Multiplexable and Biocomputational Virus Detection by CRISPR-Cas9-Mediated Strand Displacement.
Rosa Márquez-CostaRoser Montagud-MartínezMaría-Carmen MarquésEliseo AlbertDavid NavarroJosé-Antonio DaròsRaúl RuizGuillermo RodrigoPublished in: Analytical chemistry (2023)
Recurrent disease outbreaks caused by different viruses, including the novel respiratory virus SARS-CoV-2, are challenging our society at a global scale; so versatile virus detection methods would enable a calculated and faster response. Here, we present a novel nucleic acid detection strategy based on CRISPR-Cas9, whose mode of action relies on strand displacement rather than on collateral catalysis, using the Streptococcus pyogenes Cas9 nuclease. Given a preamplification process, a suitable molecular beacon interacts with the ternary CRISPR complex upon targeting to produce a fluorescent signal. We show that SARS-CoV-2 DNA amplicons generated from patient samples can be detected with CRISPR-Cas9. We also show that CRISPR-Cas9 allows the simultaneous detection of different DNA amplicons with the same nuclease, either to detect different SARS-CoV-2 regions or different respiratory viruses. Furthermore, we demonstrate that engineered DNA logic circuits can process different SARS-CoV-2 signals detected by the CRISPR complexes. Collectively, this CRISPR-Cas9 R-loop usage for the molecular beacon opening (COLUMBO) platform allows a multiplexed detection in a single tube, complements the existing CRISPR-based methods, and displays diagnostic and biocomputing potential.
Keyphrases
- crispr cas
- genome editing
- sars cov
- nucleic acid
- loop mediated isothermal amplification
- real time pcr
- label free
- respiratory syndrome coronavirus
- single molecule
- circulating tumor
- cell free
- quantum dots
- drug delivery
- dna methylation
- candida albicans
- risk assessment
- human health
- gold nanoparticles
- genetic diversity
- transcription factor
- sensitive detection
- climate change