Accelerated RNA detection using tandem CRISPR nucleases

Tina Y Liu; Gavin J Knott; Dylan C J Smock; John J Desmarais; Sungmin Son; Abdul Bhuiya; Shrutee Jakhanwal; Noam Prywes; Shreeya Agrawal; María Díaz de León Derby; Neil A Switz; Maxim Armstrong; Andrew R Harris; Emeric J Charles; Brittney W Thornton; Parinaz Fozouni; Jeffrey Shu; Stephanie I Stephens; G Renuka Kumar; Chunyu Zhao; Amanda Mok; Anthony T Iavarone; Arturo M Escajeda; Roger McIntosh; Shin E Kim; Eli J Dugan; IGI Testing Consortium; Katherine S Pollard; Ming X Tan; Melanie Ott; Daniel A Fletcher; Liana F Lareau; Patrick D Hsu; David F Savage; Jennifer A Doudna

doi:10.1101/2021.03.19.21253328

Accelerated RNA detection using tandem CRISPR nucleases

medRxiv [Preprint]. 2021 Mar 24:2021.03.19.21253328. doi: 10.1101/2021.03.19.21253328.

Authors

Tina Y Liu^{1

2}, Gavin J Knott^{1

2

3}, Dylan C J Smock^{1

2}, John J Desmarais¹, Sungmin Son⁴, Abdul Bhuiya^{4

5}, Shrutee Jakhanwal^{1

2}, Noam Prywes², Shreeya Agrawal^{2

4}, María Díaz de León Derby^{4

5}, Neil A Switz⁶, Maxim Armstrong⁷, Andrew R Harris⁴, Emeric J Charles¹, Brittney W Thornton¹, 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 Mok¹³, Anthony T Iavarone¹⁴, Arturo M Escajeda¹⁵, Roger McIntosh¹⁵, Shin E Kim^{1

2}, Eli J Dugan^{1

2}; IGI Testing Consortium; Katherine S Pollard^{8

12

16}, Ming X Tan¹⁵, Melanie Ott^{8

11}, Daniel A Fletcher^{4

5

12}, Liana F Lareau^{2

4}, Patrick D Hsu^{2

4}, David F Savage^{1

2}, Jennifer A Doudna^{1

2

7

8

17

18

19

20}

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
² Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
³ Monash Biomedicine Discovery Institute, Department of Biochemistry & Molecular Biology, Monash University, VIC 3800, Australia.
⁴ Department of Bioengineering, University of California, Berkeley, CA, USA.
⁵ UC Berkeley-UC San Francisco Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
⁶ Department of Physics and Astronomy, San José State University, San José, CA, USA.
⁷ Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
⁸ Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, USA.
⁹ Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA 94143, USA.
¹⁰ Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA.
¹¹ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
¹² Chan-Zuckerberg Biohub, San Francisco, CA, USA.
¹³ Center for Computational Biology, University of California, Berkeley, Berkeley, CA, USA.
¹⁴ QB3/Chemistry Mass Spectrometry Facility, University of California, Berkeley, Berkeley, CA, USA.
¹⁵ Wainamics Inc., Pleasanton, CA, USA.
¹⁶ Department of Epidemiology & Biostatistics, University of California, San Francisco, CA, USA.
¹⁷ Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, USA.
¹⁸ Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA.
¹⁹ Gladstone Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA, USA.
²⁰ California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA.

Abstract

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 recognition of RNA that triggers cleavage and release of a fluorescent reporter molecule^1,2, but long reaction times hamper sensitivity and speed when applied to point-of-care testing. 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 RNA copies/microliter in 20 minutes. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that detected SARS-CoV-2 RNA from nasopharyngeal samples with PCR-derived Ct values up to 29 in microfluidic chips, using a compact imaging system. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables direct RNA detection in a format amenable to point-of-care infection diagnosis, as well as to a wide range of other diagnostic or research applications.

Publication types

Preprint

Abstract

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