- Liu, Tina Y;
- Knott, Gavin J;
- Smock, Dylan CJ;
- Desmarais, John J;
- Son, Sungmin;
- Bhuiya, Abdul;
- Jakhanwal, Shrutee;
- Prywes, Noam;
- Agrawal, Shreeya;
- de León Derby, María Díaz;
- Switz, Neil A;
- Armstrong, Maxim;
- Harris, Andrew R;
- Charles, Emeric J;
- Thornton, Brittney W;
- Fozouni, Parinaz;
- Shu, Jeffrey;
- Stephens, Stephanie I;
- Kumar, G Renuka;
- Zhao, Chunyu;
- Mok, Amanda;
- Iavarone, Anthony T;
- Escajeda, Arturo M;
- McIntosh, Roger;
- Kim, Shin E;
- Dugan, Eli J;
- Consortium, IGI Testing;
- Pollard, Katherine S;
- Tan, Ming X;
- Ott, Melanie;
- Fletcher, Daniel A;
- Lareau, Liana F;
- Hsu, Patrick D;
- Savage, David F;
- Doudna, Jennifer A
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 recognition of RNA that triggers cleavage and release of a fluorescent reporter molecule 1,2 , but long reaction times hamper sensitivity and speed when applied to point-of-care testing. 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 RNA copies/microliter in 20 minutes. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that detected SARS-CoV-2 RNA from nasopharyngeal samples with PCR-derived Ct values up to 29 in microfluidic chips, using a compact imaging system. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables direct RNA detection in a format amenable to point-of-care infection diagnosis, as well as to a wide range of other diagnostic or research applications.