Journal: Mammalian genome : official journal of the International Mammalian Genome Society
Article Title: CRISPR-based strategies for studying regulatory elements and chromatin structure in mammalian gene control
doi: 10.1007/s00335-017-9727-2
Figure Lengend Snippet: CRISPR/dCas9-based epigenome-editing strategies for studying regulatory genomic elements and chromatin dynamics. a Block transcription factor-binding sites on an enhancer. The binding of dCas9s to the transcription factor-binding sites causes a steric block that interferes with the binding of transcription factors and chromatin loop formation between promoter and enhancer, resulting in decreased target gene expression. b Repress enhancer activity. dCas9-KRAB can repress target gene expression by preventing the transcriptional complex formation, inducing H3K9 trimethylation at the enhancer, and recruiting other histone repressive modifiers. c Promote enhancer activity. dCas9-VP64 can promote transcriptional activation by recruiting multiple components of the transcription pre-initiation complex, as well as histone acetyltransferases to promote acetylation of histone H3K27 at targeted enhancers. d CLOuD9-mediated chromatin loop reorganization. Addition of abscisic acid brings two complementary dSpCas9-PYL1 and dSaCas9-ABI1 into proximity by dimerization of PYL1 and ABI1 domains, and lead to chromatin structure remodeling. Removal of abscisic acid restores the endogenous chromatin conformation. e Chromatin opening. Two SpdCas9s bind the proximal target sites of FnCas9 to induce the chromatin opening and to enable the FnCas9 nuclease to access and cleave the otherwise inaccessible target sites. f Chromatin dynamics tracking. CRISPRainbow and multicolor CRISPR utilize different fluorescent-tagged dCas9-sgRNAs (red, green, and blue) to allow multiplexed labeling of chromatin loci for tracking chromatin dynamics in a spatial and temporal manner, visualizing endogenous genomic loci in live cells. Co-localization of red, green, and blue fluorescents indicates a close physical interaction between TADs from different chromosomes. g Chromatin loop visualization. In the case of studying cis/trans-acting regulatory elements within a TAD, CRISPRainbow and multicolor CRISPR allow tracking the dynamic interaction of enhancers or silencers with a specific promoter for transcriptional regulation. When the dCas9-blue-labeled enhancer is in close contact with the dCas9-red-labeled promoter, gene A is activated and pink fluorescent can be visualized. Similarly, when the dCas9-green-labeled silencer is in close contact with the dCas9-red-labeled promoter, gene B is activated and yellow fluorescent can be visualized. h Telomere dynamics tracking. The combined use of dCas9-eGFP imaging and DNA sequential FISH probes enables multiplexed visualization of telomere dynamics and subtelomeric regions. i Chromosome painting. dCas9-eGFP or dCas9-mCherry with hundreds of different sgRNAs can be used to paint an entire chromosome in order to visualize a desired chromosomal territory, spatial arrangements of sister chromatids, and to track the movement of a particular chromosome in dividing live cells. FISH fluorescence in situ hybridization, KRAB Krüppel-associated box, SNP single nucleotide polymorphism, TAD topologically associated domain, TSS transcription initiation site, VP64 multiple repeats of the herpes simplex VP16 transactivation domain. (Color figure online)
Article Snippet: , telomere , Combined CRISPR imaging and DNA sequential FISH , Multiplexed visualization of telomere dynamics, and identified 12 unique subtelomeric regions , Mouse embryonic (ESCs) , Takei et al. (2017).
Techniques: CRISPR, Blocking Assay, Binding Assay, Targeted Gene Expression, Activity Assay, Activation Assay, Labeling, Imaging, Fluorescence, In Situ Hybridization
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