CRISPR/dCas9-mediated biosensor for detection of tick-borne diseases

Abstract

Rapid and highly sensitive detection of biomolecules is greatly needed for pathogen diagnosis in clinical samples, but the method needs to be significantly improved in terms of sensitivity and specificity for actual use in clinical settings. Here, we report the development of an improved molecular diagnostics tool that utilizes CRISPR/dCas9mediated biosensor that couples a nuclease inactivated Cas9 (dCas9) and single microring resonator biosensor, enables label-free and real-time detection of pathogenic DNA and RNA. We addressed the clinical utility of this CRISPR/dCas9-mediated biosensor in tick-borne illnesses including scrub typhus (ST) and severe fever with thrombocytopenia syndrome (SFTS), whose clinical presentations are too similar to be easily differentiated. By using CRISPR/dCas9-mediated biosensor, we achieved single molecule sensitivity for the detection of ST (0.54 aM) and SFTS (0.63 aM); this detection sensitivity is 100 times more sensitive than that of RT-PCR assay. Finally, CRISPR/dCas9-mediated biosensor was able to clearly distinguish between ST and SFTS in serum samples within 20 min. We believe that CRISPR/dCas9-mediated biosensor will be useful for rapid and accurate molecular diagnostic tool that is suitable for immediate clinical applications.

Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

“…With this method, genetically very similar tick-borne diseases could be distinguished in serum samples at the single molecule level within 20 min. The detection sensitivity of around 0.6 aM is about 100 times lower than that of conventional RT-PCR assays ( Koo et al, 2018a ).…”

“…c) Cross-section view of the chip in the state of assay/reagent loading, sliding, and readout. Reproduced with permission from ( Koo et al, 2018a ). Copyright 2019 American Chemical Society.…”

“…dCas9 was also utilized in a single microring resonator (SMR) biosensor, enabling real-time detection of both DNA and RNA samples ( Koo et al, 2018a ). A SMR is a set of waveguides of which one is a closed loop.…”

Dead Cas Systems: Types, Principles, and Applications

“…Creating cellular recorders with dCas systems like CAMERA [171] and DOMINO [172] allows writing incoming events and their parameters for detailed investigations of signaling cascades and other biological processes. CRISPR-driven evolution [132], CRISPR-diagnostics [173,174], CRISPR-biosensors [175], and many more create the foundations for the new technological era.…”

Architectured Therapeutic and Diagnostic Nanoplatforms for Combating SARS-CoV-2: Role of Inorganic, Organic, and Radioactive Materials

“…The sensitivity of the assay was highly improved compared to that of traditional RT-PCR. 67 The detection limit of these methods could reach the attomole range, that is, even single molecules can be detected.…”