Browse
Submit Data
Databases
API
Help

Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

13 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

RNA-guided Location and Function of the Cas9 CRISPR Endonuclease in Mammalian Cells

ABSTRACT: The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to 5’NGG3’ protospacer adjacent motifs (PAM). Examination of dmCas9 binding sites using two Trp53 targeting sgRNAs in Arf -/- MEF cell line (mouse).

ORGANISM(S): Mus musculus

SUBMITTER: Jerry Pelletier

PROVIDER: E-GEOD-57650 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Json Xml

Publications

Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage.

Cencic Regina R Miura Hisashi H Malina Abba A Robert Francis F Ethier Sylvain S Schmeing T Martin TM Dostie Josée J Pelletier Jerry J

PloS one 20141002 10

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive ...[more]

PMID: 25275497

Similar Datasets

CRISPR enhancer scan to detect critical elements for the regulation of EVI1 in a translocated enhancer

Project description:To uncover, in an unbiased fashion, which elements of the 18 kb translocated region control EVI1 transcription, we devised a CRISPR/Cas9-based enhancer scanning approach. We considered all possible sgRNA target sites containing a canonical Cas9 PAM site (NGG) on both strands of the minimal 18 kb translocated region. Deep-sequencing libraries were generated by PCR amplification of sgRNA guide strands using primers that tag the product with standard Illumina adapters and a 4 bp sample barcode in a 2 step-PCR protocol.

2021-07-26 | E-MTAB-9948 | biostudies-arrayexpress

Genome-wide profiling of MED1 and RUNX1 binding in MED1 wild-type and knockout human pre-B ALL cell line 697 (ChIP-seq)

Project description:CRISPR/Cas9 system was used to generate mediator complex subunit 1 (MED1) knockout human pre-B ALL cell line 697. ChIP-seq was performed to identify genomic regions responsible for recruitment of MED1 and RUNX1.

2020-05-13 | E-MTAB-8177 | biostudies-arrayexpress

Classifying leukemia types with chromatin conformation data (ChIP-Seq)

Project description:Background: Although genetic or epigenetic alterations have shown to affect the three-dimensional organization of genomes, the utility of chromatin conformation in the classification of human disease has never been addressed. Results: Here, we explore whether chromatin conformation can be used to classify human leukemia. We map the conformation of the HOXA gene cluster in a panel of cell lines with 5C chromosome conformation capture technology, and use the data to train and test a support vector machine classifier named 3D-SP. We show that 3D-SP is able to accurately distinguish leukemias expressing MLL-fusion proteins from those expressing only wild-type MLL, and that it can also classify leukemia subtypes according to MLL fusion partner, based solely on 5C data. Conclusions: Our study provides the first proof-of-principle demonstration that chromatin conformation contains the information value necessary for classification of leukemia subtypes. Examination of CTCF and RAD21 binding sites in THP-1 cell.

2014-02-27 | E-GEOD-55407 | biostudies-arrayexpress

Gene expression profiling of a model of CRISPR-mediated insulator disruption in the MMP locus in triple-negative breast cancer cell lines

Project description:Using CRISPR-Cas9 technology, we stably disrupted an insulator element in two different TNBC cell lines. The aim of this experiment was to characterize the transcriptomic profile using RNA-seq. For this experiment, we selected three biological replicates of MDA-MB-231 (WT and IE8dis).

2023-08-31 | E-MTAB-12823 | biostudies-arrayexpress

Chromatin accessibility of a model of CRISPR-mediated insulator disruption in the MMP locus in triple-negative breast cancer cell lines

Project description:Using CRISPR-Cas9 technology, we stably disrupted an insulator element in two different TNBC cell lines. The aim of this experiment was to characterize the chromatin accessibility profile using ATAC-seq. For this experiment, we selected three biological replicates of MDA-MB-231 (WT and IE8dis) and MDA-MB-436 (WT and IE8dis).

2023-08-31 | E-MTAB-12821 | biostudies-arrayexpress

Lineage recording in human cerebral organoids (iTracer-perturb)

Project description:Induced pluripotent stem cell (iPSC) derived organoid systems provide models to study human organ development. Single-cell transcriptome sequencing enables highly-resolved descriptions of cell state heterogeneity within these systems and computational methods can reconstruct developmental trajectories. However, new approaches are needed to directly measure lineage relationships in these systems. Here we establish an inducible dual channel lineage recorder, iTracer, that couples reporter barcodes, inducible CRISPR/Cas9 scarring, and single-cell transcriptomics to analyze state and lineage relationships in iPSC-derived systems. This data set include the iTracer-perturb data of one cerebral organoid with simultaneous TSC2 perturbation and lineage recording.

2021-11-03 | E-MTAB-10971 | biostudies-arrayexpress

Chromatin architecture profiling of a model of CRISPR-mediated insulator disruption in the MMP locus in triple-negative breast cancer cell lines

Project description:Using CRISPR-Cas9 technology, we stably disrupted an insulator element in two different TNBC cell lines. The aim of this experiment was to characterize the chromatin architecture profile using Hi-C. For this experiment, we selected three biological replicates of MDA-MB-231 (WT and IE8dis).

2023-08-31 | E-MTAB-12825 | biostudies-arrayexpress

Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease

Project description:RNA-guided genome editing with the CRISPR-Cas9 system has great potential for basic and clinical research, but the determinants of targeting specificity and the extent of off-target cleavage remain insufficiently understood. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) in HEK293T cells, in combination with 12 different single guide RNAs (sgRNAs). The number of off-target sites bound by dCas9 varied from ~10 to >1,000 depending on the sgRNA. Analysis of off-target binding sites showed the importance of the PAM-proximal region of the sgRNA guiding sequence and that dCas9 binding sites are enriched in open chromatin regions. When targeted with catalytically active Cas9, some off-target binding sites had indels above background levels in a region around the ChIP-seq peak, but generally at lower rates than the on-target sites. Our results elucidate major determinants of Cas9 targeting, and we show that ChIP-seq allows unbiased detection of Cas9 binding sites genome-wide 1.sgRNA1-6 binding sites were identified with ChipSeq by using HA antibody (there are 2 replicates for sgRNA1-3, one sample for sgRNA4-6,one control without sgRNA) 2.PCR products which amplifies " off-target genomic sites" were deep sequenced in the presence of WT Cas9+sgRNA or WT Cas9 alone( unique adaptor was used for each sgRNA and mixed for multiplex run)

2014-05-18 | E-GEOD-55887 | biostudies-arrayexpress

Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish (Run6)

Project description:Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish. Each loci has a control, where no Cas9/sgRNA were injected. This is amplicon sequencing with Illumina, after PCR amplification. Data was processed with ampliCan R package version 1.1.1.

2017-12-25 | E-MTAB-6310 | biostudies-arrayexpress

Analysis of Cas9/sgRNA mutagenic activity at a variety of loci in zebrafish (Run 9)

2017-12-15 | E-MTAB-6357 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

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