Similar Datasets
Project description:Systematic Identification of Transcription Factors Driving Cell Fate Conversion using CRISPR Activation Screens
| PRJNA487076 | ENA
Project description:Comprehensive identification of factors that can specify neuronal fate could provide valuable insights into lineage specification and reprogramming, but systematic interrogation of transcription factors, and their interactions with each other, has proven technically challenging. We developed a CRISPR activation (CRISPRa) approach to systematically identify regulators of neuronal-fate specification. We activated expression of all endogenous transcription factors and other regulators via a pooled CRISPRa screen in embryonic stem cells, revealing genes including epigenetic regulators such as Ezh2 that can induce neuronal fate. Systematic CRISPR-based activation of factor pairs allowed us to generate a genetic interaction map for neuronal differentiation, with confirmation of top individual and combinatorial hits as bona fide inducers of neuronal fate. Several factor pairs could directly reprogram fibroblasts into neurons, which shared similar transcriptional programs with endogenous neurons. This study provides an unbiased discovery approach for systematic identification of genes that drive cell-fate acquisition.
| S-EPMC6214761 | biostudies-literature
Project description:Regulation of cytokine production in stimulated T cells can be disrupted in autoimmunity, immunodeficiencies, and cancer. Systematic discovery of stimulation-dependent cytokine regulators requires both loss-of-function and gain-of-function studies, which have been challenging in primary human cells. We now report genome-wide CRISPR activation (CRISPRa) and interference (CRISPRi) screens in primary human T cells to identify gene networks controlling interleukin 2 and interferon gamma production. Arrayed CRISPRa confirmed key hits and enabled multiplexed secretome characterization, revealing reshaped cytokine responses. Coupling CRISPRa screening with single-cell RNA-seq enabled deep molecular characterization of screen hits, revealing how perturbations tuned T cell activation and promoted cell states characterized by distinct cytokine expression profiles. Together, these screens reveal genes that reprogram critical immune cell functions, which could inform the design of immunotherapies.
2021-05-13 | GSE174255 | GEO
Project description:Regulation of cytokine production in stimulated T cells can be disrupted in autoimmunity, immunodeficiencies, and cancer. Systematic discovery of stimulation-dependent cytokine regulators requires both loss-of-function and gain-of-function studies, which have been challenging in primary human cells. We now report genome-wide CRISPR activation (CRISPRa) and interference (CRISPRi) screens in primary human T cells to identify gene networks controlling interleukin 2 and interferon gamma production. Arrayed CRISPRa confirmed key hits and enabled multiplexed secretome characterization, revealing reshaped cytokine responses. Coupling CRISPRa screening with single-cell RNA-seq enabled deep molecular characterization of screen hits, revealing how perturbations tuned T cell activation and promoted cell states characterized by distinct cytokine expression profiles. Together, these screens reveal genes that reprogram critical immune cell functions, which could inform the design of immunotherapies.
2022-02-04 | GSE190846 | GEO
Project description:All bulk CRISPR based screens CD2 and B2M CRISPRi tiling screens (primary human CD8 T cells), IL2RA CRISPRa tiling screens (Jurkats), CRISPRi/a TF screens (primary human CD8 T cells), and CRISPR TFome KO (primary human T cells)
2023-08-31 | GSE241933 | GEO
Project description:CD8+ cytotoxic T cells (CTLs) orchestrate antitumour immunity and exhibit inherent heterogeneity1,2, with precursor exhausted T (Tpex) cells but not terminally exhausted T (Tex) cells capable of responding to existing immunotherapies3-7. The gene regulatory network that underlies CTL differentiation and whether Tex cell responses can be functionally reinvigorated are incompletely understood. Here we systematically mapped causal gene regulatory networks using single-cell CRISPR screens in vivo and discovered checkpoints for CTL differentiation. First, the exit from quiescence of Tpex cells initiated successive differentiation into intermediate Tex cells. This process is differentially regulated by IKAROS and ETS1, the deficiencies of which dampened and increased mTORC1-associated metabolic activities, respectively. IKAROS-deficient cells accumulated as a metabolically quiescent Tpex cell population with limited differentiation potential following immune checkpoint blockade (ICB). Conversely, targeting ETS1 improved antitumour immunity and ICB efficacy by boosting differentiation of Tpex to intermediate Tex cells and metabolic rewiring. Mechanistically, TCF-1 and BATF are the targets for IKAROS and ETS1, respectively. Second, the RBPJ-IRF1 axis promoted differentiation of intermediate Tex to terminal Tex cells. Accordingly, targeting RBPJ enhanced functional and epigenetic reprogramming of Tex cells towards the proliferative state and improved therapeutic effects and ICB efficacy. Collectively, our study reveals that promoting the exit from quiescence of Tpex cells and enriching the proliferative Tex cell state act as key modalities for antitumour effects and provides a systemic framework to integrate cell fate regulomes and reprogrammable functional determinants for cancer immunity.
