Optogenetic Control of Background Fluorescence Reduction for CRISPR-Based Genome Imaging.

Abstract

The CRISPR/dCas9 system has become an essential tool for live-cell imaging of genomic loci, but it has limited applications in imaging low-/non-repetitive genomic loci due to the strong nuclear background noise emerging from many untargeted fluorescent modules. Here, we propose an optogenetically controlled background fluorescence reduction strategy that combines the CRISPR-SunTag system with a light-inducible nuclear export tag (LEXY). Utilizing the SunTag system, multiple copies of LEXY-tagged sfGFP were recruited to the C-terminal dCas9, recognizing the target genomic loci. As the nuclear export sequence at the C-terminal LEXY could be exposed to pulsed blue light irradiation, the untargeted nuclear labeling modules were light controllably transferred to the cytoplasm. Consequently, genomic loci containing as few as nine copies of repeats were clearly visualized, and a significant increase in the signal-to-noise ratio was achieved. This simple and controllable method is expected to have a wide range of applications in cell biology.