Three-dimensional structures of the genome are dynamic and heterogeneous. Live-cell genome imaging has become a powerful way to reveal these properties. Previous live-cell genome imaging methods were either restricted to repetitive sequences, required insertion of long arrays of artificial tandem repeats at target loci, or the co-transfection of tens of target-specific plasmids. A few years ago, we developed a modified CRISPR-Cas platform for epigenetic editing called Casilio by combining CRISPR-dCas9 with Pumilio RNA binding domain-fused effectors, in which modified sgRNAs containing multiple copies of diverse Pumilio binding sites enable the simultaneous recruitment of effectors at different targets, as well as the multimerization of effector molecules at each target. We exploit this multimerization capability to enhance signal-to-noise ratio of genome imaging by recruiting 15 to 25 copies of PUF-fused fluorescent protein to sgRNA, enabling one sgRNA to label a nonrepetitive genomic locus. Furthermore, we track the dynamic 3D locations of three reference points along a chromatin loop using three different fluorescent proteins fused to orthogonal PUF domains and matching sgRNAs. In addition to imaging enhancer-promoter loops, this one-guide-per-locus capability allows for the imaging of extrachromosomal DNA molecules using unique junctional sequences formed during their circularization. We propose the potential of combining Casilio’s epigenetic editing and imaging capabilities to study how epigenetic modifications affect chromatin dynamics on-the-fly.
 

Imaging,CRISPR,epigenetics,epigenetic editing,3d genome,single cell 3D genome,live-cell imaging