Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A microRNA-inducible CRISPR-Cas9 platform serves as microRNA sensors and cell type specific genome regulation tools

ABSTRACT: microRNAs (miRNAs) are an important class of small regulatory noncoding RNAs expressed by plants, animals and viruses. However, measuring miRNA activity in vivo remains a big challenge. Using a miRNA-mediated sgRNA releasing strategy and dCas9-VPR to drive a transgene RFP expression, we create a miRNA sensor that can faithfully measure miRNA activity at cellular levels and show that it can be used to monitor the differentiation status of stem cells. When sgRNAs are designed to target endogenous locus, we show this system can be adopted to achieve cell type specific activation of endogenous genes. Furthermore, when dCas9 is fused with a transcriptional repressor or a base editor, we show this system can be used to repress the expression of endogenous genes or mutate specific DNA bases of chromosome depending on the expression of cell type-specific miRNAs. Finally, by a miRNA sensor library screening, we discovered a previously undefined layer of heterogeneity associated with miR-21a activity in mouse embryonic stem cells. Together, these results highlight the utility of a miRNA-induced CRISPR-Cas9 system as miRNA sensors and cell type-specific genome regulation tools.

ORGANISM(S): Mus musculus

PROVIDER: GSE122683 | GEO | 2019/01/16

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Histone exchange sensors reveal variant specific dynamics in mouse embryonic stem cells

Project description:Eviction of histones from nucleosomes and their exchange with newly synthesized or alternative variants is a central mechanism shaping the epigenome. Here, we implemented a recently established sensor system that enables measuring genome-wide incorporation and exchange of canonical and non-canonical histone variants in mouse embryonic stem cells. While exchange of all measured variants scales with transcription, variant-specific patterns are associated with transcription elongation and Polycomb binding. We found considerable exchange rates of canonical H3.1 and H2B in heterochromatin and repeat elements where non-canonical H3.3 is stably incorporated but not exchanged. This unexpected association between H3.3 incorporation and the exchange of H3.1 and H2B is also evident in active promoters and enhancers, which we validated using HIRA knockout cells harboring the sensor system. The sensor system provides a powerful experimental framework with quantitative readout, facilitating functional studies of histone dynamics and epigenetic gene regulation in vivo.

2023-05-24 | GSE213076 | GEO

Endogenous miRNA-200b/c activity and regulation explored by a functional dual fluorescence reporter

Project description:Since their discovery, microRNAs (miRNA)s have been widely studied in almost every field of biology and medicine, leading to the identification of important gene regulation circuits and cellular mechanisms. However, investigations are generally focused on the analysis of their downstream targets and biological functions in overexpression and knockdown approaches, while miRNAs endogenous levels and activity remain poorly investigated. Here, we used the cellular plasticity-regulating process of epithelial-to-mesenchymal transition (EMT) as a model to show the efficacy of a plasmid-based fluorescent sensor to separate cells with distinct EMT signatures, based on miR-200b/c activity. The system was further combined with a CRISPR-Cas9 screening platform to unbiasedly identify miR-200b/c upstream regulating genes. The sensor allows to infer miRNAs fundamental biological properties, as profiling of sorted cells indicated miR-200b/c as a molecular switch between EMT differentiation and proliferation, and suggested a role for metabolic enzymes in miR-200/EMT regulation. Analysis of miRNAs’ endogenous levels and activity could lead to a better comprehension of their biological role in physiology and disease.

