Similar Datasets
Project description:Although CRISPR-Cas technology has revolutionized functional genomics, the systematic exploration of the role of individual exons for critical cellular phenotypes is lagging, limiting our understanding of genome regulation. To overcome this constraint, we have optimized and applied massively parallel exon deletion and splice site mutation screens in human cell lines identifying thousands of exons required for cell fitness. Fitness-promoting exons are enriched in essential and highly expressed genes and frequently overlap protein domains and interaction interfaces. In contrast, fitness-suppressing exons that are enriched in low-expressed, non-essential genes and tend to overlap intrinsically disordered regions. In-depth mechanistic investigation of a screen hit, TAF5 alternative exon-8, reveals that its inclusion controls the assembly of the TFIID general transcription initiation complex regulating gene expression outputs. Collectively, by applying orthogonal exon perturbation screening strategies we have generated a resource of phenotypically important exons and uncovered mechanisms that control gene expression and cell fitness.
2024-06-24 | GSE244337 | GEO
Project description:Although CRISPR-Cas technology has revolutionized functional genomics, the systematic exploration of the role of individual exons for critical cellular phenotypes is lagging, limiting our understanding of genome regulation. To overcome this constraint, we have optimized and applied massively parallel exon deletion and splice site mutation screens in human cell lines identifying thousands of exons required for cell fitness. Fitness-promoting exons are enriched in essential and highly expressed genes and frequently overlap protein domains and interaction interfaces. This contrasts fitness-suppressing exons that are enriched in low-expressed, non-essential genes and tend to overlap intrinsically disordered regions. In-depth mechanistic investigation of a screen hit, the alternative exon-8 in TAF5, reveals that its inclusion controls the assembly of the TFIID general transcription initiation complex regulating gene expression outputs. Collectively, by applying orthogonal exon perturbation screening strategies we have interrogated phenotypically important exons at genome-scale and uncovered mechanisms that control gene expression and cell fitness.
2023-08-30 | MSV000092798 | MassIVE
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 | GSE244373 | 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
Project description:Although CRISPR-Cas technology has revolutionized functional genomics, the systematic exploration of the role of individual exons for critical cellular phenotypes is lagging, limiting our understanding of genome regulation. To overcome this constraint, we have optimized and applied massively parallel exon deletion and splice site mutation screens in human cell lines identifying thousands of exons required for cell fitness. Fitness-promoting exons are enriched in essential and highly expressed genes and frequently overlap protein domains and interaction interfaces. In contrast, fitness-suppressing exons that are enriched in low-expressed, non-essential genes and tend to overlap intrinsically disordered regions. In-depth mechanistic investigation of a screen hit, TAF5 alternative exon-8, reveals that its inclusion controls the assembly of the TFIID general transcription initiation complex regulating gene expression outputs. Collectively, by applying orthogonal exon perturbation screening strategies we have generated a resource of phenotypically important exons and uncovered mechanisms that control gene expression and cell fitness.
2024-06-24 | GSE244340 | GEO
Project description:Although CRISPR-Cas technology has revolutionized functional genomics, the systematic exploration of the role of individual exons for critical cellular phenotypes is lagging, limiting our understanding of genome regulation. To overcome this constraint, we have optimized and applied massively parallel exon deletion and splice site mutation screens in human cell lines identifying thousands of exons required for cell fitness. Fitness-promoting exons are enriched in essential and highly expressed genes and frequently overlap protein domains and interaction interfaces. In contrast, fitness-suppressing exons that are enriched in low-expressed, non-essential genes and tend to overlap intrinsically disordered regions. In-depth mechanistic investigation of a screen hit, TAF5 alternative exon-8, reveals that its inclusion controls the assembly of the TFIID general transcription initiation complex regulating gene expression outputs. Collectively, by applying orthogonal exon perturbation screening strategies we have generated a resource of phenotypically important exons and uncovered mechanisms that control gene expression and cell fitness.
