Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System

Chen, Baohui; Gilbert, Luke A.; Cimini, Beth A.; Schnitzbauer, Joerg; Zhang, Wei; Li, Gene-Wei; Park, Jason; Blackburn, Elizabeth H.; Weissman, Jonathan S.; Qi, Lei S.; Huang, Bo

doi:10.1016/j.cell.2013.12.033

Cited by 390 publications

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“…Constrained motion could reflect hindrance imposed by physical attachment, a modification in the compaction parameters of the fiber or a combination of both. FROS-labeled loci in mammalian cells were also reported to be less dynamic [65] when next to the nuclear envelope, whereas Cas9-GFP or TALE-GFP marked telomeres tended to be rather mobile within the nuclear lumen in human cells [15]. Motion of randomly labeled sites [65] or of fluorescent ANCHOR tags next to specific genes (my group's unpublished data) is heterogeneous within a cell population, in which their localization with respect to nuclear compartments is likely to vary.…”

Section: Flexibility and Confinement Defined By Fiber Motionmentioning

confidence: 98%

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Chromosome dynamics and folding in eukaryotes: Insights from live cell microscopy

Bystricky

2015

FEBS Letters

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a b s t r a c tHow chromosomes are folded and how this folding relates to function remain fundamental questions. Answering them is rendered difficult by the stochasticity of chromatin fiber motion which inevitably results in heterogeneity of the populations analyzed. Even if single cell analyses are beginning to yield precious insights, how can we determine whether a snapshot of position is related to function of the probed locus or cell-type? Fluorescence labeling of DNA at single or multiple loci allows determination of their position relative to nuclear landmarks and to each other, enabling us to derive physical parameters of the underlying chromatin fiber. Here I review the contribution of quantitative spatial and temporal analysis of labeled DNA to our understanding of chromosome conformation in different cell types, highlighting live cell imaging techniques and large scale geometrical analysis of multiple loci in 3D.

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Section: Flexibility and Confinement Defined By Fiber Motionmentioning

confidence: 98%

“…Consequently, use of these systems has so far been restricted largely to labeling of naturally repeated sequences e.g. telomeres [35]. Suntag is a non-invasive technique but appears thus far to be reserved for selected proteins that retain their function within the construct [19].…”

Section: Visualizing Dna In Living Eukaryotic Cellsmentioning

confidence: 99%

Chromosome dynamics and folding in eukaryotes: Insights from live cell microscopy

Bystricky

2015

FEBS Letters

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“…Two innovative guide RNA designs have been reported to dramatically enhance the frequency of targeted mutagenesis. One modified sgRNA (F 1 E) with an extended Cas9 binding hairpin structure and the removal of a potential Polymerase III terminator exhibits improved activity in both mammalian cells and C. elegans (Chen et al, 2013a;Ward, 2015). The other design of sgRNAs, with a GG motif at the 3 0 end of protospacers, also significantly enhances the frequency of genome editing by Cas9 in C. elegans (Farboud and Meyer, 2015).…”

Section: Increasing Crispr-cas9 Editing Efficiencymentioning

confidence: 99%

The application of somatic CRISPR‐Cas9 to conditional genome editing in Caenorhabditis elegans

2016

Genesis

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Summary: Forward and reverse genetic approaches have been well developed in the nematode Caenorhabditis elegans; however, efficient genetic tools to generate conditional gene mutations are still in high demand. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR-Cas9) system for genome modification has provided an additional tool for C. elegans researchers to achieve simple and efficient conditional targeted mutagenesis. Here, we review recent advances in the somatic expression of Cas9 endonuclease for conditional gene editing. We present some practical considerations for improving the efficiency and reducing the off-target effects of somatic CRISPR-Cas9 and highlight a strategy to analyze somatic mutation at single-cell resolution. Finally, we outline future applications and consider challenges for this emerging genome editing platform that will need to be addressed in the future. genesis 54:170-181,

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“…A recent exciting development in labeling specific gene loci is the use of TALEs, (DNA binding proteins without endonuclease activity) or the CRISPR/dCas9 (deactivated Cas9 without the endonuclease activity) fused with fluorescent proteins. While the TALEs have been shown to be efficient in labeling repetitive sequences [69], CRISPR/dCas9 has also been used for labeling non-repetitive sequences [70]. Both of these labeling approaches are compatible with super-resolution microscopy (STED and SMLM) depending on the fluorescent protein used.…”

Section: Labeling Of Dnamentioning

confidence: 99%

Advanced microscopy methods for visualizing chromatin structure

Lakadamyali

Cosma

2015

FEBS Letters

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a b s t r a c tIn the recent years it has become clear that our genome is not randomly organized and its architecture is tightly linked to its function. While genomic studies have given much insight into genome organization, they mostly rely on averaging over large populations of cells, are not compatible with living cells and have limited resolution. For studying genome organization in single living cells, microscopy is indispensable. In addition, the visualization of biological structures helps to understand their function. Up to now, fluorescence microscopy has allowed us to probe the larger scale organization of chromosome territories in the micron length scales, however, the smaller length scales remained invisible due to the diffraction limited spatial resolution of fluorescence microscopy. Thanks to the advent of super-resolution microscopy methods, we are finally starting to be able to probe the nanoscale organization of chromatin in vivo and these methods have the potential to greatly advance our knowledge about chromatin structure and function relationship.

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Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System

Cited by 390 publications

References 0 publications

Chromosome dynamics and folding in eukaryotes: Insights from live cell microscopy

Chromosome dynamics and folding in eukaryotes: Insights from live cell microscopy

The application of somatic CRISPR‐Cas9 to conditional genome editing in Caenorhabditis elegans

Advanced microscopy methods for visualizing chromatin structure

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