2020-12-01 | GSE154429 | GEO

Rescuing voluntary motor behavior in a Parkinson´s disease model by aCRISPR mediated reprogramming of striatal astrocytes into induced GABAergic neurons

Project description:Cell replacement strategies of affected cells have been performed in clinical studies of Parkinson´s disease with variable outcome. Conversion of endogenous glial cells into functional neurons might represent a more reliable alternative, thus here we developed a knock-in mouse line carrying a dual dCas9 transactivator system (dCAM), which allowed the conditional activation of the endogenous genes Ascl1, Lmx1a, and Nr4a2 in striatal astrocytes in vivo by delivering sgRNAs specific for their endogenous loci. In order to move this successful approach toward translation, we established an AAV based activator system carrying intein-split-dCas9-VP64, SAM (AAV-dCAS) activators, and the specific sgRNAs targeting transcription factor combinations. Both systems, the dCAM and the AAV-dCAS approach were successful in reprogramming of striatal astrocytes into GABAergic neurons, which functionally integrate into striatal circuits as evidenced by the alleviation of specific motor behaviors in a 6-OHDA Parkinson’s disease model. Thus, the AAV-dCAS approach may enable clinical therapies for Parkinson´s disease by reprogramming striatal astrocytes.

2022-03-15 | GSE149872 | GEO

PRMT1-mediated methylation of the Large Drosha Complex regulates microRNA biogenesis

Project description:MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Large Drosha Complex (LDC). Through high resolution mass spectrometry (MS) analysis we discovered that the LDC is extensively methylated, with 82 distinct methylated sites associated to 16 out of 23 subunits of the LDC. The majority of these modifications occurs on arginine (R)- residues (61), leading to 86 methylation events, while 29 lysine (K)-methylation events occurs on 21 sites of the complex. Interestingly, the depletion and pharmacological inhibition of PRMT1 lead to a widespread alteration of the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the specific impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the ILF3 subunit of the complex is linked to its diminished binding to the target pri-miRNAs. Overall, our study uncovers a previously uncharacterized role of R-methylation in the regulation of the LDC activity in mammalian cells, thus effecting global miRNA levels.

2019-10-23 | PXD011617 | Pride

An evolutionary mechanism to assimilate new nutrient sensors into the mTORC1 pathway

Project description:Animals sense and respond to nutrient availability in their environments, a task coordinated in part by the mTOR complex 1 (mTORC1) pathway. mTORC1 regulates growth in response to nutrients and, in mammals, senses specific amino acids through specialized sensors that bind the GATOR1/2 signaling hub. Given that animals can occupy diverse niches, we hypothesized that the pathway might evolve distinct sensors in different metazoan phyla. Whether such customization occurs—and how the mTORC1 pathway might capture new inputs—is unknown. Here, we identify the Drosophila melanogaster protein Unmet expectations (CG11596) as a species-restricted methionine sensor that directly binds the fly GATOR2 complex in a fashion antagonized by S-adenosylmethionine (SAM). We find that in Dipterans GATOR2 rapidly evolved the capacity to bind Unmet and to thereby repurpose a previously independent methyltransferase as a SAM sensor. Thus, the modular architecture of the mTORC1 pathway allows it to co-opt preexisting enzymes to expand its nutrient sensing capabilities, revealing a mechanism for conferring evolvability on an otherwise conserved system.

2024-03-21 | PXD050288 | Pride

A Daphnane Diterpenoid Isolated from Wikstroemia polyantha induces an inflammatory response and modulates miRNA activity

Project description:MicroRNAs (miRNAs) are endogenously expressed single-stranded ~21M-bM-^@M-^S23 nucleotide RNAs that inhibit gene expression post-transcriptionally by binding imperfectly to elements usually within the 3M-bM-^@M-2untranslated region (3M-bM-^@M-2UTR) of mRNAs. Small interfering RNAs (siRNAs) mediate site-specific cleavage by binding with perfect complementarity to RNA. Here, a cell-based miRNA reporter system was developed to screen for compounds from marine and plant extracts that inhibit miRNA or siRNA activity. The daphnane diterpenoid genkwanine M (GENK) isolated from the plant Wikstroemia polyantha induces an early inflammatory response and can moderately inhibit miR-122 activity in the liver Huh-7 cell line. GENK does not alter miR-122 levels nor does it directly inhibit siRNA activity in an in vitro cleavage assay. Finally, we demonstrate that GENK can inhibit HCV infection in Huh-7 cells. In summary, the development of the cell-based miRNA sensor system should prove useful in identifying compounds that affect miRNA/siRNA activity. Control (no drug treatment): 1 hour and 4 hour samples, 3 replicates each; Drug-treated: 1 hour and 4 hours samples, 3 replicates each

