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ChIP-seq analysis of dCas9 chromatin occupancy by dCas9/sgRNA-mediated CAPTURE

ABSTRACT: ChIP-seq analysis of dCas9 chromatin occupancy by dCas9/sgRNA-mediated CAPTURE

PROVIDER: PRJNA387472 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR5584120.fastq.gz	Fastqsanger.gz
	SRR5584130.fastq.gz	Fastqsanger.gz
	SRR5584140.fastq.gz	Fastqsanger.gz
	SRR5584150.fastq.gz	Fastqsanger.gz
	SRR5584160.fastq.gz	Fastqsanger.gz

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ChIP-seq analysis of dCas9 chromatin occupancy by dCas9/sgRNA-mediated CAPTURE

Project description:Cis-regulatory elements (CREs) are commonly recognized by correlative chromatin features, yet the molecular composition of the vast majority of CREs in chromatin remains unknown. Here we describe a CRISPR affinity purification in situ of regulatory elements (CAPTURE) approach to unbiasedly identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated endonuclease-deficient Cas9 protein and sequence-specific guide RNAs, we show high-resolution and selective isolation of chromatin interactions at a single copy genomic locus. Purification of human telomeres using CAPTURE identifies known and new telomeric factors. In situ capture of individual constituents of the enhancer cluster controlling human β-globin genes establishes evidence for composition-based hierarchical organization of enhancer structure. Furthermore, unbiased analysis of chromatin interactions at disease-associated cis-elements and developmentally controlled super-enhancers reveals spatial features causally regulate gene transcription. Thus, comprehensive analysis of locus-specific regulatory composition provides mechanistic insight into genome structure and function in development and disease.

2017-08-24 | GSE99177 | GEO

ChIP-seq analysis of dCas9 chromatin occupancy in HeLa cells expressing LAMPS

Project description:Significant enrichment of dCas9 chromatin occupancy at the targeted HS2 enhancer was observed in cells expressing LAMPS under the dark condition and after blue light illumination.

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ChIP-seq analysis of dCas9 chromatin occupancy in K562 cells by CAPTURE2.0

Project description:The spatiotemporal control of 3D chromatin structure is fundamental for gene regulation, yet it remains challenging to obtain high-resolution chromatin interacting profiles at cis-regulatory elements (CREs) by chromatin conformation capture (3C)-based methods. Here, we describe the redesigned dCas9-based CAPTURE method for multiplexed, high-throughput and high-resolution analysis of locus-specific chromatin interactions. Using C-terminally biotinylated dCas9, endogenous biotin ligase and pooled sgRNAs, the new system enables quantitative analysis of the spatial configuration of a few to hundreds of enhancers or promoters in a single experiment, enabling systematic comparisons across CREs within and between gene clusters. Multiplexed analyses of erythroid super-enhancers (SEs) reveal SE hierarchical structure and distinct modes of SE-gene interactions. Multiplexed capture of temporal dynamics of promoter-centric interactions establishes the instructive function of enhancer-promoter looping in transcriptional regulation during lineage differentiation. These applications illustrate the ability of multiplexed CAPTURE for decoding the organizational principles of genome structure and function.

2020-02-20 | GSE139112 | GEO

Comparison of dual sgRNA library versus Dolcetto by Perturb-seq

Project description:CRISPR interference (CRISPRi) genetic screens use programmable repression of gene expression to systematically explore questions in cell biology and genetics. However, wider adoption of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and lack of consensus on the choice of CRISPRi effector proteins. Here, we address these challenges to present next-generation CRISPRi sgRNA libraries and effectors. First, we combine empiric sgRNA selection with a dual sgRNA library design to generate an ultra-compact, highly active CRISPRi sgRNA library. Next, we rigorously compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an optimal balance between strong on-target knockdown and minimal nonspecific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines which stably express Zim3-dCas9 and demonstrate robust on-target knockdown across these cell lines. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.

