A CRISPR/Cas12a-based label-free fluorescent method for visual signal output

Abstract

CRISPR/Cas12a, as a powerful and programmable biosensing tool, has brought great convenience in visual detection of the target in a sequence-specific mode. Though fluorescent output is the predominant way of present visual methods, this strategy highly depends on cleaving fluorophore-/quencher-labeled oligonucleotides. Few research focuses on realizing fluorescent visual detection without DNA-labeling. Herein, we have proposed a CRISPR/Cas12a-based label-free fluorescent visual detection strategy. The basic principle of this strategy is that G-quadruplex can enhance the fluorescent emission of fluorescent ligands while dsDNA target-activated Cas12a make them impotent by disrupting the higher-ordered structure. After comparing four kinds of G-quadruplex-specific ligands, we selected Thioflavin T (ThT) to achieve the visual observation goal. The application feasibility was confirmed by combining with loop-mediated isothermal amplification (LAMP) for detection of Vibrio parahaemolyticus ( V. parahaemolyticus ), and it demonstrated a high sensitivity (1.36 × 10 2 copies) and specificity (among 12 kinds of bacteria). The method displayed similar performance as the real-time PCR for detection of real samples yet had lower requirement on the instrument. The signal output format could also distinguish Salmo salar from other seven kinds of sequence-similar Salmonidae. The findings and strategy proposed in this manuscript will expand the application of CRISPR/Cas12a-based label-free fluorescent analysis. • A label-free and visible fluorescent reporter of CRISPR/Cas12a has been developed. • CRISPR/Cas12a makes G-quadruplex impotent to enhance the fluorescence of ThT. • ThT is better than NMM to visually probe the cleavage of G-quadruplex. • Combined with LAMP, V. parahaemolyticus was detected specifically and sensitively.