Ontology highlight
ABSTRACT:
PROVIDER: PRJNA889818 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Massively parallel base editing to map variant effects in human hematopoiesis
| PRJNA889434 | ENA
Project description:Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow rich phenotyping through single-cell RNA sequencing readouts, and separately, characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.
2023-05-02 | GSE215253 | GEO
Project description:The majority of variants associated with complex traits and common diseases identified by genome-wide association studies (GWAS) map to noncoding regions of the genome with unknown regulatory effects. By leveraging ancestrally diverse biobank-scale GWAS data, massively parallel CRISPR screens and single cell transcriptomic and proteomic sequencing, we discovered target genes of noncoding variants for blood trait loci. For 91 GWAS loci, we identified 124 target genes in cis, which were often — but not always — the closest genes to the fine-mapped variant. Using precise variant insertion via base editing, we connect specific variants with gene expression changes. We also identified trans-effect networks of noncoding loci when cis target genes encoded transcription factors or microRNAs, such as GFI1B and miR-142. Trans-regulatory networks were themselves enriched for fine-mapped GWAS variants, demonstrating polygenic contributions to complex traits. Co-expression clustering of GFI1B trans-target genes identifies gene networks specific to different blood cell fates and differentiation stages. This platform will enable massively parallel assays to characterize the target genes and mechanisms of human noncoding variants in both cis and trans.
2021-04-08 | GSE171452 | GEO
Project description:Massively parallel assessment of human variants with base editor screens
| PRJNA638656 | ENA
Project description:We performed a massively parallel screen in human HAP1 cells to identify loss-of-function missense variants in the key DNA mismatch repair factor MSH2. Resulting variant loss-of-function (LOF) scores are strongly concordant with previous functional evidence and available variant classification.
2020-12-23 | GSE162130 | GEO
Project description:Activating Transcription Factor 4 (ATF4) is a transcription factor that regulates cellular responses to nutrient deficiency, endoplasmic reticulum stress and oxidative stress. At the organism level, it is implicated in processes such as hematopoiesis, skeletogenesis, eye development, memory, muscle atrophy, and carbohydrate and lipid metabolism. Here, we carried out a massively parallel reporter assay (MPRA) to identify allelic regulatory effects of human genetic variants that reside in ATF4 binding sites identified in ChIP-Seq experiments.
2023-12-01 | GSE225216 | GEO
Project description:An ability to map the global interactions of a chemical entity with chromatin genome-wide could provide new insights into the mechanisms by which a small molecule perturbs cellular functions. we developed a method that uses chemical derivatives and massively parallel DNA sequencing (Chem-Seq) to identify the sites bound by small chemical molecules throughout the human genome. We developed in vivo and in vitro Chem-Seq protocols with a biotinylated derivative of small molecules. In the in vivo protocol, Cells were first treated with biotinylated ligand and cross-linked with formaldehyde at the same time. Cells were then lysed, sonicated to shear the DNA, and streptavidin beads were used to isolate biotinylated ligand and associated chromatin fragments. We then used massively parallel sequencing to identify the enriched DNA fragments, and mapped these sequences to the genome. In in vitrol protocol, MM1.S cells were fixed and the derived sonicated lysate incubated with biotinylated drug to enrich for bound chromatin regions in vitro. We then used massively parallel sequencing to identify the enriched DNA fragments, and mapped these sequences to the genome.
2013-05-31 | E-GEOD-44098 | biostudies-arrayexpress
Project description:Background: The identification of causal variants responsible for disease associations from genome-wide association studies (GWAS) facilitates functional understanding of the disease mechanisms implicated by GWAS. One of the earliest GWAS associations to COPD spans an intragenic region within FAM13A, but the causal variants at this loci have not yet been identified. Massively parallel reporter assays (MPRA) can be used to prioritize functional regulatory variants in a high-throughput manner. Methods: We used an integrated approach using fine-mapping in over 10,000 subjects from COPD GWAS studies, two MPRA experiments, traditional reporter assays, chromatin conformation capture, and CRISPR-based gene editing to characterize COPD-associated regulatory variants in FAM13A in human bronchial epithelial cell lines. Results: Conditional genetic association and fine mapping analyses identified two independent COPD association signals in FAM13A. MPRA identified 45 common functional regulatory variants, and six COPD-associated putative functional variants were prioritized for further functional investigation. Three variants demonstrated significant activity in traditional reporter assays, and one variant, rs2013701, was selected for further testing in the endogenous genomic context based on a direction of effect consistent with postulated mechanisms of FAM13A-mediated COPD susceptibility. CRISPR-based genome editing for this variant confirmed allele-specific effects on FAM13A expression and altered rates of cellular proliferation, providing multiple levels of functional characterization for this COPD-associated variant. Conclusions: Comprehensive screening for regulatory variants near FAM13A identified the presence of extensive functional regulatory variation within a 250kb window of FAM13A in HBECs. Focused functional evaluation of the COPD-associated functional variants in LD with the two independent association signals in this region prioritized the common variant rs2013701, for which multiple parallel lines of functional evidence confirm allelic effects on FAM13A regulation.
2019-01-19 | GSE109452 | GEO
Project description:Base Editing has been touted the most intelligent and precise application of the CRISPR platform so far, merging the simplicity of RNA-guided nucleases with deaminases that allow for the programmable generation of single base substitutions - without introduction of double-strand breaks. Even though the two-component system has been expected to cause off-target substitutions, studies involving cytosine base editors (CBEs) showed that in most cases, relatively few single base off-targets could be detected on DNA. We introduce the concept of multi-dimensional off-targeting, presenting an extensive amount of RNA cytidines being edited by DNA base editors. Epitranscriptomic off-target effects affected different cell lines and were independent of the guide RNAs used, suggesting Cas9-independent activity of the cytidine deaminase rAPOBEC1 on single-stranded RNA. With the help of protein engineering, we developed CBE variants with massively reduced inadvertent mutation of RNA that preserve and enhance DNA base editing capabilities.
2019-04-01 | GSE129131 | GEO