Clow Patricia A, Du Menghan, Jillette Nathaniel, Taghbalout Aziz, Zhu Jacqueline J, Cheng Albert W
The Jackson Laboratory for Genomic Medicine, Farmington, CT, 06032, USA.
Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, CT, 06030, USA.
Nat Commun. 2022 Apr 6;13(1):1871. doi: 10.1038/s41467-022-29343-z.
Three-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and are ineffective in labeling non-repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method that allows for a nonrepetitive genomic locus to be labeled using one guide RNA. We construct Casilio dual-color probes to visualize the dynamic interactions of DNA elements in single live cells in the presence or absence of the cohesin subunit RAD21. Using a three-color palette, we track the dynamic 3D locations of multiple reference points along a chromatin loop. Casilio imaging reveals intercellular heterogeneity and interallelic asynchrony in chromatin interaction dynamics, underscoring the importance of studying genome structures in 4D.
基因组的三维（3D）结构是动态的、异质的且具有重要功能。活细胞成像已成为染色质动力学追踪的主要方法。然而，现有的基于CRISPR和TALE的基因组标记技术因操作繁琐而受到阻碍，并且在标记非重复序列方面效率低下。在此，我们报告了一种基于CRISPR/Casilio的通用成像方法，该方法允许使用一个导向RNA标记非重复基因组位点。我们构建了Casilio双色探针，以可视化在存在或不存在黏连蛋白亚基RAD21的情况下单个活细胞中DNA元件的动态相互作用。使用三色组合，我们追踪沿着染色质环的多个参考点的动态3D位置。Casilio成像揭示了染色质相互作用动力学中的细胞间异质性和等位基因间异步性，强调了在四维空间中研究基因组结构的重要性。
