Sequence-specific capture and concentration of viral RNA by type III CRISPR system enhances diagnostic

Anna Nemudraia, Artem Nemudryi, Murat Buyukyoruk, Andrew M. Scherffius, Trevor Zahl, Tanner Wiegand, Shishir Pandey, Joseph E. Nichols, Laina N. Hall, Aidan McVey, Helen H. Lee, Royce A. Wilkinson, Laura R. Snyder, Joshua D. Jones, Kristin S. Koutmou, Andrew Santiago-Frangos () and Blake Wiedenheft ()
Additional contact information
Anna Nemudraia: Montana State University
Artem Nemudryi: Montana State University
Murat Buyukyoruk: Montana State University
Andrew M. Scherffius: Montana State University
Trevor Zahl: Montana State University
Tanner Wiegand: Montana State University
Shishir Pandey: Montana State University
Joseph E. Nichols: Montana State University
Laina N. Hall: Montana State University
Aidan McVey: Montana State University
Helen H. Lee: Montana State University
Royce A. Wilkinson: Montana State University
Laura R. Snyder: University of Michigan
Joshua D. Jones: University of Michigan
Kristin S. Koutmou: University of Michigan
Andrew Santiago-Frangos: Montana State University
Blake Wiedenheft: Montana State University

Nature Communications, 2022, vol. 13, issue 1, 1-12

Abstract: 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.

Date: 2022
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Persistent link: https://EconPapers.repec.org/RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-35445-5

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DOI: 10.1038/s41467-022-35445-5

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