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1.
Nat Commun ; 13(1): 7762, 2022 12 15.
Article in English | MEDLINE | ID: covidwho-2160212

ABSTRACT

Type-III CRISPR-Cas systems have recently been adopted for sequence-specific detection of SARS-CoV-2. Here, we repurpose the type III-A CRISPR complex from Thermus thermophilus (TtCsm) for programmable capture and concentration of specific RNAs from complex mixtures. The target bound TtCsm complex generates two cyclic oligoadenylates (i.e., cA3 and cA4) that allosterically activate ancillary nucleases. We show that both Can1 and Can2 nucleases cleave single-stranded RNA, single-stranded DNA, and double-stranded DNA in the presence of cA4. We integrate the Can2 nuclease with type III-A RNA capture and concentration for direct detection of SARS-CoV-2 RNA in nasopharyngeal swabs with 15 fM sensitivity. Collectively, this work demonstrates how type-III CRISPR-based RNA capture and concentration simultaneously increases sensitivity, limits time to result, lowers cost of the assay, eliminates solvents used for RNA extraction, and reduces sample handling.


Subject(s)
COVID-19 , CRISPR-Cas Systems , RNA, Viral , Humans , COVID-19/diagnosis , DNA , Endonucleases/metabolism , RNA, Viral/isolation & purification , SARS-CoV-2 , Thermus thermophilus
2.
Methods ; 205: 1-10, 2022 09.
Article in English | MEDLINE | ID: covidwho-1882634

ABSTRACT

Polymerase Chain Reaction (PCR) is the reigning gold standard for molecular diagnostics. However, the SARS-CoV-2 pandemic reveals an urgent need for new diagnostics that provide users with immediate results without complex procedures or sophisticated equipment. These new demands have stimulated a tsunami of innovations that improve turnaround times without compromising the specificity and sensitivity that has established PCR as the paragon of diagnostics. Here we briefly introduce the origins of PCR and isothermal amplification, before turning to the emergence of CRISPR-Cas and Argonaute proteins, which are being coupled to fluorimeters, spectrometers, microfluidic devices, field-effect transistors, and amperometric biosensors, for a new generation of nucleic acid-based diagnostics.


Subject(s)
Argonaute Proteins , CRISPR-Cas Systems , Nucleic Acid Amplification Techniques , Polymerase Chain Reaction , Argonaute Proteins/genetics , CRISPR-Cas Systems/genetics , Humans , Nucleic Acid Amplification Techniques/methods
3.
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.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , CRISPR-Cas Systems/genetics , RNA, Viral/metabolism , COVID-19/virology , Colorimetry , Humans , Molecular Diagnostic Techniques , Nasopharynx/virology , Nucleic Acid Amplification Techniques , RNA, Guide, Kinetoplastida/metabolism , RNA, Viral/chemistry , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism
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