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

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

PROVIDER: PRJNA565971 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10132970_1.fastq.gz	Fastqsanger.gz
	SRR10132970_2.fastq.gz	Fastqsanger.gz
	SRR10132971_1.fastq.gz	Fastqsanger.gz
	SRR10132971_2.fastq.gz	Fastqsanger.gz
	SRR10132972_1.fastq.gz	Fastqsanger.gz

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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: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.

| S-EPMC8565776 | biostudies-literature

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

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

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Intergenic non-coding transcription utilises an RNA-based mechanism to drive transcriptional interference

Project description:Recent studies have revealed that eukaryotic genomes are pervasively transcribed by RNA polymerase II, producing a plethora of non-coding RNAs which frequently overlap protein-coding genes. Despite the fact that overlapping transcription is well known to repress transcription initiation from downstream promoters, the mechanism behind this phenomenon has remained elusive. It was proposed that transcriptional interference relies on the act of transcription rather than the RNA itself to block access of the transcription machinery to downstream promoters. Here, we demonstrate that an RNA and chromatin-dependent mechanism is responsible for transcriptional interference in fission yeast. This relies on co-transcriptional recruitment of the histone deacetylase Clr3 to nascent non-coding RNA, leading to formation of hypoacetylated chromatin and repression of the downstream protein-coding gene. Our findings highlight the unexpected requirement for RNA as well as trans-acting protein factors in mediating the repressive effects imposed on gene expression through overlapping non-coding transcription.

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CRISPRi Screen of Long Non-coding RNAs Identifies LINC03045 Regulating Glioblastoma Invasion

Project description:Introduction: Glioblastoma (GBM) invasion studies have focused on coding genes, while few studies evaluate long non-coding RNAs (lncRNAs), transcripts without protein-coding potential, for role in GBM invasion. We leveraged CRISPR-interference (CRISPRi) to evaluate invasive function of GBM-associated lncRNAs in an unbiased functional screen, characterizing and exploring the mechanism of identified candidates. Methods: We implemented a CRISPRi lncRNA loss-of-function screen evaluating association of lncRNA knockdown (KD) with invasion capacity in Matrigel. Top screen candidates were validated using CRISPRi and oligonucleotide(ASO)-mediated knockdown in three tumor lines. Clinical relevance of candidates was assessed via The Cancer Genome Atlas(TCGA) and Genotype-Tissue Expression(GTEx) survival analysis. Mediators of lncRNA effect were identified via differential expression analysis following lncRNA KD and assessed for tumor invasion using knockdown and rescue experiments. Results: Forty-eight lncRNAs were significantly associated with 33-83% decrease in invasion (p<0.01) upon knockdown. The top candidate, LINC03045, identified from effect size and p-value, demonstrated 82.7% decrease in tumor cell invasion upon knockdown, while LINC03045 expression was significantly associated with patient survival and tumor grade(p<0.0001). RNAseq analysis of LINC03045 knockdown revealed that WASF3, previously implicated in tumor invasion studies, was highly correlated with lncRNA expression, while WASF3 KD was associated with significant decrease in invasion. Finally, WASF3 overexpression demonstrated rescue of invasive function lost with LINC03045 KD. Conclusion: CRISPRi screening identified LINC03045, a previously unannotated lncRNA, as critical to GBM invasion. Gene expression is significantly associated with tumor grade and survival. RNA-seq and mechanistic studies suggest that this novel lncRNA may regulate invasion via WASF3.

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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.

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STAT3-dependent long non-coding RNA Lncenc1 contributes to ES cells pluripotency

Project description:The self-renewal of Embryonic Stem Cells (ESCs) relies on a complex interplay of pluripotency transcription factors and their targets. LIF-activated STAT3 is a key player in regulating stemness by a number of mechanisms, including the transcriptional induction of pluripotency factors and the maintenance of a stern–like epigenetic landscape. However, not much is known about how STAT3 may be involved in the regulation of stem-cell specific non coding RNAs, an important player in the balance between pluripotency and differentiation. Here we identify in mouse ESCs a STAT3-dependent long non coding RNA, Lncenc1, which is required for pluripotency maintenance and mostly localizes to the cytoplasm. Lncenc1 acts as a positive feedback regulator of the LIF-STAT3 axis by competing for the binding of microRNA-128 to the 3’UTR of the core pluripotency factor Klf4 mRNA, enhancing its expression. Our results unveil a novel mechanism for LIF-STAT3-mediated pluripotency.

2023-09-27 | GSE221670 | GEO

CRISPR Screen: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

Project description:To investigate the genes and pathways involved in X-chromosome reactivation and pluripotency acquisition, we generated a whole-genome CRISPR knockout screen during female NPC reprogramming into iPSCs. We analyzed the gRNA abundance in NPCs and three different reprogramming populations: non-pluripotent, early pluripotent and late pluripotent. Pair-wise comparisons of gRNA frequencies in different populations were done to identify genes and pathways involved in cell survival in reprogramming conditions, colony formation, early pluripotency acquisition and late pluripotency/X-chromosome reactivation.

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Seb1 is recruited co-transcriptionally to non-coding regions to drive transcriptional interference

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