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Accelerated RNA detection using tandem CRISPR nucleases.
Liu, Tina Y; Knott, Gavin J; Smock, Dylan C J; Desmarais, John J; Son, Sungmin; Bhuiya, Abdul; Jakhanwal, Shrutee; Prywes, Noam; Agrawal, Shreeya; Díaz de León Derby, María; Switz, Neil A; Armstrong, Maxim; Harris, Andrew R; Charles, Emeric J; Thornton, Brittney W; Fozouni, Parinaz; Shu, Jeffrey; Stephens, Stephanie I; Kumar, G Renuka; Zhao, Chunyu; Mok, Amanda; Iavarone, Anthony T; Escajeda, Arturo M; McIntosh, Roger; Kim, Shineui; Dugan, Eli J; Pollard, Katherine S; Tan, Ming X; Ott, Melanie; Fletcher, Daniel A; Lareau, Liana F; Hsu, Patrick D; Savage, David F; Doudna, Jennifer A.
Affiliation
  • Liu TY; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
  • Knott GJ; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
  • Smock DCJ; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
  • Desmarais JJ; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
  • Son S; Monash Biomedicine Discovery Institute, Department of Biochemistry & Molecular Biology, Monash University, Victoria, Australia.
  • Bhuiya A; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
  • Jakhanwal S; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
  • Prywes N; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
  • Agrawal S; Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
  • Díaz de León Derby M; Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
  • Switz NA; UC Berkeley, UC San Francisco Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
  • Armstrong M; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
  • Harris AR; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
  • Charles EJ; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
  • Thornton BW; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
  • Fozouni P; Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
  • Shu J; Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
  • Stephens SI; UC Berkeley, UC San Francisco Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
  • Kumar GR; Department of Physics and Astronomy, San José State University, San José, CA, USA.
  • Zhao C; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
  • Mok A; Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA.
  • Iavarone AT; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
  • Escajeda AM; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
  • McIntosh R; Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, USA.
  • Kim S; Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA.
  • Dugan EJ; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
  • Pollard KS; Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, USA.
  • Tan MX; Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
  • Ott M; Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, USA.
  • Fletcher DA; Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
  • Lareau LF; Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, USA.
  • Hsu PD; Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
  • Savage DF; Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, USA.
  • Doudna JA; Chan-Zuckerberg Biohub, San Francisco, CA, USA.
Nat Chem Biol ; 17(9): 982-988, 2021 09.
Article in En | MEDLINE | ID: mdl-34354262
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 (Ct) 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.
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: RNA, Viral / CRISPR-Cas Systems / SARS-CoV-2 / COVID-19 Type of study: Diagnostic_studies Limits: Humans Language: En Journal: Nat Chem Biol Journal subject: BIOLOGIA / QUIMICA Year: 2021 Document type: Article Affiliation country: United States Country of publication: United States
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: RNA, Viral / CRISPR-Cas Systems / SARS-CoV-2 / COVID-19 Type of study: Diagnostic_studies Limits: Humans Language: En Journal: Nat Chem Biol Journal subject: BIOLOGIA / QUIMICA Year: 2021 Document type: Article Affiliation country: United States Country of publication: United States