Live-cell CRISPR imaging in plants reveals dynamic telomere movements.
Live-cell CRISPR imaging in plants reveals dynamic telomere movements.
复制标题
DOI：
10.1111/tpj.13601
复制
发表时间：
2017-08
期刊：
影响因子：
--
通讯作者：
Houben A
中科院分区：
文献类型：
--
作者：
Dreissig S;Schiml S;Schindele P;Weiss O;Rutten T;Schubert V;Gladilin E;Mette MF;Puchta H;Houben A
阐明细胞核内基因组的时空组织对于我们理解发育和环境变化过程中基因和非编码序列的调控至关重要。新兴的染色质成像技术有望弥合测序研究（揭示基因组信息）与成像研究（提供定义的基因组区域的空间和时间信息）之间的长期差距。在这里，我们展示了这样一种成像技术，该技术基于细菌成簇的规则间隔短回文重复序列（CRISPR）-CRISPR相关蛋白9（Cas9）的两种直系同源物。通过将eGFP/mRuby 2融合到化脓性链球菌和金黄色葡萄球菌Cas9的催化失活版本，我们显示了本氏烟草活叶细胞中端粒重复序列的稳健可视化。通过跟踪CRISPR-dCas 9可视化的端粒动态，我们揭示了在间期30分钟内高达2 μm的动态端粒运动。此外，我们表明CRISPR-dCas 9可以与荧光标记的蛋白质结合，以可视化体内DNA-蛋白质相互作用。通过同时使用两个dCas 9直系同源物，我们为活植物细胞中多个基因组位点的成像铺平了道路。CRISPR成像有可能显着提高我们对活植物细胞中染色体动态的理解。可视化基因组的时空组织可以提高我们对染色质结构和功能如何相互交织的理解。我们开发了一个工具包，基于荧光标记的CRISPR-dCas 9在活植物细胞中可视化定义的基因组序列，并证明了其揭示动态端粒运动的能力。
Elucidating the spatiotemporal organization of the genome inside the nucleus is imperative to our understanding of the regulation of genes and non‐coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long‐standing gap between sequencing studies, which reveal genomic information, and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR associated protein 9 (Cas9). By fusing eGFP/mRuby2 to catalytically inactive versions of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR–dCas9, we reveal dynamic telomere movements of up to 2 μm over 30 min during interphase. Furthermore, we show that CRISPR–dCas9 can be combined with fluorescence‐labelled proteins to visualize DNA–protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for the imaging of multiple genomic loci in live plants cells. CRISPR imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells. Visualizing the spatio‐temporal organisation of the genome can improve our understanding of how chromatin structure and function are intertwined. We developed a toolkit to visualize defined genomic sequences in living plant cells based on fluorescence‐tagged CRISPR‐dCas9 and demonstrated its ability to reveal dynamic telomere movements.
登录
查看更多内容
DOI：
10.1083/jcb.201604115
发表时间：
2016-08-29
期刊：
The Journal of cell biology
影响因子：
--
作者：
Ma H;Tu LC;Naseri A;Huisman M;Zhang S;Grunwald D;Pederson T
通讯作者：
Pederson T
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
5.1
作者：
Belhaj K;Chaparro-Garcia A;Kamoun S;Nekrasov V
通讯作者：
Nekrasov V
影响因子：
11.4
作者：
Cryderman, DE;Morris, EJ;Wallrath, LL
通讯作者：
Wallrath, LL
DOI：
10.1073/pnas.1420024112
发表时间：
2015-03-10
影响因子：
11.1
作者：
Ma, Hanhui;Naseri, Ardalan;Pederson, Thoru
通讯作者：
Pederson, Thoru