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Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency

ABSTRACT: Although many long non-coding RNAs (lncRNAs) exhibit lineage-specific expression, the vast majority remain functionally uncharacterized in the context of development. Here, we report the first described human embryonic stem cell (hESC) lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers, facilitating the modulation of lncRNA expression during early development. We performed an unbiased, genome-wide CRISPRi screen targeting thousands of lncRNA loci expressed during endoderm differentiation. While dozens of lncRNA loci were required for proper differentiation, most differentially expressed lncRNAs were not, supporting the necessity for functional screening instead of relying solely on gene expression analyses. In parallel, we developed a clustering approach to infer mechanisms of action of lncRNA hits based on a variety of genomic features. We subsequently identified and validated FOXD3-AS1 as a functional lncRNA essential for pluripotency and differentiation. Taken together, the cell lines and methodology described herein can be adapted to discover and characterize novel regulators of differentiation into any lineage.

SUBMITTER: Haswell J

PROVIDER: S-EPMC8565776 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency

Project description:Systematic identification of factors that regulate human embryonic stem cell (hESC) differentiation can provide valuable insights into early development and disease. However, effective modulation of gene expression during differentiation has proven technically challenging. Here, we report the first described hESC lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers. We subsequently performed an unbiased, genome-wide CRISPRi screen targeting long non-coding RNAs during endoderm differentiation. From our screen, we identified known factors, as well as novel lncRNAs required for differentiation. Surprisingly, lncRNA expression was not predictive of enrichment scores, supporting the necessity for functional screening instead of relying on gene expression analysis. Further downstream analysis of two hits, RP11-222K16.2 and FOXD3-AS1, revealed two lncRNAs essential for differentiation and pluripotency, respectively. Taken together, the cell lines and screen methodology described herein can be adapted to discover novel regulators of differentiation into any lineage.

2020-03-01 | GSE137584 | GEO

Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency

Project description:Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency

| PRJNA565971 | ENA

A Genome-Wide CRISPR Screen Identifies Factors Regulating Pluripotency Exit in Mouse Embryonic Stem Cells.

Project description:Pluripotency maintenance and exit in embryonic stem cells is a focal topic in stem cell biology. However, the effects of screening under very stringent culture conditions (e.g., differentiation medium, no leukemia inhibitory factor, no chemical inhibitors such as PD0325901 and CHIR99021, and no feeder cells) and of prolonging culture for key factors that regulate pluripotency exit, have not yet been reported. Here, we used a genome-wide CRISPR library to perform such a screen in mouse embryonic stem cells. Naïve NANOG-GFP mESCs were first transfected with a mouse genome-wide CRISPR knockout library to obtain a mutant mESCs library, followed by screening for two months in a strict N2B27 differentiation medium. The clones that survived our stringent screening were analyzed to identify the inserted sgRNAs. In addition to identifying the enriched genes that were reported in previous studies (Socs3, Tsc1, Trp53, Nf2, Tcf7l1, Csnk1a1, and Dhx30), we found 17 unreported genes, among which Zfp771 and Olfr769 appeared to be involved in pluripotency exit. Furthermore, Zfp771 knockout ESCs showed a differentiation delay in embryonic chimera experiments, indicating Zfp771 played an important role in pluripotency exit. Our results show that stringent screening with the CRISPR library can reveal key regulators of pluripotency exit.

| S-EPMC9331787 | biostudies-literature

Genome-Wide CRISPR Screen Identifies Host Factors Required by Toxoplasma gondii Infection.

Project description:Toxoplasma gondii are obligate intracellular protoza, and due to their small genome and limited encoded proteins, they have to exploit host factors for entry, replication, and dissemination. Such host factors can be defined as host dependency factors (HDFs). Though HDFs are inessential for cell viability, they are critical for pathogen infection, and potential ideal targets for therapeutic intervention. However, information about these HDFs required by T. gondii infection is highly deficient. In this study, the genes of human foreskin fibroblast (HFF) cells were comprehensively edited using the lentiviral CRISPR-Cas9-sgRNA library, and then the lentivirus-treated cells were infected with T. gondii at multiplication of infection 1 (MOI = 1) for 10 days to identify HDFs essential for T. gondii infection. The survival cells were harvested and sent for sgRNA sequencing. The sgRNA sequence matched genes or miRNAs were potential HDFs. Some cells in the lentivirus-treated group could survive longer than those in the untreated control group after T. gondii infection. From a pool of 19,050 human genes and 1,864 human pri-miRNAs, 1,193 potential HDFs were identified, including 1,183 genes and 10 pri-miRNAs (corresponding with 17 mature miRNAs). Among them, seven genes and five mature miRNAs were validated with siRNAs, miRNA inhibitors, and mimics, respectively. Bioinformatics analysis revealed that, among the 1,183 genes, 53 potential HDFs were associated with regulation of host actin cytoskeleton and 23 potential HDFs coded immune negative regulators. This result indicated that actin dynamics were indispensable for T. gondii infection, and some host immune negative regulators may be involved in disarming host defenses. Our findings contribute to the current limited knowledge about host factors required by T. gondii infection and provide us with new targets for medication therapy and vaccine exploitation.

| S-EPMC6987080 | biostudies-literature

Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation.

