wide range of other diagnostic or research applications.

中文翻译：

使用串联 CRISPR 核酸酶加速 RNA 检测

直接、无扩增的 RNA 检测有可能通过对人类或环境样本进行简单的现场分析来改变分子诊断。CRISPR-Cas 核酸酶提供可编程的 RNA 引导的 RNA 识别，可触发荧光报告分子的切割和释放，但反应时间长会阻碍其检测灵敏度和速度。在这里，我们表明无关的 CRISPR 核酸酶可以串联部署以提供直接 RNA 传感和快速信号生成，从而能够在 20 分钟内对每微升 RNA 约 30 个分子进行稳健检测。C _t ) 值高达 33，使用紧凑型检测器。这种快速集成核酸酶串联检测 (FIND-IT) 方法能够以一种适用于即时感染诊断以及广泛的其他诊断或研究应用的形式进行灵敏、直接的 RNA 检测。

更新日期：2021-08-05

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