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

• Published in: Nature communications Vol. 13; no. 1; pp. 7762 - 12
• Main Authors: Nemudraia, Anna, Nemudryi, Artem, Buyukyoruk, Murat, Scherffius, Andrew M., Zahl, Trevor, Wiegand, Tanner, Pandey, Shishir, Nichols, Joseph E., Hall, Laina N., McVey, Aidan, Lee, Helen H., Wilkinson, Royce A., Snyder, Laura R., Jones, Joshua D., Koutmou, Kristin S., Santiago-Frangos, Andrew, Wiedenheft, Blake
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 15.12.2022; Nature Publishing Group; Nature Portfolio
• Subjects: 38/88; 45/23; 45/71; 45/90; 631/326/2521; 631/326/596/4130; 631/337/4041; 631/61; 82/16; 82/83; 96/34; COVID-19 - diagnosis; CRISPR; CRISPR-Cas Systems; Deoxyribonucleic acid; Diagnostic systems; DNA; Double-stranded RNA; Endonucleases - metabolism; Humanities and Social Sciences; Humans; multidisciplinary; Nuclease; Nucleic acids; Nucleotide sequence; Ribonucleic acid; RNA; RNA, Viral - isolation & purification; SARS-CoV-2; Science; Science (multidisciplinary); Sensitivity; Severe acute respiratory syndrome coronavirus 2; Single-stranded DNA; Thermus thermophilus
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-35445-5

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. Many viral diagnostic approaches require nucleic acid extraction and amplification prior to detection. Here, the authors develop a method based on type III CRISPR systems which allows sequence specific capture, concentration, and detection of viral RNA directly from patient samples.