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

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

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