Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic.
Cell Rep Med
; 2(6): 100319, 2021 06 15.
Article
in English
| MEDLINE | ID: covidwho-1244849
ABSTRACT
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.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
RNA, Viral
/
CRISPR-Cas Systems
/
COVID-19 Nucleic Acid Testing
/
COVID-19
Type of study:
Diagnostic study
/
Prognostic study
Limits:
Humans
Language:
English
Journal:
Cell Rep Med
Year:
2021
Document Type:
Article
Affiliation country:
J.xcrm.2021.100319
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