Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy.

Autor:	Fozouni P; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA., Son S; Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA., Díaz de León Derby M; Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA; UC Berkeley-UC San Francisco Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA., Knott GJ; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Monash Biomedicine Discovery Institute, Department of Biochemistry & Molecular Biology, Monash University, VIC 3800, Australia., Gray CN; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA., D'Ambrosio MV; Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA., Zhao C; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA., Switz NA; Department of Physics and Astronomy, San José State University, San Jose, CA 95192, USA., Kumar GR; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA., Stephens SI; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA., Boehm D; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA., Tsou CL; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA., Shu J; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA., Bhuiya A; Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA; UC Berkeley-UC San Francisco Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA., Armstrong M; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA., Harris AR; Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA., Chen PY; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA., Osterloh JM; J. David Gladstone Institutes, San Francisco, CA 94158, USA., Meyer-Franke A; J. David Gladstone Institutes, San Francisco, CA 94158, USA., Joehnk B; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA., Walcott K; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA., Sil A; Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA., Langelier C; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA; Division of Infectious Diseases, University of California, San Francisco, San Francisco, CA 94143, USA., Pollard KS; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA; Institute for Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Epidemiology and Biostatistics and Institute of Computational Health Sciences, University of California, San Francisco, San Francisco, CA 94143, USA., Crawford ED; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA., Puschnik AS; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA., Phelps M; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA., Kistler A; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA., DeRisi JL; Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA; Division of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94143, USA., Doudna JA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA., Fletcher DA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA; UC Berkeley-UC San Francisco Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA; Biophysics Program, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA 94720, USA; Division of Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. Electronic address: fletch@berkeley.edu., Ott M; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: melanie.ott@gladstone.ucsf.edu.
Jazyk:	angličtina
Zdroj:	Cell [Cell] 2021 Jan 21; Vol. 184 (2), pp. 323-333.e9. Date of Electronic Publication: 2020 Dec 04.
DOI:	10.1016/j.cell.2020.12.001
Abstrakt:	The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved ∼100 copies/μL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2. Competing Interests: Declaration of interests P.F., S.S., G.J.K., J.A.D., D.A.F., and M.O. have filed patent applications related to this work. The Regents of the University of California have patents issued and pending for CRISPR technologies on which J.A.D. is an inventor. J.A.D. is a cofounder of Caribou Biosciences, Editas Medicine, Scribe Therapeutics, Intellia Therapeutics, and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Caribou Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Mammoth Biosciences, Synthego, Algen Biotechnologies, Felix Biosciences, and Inari. J.A.D. is a director at Johnson & Johnson and has research projects sponsored by Biogen, Pfizer, AppleTree Partners, and Roche. (Copyright © 2020 Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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