Gene

Quantum mechanics

Oligonucleotide

Molecular biology

Biology

Molecular probe

Materials science

Genome editing

Small molecule

Molecular beacon

Trans-activating crRNA

CRISPR

Biochemistry

Fluorophore

Aptamer

Computational biology

Biosensor

Combinatorial chemistry

Physics

Nanotechnology

Nucleic acid

Fluorescence

Chemistry

Biophysics

Biosensors and Bioelectronics

Molecular diagnostics are vital for the identification, prevention, and treatment of numerous diseases and are of particular demand in point-of-care (POC) settings. Nevertheless, most reported biosensors based on the CRISPR-Cas system have focused on nucleic-acid targets. Here, we report a versatile diagnostic strategy for small molecules called Molecular Radar (Random Molecular Aptamer-Dependent CRISPR-Assist Reporter), The workflow is simple, convenient, and rapid (conducted at 37 °C in under 25 min), indicating the substantial potential of the proposed assay could be adapted into a biosensor for POC settings and on-site molecular diagnostics. This strategy is based on the CRISPR Cas12a-assisted fluorescence reporter system that consists of Cas12a, CRISPR RNA (crRNA), a single-stranded DNA (ssDNA) probe labeled with a fluorophore at the 5′ end and a quencher at the 3’ end (F-Q probe), and a single-stranded DNA aptamer for the target molecule. In the presence of a target molecule, the aptamer binds to this small molecule with high specificity and affinity, resulting in a decrease of aptamer hybridized to the crRNA-Cas12a duplex. This decrease in activated Cas12a leads to a significant reduction in fluorescence signal. In this study, adenosine-5ˊ-triphosphate (ATP) was selected as model target molecule and an ATP detect method was developed with high specificity and sensitivity with a linear range from 25 to 500 μM and a detection limit of 104 nM. Moreover, the particular characteristics of CRISPR-Cas12a that we report here for the first time have enriched our understanding of Cas12a and provided guidance for further research on CRISPR-Cas12a-based biosensors.

Aptamer assisted CRISPR-Cas12a strategy for small molecule diagnostics