CRISPR/Cas9-Based RGEN-ISL Allows the Simultaneous and Specific Visualization of Proteins, DNA Repeats, and Sites of DNA Replication
CRISPR/Cas9-Based RGEN-ISL Allows the Simultaneous and Specific Visualization of Proteins, DNA Repeats, and Sites of DNA Replication
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DOI：
10.1159/000502600
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发表时间：
2019-11-01
影响因子：
1.7
通讯作者：
Houben, Andreas
中科院分区：
文献类型：
--
作者：
Nemeckova, Alzbeta;Waesch, Christina;Houben, Andreas
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交织在一起的理解。在这里，我们描述了基于CRISPR/ cas9的rna引导内切酶原位标记（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，分别可视化原位特异性重复序列、组蛋白标记和DNA复制位点。采用超分辨显微镜比较非变性RGEN-ISL和标准变性FISH对染色质结构的影响。三维结构照明显微镜显示变性和醋酸固定损伤并使染色质变平。RGEN-ISL对不同方法组合的广泛适应性有可能推动染色体生物学领域的发展。
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.