Dataset Information

Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis.

ABSTRACT: Single-cell transcriptomics provide a systematic map of gene expression in different human cell types. The next challenge is to systematically understand cell-type-specific gene function. The integration of CRISPR-based functional genomics and stem cell technology enables the scalable interrogation of gene function in differentiated human cells. Here we present the first genome-wide CRISPR interference and CRISPR activation screens in human neurons. We uncover pathways controlling neuronal response to chronic oxidative stress, which is implicated in neurodegenerative diseases. Unexpectedly, knockdown of the lysosomal protein prosaposin strongly sensitizes neurons, but not other cell types, to oxidative stress by triggering the formation of lipofuscin, a hallmark of aging, which traps iron, generating reactive oxygen species and triggering ferroptosis. We also determine transcriptomic changes in neurons after perturbation of genes linked to neurodegenerative diseases. To enable the systematic comparison of gene function across different human cell types, we establish a data commons named CRISPRbrain.

SUBMITTER: Tian R

PROVIDER: S-EPMC8254803 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis.

Tian Ruilin R Abarientos Anthony A Hong Jason J Hashemi Sayed Hadi SH Yan Rui R Dräger Nina N Leng Kun K Nalls Mike A MA Singleton Andrew B AB Xu Ke K Faghri Faraz F Kampmann Martin M

Nature neuroscience 20210524 7

Single-cell transcriptomics provide a systematic map of gene expression in different human cell types. The next challenge is to systematically understand cell-type-specific gene function. The integration of CRISPR-based functional genomics and stem cell technology enables the scalable interrogation of gene function in differentiated human cells. Here we present the first genome-wide CRISPR interference and CRISPR activation screens in human neurons. We uncover pathways controlling neuronal respo ...[more]

PMID: 34031600

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Project description:Antibiotic resistance and tolerance remain a major problem for the treatment of staphylococcal infections. Identifying genes that influence antibiotic susceptibility could open the door to novel antimicrobial strategies, including targets for new synergistic drug combinations. Here, we developed a genome-wide CRISPR interference library for Staphylococcus aureus, demonstrated its use by quantifying gene fitness in different strains through CRISPRi-seq, and used it to identify genes that modulate susceptibility to the lipoglycopeptide dalbavancin. By exposing the library to sublethal concentrations of dalbavancin using both CRISPRi-seq and direct selection methods, we not only found genes previously reported to be involved in antibiotic susceptibility but also identified genes thus far unknown to affect antibiotic tolerance. Importantly, some of these genes could not have been detected by more conventional transposon-based knockout approaches because they are essential for growth, stressing the complementary value of CRISPRi-based methods. Notably, knockdown of a gene encoding the uncharacterized protein KapB specifically sensitizes the cells to dalbavancin, but not to other antibiotics of the same class, whereas knockdown of the Shikimate pathway showed the opposite effect. The results presented here demonstrate the promise of CRISPRi-seq screens to identify genes and pathways involved in antibiotic susceptibility and pave the way to explore alternative antimicrobial treatments through these insights.IMPORTANCEAntibiotic resistance is a challenge for treating staphylococcal infections. Identifying genes that affect how antibiotics work could help create new treatments. In our study, we made a CRISPR interference library for Staphylococcus aureus and used this to find which genes are critical for growth and also mapped genes that are important for antibiotic sensitivity, focusing on the lipoglycopeptide antibiotic dalbavancin. With this method, we identified genes that altered the sensitivity to dalbavancin upon knockdown, including genes involved in different cellular functions. CRISPRi-seq offers a means to uncover untapped antibiotic targets, including those that conventional screens would disregard due to their essentiality. This paves the way for the discovery of new ways to fight infections.

Dataset Information

Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis.

Publications

Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis.

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