Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Pervasive off-target activity in CRISPR-interference (CRISPRi) system [ChIP-seq]

ABSTRACT: CRISPR/Cas system and its dCas derivatives have been widely and effectively used to alter the intended genomic target. However, the presence of off-targets due to unintended binding of sgRNAs to sequences that closely resemble the target sequence, is still a major challenge. Here, we utilized a genome-wide sgRNA library for the dCas9-KRAB CRISPRi system to investigate the presence of off-target activity and its effects on gene expression. Our study provides strong evidence that CRISPRi off-targets affect the transcriptome of a cell extensively. We also highlight that mis-matches to the target DNA are tolerated while sgRNA binding and the length of the contiguous matches in the PAM-proximal region is one of the most predictive features of binding, along with open chromatin and the DNA-RNA hybrid energy. Additionally, we provide a curated random forest model that can be used to predict the off-target activity in the dCas systems.

ORGANISM(S): Homo sapiens

PROVIDER: GSE252978 | GEO | 2024/10/11

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Pervasive off-target activity in CRISPR-interference (CRISPRi) system [RNA-seq]

Project description:CRISPR/Cas system and its dCas derivatives have been widely and effectively used to alter the intended genomic target. However, the presence of off-targets due to unintended binding of sgRNAs to sequences that closely resemble the target sequence, is still a major challenge. Here, we utilized a genome-wide sgRNA library for the dCas9-KRAB CRISPRi system to investigate the presence of off-target activity and its effects on gene expression. Our study provides strong evidence that CRISPRi off-targets affect the transcriptome of a cell extensively. We also highlight that mis-matches to the target DNA are tolerated while sgRNA binding and the length of the contiguous matches in the PAM-proximal region is one of the most predictive features of binding, along with open chromatin and the DNA-RNA hybrid energy. Additionally, we provide a curated random forest model that can be used to predict the off-target activity in the dCas systems.

2024-10-11 | GSE252977 | GEO

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

Comparison of non-specific transcriptional effects of CRISPRi effector proteins by RNA-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.

2022-06-03 | GSE205147 | GEO

Comparison of dual sgRNA library versus Dolcetto by Perturb-seq

2022-06-04 | GSE205310 | GEO

Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity [ChIP-Seq]

Project description:Over the last decade, gene-silencing mediated by dCas9 binding to transcribed regions or their promoters, a strategy referred to as CRISPRi, has emerged as a powerful tool in bacterial genetics. While this strategy has already been broadly adopted, several studies have reported experimental setups in which dCas9 expression was toxic. In particular, guide RNAs that share specific PAM-proximal sequence motifs were shown to be toxic to E. coli. Here we demonstrate that this toxicity is caused by off-target binding of dCas9 to the promoter of essential genes. Silencing of off-target genes can occur with as little as 4nt of identity between the PAM-proximal sequence and the off-target position. This phenomenon only occurs in some promoter sequences but does not appear to be constrained to any specific PAM-proximal sequence. Accordingly, screens performed in various strains of E. coli and related species shows that the nature of toxic guide RNAs changes together with the evolution of the sequence of off-target positions. These results highlight the importance of relying on several guide RNAs targeting the same gene when performing CRISPRi experiments in bacteria in order to avoid any possible confounding results due to off-target binding.

2022-11-04 | GSE217004 | GEO

Cas9 off-target binding to the promoter of bacterial genes leads to silencing and toxicity [RNA-Seq]

2022-10-25 | GSE216032 | GEO

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

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

Project description: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).

2014-08-29 | E-GEOD-57650 | biostudies-arrayexpress

Expanding the Scope of Bacterial CRISPR Activation with PAM-flexible dCas9 Variants

Project description:CRISPR-Cas transcriptional tools have been widely applied for programmable regulation of complex biological networks. In comparison to eukaryotic systems, bacterial CRISPR activation (CRISPRa) has stringent target site requirements for effective gene activation. While genes may not always have an NGG protospacer adjacent motif (PAM) at the appropriate position, PAM-flexible dCas9 variants can expand the range of targetable sites. Here we systematically evaluate a panel of PAM-flexible dCas9 variants for their ability to activate bacterial genes. We observe that dxCas9-NG provides a high dynamic range of gene activation for sites with NGN PAMs while dSpRY permits modest activity across almost any PAM. Similar trends were observed for heterologous and endogenous promoters. For all variants tested, improved PAM-flexibility comes with the tradeoff that CRISPRi-mediated gene repression becomes less effective. Weaker CRISPR interference (CRISPRi) gene repression can be partially rescued by expressing multiple sgRNAs to target many sites in the gene of interest. Our work provides a framework to choose the most effective dCas9 variant for a given set of gene targets, which will further expand the utility of CRISPRa/i gene regulation in bacterial systems.

2022-11-15 | GSE217559 | GEO

Genome-wide identification of CRISPR/Cas9 off-targets in human genome

Project description:By a robust unbiased ChIP-seq approach, we demonstrated that CRISPR/Cas9 had crRNA-specific off-target binding activities in human genome. However, most of those binding off-targets could not be efficiently cleaved both in vivo and in vitro which suggested the cleavage off-target activity of CRISPR/Cas9 in human genome is very limited. We provided a valuable tool to further investigate the molecular mechanism of CRISPR/Cas9 and to optimize its in vivo targeting sgRNA binding sites were identified with ChipSeq by using GFP antibody (there are 2 replicates for egfa-t1 sgRNA,emx1 sgRNA and control without sgRNA in Hek293T cells, one egfa-t1 sgRNA,emx1 sgRNA and control without sgRNA in HeLaS3 cells)

2014-06-18 | E-GEOD-58511 | biostudies-arrayexpress

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