A Rationally Designed CRISPR/Cas12a Assay Using a Multimodal Reporter for Various Readouts

The CRISPR/Cas systems offer a programmable platformfor nucleicacid detection, and CRISPR/Cas-based diagnostics (CRISPR-Dx) havedemonstrated the ability to target nucleic acids with greater accuracyand flexibility. However, due to the configuration of the reporterand the underlying labeling mechanism, almost all reported CRISPR-Dxrely on a single-option readout, resulting in limitations in end-pointresult readouts. This is also associated with high reagent consumptionand delays in diagnostic reports due to protocol differences. Herein,we report for the first time a rationally designed Cas12a-based multimodaluniversal reporter (CAMURE) with improved sensitivity that harnessesa dual-mode reporting system, facilitating options in end-point readouts.Through systematic configurations and optimizations, our novel universalreporter achieved a 10-fold sensitivity enhancement compared to theDETECTR reporter. Our unique and versatile reporter could be pairedwith various readouts, conveying the same diagnostic results. We appliedour novel reporter for the detection of staphylococcal enterotoxinA due to its high implication in staphylococcal food poisoning. Integratedwith loop-mediated isothermal amplification, our multimodal reporterachieved 10 CFU/mL sensitivity and excellent specificity using a real-timefluorimeter, in-tube fluorescence, and lateral flow strip readouts.We also propose, using artificially contaminated milk samples, a fast(2-5 min) Triton X-100 DNA extraction approach with a comparableyield to the commercial extraction kit. Our CAMURE could be leveragedto detect all gene-encoding SEs by simply reprogramming the guideRNA and could also be applied to the detection of other infectionsand disease biomarkers.