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.

Fluorescent Zn 2+ sensors play a pivotal role in zinc biology, but their application in complex media such as blood serum or plate reader-based cellular assays is hampered by autofluorescence and light scattering. Bioluminescent sensor proteins provide an attractive alternative to fluorescent sensors for these applications, but the only bioluminescent sensor protein developed so far, BLZinCh, has a limited sensor response and a suboptimal Zn 2+ affinity. In this work, we expanded the toolbox of bioluminescent Zn 2+ sensors by developing two new sensor families that show a large change in the emission ratio and cover a range of physiologically relevant Zn 2+ affinities. The LuZi platform relies on competitive complementation of split NanoLuc luciferase and displays a robust, 2-fold change in red-to-blue emission, allowing quantification of free Zn 2+ between 2 pM and 1 nM. The second platform was developed by replacing the long flexible GGS linker in the original BLZinCh sensor by rigid polyproline linkers, yielding a series of BLZinCh-Pro sensors with a 3–4-fold improved ratiometric response and physiologically relevant Zn 2+ affinities between 0.5 and 1 nM. Both the LuZi and BLZinCh-Pro sensors allowed the direct determination of low nM concentrations of free Zn 2+ in serum, providing an attractive alternative to more laborious and/or indirect approaches to measure serum zinc levels. Furthermore, the genetic encoding of the BLZinCh-Pro sensors allowed their use as intracellular sensors, where the sensor occupancy of 40–50% makes them ideally suited to monitor both increases and decreases in intracellular free Zn 2+ concentration in simple, plate reader-based measurements, without the need for fluorescence microscopy.

Nucleic acid detection methods based on 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 post-amplification 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. Whereas LUNAS itself features a detection limit of ~1 pM for dsDNA targets, the LUNAS platform is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a single-pot assay. We designed a one-pot RT-RPA-LUNAS assay for detecting SARS-CoV-2 RNA without the need for RNA isolation and demonstrated the diagnostic performance for COVID-19 patient nasopharyngeal swab samples using a digital camera to record the ratiometric signal. Detection of SARS-CoV-2 from samples with viral RNA loads of ~200 cp/microL was achieved within ~20 minutes, showing that RPA-LUNAS is attractive for point-of-care diagnostic applications.