CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci.
CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci.
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DOI：
10.1038/ncomms11707
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发表时间：
2016-05-25
影响因子：
16.6
通讯作者：
Skok JA
中科院分区：
文献类型：
--
作者：
Fu Y;Rocha PP;Luo VM;Raviram R;Deng Y;Mazzoni EO;Skok JA
允许特定位点和核结构可视化的成像系统对于研究细胞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是高度相关的。在这里，我们提出了一个实时成像系统的基因组区域的靶向检测。我们的方法涉及产生病毒RNA（MS2和PP7）和单向导RNA（sgRNA）的嵌合转录物，当与切割缺陷型Cas9共表达时，它们可以将荧光标记的病毒RNA结合蛋白（MCP和PCP）募集到特定的基因组位点。这允许靶基因座的快速、稳定、低背景可视化。我们证明了我们的方法的效率和灵活性，同时标记的主要和次要的卫星区域，以及两个单独的位点上的小鼠12号染色体。该系统提供了一个工具，双色标记，这是重要的染色质相互作用的动态跟踪和验证表观遗传过程中确定的固定细胞。 将Cas9靶向特定基因组位点的能力为基因组编辑以外的应用提供了潜力。在这里，作者使用失活的Cas9和嵌合向导RNA来可视化活细胞中的单个重复基因座。
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. The ability to target Cas9 to specific genomic loci offers the potential for applications beyond genome editing. Here the authors use deactivated Cas9 and chimeric guide RNAs to visualise individual repetitive loci in living cells.