Santiago-Frangos, AndrewHall, Laina N.Nemudraia, AnnaNemudryi, ArtemKrishna, PushyaWiegand, TannerWilkinson, Royce A.Snyder, Deann T.Hedges, Jodi F.Cicha, CalvinLee, Helen H.Graham, AvaJutila, Mark A.Taylor, Matthew P.Wiedenheft, Blake2022-08-302022-08-302021-06Santiago-Frangos, A., Hall, L. N., Nemudraia, A., Nemudryi, A., Krishna, P., Wiegand, T., ... & Wiedenheft, B. (2021). Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic. Cell Reports Medicine, 2(6), 100319.2666-3791https://scholarworks.montana.edu/handle/1/17034There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons, and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design ten guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 107 copies/μL for a single guide RNA to 106 copies/μL for ten guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/μL and is completed using patient samples in less than 30 min.en-UScc-byhttps://creativecommons.org/licenses/by/4.0/sars cov 2Article