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Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control [sgRNA]

ABSTRACT: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control [sgRNA]

PROVIDER: PRJNA1032904 | ENA |

REPOSITORIES: ENA

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Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

Project description:Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

| PRJNA1032902 | ENA

Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

Project description:Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

| PRJNA1032057 | ENA

Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

Project description:Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

| PRJNA1032905 | ENA

Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control [sgRNA]

Project description:The translation of mRNA into proteins in multicellular organisms needs to be carefully tuned to changing proteome demands in development and differentiation, and defects in translation often have a disproportionate impact in distinct cell types. Here we used inducible CRISPR interference screens to compare the essentiality of genes with functions in mRNA translation in human induced pluripotent stem cells (hiPSC) and hiPSC-derived neural and cardiac cells. We find that core components of the mRNA translation machinery are broadly essential, but the consequences of perturbing translation-coupled quality control factors are highly cell type dependent. Human stem cells critically depend on pathways that detect and rescue slow or stalled ribosomes, and on the E3 ligase ZNF598 to resolve a novel type of ribosome collisions at translation start sites on endogenous mRNAs with highly efficient initiation. Our findings underscore the importance of cell identity for deciphering the molecular mechanisms of translational control in metazoans.

2025-07-07 | GSE246415 | GEO

Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

Project description:The synthesis of proteins in multicellular organisms needs to be carefully tuned to changing proteome demands during development, and defects in this process often have a disproportionate impact in specific cell types and tissues. Here, we compared the essentiality of 262 genes from the mRNA translation machinery by inducible CRISPRi screens in HEK293 cells, human induced pluripotent stem cells (hiPSC) and hiPSC-derived neuronal progenitor cells, neurons, and cardiomyocytes. While ribosomal proteins and core translation factors were broadly essential, the cellular responses and phenotypic consequences of perturbing translation-dependent quality control pathways were highly dependent on the cellular context. By profiling ribosome occupancy in cells that are particularly sensitive to ZNF598 loss, we discovered a previously unappreciated role of this protein in detecting ribosome collisions during translation initiation. Our findings illuminate the importance of cell identity for deciphering the molecular mechanisms of translational control in metazoans.

2025-07-07 | GSE246212 | GEO

Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

2025-07-07 | GSE246418 | GEO

Comparison of dual sgRNA library versus Dolcetto by Perturb-seq

Project description:CRISPR interference (CRISPRi) genetic screens use programmable repression of gene expression to systematically explore questions in cell biology and genetics. However, wider adoption of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and lack of consensus on the choice of CRISPRi effector proteins. Here, we address these challenges to present next-generation CRISPRi sgRNA libraries and effectors. First, we combine empiric sgRNA selection with a dual sgRNA library design to generate an ultra-compact, highly active CRISPRi sgRNA library. Next, we rigorously compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an optimal balance between strong on-target knockdown and minimal nonspecific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines which stably express Zim3-dCas9 and demonstrate robust on-target knockdown across these cell lines. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.

2022-06-04 | GSE205310 | GEO

dHyperCas12a enables multiplexed CRISPRi screens

Project description:dHyperCas12a enables multiplexed CRISPRi screens

| PRJNA1166678 | ENA

Comparison of non-specific transcriptional effects of CRISPRi effector proteins by RNA-seq

2022-06-03 | GSE205147 | GEO

Multiplexed Perturbation Enables Scalable Pooled Screens

Project description:CRISPR-based genetic perturbation screens have revolutionized the ability to link genes to cellular phenotypes with unprecedented precision and scale. However, conventional pooled CRISPR screens require large cell numbers to achieve adequate sgRNA representation, posing technical and financial challenges. Here, we investigate the impact of multiplexed sgRNA delivery via high multiplicity of infection (MOI) in pooled CRISPR interference (CRISPRi) screens as a strategy to enhance screening efficiency while reducing cell numbers. We systematically evaluate screen performance across varying MOIs, assessing the effects of multiplexing on knockdown efficiency, sgRNA representation, and collision rates. Our data demonstrate that sgRNA multiplexing (MOI 2.5-10) can maintain screen performance while enabling significant reductions in cell requirements. We further apply these optimized conditions to conduct a genome-wide CRISPR screen for regulators of intracellular adhesion molecule ICAM-1, successfully identifying novel candidates using as few as half a million cells. This study provides a framework for adopting multiplexed sgRNA strategies to streamline CRISPR screening applications in resource-limited settings.

2026-01-16 | GSE302335 | GEO

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