2012-06-28 | E-GEOD-39002 | biostudies-arrayexpress

Defining the contribution of microRNA-specific slicing Argonautes in animals

Project description:microRNAs regulate gene expression through interaction with an Argonaute protein family member. While some members of this protein family retain an enzymatic activity capable of cleaving RNA molecules complementary to Argonaute-bound small RNAs, the role of the slicing activity in the canonical microRNA pathway is still unclear in animals. To address the importance of slicing Argonautes in animals, we created Caenorhabditis elegans mutant strains carrying catalytically dead endogenous ALG-1 and ALG-2, the only two slicing Argonautes essential for the miRNA pathway in this animal model. We observe that the loss of ALG-1 and ALG-2 slicing activity affects overall animal fitness and causes phenotypes reminiscent of miRNA defects only when grown and maintained at restrictive temperature. Furthermore, the analysis of global miRNA expression show that the catalytic activity of ALG-1 and ALG-2 differentially regulate the level of specific subsets of miRNAs in young adults. We also demonstrate that altering the slicing activity of those miRNA-specific Argonautes does not result in any defect in the production of canonical miRNAs. Together, these data support that the slicing activity of miRNA-specific Argonautes functions to maintain levels of a set of miRNAs for optimal viability and fitness in animals particularly exposed to specific growing conditions.

2024-03-15 | GSE222733 | GEO

A Daphnane Diterpenoid Isolated from Wikstroemia polyantha induces an inflammatory response and modulates miRNA activity

Project description:MicroRNAs (miRNAs) are endogenously expressed single-stranded ~21–23 nucleotide RNAs that inhibit gene expression post-transcriptionally by binding imperfectly to elements usually within the 3′untranslated region (3′UTR) of mRNAs. Small interfering RNAs (siRNAs) mediate site-specific cleavage by binding with perfect complementarity to RNA. Here, a cell-based miRNA reporter system was developed to screen for compounds from marine and plant extracts that inhibit miRNA or siRNA activity. The daphnane diterpenoid genkwanine M (GENK) isolated from the plant Wikstroemia polyantha induces an early inflammatory response and can moderately inhibit miR-122 activity in the liver Huh-7 cell line. GENK does not alter miR-122 levels nor does it directly inhibit siRNA activity in an in vitro cleavage assay. Finally, we demonstrate that GENK can inhibit HCV infection in Huh-7 cells. In summary, the development of the cell-based miRNA sensor system should prove useful in identifying compounds that affect miRNA/siRNA activity.

2012-06-29 | GSE39002 | GEO

Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system

Project description:Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3 to 4 sgRNAs binding to the proximal promoters suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system. We used microarray to assay the gene expression changes after transfection of dCas9VP160 with the different sgRNAs

2013-08-10 | E-GEOD-49701 | biostudies-arrayexpress

Chemical Sensors Detect and Resolve Proteome Aggregation in Peripheral Neuropathy induced by Chemotherapeutic Agents

Project description:Neuropathic pain, a consequence of somatosensory nervous system injury or disease, is commonly associated with cancer and its treatments, including chemotherapy-induced peripheral neuropathy (CIPN). However, the mechanisms underlying CIPN-induced proteome aggregation remain elusive due to limited detection tools. Here, we present a combined approach employing fluorescence imaging and proteomic sensors to comprehensively study proteome aggregation in neuronal cells subjected to CIPN. The AggStain imaging probe sensitively detected proteome aggregation, while the AggLink proteomic sensor captured and profiled aggregated proteins. This integrative sensor system offers novel insights into proteome aggregation dynamics in CIPN and highlights its potential for broader applications in assessing proteome stability under various conditions.

2023-10-09 | PXD045972 |

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