The higher-order structural organization and dynamics of the chromosomes play a central role in gene regulation. To explore this structure–function relationship, it is necessary to directly visualize genomic elements in living cells. Genome imaging based on the CRISPR system is a powerful approach but has limited applicability due to background signals and nonspecific aggregation of fluorophores within nuclei. To address this issue, we developed a novel visualization scheme combining tripartite fluorescent proteins with the SunTag system and demonstrated that it strongly suppressed background fluorescence and amplified locus-specific signals, allowing long-term tracking of genomic loci. We integrated the multicomponent CRISPR system into stable cell lines to allow quantitative and reliable analysis of dynamic behaviors of genomic loci. Due to the greatly elevated signal-to-background ratio, target loci with only small numbers of sequence repeats could be successfully tracked, even under a conventional fluorescence microscope. This feature enables the application of CRISPR-based imaging to loci throughout the genome and opens up new possibilities for the study of nuclear processes in living cells.

中文翻译：

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使用基于分离荧光团的改进 CRISPR 系统对基因组位点进行背景抑制实时可视化。

染色体的高阶结构组织和动力学在基因调控中起着核心作用。为了探索这种结构-功能关系，有必要直接可视化活细胞中的基因组元素。基于 CRISPR 系统的基因组成像是一种强大的方法，但由于背景信号和细胞核内荧光团的非特异性聚集，其适用性有限。为了解决这个问题，我们开发了一种新的可视化方案，将三方荧光蛋白与 SunTag 系统相结合，并证明它强烈抑制了背景荧光并放大了位点特异性信号，允许长期跟踪基因组位点。我们将多组分 CRISPR 系统集成到稳定的细胞系中，以便对基因组位点的动态行为进行定量和可靠的分析。由于信噪比大大提高，即使在传统的荧光显微镜下，也可以成功跟踪只有少量序列重复的目标基因座。这一特性使基于 CRISPR 的成像能够应用于整个基因组的位点，并为研究活细胞的核过程开辟了新的可能性。