CRISPR-mediated imaging of three-dimensional genomic loci in live cells*

The three-dimensional (3D) organization and dynamics of eukaryotic genome play significant roles in regulating gene transcription and cellular function. Conventional CRISPR-based imaging approach is powerful for imaging specific chromatin regions in live cells but relies on laborious genome engineering. We recently developed a versatile imaging technique, LiveFISH, which visualizes genomic element dynamics in live cells by delivering in vitro assembled fluorescent ribonucleoproteins (fRNPs) of fluorophore-labeled guide RNAs and purified nuclease-deactivated dCas9. However, it was mostly applicable to imaging repetitive genomic regions. Here we report an improved LiveFISH method that could target non-repetitive genomic loci of choice. Our method integrates computational guide RNA probe design, chemical fluorophore labeling, and high-quality cell delivery platforms. We characterized quantitively the dependence of imaging quality on a series of probe design parameters and applied this improved LiveFISH technology to track 3D dynamics and organization of enhancers and promoters of different genes with high localization precision. The platform can work potentially in diverse cell types to study the causality between the 3D genome and gene regulation.