Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Proteome of Cas9- and dCas9-expressing Lucilia cuprina larvae reared on vs. off doxycycline

ABSTRACT: We analyzed the proteomes of transgenic Cas9- and dCas9-expressing larvae of the Australian sheep blowfly, Lucilia cuprina. We measured abundance of red fluorescent protein (dsRed) in our samples in order to test Cas9 and dCas9 activity. Flies from two transgenic strains, DR7#2;Cas9-ryt and DR7#2;dCas9-ryt/+, were reared on or off 25 ug/mL doxycycline. Removal of doxycycline from the diet of these transgenic flies should activate the expression of Cas9 or dCas9 genes. Flies reared on doxycycline served as our control groups, as these flies should express no (or minimal amounts of) Cas9 or dCas9. Four replicate samples were obtained from each experimental and control group, for a total of 16 samples across this experiment. Each replicate consisted of a single third instar (wandering stage) larvae. Whole-tissue samples were flash-frozen in liquid nitrogen and stored at -80°C prior to sample preparation for 1D-LC-MS/MS analysis.

INSTRUMENT(S):

ORGANISM(S): Lucilia Cuprina

TISSUE(S): Whole Body

SUBMITTER: Olivia Mathieson

LAB HEAD: Manuel Kleiner

PROVIDER: PXD071864 | Pride | 2026-04-27

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	FileAndSampleDescriptionTable_BlowflyProteome.txt	Txt
	OM016_C01_500ng_70min.msf	Msf
	OM016_C01_500ng_70min.raw	Raw
	OM016_C02_500ng_70min.msf	Msf
	OM016_C02_500ng_70min.raw	Raw

Items per page:

1 - 5 of 35

Similar Datasets

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 gRNA engineering for selectable genome editing, activation and repression

Project description:RNA-Seq after Cas9-gRNA transfection with different length gRNAs we performed PolyA Selection and RNA-Seq on cells transfected with dCas9-VPR and a gRNA of each length (20nt, 16nt, or 14nt) targeting ACTC1, MIAT, or HBG1/2

2015-07-15 | E-GEOD-70694 | biostudies-arrayexpress

Keap1 is an endogenous dCas9 suppressor inhibiting CRISPRa and CRISPRi efficiency

Project description:As a potent and accurate genome-editing tool, CRISPR-Cas9 has been widely used in biomedical research and evaluated as gene therapy in treating human diseases. Although distinct engineered Cas9s, dCas9s and additional endonucleases have been identified, as these bacterial enzymes do not naturally express in mammalian cells, whether and how bacterial Cas9 proteins are regulated by mammalian hosts remains poorly understood. Here, we identified Keap1 as an endogenous E3 ligase that targets Cas9/dCas9/Fanzor1 for ubiquitination and degradation. Cas9-“ETGE” mutants evading Keap1 recognition displayed enhanced gene editing ability in cells. dCas9-“ETGE” mutants displayed extended protein half-life on chromatin, leading to significantly improved CRISPa and CRISPRi efficacy. Cas9 binding to Keap1 also inactivate Keap1 function via competing with Keap1 substrates or binding partners, while engineered Cas9 mutants showed less perturbation. Thus, our study reveals a mammalian specific Cas9 regulation and provides new Cas9 designs not only with enhanced gene regulatory capacity but also with minimal effects on disrupting endogenous Keap1 signaling.

2025-01-20 | PXD050935 | Pride

Differential phosphoproteomics upon chitin-treatment in Marchantia polymorpha

Project description:Pattern-recognition receptor (PRR)-triggered immunity (PTI) plays a pivotal role in plant immunity to ward off a wide range of pathogenic microbes. The model liverwort Marchantia polymorpha is gaining popularity in investigating the evolution of plant-microbe interactions. The M. polymorpha is capable of triggering defense-related gene expression by sensing components in bacterial and fungal extracts, suggesting existence of PTI in this plant model. However, the molecular components that would form PTI in M. polymorpha have not yet been described. We show that, in M. polymorpha, lysin motif (LysM) receptor-like kinase (LYK) MpLYK1 and LYK-related (LYR) MpLYR, among four LysM receptor homologs, are required for sensing chitin and peptidoglycan (PGN) fragments and thereby triggering a series of immune responses. Phosphoproteomic analysis of M. polymorpha in response to chitin treatment comprehensively identified regulatory proteins that would shape LysM-mediated PTI. The identified proteins covered homologs of well-described PTI components in angiosperms as well as proteins whose roles in PTI are not yet determined including the blue-light receptor phototropin MpPHOT. We revealed that MpPHOT is required for a negative feedback of defense-related gene expression during PTI. Taken together, this study provides the basic framework of LysM-mediated PTI in M. polymorpha and demonstrates the utility of M. polymorpha as a plant model for discovering novel or fundamental molecular mechanisms underlying PRR-triggered immune signaling in plants.

