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

ABSTRACT: 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.

SUBMITTER: van der Veer HJ 

PROVIDER: S-EPMC10141630 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature