Fig. 3: Casilio live-cell imaging of chromatin interactions and validation.

a Casilio probes for visualizing chromatin interactions mediated by cohesin. Locus A (5’ anchor) labeled by gRNA-15xPBSc/Clover-PUFc and locus B (3’ anchor) labeled by gRNA-15xPBS9R/PUF9R-iRFP670. MASP1-BCL6 loop with anchors 367 kb apart. Arc height is proportional to number of ChIA-PET sequencing reads. b Representative time-lapse images of MASP1-BCL6 loop anchors, locus A near MASP1 (Clover, green, stealth arrowhead) and locus B near BCL6 (iRFP670, red, triangle arrowhead), in ARPE-19 cells. Image strips show two allelic pairs at indicated time points. Whole nucleus (outlined) at time zero is shown on left. See Supplementary Video 1. Whole nucleus scale bar, 5 μm. Time-lapse scale bars, 1 μm. Experiment repeated 8 times. c Pairwise 3D distances of fluorescent foci in b over time. See Supplementary Fig. 1 for all distance plots of imaged nuclei (n = 20 nuclei; 33 pairs). d Schematic of competitor experiment. “Cold” competitor gRNA without Pumilio binding sites (PBS) for fluorescent protein recruitment binds to sequence overlapping test target. e Competitor experiments for validating MASP1-BCL6. Column plot shows percentages of nuclei with indicated numbers of Clover (green) spots (nucleus counts on top of columns) in ARPE-19 cells co-transfected with Clover-PUFc/A-15xPBSc, PUF9R-iRFP670/B-15xBPS9R and either a non-competing GAL4 gRNA (black columns) or a gRNA competing with corresponding A locus of the loop (patterned columns). Representative images in non-competed or locus A-competed samples (Clover, green, stealth arrowheads; iRFP670, red, triangle arrowheads). Scale bars, 5 μm. Experiment repeated 3 times. f Imaging of downstream region B–R equidistant and adjacent to MASP1-BCL6 loop (A–B) with locus R at 312 kb 3’ of locus B. Arc height is proportional to number of ChIA-PET sequencing reads. g Representative image of co-labeling of locus B (Clover, green, stealth arrowheads) and locus R (iRFP670, red, triangle arrowheads) in ARPE-19 cells (n = 10 nuclei; 14 pairs). Scale bar, 5 μm. Experiment repeated 3 times. h Boxplot showing pairwise 3D distances of A–B pair (n = 2347 measurements; mean = 1.36 μm; median = 0.97 μm) and B–R pair (n = 666 measurements; mean = 1.81 μm; median = 1.40 μm) over time. Boxplot displays median line, mean x-marked, interquartile range boxes, and min to max whiskers. ***Mann–Whitney test two-sided p = 6.01 × 10⁻²⁹ < 0.00001. Source data are provided as a Source Data file.