Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic

• Main Authors: Andrew Santiago-Frangos, Laina N. Hall, Anna Nemudraia, Artem Nemudryi, Pushya Krishna, Tanner Wiegand, Royce A. Wilkinson, Deann T. Snyder, Jodi F. Hedges, Calvin Cicha, Helen H. Lee, Ava Graham, Mark A. Jutila, Matthew P. Taylor, Blake Wiedenheft
• Format: Article
• Language: English
• Published: Elsevier 2021-06-01
• Series: Cell Reports Medicine
• Subjects: CRISPR-Cas; type III; SARS-CoV-2; viral diagnostics; CRISPR Dx; COVID-19
• Online Access: http://www.sciencedirect.com/science/article/pii/S2666379121001622

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 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.