Project description:Macroautophagy is an intracellular degradation process that requires multiple autophagy-related (ATG) genes. In this study, we performed a genome-wide screen using the autophagic flux reporter GFP-LC3-RFP and identified TMEM41B as a novel ATG gene. TMEM41B is a multispanning membrane protein localized in the endoplasmic reticulum (ER). It has a conserved domain also found in vacuole membrane protein 1 (VMP1), another ER multispanning membrane protein essential for autophagy, yeast Tvp38, and the bacterial DedA family of putative half-transporters. Deletion of TMEM41B blocked the formation of autophagosomes at an early step, causing accumulation of ATG proteins and small vesicles but not elongating autophagosome-like structures. Furthermore, lipid droplets accumulated in TMEM41B-knockout (KO) cells. The phenotype of TMEM41B-KO cells resembled those of VMP1-KO cells. Indeed, TMEM41B and VMP1 formed a complex in vivo and in vitro, and overexpression of VMP1 restored autophagic flux in TMEM41B-KO cells. These results suggest that TMEM41B and VMP1 function together at an early step of autophagosome formation.

| S-EPMC6219718 | biostudies-literature

A genome-wide RNA interference screen identifies caspase 4 as a factor required for tumor necrosis factor alpha signaling.

Project description:Tumor necrosis factor alpha (TNF-α) is a potent inflammatory cytokine secreted upon cellular stress as well as immunological stimuli and is implicated in the pathology of inflammatory diseases and cancer. The therapeutic potential of modifying TNF-α pathway activity has been realized in several diseases, and antagonists of TNF-α have reached clinical applications. While much progress in the understanding of signaling downstream of the TNF-α receptor complex has been made, the compendium of factors required for signal transduction is still not complete. In order to find novel regulators of proinflammatory signaling induced by TNF-α, we conducted a genome-wide small interfering RNA screen in human cells. We identified several new candidate modulators of TNF-α signaling, which were confirmed in independent experiments. Specifically, we show that caspase 4 is required for the induction of NF-κB activity, while it appears to be dispensable for the activation of the Jun N-terminal protein kinase signaling branch. Taken together, our experiments identify caspase 4 as a novel regulator of TNF-α-induced NF-κB signaling that is required for the activation of IκB kinase. We further provide the genome-wide RNA interference data set as a compendium in a format compliant with minimum information about an interfering RNA experiment (MAIRE).

| S-EPMC3422000 | biostudies-literature

Genome-wide CRISPR interference screen identifies long non-coding RNA DARS1-AS1 as a novel regulator of glioblastoma tumorigenesis.

Project description:There are over 15,000 long (>200 nucleotides) noncoding RNA (lncRNAs) genes in the human genome, but only few of them has been functionally characterized. Emerging evidence has shown that long non-coding RNAs (lncRNAs) modulate diverse biological processes and mediate tumor-promoting/suppressing effects and serve as independent diagnostic/prognostic biomarkers in cancer. However, the role and function mechanism of the lncRNA/RBP-mediated regulatory circuits in determining the functional characteristics of GSCs underlying GBM pathogenesis remains poorly understood. To fill this gap, we combined CRISPR (clustered regularly interspaced short palindromic repeat) interference (CRISPRi), with large-scale computational analysis of The Cancer Genome Atlas (TCGA) to systematically identify the functional lncRNAs with clinically relevant expression in GBM.

2023-08-09 | GSE213046 | GEO

Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation.

Project description:Protein misfolding and the formation of aggregates are increasingly recognized components of the pathology of human genetic disease and hallmarks of many neurodegenerative disorders. As exemplified by polyglutamine diseases, the propensity for protein misfolding is associated with the length of polyglutamine expansions and age-dependent changes in protein-folding homeostasis, suggesting a critical role for a protein homeostatic buffer. To identify the complement of protein factors that protects cells against the formation of protein aggregates, we tested transgenic Caenorhabditis elegans strains expressing polyglutamine expansion yellow fluorescent protein fusion proteins at the threshold length associated with the age-dependent appearance of protein aggregation. We used genome-wide RNA interference to identify genes that, when suppressed, resulted in the premature appearance of protein aggregates. Our screen identified 186 genes corresponding to five principal classes of polyglutamine regulators: genes involved in RNA metabolism, protein synthesis, protein folding, and protein degradation; and those involved in protein trafficking. We propose that each of these classes represents a molecular machine collectively comprising the protein homeostatic buffer that responds to the expression of damaged proteins to prevent their misfolding and aggregation.

| S-EPMC404057 | biostudies-literature

A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes.

Project description:Apicomplexan parasites are leading causes of human and livestock diseases such as malaria and toxoplasmosis, yet most of their genes remain uncharacterized. Here, we present the first genome-wide genetic screen of an apicomplexan. We adapted CRISPR/Cas9 to assess the contribution of each gene from the parasite Toxoplasma gondii during infection of human fibroblasts. Our analysis defines ?200 previously uncharacterized, fitness-conferring genes unique to the phylum, from which 16 were investigated, revealing essential functions during infection of human cells. Secondary screens identify as an invasion factor the claudin-like apicomplexan microneme protein (CLAMP), which resembles mammalian tight-junction proteins and localizes to secretory organelles, making it critical to the initiation of infection. CLAMP is present throughout sequenced apicomplexan genomes and is essential during the asexual stages of the malaria parasite Plasmodium falciparum. These results provide broad-based functional information on T. gondii genes and will facilitate future approaches to expand the horizon of antiparasitic interventions.

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A CRISPR screen identifies a pathway required for paraquat-induced cell death.

Project description:Paraquat, a herbicide linked to Parkinson's disease, generates reactive oxygen species (ROS), which causes cell death. Because the source of paraquat-induced ROS production remains unknown, we conducted a CRISPR-based positive-selection screen to identify metabolic genes essential for paraquat-induced cell death. Our screen uncovered three genes, POR (cytochrome P450 oxidoreductase), ATP7A (copper transporter), and SLC45A4 (sucrose transporter), required for paraquat-induced cell death. Furthermore, our results revealed POR as the source of paraquat-induced ROS production. Thus, our study highlights the use of functional genomic screens for uncovering redox biology.

| S-EPMC5698099 | biostudies-literature

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