Multiplexable and Biocomputational Virus Detection by CRISPR-Cas9-Mediated Strand Displacement

Recurrent disease outbreaks caused by different viruses,includingthe novel respiratory virus SARS-CoV-2, are challenging our societyat a global scale; so versatile virus detection methods would enablea calculated and faster response. Here, we present a novel nucleicacid detection strategy based on CRISPR-Cas9, whose mode of actionrelies on strand displacement rather than on collateral catalysis,using the Streptococcus pyogenes Cas9 nuclease. Givena preamplification process, a suitable molecular beacon interactswith the ternary CRISPR complex upon targeting to produce a fluorescentsignal. We show that SARS-CoV-2 DNA amplicons generated from patientsamples can be detected with CRISPR-Cas9. We also show that CRISPR-Cas9allows the simultaneous detection of different DNA amplicons withthe same nuclease, either to detect different SARS-CoV-2 regions ordifferent respiratory viruses. Furthermore, we demonstrate that engineeredDNA logic circuits can process different SARS-CoV-2 signals detectedby the CRISPR complexes. Collectively, this CRISPR-Cas9 R-loop usagefor the molecular beacon opening (COLUMBO) platform allows a multiplexeddetection in a single tube, complements the existing CRISPR-basedmethods, and displays diagnostic and biocomputing potential.

Automated summary

This study presents a novel nucleic acid detection strategy based on CRISPR-Cas9, which enables the detection of different viruses, including the novel respiratory virus SARS-CoV-2. The results show that CRISPR-Cas9 allows the simultaneous detection of different DNA amplicons, and engineered DNA logic circuits can process the detected signals. This platform has the potential for diagnosis and biocomputing.