2023-11-06 | PXD042084 | Pride

Transcription profiling of human nasal brushing cells from 19 cystic fibrosis (CF) patients and 19 healthy controls

Project description:We have compared gene expression in human nasal brushing cells from 19 cystic fibrosis (CF) patients and 19 healthy controls using a 5.2K cDNA microarray. Our aim is to identify new disease biomarkers for the Cystic Fibrosis Gene Therapy Consortium. These markers will be used to report more effectively on the response to the administration of gene therapy in vivo. Cystic Fibrosis is a recessive genetic disease caused by mutations in the cystic fibrosis conductance regulator (CFTR) gene which encodes a chloride ion channel. The most common mutation is the ∆F508 mutation, present on 70% of CF chromosomes in Caucasian populations. The disease affects many organs in the body such as the pancreas, liver, sweat glands, small intestine and reproductive tracts but is most commonly associated with progressive, inflammatory lung disease. The current average life expectancy of CF patients is 35 years. Gene therapy is being developed as a treatment for CF airway disease, however, means of measuring the efficiency and efficacy of gene therapy in vivo are lacking. This is mainly due to the difficulty in measuring the chloride conductance of CFTR in cells and tissues. Furthermore, clinical assays for measuring improvements in lung function are insensitive. Surrogate markers of inflammation and CFTR function will therefore be important for the effective assessment of gene therapy in vivo. We have analysed gene expression in human nasal epithelium as this is considered an accessible surrogate for the conducting airways where disease manifests in the majority of patients. Additionally, this tissue will be sampled in clinical trials.

2011-04-30 | E-MEXP-436 | biostudies-arrayexpress

CRISPR Display: A modular method for locus-specific targeting of long noncoding RNAs and synthetic RNA devices in vivo [RNA-Seq]

Project description:Noncoding RNAs (ncRNAs) comprise an important class of natural regulators that mediate a vast array of biological processes, including the modulation of chromatin architecture. Moreover, artificial ncRNAs have revealed that the functional capabilities of RNA are extremely broad. To further investigate and harness these capabilities, we developed CRISPR-Display ("CRISP-Disp"), a targeted localization strategy that uses Cas9 to deploy large RNA cargos to specific DNA loci. We demonstrate that exogenous RNA domains can be functionally appended onto the CRISPR scaffold at multiple insertion points, allowing the construction of Cas9 complexes with RNAs nearing one kilobase in length, with structured RNAs, protein-binding cassettes, artificial aptamers and pools of random sequences. CRISP-Disp also allows the simultaneous multiplexing of disparate functions at multiple targets. We anticipate that this technology will provide a powerful method with which to ectopically localize functional RNAs and ribonuceloprotein complexes at specified genomic loci. Whole cell poly(A) selected RNA seq, from HEK293FT cells bearing lentivirally-integrated Gaussia and Cypridina luciferase reporter loci. Cells were transiently transfected with dCas9~VP64 alone, or with dCas9~VP and one of several modified sgRNAs,each targeting the Gaussia reporter.

2015-05-11 | E-GEOD-66755 | biostudies-arrayexpress

Heterozygous mutation of NF-B innate immune response transcription factor Relish increases survival following traumatic brain injury in Drosophila

Project description:Traumatic brain injury (TBI) pathologies are caused by primary and secondary injuries. Primary injuries result from physical damage to the brain, and secondary injuries arise from cellular responses to primary injuries. A characteristic cellular response is sustained activation of inflammatory pathways commonly mediated by NF-B transcription factors. Using a Drosophila melanogaster TBI model, we previously found that the main proximal transcriptional response to primary injuries is triggered by activation of Toll and Imd innate immune response pathways that engage NF-B factors Dif and Relish (Rel), respectively. Here, we monitor the abundance of Rel protein by mass spectrometry (MS) and observe that Rel increases in fly heads at 4-8 h after TBI. To investigate the necessity of Rel for secondary injuries, we generated a null allele, Reldel, by CRISPR/Cas9 editing. Heterozygous but not homozygous Reldel mutation reduced mortality at 24 h after TBI and increased the lifespan of injured flies. Additionally, the effect of heterozygous Reldel mutation on mortality was modulated by genetic background and diet. To identify genes that facilitate effects of heterozygous Reldel mutation on TBI outcomes, we compared genome-wide mRNA expression profiles of uninjured and injured +/+, +/Reldel, and Reldel/Reldel flies at 4 h following TBI. Only a few genes changed expression more than two-fold in +/Reldel flies relative to +/+ and Reldel/Reldel flies, and they were not canonical innate immune response genes. Therefore, Rel is necessary for TBI-induced secondary injuries but in complex ways involving Rel gene dose, genetic background, diet, and possibly small changes in expression of innate immune response genes.

