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

Parinaz Fozouni; Sungmin Son; María Díaz de León Derby; Gavin J Knott; Carley N Gray; Michael V D'Ambrosio; Chunyu Zhao; Neil A Switz; G Renuka Kumar; Stephanie I Stephens; Daniela Boehm; Chia-Lin Tsou; Jeffrey Shu; Abdul Bhuiya; Maxim Armstrong; Andrew R Harris; Pei-Yi Chen; Jeannette M Osterloh; Anke Meyer-Franke; Bastian Joehnk; Keith Walcott; Anita Sil; Charles Langelier; Katherine S Pollard; Emily D Crawford; Andreas S Puschnik; Maira Phelps; Amy Kistler; Joseph L DeRisi; Jennifer A Doudna; Daniel A Fletcher; Melanie Ott

doi:10.1016/j.cell.2020.12.001

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

Cell. 2021 Jan 21;184(2):323-333.e9. doi: 10.1016/j.cell.2020.12.001. Epub 2020 Dec 4.

Authors

Parinaz Fozouni¹, Sungmin Son², María Díaz de León Derby³, Gavin J Knott⁴, Carley N Gray⁵, Michael V D'Ambrosio², Chunyu Zhao⁶, Neil A Switz⁷, G Renuka Kumar⁵, Stephanie I Stephens⁵, Daniela Boehm⁵, Chia-Lin Tsou⁵, Jeffrey Shu⁵, Abdul Bhuiya³, Maxim Armstrong⁸, Andrew R Harris², Pei-Yi Chen⁵, Jeannette M Osterloh⁹, Anke Meyer-Franke⁹, Bastian Joehnk¹⁰, Keith Walcott¹⁰, Anita Sil¹¹, Charles Langelier¹², Katherine S Pollard¹³, Emily D Crawford¹⁴, Andreas S Puschnik⁶, Maira Phelps⁶, Amy Kistler⁶, Joseph L DeRisi¹⁵, Jennifer A Doudna¹⁶, Daniel A Fletcher¹⁷, Melanie Ott¹⁸

Affiliations

¹ 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.
² Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA.
³ 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.
⁴ 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.
⁵ J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
⁶ Chan Zuckerberg Biohub, San Francisco, CA 94158, USA.
⁷ Department of Physics and Astronomy, San José State University, San Jose, CA 95192, USA.
⁸ Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
⁹ J. David Gladstone Institutes, San Francisco, CA 94158, USA.
¹⁰ Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA.
¹¹ 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.
¹² Chan Zuckerberg Biohub, San Francisco, CA 94158, USA; Division of Infectious Diseases, University of California, San Francisco, San Francisco, CA 94143, USA.
¹³ 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.
¹⁴ Chan Zuckerberg Biohub, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA.
¹⁵ 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.
¹⁶ 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.
¹⁷ 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.
¹⁸ 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.

Abstract

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.

Keywords: COVID-19; CRISPR Dx; CRISPR-Cas13; SARS-CoV-2; mobile phone microscopy; point-of-care diagnostics.

Publication types

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

MeSH terms

Animals
COVID-19 Nucleic Acid Testing / economics
COVID-19 Nucleic Acid Testing / instrumentation
COVID-19 Nucleic Acid Testing / methods*
CRISPR-Cas Systems
Cell Line
Cell Phone / instrumentation*
Coronavirus Nucleocapsid Proteins / genetics
Humans
Nasopharynx / virology
Optical Imaging / instrumentation
Optical Imaging / methods*
Phosphoproteins / genetics
Point-of-Care Testing
RNA Interference
RNA, Viral / analysis*
RNA, Viral / genetics
Sensitivity and Specificity
Viral Load / economics
Viral Load / instrumentation
Viral Load / methods*

Substances

Coronavirus Nucleocapsid Proteins
Phosphoproteins
RNA, Viral
nucleocapsid phosphoprotein, SARS-CoV-2

Abstract

Publication types

MeSH terms

Substances

Grants and funding