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; Shineui 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:10.1038/s41589-021-00842-2

Accelerated RNA detection using tandem CRISPR nucleases

Nat Chem Biol. 2021 Sep;17(9):982-988. doi: 10.1038/s41589-021-00842-2. Epub 2021 Aug 5.

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, Shineui 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

17}, Liana F Lareau^{2

4}, Patrick D Hsu^{18

19

20

21

22}, David F Savage^{23

24}, Jennifer A Doudna^{25

26

27

28

29

30

31}

Collaborators

IGI Testing Consortium:
Jennifer R Hamilton, Enrique Lin-Shiao, Elizabeth C Stahl, Connor A Tsuchida, Erica A Moehle, Petros Giannikopoulos, Matthew McElroy, Shana McDevitt, Arielle Zur, Iman Sylvain, Alison Ciling, Madeleine Zhu, Clara Williams, Alisha Baldwin

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, 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, Victoria, Australia.
⁴ Department of Bioengineering, University of California, Berkeley, 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, USA.
¹⁰ Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
¹¹ Department of Medicine, University of California, San Francisco, San Francisco, CA, 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, San Francisco, CA, USA.
¹⁷ California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA.
¹⁸ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu.
¹⁹ Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu.
²⁰ Center for Computational Biology, University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu.
²¹ California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu.
²² Berkeley Stem Cell Center, University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu.
²³ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA. dsavage@berkeley.edu.
²⁴ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. dsavage@berkeley.edu.
²⁵ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.
²⁶ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.
²⁷ Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. doudna@berkeley.edu.
²⁸ California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.
²⁹ Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.
³⁰ Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.
³¹ Gladstone Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA, USA. doudna@berkeley.edu.

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 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.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

MeSH terms

COVID-19 / genetics*
CRISPR-Cas Systems / genetics*
Humans
RNA, Viral / genetics*
Reverse Transcriptase Polymerase Chain Reaction
SARS-CoV-2 / genetics*

Substances

RNA, Viral

Abstract

Publication types

MeSH terms

Substances

Grants and funding