High throughput CRISPR perturbation screens identify epigenetic regulators impacting primordial germ cell development [RNA-seq]
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ABSTRACT:
PROVIDER: PRJNA1080592 | ENA |
REPOSITORIES: ENA
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Project description:High throughput CRISPR perturbation screens identify epigenetic regulators impacting primordial germ cell development [CRISPRi]
| PRJNA1080593 | ENA
Project description:There is concern that environmental factors underlie reported declines in fertility and reproductive parameters such as the number and quality of sperm and eggs. One possible mechanism is the perturbation of epigenetic landscapes in the germline. To explore this possibility, we utilized a highly scalable in vitro platform for the differentiation of embryonic stem cells (ESCs) into primordial germ cell-like cells (PGCLCs) to conduct a CRISPRi screen of epigenetic-related genes to identify those that specifically perturb the efficiency of PGCLC formation. This platform leveraged an ESC line containing 4 inducible transcription factors (Prdm1, Prdm14, Tfap2c, and Nanog) and a PGC reporter (Stella-eGFP) that enabled highly efficient, cytokine-free differentiation and quantification en masse. A total of 701 genes with distinct sgRNAs were screened, and 53 genes were identified that decreased the efficiency of PGCLC formation. NCOR2, a transcriptional repressor that acts via recruitment of Class I and Class IIa histone deacetylases (HDACs) to gene targets, was particularly potent in suppressing PGCLC differentiation. Consistent with evidence that histone deacetylation is crucial for germline differentiation, we found that HDAC inhibitors (HDACi), including the anti-convulsive drug valproic acid (VPA) and widely-available dietary supplement sodium butyrate (SB), also suppressed ESC>PGCLC differentiation. Transcriptome analyses of HDACi-treated, differentiating ESC>PGCLC cultures revealed suppression of germline-associated pathways and enhancement of somatic pathways. Additionally, exposure of developing mouse embryos to SB or VPA caused hypospermatogenesis. This work demonstrates the feasibility of conducting large scale functional screens of genes, chemical agents and possibly genetic variants that impact germline development and epigenome.
2025-09-04 | GSE259289 | GEO
Project description:There is concern that environmental factors underlie reported declines in fertility and reproductive parameters such as the number and quality of sperm and eggs. One possible mechanism is the perturbation of epigenetic landscapes in the germline. To explore this possibility, we utilized a highly scalable in vitro platform for the differentiation of embryonic stem cells (ESCs) into primordial germ cell-like cells (PGCLCs) to conduct a CRISPRi screen of epigenetic-related genes to identify those that specifically perturb the efficiency of PGCLC formation. This platform leveraged an ESC line containing 4 inducible transcription factors (Prdm1, Prdm14, Tfap2c, and Nanog) and a PGC reporter (Stella-eGFP) that enabled highly efficient, cytokine-free differentiation and quantification en masse. A total of 701 genes with distinct sgRNAs were screened, and 53 genes were identified that decreased the efficiency of PGCLC formation. NCOR2, a transcriptional repressor that acts via recruitment of Class I and Class IIa histone deacetylases (HDACs) to gene targets, was particularly potent in suppressing PGCLC differentiation. Consistent with evidence that histone deacetylation is crucial for germline differentiation, we found that HDAC inhibitors (HDACi), including the anti-convulsive drug valproic acid (VPA) and widely-available dietary supplement sodium butyrate (SB), also suppressed ESC>PGCLC differentiation. Transcriptome analyses of HDACi-treated, differentiating ESC>PGCLC cultures revealed suppression of germline-associated pathways and enhancement of somatic pathways. Additionally, exposure of developing mouse embryos to SB or VPA caused hypospermatogenesis. This work demonstrates the feasibility of conducting large scale functional screens of genes, chemical agents and possibly genetic variants that impact germline development and epigenome.
