Shuttle vector pSP189
Ontology highlight
ABSTRACT:
PROVIDER: PRJDB13753 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| DRR385777_1.fastq.gz | Fastqsanger.gz | |||
| DRR385777_2.fastq.gz | Fastqsanger.gz | |||
| DRR385778_1.fastq.gz | Fastqsanger.gz | |||
| DRR385778_2.fastq.gz | Fastqsanger.gz | |||
| DRR385779_1.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 176 |
Similar Datasets
Project description:CRISPR tiling screens have advanced the identification and characterization of regulatory sequences but are limited by low resolution arising from the indirect readout of editing via guide RNA sequencing. This study introduces CRISPR-CLEAR, an end-to-end experimental assay and computational pipeline, which leverages targeted sequencing of CRISPR-introduced alleles at the endogenous target locus following dense base-editing mutagenesis. This approach enables the dissection of regulatory elements at nucleotide resolution, facilitating a direct assessment of genotype-phenotype effects.
2024-10-03 | GSE278581 | GEO
Project description:Safety assessment in retroviral vector-mediated gene therapy remains challenging. In clinical trials for different blood and immune disorders, insertional mutagenesis led to myeloid and lymphoid leukemia. We previously developed the In Vitro Immortalization Assay (IVIM) and Surrogate Assay for Genotoxicity Assessment (SAGA) for pre-clinical genotoxicity prediction of integrating vectors. Murine hematopoietic stem and progenitor cells (mHSPC) transduced with mutagenic vectors acquire a proliferation advantage under limiting dilution (IVIM) and activate stem cell- and cancer-related transcriptional programs (SAGA). However, both assays present an intrinsic myeloid bias due to culture conditions. To detect lymphoid mutants, we differentiated mHSPC to mature T cells and analyzed their phenotype, insertion site pattern, and gene expression changes after transduction with retroviral vectors. Mutagenic vectors induced a block in differentiation at an early progenitor stage (double-negative 2) compared to fully differentiated untransduced mock cultures. Arrested samples harbored high-risk insertions close to Lmo2, frequently observed in clinical trials with severe adverse events. Lymphoid insertional mutants displayed a unique gene expression signature identified by the machine learning algorithm of SAGA. The gene expression-based highly sensitive molecular readout will broaden our understanding of vector-induced oncogenicity and help in pre-clinical prediction of retroviral genotoxicity.
2023-09-21 | GSE241155 | GEO
Project description:Safety assessment in retroviral vector-mediated gene therapy remains challenging. In clinical trials for different blood and immune disorders, insertional mutagenesis led to myeloid and lymphoid leukemia. We previously developed the In Vitro Immortalization Assay (IVIM) and Surrogate Assay for Genotoxicity Assessment (SAGA) for pre-clinical genotoxicity prediction of integrating vectors. Murine hematopoietic stem and progenitor cells (mHSPC) transduced with mutagenic vectors acquire a proliferation advantage under limiting dilution (IVIM) and activate stem cell- and cancer-related transcriptional programs (SAGA). However, both assays present an intrinsic myeloid bias due to culture conditions. To detect lymphoid mutants, we differentiated mHSPC to mature T cells and analyzed their phenotype, insertion site pattern, and gene expression changes after transduction with retroviral vectors. Mutagenic vectors induced a block in differentiation at an early progenitor stage (double-negative 2) compared to fully differentiated untransduced mock cultures. Arrested samples harbored high-risk insertions close to Lmo2, frequently observed in clinical trials with severe adverse events. Lymphoid insertional mutants displayed a unique gene expression signature identified by the machine learning algorithm of SAGA. The gene expression-based highly sensitive molecular readout will broaden our understanding of vector-induced oncogenicity and help in pre-clinical prediction of retroviral genotoxicity.
2023-09-21 | GSE241146 | GEO
Project description:To assay Rnf149 function during early cardiopharyngeal specification, bulk RNAseq was performed to profile the FACS-purified cardiopharyngeal lineage cells isolated from20 hpf Ciona larvae with CRISPR/Cas9-mediated Rnf149 mutagenesis.
2022-03-07 | GSE171152 | GEO
Project description:To assay Tcf functions during early cardiopharyngeal specification, bulk RNAseq were performed to profile the FACS-purified cardiopharyngeal lineage cells isolated from 15 and 18hpf Ciona larvae with CRISPR/Cas9-mediated Tcf mutagenesis.
2018-12-04 | GSE123295 | GEO
Project description:A highly metastatic breast cancer cell line, 4T1, was used to generate stable Wnt5a expressing and vector only control cells. Cells were generated using lentivirus infection and selection with blasticidin. Expression of Wnt5a was confirmed using western blot. Cell behaviour was characterized. Wnt5a expressing cells exhibited reduced migration in a transwell assay and reduced metastasis in a tail vein injection assay. Growth was not significantly affected.
2013-06-26 | GSE41792 | GEO
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:This experiment aims at analyzing crossover distribution genome-wise, in the fission yeast. S. pombe strains PR109 (h- leu1-32 ura4-D18) and PR110 (h+ leu1-32 ura4-D18) were used for three successive rounds of mutagenesis with Ethylmethane Sulfonate Mutagenesis. Five independent clones of the first round of mutagenesis were at the root of two subsequent similar rounds of mutagenesis. Each clone used was checked for its ability to mate and sporulate. Eventually, five mutagenized clones from each of the PR109 and PR110 backgrounds were sequenced to identify de novo mutations and determine the optimal combinations of mutation patterns for recombination analyses.
2023-03-03 | E-MTAB-12514 | biostudies-arrayexpress
Project description:In this study we use massively parallel combinatorial mutagenesis, a kinetic selection assay, and machine learning to better understand the nucleation reaction of amyloid beta (Aꞵ42), the protein that aggregates as a hallmark of Alzheimer’s disease (AD) and is mutated to cause familial AD.
2024-07-26 | GSE269461 | GEO