2020-12-04 | PXD021869 | Pride

Potent and targeted activation of HIV-1 using the CRISPR/Cas9 activator Complex

Project description:Integration of the HIV-1 provirus in the host genome ensures a persistent supply of latently infected cells. This latent reservoir is recalcitrant to antiretroviral therapy (ART) making lifelong treatment the only option for patients. The â??shock and killâ?? strategy aims to eradicate latent HIV by reactivating proviral gene expression followed by ART treatment. Gene specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising small guide RNAs (sgRNAs) with a nuclease deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64). Â We engineered this system to target 23 sites within the LTR promoter of HIV-1 and identified a â??hotspotâ?? for activation. We studied the functionality of activating sgRNAs to transcriptionally modulate the latent proviral genome across multiple differentÂ in vitroÂ latency cell models including several J-Lat, ACH2 J1.1 and the CEM T cell model comprising a single clonal population of integrated mCherry-IRES-Tat from a full-length HIV LTR (LChIT).Â While we observed variable responses of latent cell models to well-characterized chemical stimuli, we detected consistent efficient activation of latent virus mediated by activator sgRNAs.Â In addition, transcriptome analysis of chemically treated cells revealed massive non-specific gene dysregulation whereas by comparison, dCas9-VP64/sgRNAs induced specific activation of the integrated provirus.Â In conclusion, we show the potential for CRISPR-mediated gene activation systems to provide enhanced efficiency and specificity in a targeted latency reactivation strategy. This represents a promising approach to a â??functional cureâ?? of HIV/AIDS. Three experimental conditions (sgRNA control, TNF treated and sgRNA against the LTR of HIV-1) were analyzed in triplicate using two sequencing lanes

2016-08-20 | E-GEOD-72259 | biostudies-arrayexpress

RNA-seq transcriptomic analyses of group B Streptococcus Cas9 variants and CRISPRi knockdown strains

Project description:Group B Streptococcus (GBS) strain CNCTC 10/84 was modified to have specific mutations to the cas9 gene: allelic exchange replacement with a chloramphenicol resistance marker (Cas9 knockout), D10A and H845A (catalytically inactive dCas9), or R1339A and R1441A (sCas9 unable to scan for protospacer adjacent motifs). Wild type (WT) and mutant strains were grown in biological triplicate samples and used for RNA purification at two growth timepoints: OD600=0.6 and OD600=1.2. Additionally, CNCTC 10/84 dCas9 and A909 dCas9 were transformed with p3015b expression plasmids expressing sgRNA cassettes designed to downregulate the cyl operon in CNCTC 10/84 dCas9 and the covR/covS two-component system in A909 dCas9. Triplicate samples were grown alongside control transformants bearing "sham" sgRNA plasmid. RNA was purified at OD600=1.2. All RNA samples were used for RNA-seq and subsequent bioinformatic analyses.

2023-05-15 | GSE226236 | GEO

Exploring the virulence gene interactome with CRISPR/dCas9 in the human malaria parasite

Project description:The var multigene family encodes clonally variant surface antigens that are key to immune evasion and pathogenesis in the human malaria parasite, Plasmodium falciparum. Epigenetics and nuclear organization regulate the var gene family in a system of mutually exclusive expression; however, few factors have been shown to play a direct role in these processes. Thus, we adapted a CRISPR-based immunoprecipitation-mass spectrometry approach for identification of novel factors associated with var genes in their natural chromatin context. A tagged, catalytically inactive Cas9 (“dCas9”) was targeted to the promoters or introns of a subset of var genes and subjected to immunoprecipitation followed by label-free LC-MS/MS. A non-targeted dCas9 served as a control.

2020-07-15 | PXD013131 | Pride

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