Ontology highlight
ABSTRACT:
PROVIDER: PRJNA675288 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13004259_1.fastq.gz | Fastqsanger.gz | |||
| SRR13004259_2.fastq.gz | Fastqsanger.gz | |||
| SRR13004260_1.fastq.gz | Fastqsanger.gz | |||
| SRR13004260_2.fastq.gz | Fastqsanger.gz | |||
| SRR13004261_1.fastq.gz | Fastqsanger.gz |
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Project description:The CRISPR-Cas9 system enables efficient sequence-specific mutagenesis for creating germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we established in vitro-assembled, fluorescent Cas9-sgRNA RNPs in stabilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. Sequence analysis of targeted loci in individual embryos reveals highly efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis reveals preliminary loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show efficient targeting of functional non-coding elements in gene-regulatory regions using saturating mutagenesis towards uncovering functional control elements in transgenic reporters and endogenous genes. Our results suggest that in vitro assembled, fluorescent Cas9-sgRNA RNPs provide a rapid reverse-genetics tool for direct and scalable loss-of-function studies beyond zebrafish applications.
2016-05-01 | E-MTAB-4143 | biostudies-arrayexpress
Project description:Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10^6 reads corresponding to >10^4 insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors. Solid tumors in mice were generated by somatic transposon mutagenesis with a PiggyBac transposon system. Insertion sites of transposons in 11 tumors and 6 non-cancerous tail controls were determined by Illumina high-throughput sequencing. Insertions were determined both on 5' and 3' sides of the transposon (PB5 and PB3, respectively). Quantitative analysis of read numbers revealed enrichment of certain insertions in tumors, but not in controls, and these enriched insertions identify candidate cancer genes.
2013-08-28 | E-GEOD-46210 | biostudies-arrayexpress
Project description:Zinc Finger Nucleases (ZFNs) facilitate precise editing of DNA enabling targeted genomic modifications in vivo. ZFNs have been employed to obtain genetically modified plants and animals, and cell-based therapies utilizing ZFNs are undergoing clinical trials. However, many ZFNs display dose-dependent toxicity presumably due to the generation of undesired double stranded breaks at off-target sites within the genome. To evaluate the parameters influencing the functional specificity of ZFNs, we compared the in vivo activity of ZFN variants targeting the zebrafish kdrl locus, which display both high on-target activity and dose-dependent toxicity. We evaluated their functional specificity by assessing lesion frequency at 141 potential off-target sites within the zebrafish genome using Illumina sequencing. Only a minority of these off-target sites displayed significant lesion frequency with kdrl ZFNs. Furthermore, we find that active off-target sites appear to be defined by the thermodynamics of zinc finger-DNA recognition. Surprisingly, we observed that the zinc finger protein specificity and the choice of the engineered dimerization domain of the FokI nuclease could independently influence the fidelity of these ZFNs. The results of this study have implications for the assessment of likely off-target sites within a genome and point to both ZFP-dependent and –independent mechanisms of potential improvement for engineering ZFNs with higher levels of precision. Examined lesions at 141 off-target sites for various treatments of ZFNs and compare to the untreated sample stage 1: raw read but missing quality values stage 2: fastq files available from SRA
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Project description:The bacterial CRISPR-Cas9 system has been widely adapted for RNA-guided genome editing and gene regulation in diverse organisms yet its in vivo target specificity is poorly understood. Here we provide the first genome-wide binding maps of nuclease-deactivated Cas9 loaded with guide RNAs in mammalian cells. We find a 5-nucleotide seed region in the guide RNA targets Cas9 to thousands of sites in the genome. Chromatin accessibility limits binding to the other hundreds of thousands sites with matching seed sequences, and consequently 70% of off-target binding sites are associated with genes. U-rich seeds have low numbers of off-target sites limited by both low guide RNA abundance and scarcity of complimentary sites in accessible chromatin. Unexpectedly, off-target sites show little evidence of cleavage, supporting a two-state model reminiscent of eukaryotic RNAi machinery where a short seed match triggers binding but extensive pairing is required for cleavage. ChIP-seq of HA-dCas9 loaded with 4 sgRNAs (Phc1-sg1, Phc1-sg2, Nanog-sg2, and Nanog-sg3) in mouse, and 2 sgRNAs in human (EMX1-sg1 and EMX1-sg3)
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Project description:Most eukaryotic transcription factors (TFs) are part of large protein families, with members of the same family (i.e. paralogous TFs) recognizing similar DNA-binding motifs but performing different regulatory functions. Many TF paralogs are co-expressed in the cell, and thus can compete for target sites across the genome. Here, we show that direct competition for DNA binding between TF paralogs is a major determinant of their genomic binding patterns. Using yeast proteins Cbf1 and Pho4 as our model system, we designed a high-throughput quantitative assay to capture the genomic binding profiles of competing TFs in a cell-free system. Our data shows that Cbf1 and Pho4 greatly influence each other’s occupancy by competing for their common putative genomic binding sites. The competition is different at different genomic sites, as dictated by the TFs' expression levels and their divergence in DNA-binding specificity and affinity. Analyses of ChIP-seq data show that the biophysical rules that dictate the competitive TF binding patterns in vitro are also followed in vivo, in the complex cellular environment. Furthermore, the Cbf1-Pho4 competition for genomic sites, as characterized in vitro using our new assay, plays a critical role in the specific activation of their target genes in the cell. Overall, our study highlights the importance of direct TF-TF competition for genomic binding and gene regulation by TF paralogs, and proposes an approach for studying this competition in a quantitative and high-throughput manner.
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Project description:Early growth response gene 1 (EGR1) has been implicated in megakaryocyte differentiation induced by PMA (phorbol 12-myristate 13-acetate). The identification of direct EGR1 target genes in global scale is critical for our understanding of how EGR1 contributes to this process. In this study, we provide a global survey on the binding location of EGR1 in the K562 cell treated by PMA using chromatin immunoprecipitation and massively parallel sequencing (ChIP-Seq). K562 is a human erythroleukemia cell line, which is situated in the common progenitor stage of megakaryocytic and erythroid lineages of the hematopoietic stem cell differentiation and its normally following differentiation is blockaded. Upon exposure to PMA stimuli, K562 cell can be induced into megakaryocytic cell, which provides a model for the study of transcriptional control networks. Over 14 000 highly confident in vivo EGR1 binding sites were identified in PMA treated K562 cell. More than 70% of these genomic sites associated with EGR1 binding were located around annotated gene regions. This whole genome study on the EGR1 targets may help a better understanding of the EGR1 regulated genes and the downstream pathway in megakaryocyte differentiation. The in vivo binding locations of EGR1 in K562 cell treated with PMA (phorbol 12-myristate 13-acetate, 10 ng/ml for 2 hours) were identified using chromatin immunoprecipitation combing with massively parallel sequencing (ChIP-Seq) based on AB SOLiD System 2.0.
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