Amplification-free detection of zoonotic viruses using Cas13 and multiple CRISPR RNAs
Year: 2025
Type: article
Abstract: Zoonotic viruses such as hantaviruses and influenza A viruses present a threat to humans and livestock. There is thus a need for methods that are rapid, sensitive, and relatively cheap to detect infections with these pathogens early. Here we use an amplification-free CRISPR-Cas13-based assay, which is simple, cheap and field-deployable, to detect the presence or absence of genomic hantavirus or influenza A virus RNA. In addition, we evaluate whether the use of multiple CRISPR RNAs (crRNAs) can improve the sensitivity of this amplification-free method. We demonstrate that for the hantaviruses Tula Virus (TULV) and Andes Virus (ANDV) a combination of two or three crRNAs provides the best sensitivity for detecting viral RNA, whereas for influenza virus RNA detection, additional crRNAs provide no benefit. We also show that the amplification-free method can be used to detect TULV and ANDV RNA in tissue culture infection samples and influenza A virus RNA in clinical nasopharyngeal swabs. In clinical samples, the Cas13 assay has an 85% agreement with RT-qPCR for identifying a positive sample. Overall, these findings indicate that amplification-free CRISPR-Cas13 detection of viral RNA has potential as a tool for rapidly detecting zoonotic virus infections.
Source: Journal of General Virology
DOI: 10.1099/jgv.0.002169
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Funders: National Science Foundation, Bill and Melinda Gates Foundation, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Deutsche Forschungsgemeinschaft, Vetenskapsrådet, Robert Koch Institut, National Institutes of Health, Centers for Disease Control and Prevention, National Institute of General Medical Sciences, National Institute of Allergy and Infectious Diseases, National Center for Advancing Translational Sciences, Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases
Awards: G1052925518, Characterisation of the molecular mechanisms of hantavirus polymerase function, G1395363761, G1674541431, Rutgers, The State University of New Jersey - to develop CRISPR cas 12/13-based multi-pathogen wastewater surveillance in Uganda, Graduate Research Fellowship Program (GRFP), G355248549, G3797236039, EXC 2155: RESIST Resolving Infection Susceptibility, Development of a highly-multiplexed CRISPR-based TB drug susceptibility test, G4733222645, G5186833673, Defective hantavirus genomes as a determinant of the host immune response and pathogenesis, G83226193, G9000009221, G9000078355, Structure and dynamics of RNA elements regulating viral aberrant RNA synthesis
OpenAlex ID: W4415819023