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 theStreptococcus pyogenesCas9 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.