Designing a Chemiluminescent Platform for Type III CRISPR-Cas Systems: An Innovative Approach to Nucleic Acid Detection

CRISPR-Cas systems serve as an adaptive immune mechanism in prokaryotes, targeting and neutralizing mobile genetic elements. These systems are categorized into two main classes and various subtypes, with Type III systems being particularly distinctive for their RNA-targeting capabilities. Using a multi-subunit effector complex guided by CRISPR RNA (crRNA), these systems recognize and degrade foreign RNA molecules. This process also activates the Cas10 subunit, initiating the production of cyclic oligoadenylate (cOA) signaling molecules. These cOA molecules then activate effector proteins equipped with sensory domains, which drive further biochemical reactions.

Type III CRISPR-Cas systems have been repurposed for innovative nucleic acid detection due to their ability to amplify signals. One established approach uses Csx1, an RNase activated by cOA, in conjunction with an RNA-targeting complex, to produce a detectable fluorescent signal. However, fluorescence-based methods rely on external light sources, complicating their use in portable diagnostic devices.

To address this limitation, a chemiluminescent (CL) detection strategy was developed. This technique employs a G-quadruplex (G4) RNA probe, which catalyzes a chemiluminescent reaction between luminol and hydrogen peroxide in the presence of hemin. When the target RNA is present, Csx1 is activated by the CRISPR-Cas complex, leading to the degradation of the G4 probe and resulting in the loss of the chemiluminescent signal. This method offers a simple, highly sensitive detection system without the need for external light sources. The chemiluminescent readout provides a practical solution for creating portable and efficient diagnostic tools, making it an ideal choice for on-site nucleic acid testing.

This work was supported by the Nano-ImmunoEra project that has received funding from the European Union’s MSCA Staff exchange Horizon Europe programme, Grant Agreement Number 101086341.