Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation

Nucleic acid detection methods based on CRISPR and isothermal
amplification
techniques show great potential for point-of-care diagnostic applications.
However, most current methods rely on fluorescent or lateral flow
assay readout, requiring external excitation or postamplification
reaction transfer. Here, we developed a bioluminescent nucleic acid
sensor (LUNAS) platform in which target dsDNA is sequence-specifically
detected by a pair of dCas9-based probes mediating split NanoLuc luciferase
complementation. LUNAS is easily integrated with recombinase polymerase
amplification (RPA), providing attomolar sensitivity in a rapid one-pot
assay. A calibrator luciferase is included for a robust ratiometric
readout, enabling real-time monitoring of the RPA reaction using a
simple digital camera. We designed an RT-RPA-LUNAS assay that allows
SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation
and demonstrated its diagnostic performance for COVID-19 patient nasopharyngeal
swab samples. Detection of SARS-CoV-2 from samples with viral RNA
loads of ∼200 cp/μL was achieved within ∼20 min,
showing that RPA-LUNAS is attractive for point-of-care infectious
disease testing.