Light-activated CRISPR-Cas12a for amplified imaging of microRNA in cell cycle phases at single-cell levels.

ABSTRACT

An ortho-nitrobenzyl phosphate ester-caged nucleic acid hairpin structure coupled to the CRISPR-Cas12a complex is introduced as a functional reaction module for the light-induced activation of the CRISPR-Cas12a (LAC12a) machinery toward the amplified fluorescence detection of microRNA-21 (miRNA-21). The LAC12a machinery is applied for the selective, in vitro sensing of miRNA-21 and for the intracellular imaging of miRNA-21 in different cell lines. The LAC12a system is used to image miRNA-21 in different cell cycle phases of MCF-7 cells. Moreover, the LAC12a machinery integrated in cells enables the two-photon laser confocal microscopy-assisted, light-stimulated spatiotemporal, selective activation of the CRISPR-Cas12a miRNA-21 imaging machinery at the single-cell level and the evaluation of relative expression levels of miRNA-21 at distinct cell cycle phases. The method is implemented to map the distribution of cell cycle phases in an array of single cells.