Browse
Submit Data
Databases
API
Help

Dataset Information

18 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Locus-specific histone deacetylation using a synthetic CRISPR-Cas9-based HDAC.

ABSTRACT: Efforts to manipulate locus-specific histone acetylation to assess their causal role in gene expression and cellular and behavioural phenotypes have been impeded by a lack of experimental tools. The Cas9 nuclease has been adapted to target epigenomic modifications, but a detailed description of the parameters of such synthetic epigenome remodellers is still lacking. Here we describe a Cas9-based histone deacetylase (HDAC) and the design principles required to achieve locus-specific histone deacetylation. We assess its range of activity and specificity, and analyse target gene expression in two different cell types to investigate cellular context-dependent effects. Our findings demonstrate that the chromatin environment is an important element to consider when utilizing this synthetic HDAC.

SUBMITTER: Kwon DY

PROVIDER: S-EPMC5437308 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Locus-specific histone deacetylation using a synthetic CRISPR-Cas9-based HDAC.

Kwon Deborah Y DY Zhao Ying-Tao YT Lamonica Janine M JM Zhou Zhaolan Z

Nature communications 20170512

Efforts to manipulate locus-specific histone acetylation to assess their causal role in gene expression and cellular and behavioural phenotypes have been impeded by a lack of experimental tools. The Cas9 nuclease has been adapted to target epigenomic modifications, but a detailed description of the parameters of such synthetic epigenome remodellers is still lacking. Here we describe a Cas9-based histone deacetylase (HDAC) and the design principles required to achieve locus-specific histone deace ...[more]

PMID: 28497787

Similar Datasets

Locus-specific histone deacetylation using a synthetic CRISPR-Cas9-based HDAC

Project description:Efforts to manipulate locus-specific histone acetylation to assess their causal role in gene expression and cellular and behavioural phenotypes have been impeded by a lack of experimental tools. The Cas9 nuclease has been adapted to target epigenomic modifications, but a detailed description of the parameters of such synthetic epigenome remodellers is still lacking. Here we describe a Cas9-based histone deacetylase (HDAC) and the design principles required to achieve locus-specific histone deacetylation. We assess its range of activity and specificity, and analyse target gene expression in two different cell types to investigate cellular context-dependent effects. Our findings demonstrate that the chromatin environment is an important element to consider when utilizing this synthetic HDAC.

2017-07-09 | GSE91043 | GEO

Vascular histone deacetylation by pharmacological HDAC inhibition.

Project description:HDAC inhibitors can regulate gene expression by post-translational modification of histone as well as nonhistone proteins. Often studied at single loci, increased histone acetylation is the paradigmatic mechanism of action. However, little is known of the extent of genome-wide changes in cells stimulated by the hydroxamic acids, TSA and SAHA. In this article, we map vascular chromatin modifications including histone H3 acetylation of lysine 9 and 14 (H3K9/14ac) using chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (ChIP-seq). Since acetylation-mediated gene expression is often associated with modification of other lysine residues, we also examined H3K4me3 and H3K9me3 as well as changes in CpG methylation (CpG-seq). RNA sequencing indicates the differential expression of ∼30% of genes, with almost equal numbers being up- and down-regulated. We observed broad deacetylation and gene expression changes conferred by TSA and SAHA mediated by the loss of EP300/CREBBP binding at multiple gene promoters. This study provides an important framework for HDAC inhibitor function in vascular biology and a comprehensive description of genome-wide deacetylation by pharmacological HDAC inhibition.

| S-EPMC4120081 | biostudies-literature

CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses.

Project description:A key goal for cell biological analyses is to assess the phenotypes that result from eliminating a target gene. Since the early 1990s, the predominant strategy utilized in human tissue culture cells has been RNA interference (RNAi)-mediated protein depletion. However, RNAi suffers well-documented off-target effects as well as incomplete and reversible protein depletion. The implementation of CRISPR/Cas9-based DNA cleavage has revolutionized the capacity to conduct functional studies in human cells. However, this approach is still underutilized for conducting visual phenotypic analyses, particularly for essential genes that require conditional strategies to eliminate their gene products. Optimizing this strategy requires effective and streamlined approaches to introduce the Cas9 guide RNA into target cells. Here we assess the efficacy of synthetic guide RNA transfection to eliminate gene products for cell biological studies. On the basis of three representative gene targets (KIF11, CENPN, and RELA), we demonstrate that transfection of synthetic single guide RNA (sgRNA) and CRISPR RNA (crRNA) guides works comparably for protein depletion as cell lines stably expressing lentiviral-delivered RNA guides. We additionally demonstrate that synthetic sgRNAs can be introduced by reverse transfection on an array. Together, these strategies provide a robust, flexible, and scalable approach for conducting functional studies in human cells.

| S-EPMC6233062 | biostudies-literature

Programmable human histone phosphorylation and gene activation using a CRISPR/Cas9-based chromatin kinase.

