Chen Baohui, Guan Juan, Huang Bo
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143; email:
Annu Rev Biophys. 2016 Jul 5;45:1-23. doi: 10.1146/annurev-biophys-062215-010830. Epub 2016 Apr 27.
The three-dimensional organization of the genome plays important roles in regulating the functional output of the genome and even in the maintenance of epigenetic inheritance and genome stability. Here, we review and compare a number of newly developed methods-especially those that utilize the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated protein 9) system-that enable the direct visualization of specific, endogenous DNA sequences in living cells. We also discuss the practical considerations in implementing the CRISPR imaging technique to achieve sufficient signal-to-background levels, high specificity, and high labeling efficiency. These DNA labeling methods enable tracking of the copy number, localization, and movement of genomic elements, and we discuss the potential applications of these methods in understanding the searching and targeting mechanism of the Cas9-sgRNA complex, investigating chromosome organization, and visualizing genome instability and rearrangement.
基因组的三维组织在调节基因组的功能输出，甚至在维持表观遗传遗传和基因组稳定性方面发挥着重要作用。在这里，我们回顾并比较了一些新开发的方法，特别是那些利用CRISPR（成簇规律间隔短回文重复序列）-Cas9（CRISPR相关蛋白9）系统的方法，这些方法能够在活细胞中直接可视化特定的内源性DNA序列。我们还讨论了实施CRISPR成像技术以实现足够的信号背景水平、高特异性和高标记效率的实际考虑因素。这些DNA标记方法能够追踪基因组元件的拷贝数、定位和移动，并且我们讨论了这些方法在理解Cas9-sgRNA复合物的搜索和靶向机制、研究染色体组织以及可视化基因组不稳定性和重排方面的潜在应用。
