Imaging and Modeling Unit, Institut Pasteur, UMR 3691 CNRS, IP CNRS, Paris, France.
IGH, University of Montpellier, CNRS, Montpellier, France.
Methods Mol Biol. 2022;2532:275-290. doi: 10.1007/978-1-0716-2497-5_13.
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.
Hi-C 和相关的基于测序的技术为理解三维基因组结构带来了更深入的认识，并发现了新型结构，如拓扑关联域（TAD）和染色质环，这些结构是由黏连蛋白介导的 DNA 挤出所形成的。然而，这些技术需要细胞固定，这排除了对染色质结构动力学的评估，并且通常仅限于群体平均值，从而掩盖了细胞间的异质性。相比之下，活细胞成像允许对染色质的时空动力学进行特征描述和量化，可能包括单个细胞中的 TAD 和环。通过插入由荧光标记结合的细菌操纵子序列的重复阵列，可以以高时空分辨率可视化特定的染色质位点。使用两种不同类型的重复序列，可以将环的两个锚定点标记为不同的颜色，从而即使它们非常接近，也可以分别跟踪它们。在这里，我们描述了一种用于平行生成许多重复阵列修复盒的多功能克隆方法，并通过 CRISPR-Cas9 将其插入人类基因组。这种方法对于研究单个人类细胞中的染色质环动力学应该是非常有用的。
