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

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

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.