Polymer simulations guide the detection and quantification of chromatin loop extrusion by imaging.

Genome-wide chromosome conformation capture (Hi-C) has revealed the organization of chromatin into topologically associating domains (TADs) and loops, which are thought to help regulate genome functions. TADs and loops are understood as the result of DNA extrusion mediated by the cohesin complex. However, despite recent efforts, direct visualization and quantification of this process in single cells remains an open challenge. Here, we use polymer simulations and dedicated analysis methods to explore if, and under which conditions, DNA loop extrusion can be detected and quantitatively characterized by imaging pairs of fluorescently labeled loci located near loop or TAD anchors in fixed or living cells. We find that under realistic conditions, extrusion can be detected and the frequency of loop formation can be quantified from fixed cell images alone, while the lifetime of loops and the speed of extrusion can be estimated from dynamic live-cell data. Our delineation of appropriate imaging conditions and the proposed analytical methods lay the groundwork for a systematic quantitative characterization of loop extrusion in fixed or living cells.

Versatile CRISPR-Based Method for Site-Specific Insertion of Repeat Arrays to Visualize Chromatin Loci in Living Cells.

Hi-C and related sequencing-based techniques have brought a detailed understanding of the 3D genome architecture and the discovery of novel structures such as topologically associating domains (TADs) and chromatin loops, which emerge from cohesin-mediated DNA extrusion. However, these techniques require cell fixation, which precludes assessment of chromatin structure dynamics, and are generally restricted to population averages, thus masking cell-to-cell heterogeneity. By contrast, live-cell imaging allows to characterize and quantify the temporal dynamics of chromatin, potentially including TADs and loops in single cells. Specific chromatin loci can be visualized at high temporal and spatial resolution by inserting a repeat array from bacterial operator sequences bound by fluorescent tags. Using two different types of repeats allows to tag both anchors of a loop in different colors, thus enabling to track them separately even when they are in close vicinity. Here, we describe a versatile cloning method for generating many repeat array repair cassettes in parallel and inserting them by CRISPR-Cas9 into the human genome. This method should be instrumental to studying chromatin loop dynamics in single human cells.