Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a further development of the CRISPR/Cas9-based RNA-guided endonuclease-in situ labeling (RGEN-ISL) method. RGEN-ISL allowed the differentiation between vertebrate-type (TTAGGG)n and Arabidopsis-type (TTTAGGG)n telomere repeats. Using maize as an example, we established a combination of RGEN-ISL, immunostaining, and EdU labeling to visualize in situ specific repeats, histone marks, and DNA replication sites, respectively. The effects of the non-denaturing RGEN-ISL and standard denaturing FISH on the chromatin structure were compared using super-resolution microscopy. 3D structured illumination microscopy revealed that denaturation and acetic acid fixation impaired and flattened the chromatin. The broad range of adaptability of RGEN-ISL to different combinations of methods has the potential to advance the field of chromosome biology.

中文翻译：

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基于 CRISPR/Cas9 的 RGEN-ISL 允许对蛋白质、DNA 重复序列和 DNA 复制位点进行同步和特异性可视化

可视化基因组的时空组织将提高我们对染色质结构和功能如何相互交织的理解。在这里，我们描述了基于 CRISPR/Cas9 的 RNA 引导的核酸内切酶原位标记 (RGEN-ISL) 方法的进一步发展。RGEN-ISL 允许区分脊椎动物型 (TTAGGG)n 和拟南芥型 (TTTAGGG)n 端粒重复序列。以玉米为例，我们建立了 RGEN-ISL、免疫染色和 EdU 标记的组合，以分别可视化原位特定重复、组蛋白标记和 DNA 复制位点。使用超分辨率显微镜比较了非变性 RGEN-ISL 和标准变性 FISH 对染色质结构的影响。3D 结构照明显微镜显示，变性和醋酸固定使染色质受损和变平。RGEN-ISL 对不同方法组合的广泛适应性具有推进染色体生物学领域的潜力。