Multiplexable and Biocomputational Virus Detection by CRISPR-Cas9-Mediated Strand Displacement
Rosa Márquez‐Costa, Roser Montagud‐Martínez, María-Carmen Marqués, Eliseo Albert, David Navarro, José‐Antonio Daròs, Raúl Ruiz, Guillermo Rodrigo
Abstract
High Resolution Image Download MS PowerPoint Slide 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.