Background-suppressed live visualization of genomic loci with an improved CRISPR system based on a split fluorophore.
Background-suppressed live visualization of genomic loci with an improved CRISPR system based on a split fluorophore.
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DOI：
10.1101/gr.260018.119
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发表时间：
2020-09
期刊：
影响因子：
7
通讯作者：
Kim H
中科院分区：
文献类型：
--
作者：
Chaudhary N;Nho SH;Cho H;Gantumur N;Ra JS;Myung K;Kim H
染色体的高级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件。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而具有有限的适用性。为了解决这个问题，我们开发了一种新的可视化方案，结合三方荧光蛋白与SunTag系统，并证明它强烈抑制背景荧光和放大基因座特异性信号，允许长期跟踪基因组位点。我们将多组分CRISPR系统整合到稳定的细胞系中，以允许定量和可靠地分析基因组位点的动态行为。由于信号背景比大大提高，即使在传统的荧光显微镜下，也可以成功地跟踪只有少量序列重复的靶基因座。这一功能使基于CRISPR的成像技术能够应用于整个基因组的基因座，并为活细胞核过程的研究开辟了新的可能性。
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.
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影响因子：
16.6
作者：
Fu Y;Rocha PP;Luo VM;Raviram R;Deng Y;Mazzoni EO;Skok JA
通讯作者：
Skok JA
影响因子：
46.9
作者：
Cabantous, S;Terwilliger, TC;Waldo, GS
通讯作者：
Waldo, GS
影响因子：
46.9
作者：
Kuscu, Cem;Arslan, Sevki;Adli, Mazhar
通讯作者：
Adli, Mazhar
DOI：
10.1126/science.aao3136
发表时间：
2018-03-02
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Gu B;Swigut T;Spencley A;Bauer MR;Chung M;Meyer T;Wysocka J
通讯作者：
Wysocka J
CRISPR-CAS9介导的基因失活的高度活性SGRNA的合理设计。
影响因子：
46.9
作者：
Doench, John G.;Hartenian, Ella;Graham, Daniel B.;Tothova, Zuzana;Hegde, Mudra;Smith, Ian;Sullender, Meagan;Ebert, Benjamin L.;Xavier, Ramnik J.;Root, David E.
通讯作者：
Root, David E.