Project description:Histone phosphorylation is a ubiquitous post-translational modification that allows eukaryotic cells to rapidly respond to environmental stimuli. Despite correlative evidence linking histone phosphorylation to changes in gene expression, establishing the causal role of this key epigenomic modification at diverse loci within native chromatin has been hampered by a lack of technologies enabling robust, locus-specific deposition of endogenous histone phosphorylation. To address this technological gap, here we build a programmable chromatin kinase, called dCas9-dMSK1, by directly fusing nuclease-null CRISPR/Cas9 to a hyperactive, truncated variant of the human MSK1 histone kinase. Targeting dCas9-dMSK1 to human promoters results in increased target histone phosphorylation and gene activation and demonstrates that hyperphosphorylation of histone H3 serine 28 (H3S28ph) in particular plays a causal role in the transactivation of human promoters. In addition, we uncover mediators of resistance to the BRAF V600E inhibitor PLX-4720 in human melanoma cells using genome-scale screening with dCas9-dMSK1. Collectively, our findings enable a facile way to reshape human chromatin using CRISPR/Cas9-based epigenome editing and further define the causal link between histone phosphorylation and human gene activation.

| S-EPMC7873277 | biostudies-literature

CRISPR-Cas9 RBM10 synthetic lethality screen

Project description:Genome-wide CRISPR-Cas9 knockout screen using TKOv1 sgRNA library was performed in isogenic RBM10-proficient and RBM10-deficient HCC827 cells.

2023-03-04 | E-MTAB-12724 | biostudies-arrayexpress

Reprogramming of Fibroblasts to Oligodendrocyte Progenitor-like Cells Using CRISPR/Cas9-Based Synthetic Transcription Factors.

Project description:Cell lineage reprogramming via transgene overexpression of key master regulatory transcription factors has been well documented. However, the poor efficiency and lack of fidelity of this approach is problematic. Synthetic transcription factors (sTFs)-built from the repurposed CRISPR/Cas9 system-can activate endogenous target genes to direct differentiation or trigger lineage reprogramming. Here we explored whether sTFs could be used to steer mouse neural stem cells and mouse embryonic fibroblasts toward the oligodendrocyte lineage. We developed a non-viral modular expression system to enable stable multiplex delivery of pools of sTFs capable of transcriptional activation of three key oligodendrocyte lineage master regulatory genes (Sox10, Olig2, and Nkx6-2). Delivery of these sTFs could enhance neural stem cell differentiation and initiated mouse embryonic fibroblast direct reprograming toward oligodendrocyte progenitor-like cells. Our findings demonstrate the value of sTFs as tools for activating endogenous genes and directing mammalian cell-type identity.

| S-EPMC6915844 | biostudies-literature

Homology-mediated end joining-based targeted integration using CRISPR/Cas9.

Project description:Targeted integration of transgenes can be achieved by strategies based on homologous recombination (HR), microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ). The more generally used HR is inefficient for achieving gene integration in animal embryos and tissues, because it occurs only during cell division, although MMEJ and NHEJ can elevate the efficiency in some systems. Here we devise a homology-mediated end joining (HMEJ)-based strategy, using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and ?800 bp of homology arms, and the targeted genome. We found no significant improvement of the targeting efficiency by the HMEJ-based method in either mouse embryonic stem cells or the neuroblastoma cell line, N2a, compared to the HR-based method. However, the HMEJ-based method yielded a higher knock-in efficiency in HEK293T cells, primary astrocytes and neurons. More importantly, this approach achieved transgene integration in mouse and monkey embryos, as well as in hepatocytes and neurons in vivo, with an efficiency much greater than HR-, NHEJ- and MMEJ-based strategies. Thus, the HMEJ-based strategy may be useful for a variety of applications, including gene editing to generate animal models and for targeted gene therapies.

