Department of Pathology, New York University School of Medicine, 550 First Avenue, Sml 311, New York, New York 10016, USA.
Department of Biology, New York University, New York, New York 10003, USA.
Nat Commun. 2016 May 25;7:11707. doi: 10.1038/ncomms11707.
Imaging systems that allow visualization of specific loci and nuclear structures are highly relevant for investigating how organizational changes within the nucleus play a role in regulating gene expression and other cellular processes. Here we present a live imaging system for targeted detection of genomic regions. Our approach involves generating chimaeric transcripts of viral RNAs (MS2 and PP7) and single-guide RNAs (sgRNAs), which when co-expressed with a cleavage-deficient Cas9 can recruit fluorescently tagged viral RNA-binding proteins (MCP and PCP) to specific genomic sites. This allows for rapid, stable, low-background visualization of target loci. We demonstrate the efficiency and flexibility of our method by simultaneously labelling major and minor satellite regions as well as two individual loci on mouse chromosome 12. This system provides a tool for dual-colour labelling, which is important for tracking the dynamics of chromatin interactions and for validating epigenetic processes identified in fixed cells.
成像系统能够可视化特定的基因座和核结构，对于研究核内的组织变化如何在调节基因表达和其他细胞过程中发挥作用具有重要意义。在这里，我们提出了一种用于靶向检测基因组区域的实时成像系统。我们的方法涉及生成病毒 RNA（MS2 和 PP7）和单指导 RNA（sgRNA）的嵌合转录本，当与无切割活性的 Cas9 共表达时，这些转录本可以将荧光标记的病毒 RNA 结合蛋白（MCP 和 PCP）招募到特定的基因组位点。这使得靶基因座能够快速、稳定、低背景地可视化。我们通过同时标记主要和次要卫星区域以及小鼠染色体 12 上的两个单独的基因座来证明我们方法的效率和灵活性。该系统提供了一种用于双色标记的工具，这对于跟踪染色质相互作用的动态以及验证固定细胞中鉴定的表观遗传过程非常重要。