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In situ CAPTURE of chromatin interactions by biotinylated dCas9

Project description:In situ CAPTURE of chromatin interactions by biotinylated dCas9

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Inhibition of uPA expression by CRISPR-dCas9 DNA methyltransferases

Project description:We report the generation of CRISPR-dCas9 DNA methyltransferases to mediate targeted DNA methylation. Using the dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B methyltransferases, we have demonstrated that these two methyltransferase can mediate targeted methylation in three human genes tested: uPA, TGFBR3, and CDKN2A in human HEK293T cells. We also showed that these methyltransferases could mediate gene inhibition. five samples co-transfected with five uPA sgRNAs and each of the four dCas9 fusions, or control transfection with pUC19 plasmid

2016-06-01 | E-GEOD-74935 | biostudies-arrayexpress

ChIP-seq analysis of dCas9 chromatin occupancy in K562 cells by CAPTURE2.0

Project description:ChIP-seq analysis of dCas9 chromatin occupancy in K562 cells by CAPTURE2.0

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ChIP-seq analysis of dCas9 chromatin occupancy in HeLa cells expressing LAMPS

Project description:ChIP-seq analysis of dCas9 chromatin occupancy in HeLa cells expressing LAMPS

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Optimizing sgRNA position markedly improves the efficiency of CRISPR/dCas9-mediated transcriptional repression.

Project description:CRISPR interference (CRISPRi) represents a newly developed tool for targeted gene repression. It has great application potential for studying gene function and mapping gene regulatory elements. However, the optimal parameters for efficient single guide RNA (sgRNA) design for CRISPRi are not fully defined. In this study, we systematically assessed how sgRNA position affects the efficiency of CRISPRi in human cells. We analyzed 155 sgRNAs targeting 41 genes and found that CRISPRi efficiency relies heavily on the precise recruitment of the effector complex to the target gene transcription start site (TSS). Importantly, we demonstrate that the FANTOM5/CAGE promoter atlas represents the most reliable source of TSS annotations for this purpose. We also show that the proximity to the FANTOM5/CAGE-defined TSS predicts sgRNA functionality on a genome-wide scale. Moreover, we found that once the correct TSS is identified, CRISPRi efficiency can be further improved by considering sgRNA sequence preferences. Lastly, we demonstrate that CRISPRi sgRNA functionality largely depends on the chromatin accessibility of a target site, with high efficiency focused in the regions of open chromatin. In summary, our work provides a framework for efficient CRISPRi assay design based on functionally defined TSSs and features of the target site chromatin.

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dCas9-based Scn1a gene activation restores inhibitory interneuron excitability and restrains epileptic crises in Dravet syndrome mice

Project description:Dravet syndrome (DS) is a severe epileptic encephalopathy caused by heterozygous loss-of-function mutations in the SCN1A gene, indicating a haploinsufficient genetic mechanism underlining this pathology. Here, we tested whether dCas9-mediated Scn1a gene activation could rescue Scn1a haploinsufficiency and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel. We screeened sgRNAs for their ability to stimulate Scn1a gene transcription in association with the dCas9 activation system. Interestingly, we identified one single sgRNA able to significantly increase Scn1a gene expression levels in cell lines as well as in primary neurons, with high specificity. Accordingly, levels of Nav1.1 protein were sufficiently augmented to potentiate firing ability of wild-type immature GABAergic interneurons. A similar effect in activating the Scn1a transcription was elicited in Dravet GABAergic interneurons rescuing their dysfunctional properties. To determine whether this approach could have therapeutic effect, we packaged adeno-associated viruses with the Scn1a-dCas9 activation system and showed their ability to ameliorate the febrile epileptic crises in DS mice. Our results pave the way for exploiting the dCas9-based gene activation as an effective and targeted approach to DS and other similar disorders resulting from altered gene dosage.

2019-03-05 | GSE111436 | GEO

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