| S-EPMC10700132 | biostudies-literature
Project description:Regulation of cytokine production in stimulated T cells can be disrupted in autoimmunity, immunodeficiencies, and cancer. Systematic discovery of stimulation-dependent cytokine regulators requires both loss-of-function and gain-of-function studies, which have been challenging in primary human cells. We now report genome-wide CRISPR activation (CRISPRa) and interference (CRISPRi) screens in primary human T cells to identify gene networks controlling interleukin 2 and interferon gamma production. Arrayed CRISPRa confirmed key hits and enabled multiplexed secretome characterization, revealing reshaped cytokine responses. Coupling CRISPRa screening with single-cell RNA-seq enabled deep molecular characterization of screen hits, revealing how perturbations tuned T cell activation and promoted cell states characterized by distinct cytokine expression profiles. Together, these screens reveal genes that reprogram critical immune cell functions, which could inform the design of immunotherapies.
2021-05-13 | GSE174284 | GEO
Project description:Regulation of cytokine production in stimulated T cells can be disrupted in autoimmunity, immunodeficiencies, and cancer. Systematic discovery of stimulation-dependent cytokine regulators requires both loss-of-function and gain-of-function studies, which have been challenging in primary human cells. We now report genome-wide CRISPR activation (CRISPRa) and interference (CRISPRi) screens in primary human T cells to identify gene networks controlling interleukin 2 and interferon gamma production. Arrayed CRISPRa confirmed key hits and enabled multiplexed secretome characterization, revealing reshaped cytokine responses. Coupling CRISPRa screening with single-cell RNA-seq enabled deep molecular characterization of screen hits, revealing how perturbations tuned T cell activation and promoted cell states characterized by distinct cytokine expression profiles. Together, these screens reveal genes that reprogram critical immune cell functions, which could inform the design of immunotherapies.
2022-02-04 | GSE190604 | GEO
Project description:Immortalised HaCaT keratinocytes were transduced with Cas9 and the CRISPR-KO v1.1 genome-wide gRNA library. The gRNA library was prepared from genomic DNA isolated 14 days post library transduction. gRNA representation will be compared to the original CRISPR-KO v1.1 library to reveal genes essential for HaCaT survival and growth.
| EGAS00001002714 | EGA
Project description:The Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide. No approved antivirals are available to date and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates. Hepatitis C virus (HCV) also remains a major medical problem, with 160 million chronically infected patients worldwide and only expensive treatments available. Despite distinct differences in their pathogenesis and modes of transmission, the two viruses share common replication strategies. A detailed understanding of the host functions that determine viral infection is lacking. Here we use a pooled CRISPR genetic screening strategy to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified endoplasmic-reticulum (ER)-associated multi-protein complexes involved in signal sequence recognition, N-linked glycosylation and ER-associated degradation. DENV replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as the crucial step requiring the OST complex. Moreover, we show that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for infection by other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects. By contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA-binding proteins and enzymes involved in metabolism. We found an unexpected link between intracellular flavin adenine dinucleotide (FAD) levels and HCV replication. This study shows notable divergence in host-depenency factors between DENV and HCV, and illuminates new host targets for antiviral therapy.
| S-EPMC4964798 | biostudies-literature