Summary There 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 10 7 copies/μL for a single guide RNA to 10 6 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. Graphical abstract Highlights Sequence-specific recognition of RNA by CRISPR Csm complex activates Cas10 Cas10 polymerizes ATP to make cyclic oligonucleotides, pyrophosphates, and protons Cas10’s rapidly amplified products are detectable in 1–30 min RT-LAMP can be coupled to T7-Csm to rapidly and sensitively detect SARS-CoV-2 RNA Recognition of a complementary target RNA by the type III CRISPR systems uniquely triggers the activation of a CRISPR-associated polymerase domain in Cas10. The polymerase generates oligoadenylates, protons, and pyrophosphates. Santiago-Frangos et al. repurposed the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2 by developing three different detection methods that rely on each of these products.
【저자키워드】 COVID-19, SARS-CoV-2, CRISPR-Cas, type III, viral diagnostics, CRISPR Dx, point-of-care diagnostics, 【초록키워드】 viruses, Multiplexing, RNA, CRISPR, amplification, RT-LAMP, sensitivity, Region, Patient, limit of detection, SARS-CoV-2 RNA, ATP, Trigger, complementary, triggers, followed by, conserved regions, Activation, Recognition, Abstract, SARS-CoV-2 genomes, complex, domain, oligonucleotides, polymerase, protons, decrease, guide RNA, IMPROVE, detect, conserved, detectable, clinically, generate, less, activate, amplified product, CRISPR-Ca, proton, 【제목키워드】 diagnostic, provide, CRISPR-Ca,