2025-09-04 | GSE259290 | GEO
Project description:Optimised, frameshift-driven design for single-cell CRISPR perturbation screens
| PRJNA851065 | ENA
Project description:Genome-Scale Exon Perturbation Screens Uncover Critical Exons for Cell Fitness
| PRJNA1022486 | ENA
Project description:As germ cell precursor, primordial germ cells (PGCs) are widely used in transgenic animal production, regenerative medicine and other fields. However, the regulation mechanism of chicken PGCs is not incomplete, which leads to the insufficient amount of chicken PGCs obtained in vitro, which seriously affects the specific application of PGCs. During PGC formation (differentiation from ESCs to PGCs), some proteins have inconsistent changes in transcription level and protein abundance. Mediating proteasome degradation is one of the most important roles of protein ubiquitination, and enrichment analysis of transcriptome and proteome also suggests an important role of ubiquitination in the process of PGCs. In order to explore the important functions and potential targets of ubiquitination, we collected chicken ESCs and PGCs cells for label free ubiquitomics analysis. This study preliminarily analyzed how ubiquitination regulates the formation of chicken PGCs, providing a theoretical basis for the subsequent research and specific application of PGCs.
2025-09-08 | PXD052543 | Pride
Project description:Transgenic StellaGFP ESCs were used to derive primordial germ cells during embryoid body (EB) differentiation, and microarry analysis used to compared FACS sorted Stella-positive cells of day 7 Ebs with the parental ESCs and Stella-negative cells of day 7 Ebs. Keywords: in vitro, primordial germ cells, embryonic stem cells, stella
2009-06-22 | GSE7948 | GEO
Project description:Genome-Scale Exon Perturbation Screens Uncover Critical Exons for Cell Fitness [CRISPR]
| PRJNA1022426 | ENA
Project description:Primordial germ cells (PGCs) are the founder cells of the germline. The ability to generate PGC-like cells (PGCLCs) from pluripotent stem cells has advanced our knowledge of gametogenesis and holds promise for developing infertility treatments. However, generating an ample supply of PGCLCs for demanding applications such as high-throughput genetic screens has been a limitation. Here, we demonstrated that simultaneous overexpressing 4 transcriptional factors – Nanog and three PGC master regulators Prdm1, Prdm14 and Tfap2c - in suspended mouse epiblast like cells (EpiLCs) and formative embryonic stem cells (ESCs) results in efficient and cost-effective production of PGCLCs. The overexpression of Nanog enhances the PGC regulatory network and suppresses differentiation of somatic lineages, enabling a significant improvement in the efficiency of PGCLC production. Transcriptomic analysis reveals that differentiated PGCLCs exhibit similarities to in vivo PGCs and are more advanced compared to cytokine-induced PGCLCs. These differentiated PGCLCs could be sustained over prolonged periods of culture, and could differentiate into spermatogonium-like cells in vitro. Importantly, the ability to produce PGCLC differentiation at scale enables biochemical and functional genomic screens to dissect mechanisms of germ cell development and infertility.
2025-09-04 | GSE256104 | GEO
Project description:CRISPR-Cas technology has transformed functional genomics, yet understanding of how individual exons differentially shape cellular phenotypes remains limited. Here, we optimized and conducted massively parallel exon deletion and splice-site mutation screens in human cell lines to identify exons that regulate cellular fitness. Fitness-promoting exons are prevalent in essential and highly expressed genes and commonly overlap with protein domains and interaction interfaces. Conversely, fitness-suppressing exons are enriched in nonessential genes, exhibiting lower inclusion levels, and overlap with intrinsically disordered regions and disease-associated mutations. In-depth mechanistic investigation of the screen-hit TAF5 alternative exon-8 revealed that its inclusion is required for assembly of the TFIID general transcription initiation complex, thereby regulating global gene expression output. Collectively, our orthogonal exon perturbation screens established a comprehensive repository of phenotypically important exons and uncovered regulatory mechanisms governing cellular fitness and gene expression.
2024-06-24 | GSE244357 | GEO