| S-EPMC5518881 | biostudies-other

Vascular histone deacetylation by pharmacological HDAC inhibition

Project description:This SuperSeries is composed of the SubSeries listed below. HDAC inhibitors are thought to regulate gene expression by post-translational modification of histone as well as non-histone proteins. Often studied at single loci, increased histone acetylation is the paradigmatic mechanism of action, however, little is known of the extent of genome-wide changes of the mammalian genome when stimulated by the hydroxamic acids, TSA and SAHA. In primary human vascular endothelial cells we map the chromatin modifications, histone H3 acetylation of lysine 9 and 14 (H3K9/14ac) using chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (ChIP-seq). Since acetylation mediated gene expression is often associated with modification of other lysine residues we also examined H3K4me3 and H3K9me3 as well as changes in CpG methylation (CpG-seq). Genome-wide mRNA sequencing indicates the differential expression of about 30% of genes, with almost equal numbers being up- and down- regulated. We observe deacetylation conferred by TSA and SAHA that are associated with decreased gene expression. Histone deacetylation is associated with the loss of p300/CBP binding at gene promoters. This study provides an important framework for HDAC inhibitor function in vascular biology and a comprehensive description of genome-wide deacetylation. Refer to individual Series

2014-02-23 | E-GEOD-37378 | biostudies-arrayexpress

CRISPR/Cas9-Based Gene Editing Using Egg Cell-Specific Promoters in Arabidopsis and Soybean.

Project description:CRISPR/Cas9-based systems are efficient genome editing tools in a variety of plant species including soybean. Most of the gene edits in soybean plants are somatic and non-transmissible when Cas9 is expressed under control of constitutive promoters. Tremendous effort, therefore, must be spent to identify the inheritable edits occurring at lower frequencies in plants of successive generations. Here, we report the development and validation of genome editing systems in soybean and Arabidopsis based on Cas9 driven under four different egg-cell specific promoters. A soybean ubiquitin gene promoter driving expression of green fluorescent protein (GFP) is incorporated in the CRISPR/Cas9 constructs for visually selecting transgenic plants and transgene-evicted edited lines. In Arabidopsis, the four systems all produced a collection of mutations in the T2 generation at frequencies ranging from 8.3 to 42.9%, with egg cell-specific promoter AtEC1.2e1.1p being the highest. In soybean, function of the gRNAs and Cas9 expressed under control of the CaMV double 35S promoter (2x35S) in soybean hairy roots was tested prior to making stable transgenic plants. The 2x35S:Cas9 constructs yielded a high somatic mutation frequency in soybean hairy roots. In stable transgenic soybean T1 plants, AtEC1.2e1.1p:Cas9 yielded a mutation rate of 26.8%, while Cas9 expression driven by the other three egg cell-specific promoters did not produce any detected mutations. Furthermore, the mutations were inheritable in the T2 generation. Our study provides CRISPR gene-editing platforms to generate inheritable mutants of Arabidopsis and soybean without the complication of somatic mutagenesis, which can be used to characterize genes of interest in Arabidopsis and soybean.

| S-EPMC7309964 | biostudies-literature

Locus specific engineering of tandem DNA repeats in the genome of Saccharomyces cerevisiae using CRISPR/Cas9 and overlapping oligonucleotides.

Project description:DNA repeats constitute a large part of genomes of multicellular eucaryotes. For a longtime considered as junk DNA, their role in genome organization and tuning of gene expression is being increasingly documented. Synthetic biology has so far largely ignored DNA repeats as regulatory elements to manipulate functions in engineered genomes. The yeast Saccharomyces cerevisiae has been a workhorse of synthetic biology, owing to its genetic tractability. Here we demonstrate the ability to synthetize, in a simple manner, tandem DNA repeats of various size by Cas9-assisted oligonucleotide in vivo assembly in this organism. We show that long tandem DNA repeats of several kilobases can be assembled in one step for different monomer size and G/C content. The combinatorial nature of the approach allows exploring a wide variety of design for building synthetic tandem repeated DNA directly at a given locus in the Saccharomyces cerevisiae genome. This approach provides a simple way to incorporate tandem DNA repeat in synthetic genome designs to implement regulatory functions.

| S-EPMC5940919 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data