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

Autor:	Santiago-Frangos A; Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA., Hall LN; Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA., Nemudraia A; Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA., Nemudryi A; Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA., Krishna P; Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA., Wiegand T; Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA., Wilkinson RA; Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA., Snyder DT; Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA., Hedges JF; Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA., Cicha C; Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA., Lee HH; Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA., Graham A; Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA., Jutila MA; Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA., Taylor MP; Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA., Wiedenheft B; Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA.
Jazyk:	angličtina
Zdroj:	Cell reports. Medicine [Cell Rep Med] 2021 Jun 15; Vol. 2 (6), pp. 100319. Date of Electronic Publication: 2021 May 27.
DOI:	10.1016/j.xcrm.2021.100319
Abstrakt:	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. Competing Interests: B.W. is the founder of SurGene and VIRIS Detection Systems. B.W., A.S.-F., A. Nemudraia, and A. Nemudryi are inventors on patent applications related to CRISPR-Cas systems and applications thereof. (© 2021 The Author(s).)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu