Accelerated RNA detection using tandem CRISPR nucleases.

Autor: Liu TY; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA., Knott GJ; 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., Smock DCJ; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA., Desmarais JJ; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA., Son S; Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA., Bhuiya A; 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., Jakhanwal S; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA., Prywes N; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA., Agrawal S; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.; 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.; UC Berkeley, UC San Francisco Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA, USA., Switz NA; Department of Physics and Astronomy, San José State University, San José, CA, USA., Armstrong M; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA., Harris AR; Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA., Charles EJ; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA., Thornton BW; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA., Fozouni P; 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., Shu J; Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, USA.; Department of Medicine, University of California, San Francisco, San Francisco, CA, USA., Stephens SI; Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, USA.; Department of Medicine, University of California, San Francisco, San Francisco, CA, USA., Kumar GR; Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, USA.; Department of Medicine, University of California, San Francisco, San Francisco, CA, USA., Zhao C; Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, USA.; Chan-Zuckerberg Biohub, San Francisco, CA, USA., Mok A; Center for Computational Biology, University of California, Berkeley, Berkeley, CA, USA., Iavarone AT; QB3/Chemistry Mass Spectrometry Facility, University of California, Berkeley, Berkeley, CA, USA., Escajeda AM; Wainamics Inc., Pleasanton, CA, USA., McIntosh R; Wainamics Inc., Pleasanton, CA, USA., Kim S; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA., Dugan EJ; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA., Pollard KS; Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, USA.; Chan-Zuckerberg Biohub, San Francisco, CA, USA.; Department of Epidemiology & Biostatistics, University of California, San Francisco, San Francisco, CA, USA., Tan MX; Wainamics Inc., Pleasanton, CA, USA., Ott M; Gladstone Institute of Virology, Gladstone Institutes, San Francisco, CA, USA.; Department of Medicine, University of California, San Francisco, San Francisco, CA, USA., Fletcher DA; 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.; Chan-Zuckerberg Biohub, San Francisco, CA, USA.; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA., Lareau LF; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.; Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA., Hsu PD; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu.; Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu.; Center for Computational Biology, University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu.; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu.; Berkeley Stem Cell Center, University of California, Berkeley, Berkeley, CA, USA. pdhsu@berkeley.edu., Savage DF; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA. dsavage@berkeley.edu.; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. dsavage@berkeley.edu., Doudna JA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. doudna@berkeley.edu.; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.; Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA. doudna@berkeley.edu.; Gladstone Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA, USA. doudna@berkeley.edu.
Jazyk: angličtina
Zdroj: Nature chemical biology [Nat Chem Biol] 2021 Sep; Vol. 17 (9), pp. 982-988. Date of Electronic Publication: 2021 Aug 05.
DOI: 10.1038/s41589-021-00842-2
Abstrakt: 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.
(© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)
Databáze: MEDLINE
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