Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Multi-omic analyses reveal minimal impact of the CRISPR-Cas9 nuclease on the host

ABSTRACT: Transcriptomics strategies were applied to explore the cellular feedback after incorporating the Cas9. HEK293T cell lysates were collected after 24h transfected with PX330-GFP-cas9-6T, PX330-GFP-cas9-luciferase, PX330-GFP-noCas9-6T, PX330-GFP-noCas9-luciferase. RNA-seq data were generated from deep sequence. We found the impact caused by Cas9 is very minor

ORGANISM(S): Homo sapiens

PROVIDER: GSE112076 | GEO | 2021/03/20

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair

Project description:Based on the hypothesis that, enhancing the local concentration of donor oligos could increase the correction rates, we generated and tested novel CRISPR-Cas9 systems, in which the DNA repair template is covalently conjugated to Cas9 (RNPD system). To validate our results from the HEK293T reporter cells, we here tested our approach at different endogenous genomic loci and in different cell types. We first targeted the human beta globin (HBB) locus in the K562 cell line, and analyzed correction- and editing frequencies using next generation sequencing (NGS). Next we targeted the Rosa26 and proprotein convertase subtilisin/kexin type 9 (Pcsk9) locus in mouse embryonic stem cells (mESCs). Here, RNPD system was always compared to Cas9 SNAP-tag fusion proteins with uncoupled donor oligos. To also directly compare the engineered RNPD system to the classical CRISPR-Cas9 system, we performed experiments where we used wild-type Cas9 with the uncoupled donor oligos as a control. We therefore targeted the fluorescent reporter locus as well as the endogenous loci HBB, empty spiracles homeobox 1 (EMX1), and C-X-C chemokine receptor type 4 (CXCR4) in HEK293T cells. Finally, we performed the analysis of three computationally predicted off-target sites of the reporter locus.

2018-06-04 | E-MTAB-6808 | biostudies-arrayexpress

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

Multi-omic analyses reveal minimal impact of the CRISPR-Cas9 nuclease on the host

Project description:Multi-omic analyses reveal minimal impact of the CRISPR-Cas9 nuclease on the host

| PRJNA439246 | ENA

Exome-seq of Jak2VF erythroid progenitors cells transduced with Dnmt3a-Cas9-GFP lentivirus vs. emp-Cas9-GFP controls 17 weeks post transplantation

Project description:Exome sequencing of erythroid progenitors from Dnmt3a-Cas9-GFP+/- vs. emp-Cas9-GFP+/- controls.

2018-10-29 | E-MTAB-7243 | biostudies-arrayexpress

Genome-Wide Specificity of DNA-Binding, Gene Regulation, and Chromatin Remodeling by TALE- and CRISPR/Cas9-Based Transcription Factors

Project description:Use DNase-seq to assess genome-wide chromation remodeling which occurred in CRISPR/Cas9 and TALE genome engineering systems Determining chromatin structural changes in transfected cells vs. the parent HEK293T cells

2015-05-28 | E-GEOD-67007 | biostudies-arrayexpress

Functional assessment of a new PBX1 variant through CRISPR-Cas9 gene editing

Project description:To assess the role of a variant of unknown significance in PBX1 (p.(Arg107Gln) by generating a HEK293T-derived cell line knockdown for PBX1 (clone 22 cells) through CRISPR-Cas9. We then transfected the KD cells or the WT HEK293T cells with a plasmid encoding either WT or Arg107Gln PBX1 proteins.

2023-02-28 | GSE215967 | GEO

The interacting protein peptide sequences of stathmin

Project description:The peptide sequences identified by mass spectrometry in the proteins immunoprecipitated from stathmin-GFP expressed HEK293T lysates with the GFP-beads.

2021-09-10 | PXD023906 | Pride

RNA-Guided Human Gene Activation by Cas9/CRISPR-Based Engineered Transcription Factors

Project description:Synthetic transcription factors can be applied in many areas of biotechnology, medicine, and basic research. In contrast to current methods based on engineering new DNA-binding proteins, we show that Cas9 fused to a transcriptional activation domain can be targeted by combinations of guide RNA molecules to induce the expression of endogenous human genes. This simple approach for targeted gene activation circumvents the need for engineering new proteins and will enable widespread synthetic gene regulation. HEK293T cells were transfected with plasmid expressing Cas9-VP64 fusion protein and a guide RNA. As a control, empty guide RNA was transfected. Gene expression was then measured using mRNA-seq, and differential expression calculated using DESeq. All experiments were performed in biological duplicates or triplicates.

2013-07-25 | E-GEOD-47114 | 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

Cas9 target DNA specificity in vitro and in vivo (S. cerevisiae)

Project description:Cas9, a CRISPR RNA-guided nuclease, has been rapidly adopted as a tool for biochemical and genetic manipulation of DNA. Although Cas9 offers remarkable specificity and versatility for genome manipulation, mis-targeted events occur. To extend the understanding of Cas9 target::homology requirements, we compared mismatch tolerance for a specific Cas9::gRNA complex in vitro and in vivo (in Saccharomyces cerevisiae). A variety of truncated and full-length gRNAs (with 17, 18, and 20 nucleotides of complementarity sequence) were used. In each case, we observed notable differences between in vitro and in vivo Cas9 cleavage specificity profiles, with a more stringent effect of mismatches on activity seen in vivo. Increased specificity of the 18 nt complementarity truncated gRNA was evident in vivo, but not in vitro. Overall, this study highlights differences in the specificity of Cas9 cleavage between controlled in vitro conditions and complex and chromatinized in vivo conditions. We adapted a previous high throughput sequencing approach (doi: 10.1093/nar/gku1102) to assess the effects of single base variants in vivo. We used a polymorphic random variant library matched to a specific trigger sequence (for which we used a segment from the C. elegans unc-22 gene, previously designated âunc-22Aâ (doi: 10.1093/nar/gku1102). Cas9 in vivo and in vitro assays were carried out with four different gRNAs incorporating sequence from the unc-22A trigger segment. The four segments incorporate 17 nt, 18 nt, 20 nt, and 20+G nt of unc-22A complementarity respectively. The retention is calculated based on PMID: 25399416. File names denote the experiment-> gRNAname_gRNAlength_(invitro or invivo)_(incubation or induction time)_IlluminaRunID.dat The files contain all normalized sequences in column one with their calculated retentions in column 2.

2016-06-10 | E-GEOD-79667 | 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