(Po)STAC (Polycistronic SunTAg modified CRISPR) enables live-cell and fixed-cell super-resolution imaging of multiple genes

Neguembor, Maria Victoria; Sebastian-Perez, Ruben; Aulicino, Francesco; Gómez-García, Pablo Aurelio; Cosma, María Pía; Lakadamyali, Melike

doi:10.1093/nar/gkx1271

Cited by 39 publications

(45 citation statements)

References 30 publications

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“…Such visualization could ultimately be informative to study transcriptional activation. We and others (Fang et al 2018;Kraus et al 2017;Neguembor et al 2018;Chen et al 2013;Chen et al 2018) have developed methods to image specific genomic sites, opening up the possibility to visualize DNA density and compaction at clutches located in specific regulatory regions in order to correlate function with the structure of DNA.…”

Section: Discussionmentioning

confidence: 99%

Nanoscale decompaction of nucleosomal DNA revealed through multi-color super-resolution microscopy

Otterstrom

Vicario

et al. 2018

Preprint

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Super-resolution imaging of histones and DNA reveals that DNA is compacted by groups of nucleosomes -clutches -at the nanoscale level and clutch compaction of DNA is affected by histone tail acetylation especially in highly folded regions containing several nearby clutches. AbstractChromatin organization plays an important role in regulating gene expression. Previously, we showed that chromatin is organized in the form of nucleosome groups or clutches. The size and nucleosome packing density of clutches decreased in hyperacetylated cells having more open chromatin. While hyperacetylation is thought to disrupt histone-DNA and inter-nucleosome interactions, its impact on higher order DNA compaction by groups of nucleosomes in vivo is not clear. To elucidate this question, we carried out two-color super-resolution imaging of histones and DNA in cells treated with the Histone Deacetylase (HDAC) inhibitor Trichostatin A (TSA). We showed that a lower percentage of DNA was associated to clutches in hyperacetylated cells, suggesting a decrease in nucleosome occupancy. We further identified the presence of "clutch" DNA within a nanoscale distance around the clutches. Upon histone hyperacetylation, the radius of the clutch DNA decreased leading to DNA release from the clutches, consistent with disruption of DNA-histone interactions. Finally, the most dramatic decompaction was observed for groups of clutches in close spatial proximity, suggesting that neighboring clutches influence each other´s DNA compaction.

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Section: Discussionmentioning

confidence: 99%

Nanoscale decompaction of nucleosomal DNA revealed through multi-color super-resolution microscopy

Otterstrom

Vicario

et al. 2018

Preprint

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“…Since our assay relies on visualisation of two neighbouring chromosomal loci (100 to 250kb apart), and suitably spaced unique repetitive sequences are not available, currently such dCas9/sgRNA methods would not be suitable to carry out the experiments we 15 described. Nonetheless, the sensitivity of dCas9/sgRNA method is improving rapidly (Maass et al, 2018;Neguembor et al, 2018) and may allow more routine visualization of nonrepetitive sequences in the near future. Analyses of a larger number of chromosome regions in various cells and in various conditions, with live-cell imaging, would broaden our knowledge about mitotic chromosome organization.…”

Section: Discussionmentioning

confidence: 99%

Live-cell imaging of marked chromosome regions reveals dynamics of mitotic chromosome resolution and compaction

Eykelenboom

Yue

Hégarat

et al. 2018

Preprint

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SummaryWhen human cells enter mitosis, chromosomes undergo substantial changes in their organization -sister chromatids are resolved and chromosomes are compacted.However we still do not fully comprehend the timing and coordination of these events. To address these, we need to evaluate both sister chromatid resolution and chromosome compaction in one assay. Here we analysed the change in configuration of marked chromosome regions over time, with high spatial and temporal resolution by live-cell imaging to judge sister resolution and compaction.This assay showed that sister chromatid resolution already begins in late G2 phase and completes in prophase after multiple attempts. Cohesins and WAPL antagonistically regulate sister chromatid resolution in late G2 and prophase whilst local enrichment of cohesin on chromosomes prevents precocious sister chromatid resolution. Moreover, the assay allowed quantitative evaluation of the timing and efficiency of condensin II and I activities in promoting sister chromatid resolution and chromosome compaction, respectively. Our real-time assay sheds new light on the dynamics of mitotic chromosome resolution and compaction.

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“…Visualizing genomic locations directly through dCas9 technologies. (a) Visualization of repetitive chromosomal regions using fluorescently labeled dCas9 [75,78,79]. The Cas9 guide RNA is designed with homology to a sequence of a repetitive chromosomal region (different repetitive regions shown as gray lines in the chromosome above) which allows accumulation of fluorescent signal at that region.…”

Section: Use Of Crispr-cas9 To Visualize Selected Chromosome Locimentioning

confidence: 99%

Zooming in on chromosome dynamics

Eykelenboom

Tanaka

2020

Cell Cycle

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Until recently, our understanding of chromosome organization in higher eukaryotic cells has been based on analyses of large-scale, low-resolution changes in chromosomes structure. More recently, CRISPR-Cas9 technologies have allowed us to "zoom in" and visualize specific chromosome regions in live cells so that we can begin to examine in detail the dynamics of chromosome organization in individual cells. In this review, we discuss traditional methods of chromosome locus visualization and look at how CRISPR-Cas9 gene-targeting methodologies have helped improve their application. We also describe recent developments of the CRISPR-Cas9 technology that enable visualization of specific chromosome regions without the requirement for complex genetic manipulation.

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(Po)STAC (Polycistronic SunTAg modified CRISPR) enables live-cell and fixed-cell super-resolution imaging of multiple genes

Cited by 39 publications

References 30 publications

Nanoscale decompaction of nucleosomal DNA revealed through multi-color super-resolution microscopy

Nanoscale decompaction of nucleosomal DNA revealed through multi-color super-resolution microscopy

Live-cell imaging of marked chromosome regions reveals dynamics of mitotic chromosome resolution and compaction

Zooming in on chromosome dynamics

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