Accelerated RNA detection using tandem CRISPR nucleases

Liu, Tina Y.; Knott, Gavin J.; Smock, Dylan C. J.; 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.; 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.