2024-06-24 | GSE262849 | GEO
Project description:While RNA-seq has enabled comprehensive quantification of alternative splicing, no correspondingly high-throughput assay exists for functionally interrogating individual isoforms. We describe pgFARM (paired guide RNAs for alternative exon removal), a CRISPR-Cas9-based method to manipulate isoforms independent of gene inactivation. This approach enabled rapid suppression of exon recognition in polyclonal settings to identify functional roles for individual exons, such as an SMNDC1 cassette exon that regulates pan-cancer intron retention. We generalized this method to a pooled screen to measure the functional relevance of 'poison' cassette exons, which disrupt their host genes' reading frames yet are frequently ultraconserved. Many poison exons were essential for the growth of both cultured cells and lung adenocarcinoma xenografts, while a subset had clinically relevant tumor-suppressor activity. The essentiality and cancer relevance of poison exons are likely to contribute to their unusually high conservation and contrast with the dispensability of other ultraconserved elements for viability.
| S-EPMC6962552 | biostudies-literature
Project description:Protein complexes are assemblies of subunits that have co-evolved to execute one or many coordinated functions in the cellular environment. Functional annotation of mammalian protein complexes is critical to understanding biological processes, as well as disease mechanisms. Here, we used genetic co-essentiality derived from genome-scale RNAi- and CRISPR-Cas9-based fitness screens performed across hundreds of human cancer cell lines to assign measures of functional similarity. From these measures, we systematically built and characterized functional similarity networks that recapitulate known structural and functional features of well-studied protein complexes and resolve novel functional modules within complexes lacking structural resolution, such as the mammalian SWI/SNF complex. Finally, by integrating functional networks with large protein-protein interaction networks, we discovered novel protein complexes involving recently evolved genes of unknown function. Taken together, these findings demonstrate the utility of genetic perturbation screens alone, and in combination with large-scale biophysical data, to enhance our understanding of mammalian protein complexes in normal and disease states.
| S-EPMC6152908 | biostudies-literature
Project description:Pluripotent stem cells are defined by their self-renewal capacity, which is the ability of the stem cells to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into any somatic cell lineage. However, understanding the mechanisms that control stem cell fitness versus the pluripotent cell identity is challenging. To investigate the interplay between these two aspects of pluripotency, we performed four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSC self-renewal conditions, and the dissolution of the primed pluripotency identity during early differentiation. Comparative analyses led to the discovery of genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further discovered a core set of factors that control both stem cell fitness and pluripotent identity, including a network of chromatin factors that safeguard pluripotency. Our unbiased and systematic screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide rich datasets for exploring pluripotent cell identity versus cell fitness, and offer a valuable model for categorizing gene function in broad biological contexts.
2024-09-05 | PXD055592 | Pride
Project description:High-throughput perturbation screens measure the phenotypes of thousands of biological samples under various conditions. The phenotypes measured in the screens are subject to substantial biological and technical variation. At the same time, in order to enable high throughput, it is often impossible to include a large number of replicates, and to randomize their order throughout the screens. Distinguishing true changes in the phenotype from stochastic variation in such experimental designs is extremely challenging, and requires adequate statistical methodology.We propose a statistical modeling framework that is based on experimental designs with at least two controls profiled throughout the experiment, and a normalization and variance estimation procedure with linear mixed-effects models. We evaluate the framework using three comprehensive screens of Saccharomyces cerevisiae, which involve 4940 single-gene knock-out haploid mutants, 1127 single-gene knock-out diploid mutants and 5798 single-gene overexpression haploid strains. We show that the proposed approach (i) can be used in conjunction with practical experimental designs; (ii) allows extensions to alternative experimental workflows; (iii) enables a sensitive discovery of biologically meaningful changes; and (iv) strongly outperforms the existing noise reduction procedures.All experimental datasets are publicly available at www.ionomicshub.org. The R package HTSmix is available at http://www.stat.purdue.edu/~ovitek/HTSmix.html.ovitek@stat.purdue.eduSupplementary data are available at Bioinformatics online.
| S-EPMC3150043 | biostudies-literature