Multicolor CRISPR labeling of chromosomal loci in human cells
Multicolor CRISPR labeling of chromosomal loci in human cells
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DOI：
10.1073/pnas.1420024112
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发表时间：
2015-03-10
影响因子：
11.1
通讯作者：
Pederson, Thoru
中科院分区：
文献类型：
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作者：
Ma, Hanhui;Naseri, Ardalan;Pederson, Thoru
基因组位点的核内定位和这些位点的动态是理解基因表达的时空调控的重要参数。最近，已经证明可以通过使用转录激活因子样效应物（TALE）以及细菌免疫性规律性散布短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）系统的修饰版本来可视化活细胞中的内源性基因组基因座。在这里，我们报告了使用来自三种细菌直系同源物的无催化活性的Cas9内切核酸酶（dCas 9）设计CRISPR的双拷贝。每对dCas 9-荧光蛋白和同源单向导RNA（sgRNA）有效地标记活人类细胞中的几个靶基因座。使用不同颜色的dCas 9-sgRNA对，可以确定不同染色体上基因座之间的核内距离。此外，可以确定同一染色体上两个基因座之间的荧光空间分辨率，并将其与染色体物理图谱上它们之间的线性距离相关，从而允许评估活细胞中这些区域的DNA压缩。
The intranuclear location of genomic loci and the dynamics of these loci are important parameters for understanding the spatial and temporal regulation of gene expression. Recently it has proven possible to visualize endogenous genomic loci in live cells by the use of transcription activator-like effectors (TALEs), as well as modified versions of the bacterial immunity clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Here we report the design of multicolor versions of CRISPR using catalytically inactive Cas9 endonuclease (dCas9) from three bacterial orthologs. Each pair of dCas9-fluorescent proteins and cognate single-guide RNAs (sgRNAs) efficiently labeled several target loci in live human cells. Using pairs of differently colored dCas9-sgRNAs, it was possible to determine the intranuclear distance between loci on different chromosomes. In addition, the fluorescence spatial resolution between two loci on the same chromosome could be determined and related to the linear distance between them on the chromosome's physical map, thereby permitting assessment of the DNA compaction of such regions in a live cell.