Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector
| Autor: | Catherine S. Adamson, Malcolm F. White, Sabine Grüschow |
|---|---|
| Přispěvatelé: | Scottish Funding Council, BBSRC, University of St Andrews. School of Biology, University of St Andrews. Biomedical Sciences Research Complex, University of St Andrews. St Andrews Bioinformatics Unit |
| Rok vydání: | 2021 |
| Předmět: |
AcademicSubjects/SCI00010
QH301 Biology Prophages CRISPR-Associated Proteins NucC NDAS Cell Line Nucleic acid secondary structure Nuclease QH301 chemistry.chemical_compound Chlorocebus aethiops Endoribonucleases Genetics RNA and RNA-protein complexes Animals Humans CRISPR Guide RNA Vero Cells Vibrio QR355 Endodeoxyribonucleases biology SARS-CoV-2 Effector COVID-19 RNA Type III Crispr Cell biology chemistry Nucleic acid biology.protein RNA Viral CRISPR-Cas Systems QR355 Virology DNA RNA detection |
| Zdroj: | Nucleic Acids Research |
| DOI: | 10.1101/2021.09.13.460032 |
| Popis: | Funding: This work was supported by grants from the Biotechnology and Biological Sciences Research Council (Grant BB/T004789/1 to MFW), Medical Research Scotland (Grant CVG-1719-2020 to MFW) and The University of St Andrews Restarting Research Funding Scheme (SARRF), funded through the Scottish Funding Council (grant reference SFC/AN/08/020) to MFW and CSA. Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo. Publisher PDF |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|