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

Anna NemudraiaArtem A NemudryiMurat BuyukyorukAndrew M ScherffiusTrevor ZahlTanner WiegandShishir PandeyJoseph E NicholsLaina N HallAidan McVeyHelen H LeeRoyce A WilkinsonLaura R SnyderJoshua D JonesKristin D KoutmouAndrew Santiago-FrangosBlake Wiedenheft
Published in: Nature communications (2022)
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., cA 3 and cA 4 ) 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 cA 4 . 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.
Keyphrases
  • type iii
  • genome editing
  • crispr cas
  • sars cov
  • nucleic acid
  • binding protein
  • respiratory syndrome coronavirus
  • high throughput
  • protein kinase
  • gene expression
  • cell free
  • dna methylation
  • label free
  • respiratory tract