利用CRISPR/Cas9对活细胞中的基因组元件进行成像
Imaging genomic elements in living cells using CRISPR/Cas9.
作者信息
Chen Baohui, Huang Bo
机构信息
Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA.
Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA; Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA.
出版信息
Methods Enzymol. 2014;546:337-54. doi: 10.1016/B978-0-12-801185-0.00016-7.
In addition to their applications in genome editing and gene expression regulation, programmable DNA recognition systems, including both CRISPR and TALE, have been recently engineered for the visualization of endogenous genomic elements in living cells. This capability greatly helps the study of genome function regulation by its physical organization and interaction with other nuclear structures. This chapter first discusses the general considerations in designing and implementing the imaging system. The subsequent sections provide detailed protocols to use the CRISPR/Cas9 system to label and image specific genomic loci, including the establishment of expression systems for dCas9-GFP and sgRNA, the procedure to label repetitive sequences of telomeres and protein-coding genes, the simultaneous expression of many sgRNAs to label a nonrepetitive locus, and the verification of signal specificity by FISH.
除了在基因组编辑和基因表达调控方面的应用外，包括CRISPR和TALE在内的可编程DNA识别系统最近已被设计用于活细胞内源性基因组元件的可视化。这种能力极大地有助于通过其物理组织以及与其他核结构的相互作用来研究基因组功能调控。本章首先讨论设计和实施成像系统的一般注意事项。随后的章节提供了使用CRISPR/Cas9系统标记和成像特定基因组位点的详细方案，包括用于dCas9-GFP和sgRNA的表达系统的建立、端粒和蛋白质编码基因重复序列的标记程序、同时表达多个sgRNA以标记非重复位点以及通过荧光原位杂交（FISH）验证信号特异性。
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