Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Minimized combinatorial CRISPR screens identify genetic interactions in autophagy

ABSTRACT: Development of the 3Cs multiplex technology for combinatorial screening and identification of genetic interactions among human autophagy genes in cell proliferation and autophagy flux.

ORGANISM(S): Homo sapiens

PROVIDER: GSE166851 | GEO | 2021/06/16

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

Minimized combinatorial CRISPR screens identify genetic interactions in autophagy

Project description:Minimized combinatorial CRISPR screens identify genetic interactions in autophagy

| PRJNA702116 | ENA

Minimized combinatorial CRISPR screens identify genetic interactions in autophagy.

Project description: Not available

| S-EPMC8191801 | biostudies-literature

Optimized metrics for combinatorial and orthogonal CRISPR screens

Project description:CRISPR-based gene perturbation enables unbiased investigations of single and combinatorial genotype-to-phenotype associations. In light of efforts to map combinatorial gene dependencies at scale, choosing an efficient and robust CRISPR-associated (Cas) nuclease is of utmost importance. Even though SpCas9 and AsCas12a are widely used for single, combinatorial, and orthogonal screenings, side-by-side comparisons remain sparse. Here, we systematically compared combinatorial SpCas9, AsCas12a, and CHyMErA in hTERT-immortalized retinal pigment epithelial cells and extracted performance-critical parameters for combinatorial and orthogonal CRISPR screens. Our analyses identified SpCas9 to be superior to enhanced and optimized AsCas12a, with CHyMErA being largely inactive in the tested conditions. Since AsCas12a contains RNA processing activity, we used arrayed dual-gRNAs to improve AsCas12a and CHyMErA applications. While this negatively influenced the effect size of combinatorial AsCas12a applications, it enhanced the performance of CHyMErA. This improved performance, however, was limited to AsCas12a dual-gRNAs, as SpCas9 gRNAs remained largely inactive. To avoid the use of hybrid gRNAs for orthogonal applications, we engineered the multiplex SpCas9-enAsCas12a system (multiSPAS) that avoids RNA processing for efficient orthogonal gene editing.

2023-05-10 | GSE215175 | GEO

GEMINI: a variational Bayesian approach to identify genetic interactions from combinatorial CRISPR screens.

Project description: Not available

| S-EPMC6624979 | biostudies-literature

An autophagy CRISPR Screen in Jurkat cells

Project description:To identify genes regulating autophagy in human T-cells (Jurkat cell line) a CRISPR screen was conducted. Populations with low autophagic flux was sorted and the contained sgRNAs analysed by next-generation sequencing. Bioinformatic analysis compared the input of the screen versus the 'autophagy low' population.

2021-01-13 | GSE147488 | GEO

Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions.

Project description: Not available

| S-EPMC5449203 | biostudies-literature

Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens.

Project description: Not available

| S-EPMC5800952 | biostudies-literature

Genome-wide CRISPR screening reveals nucleotide synthesis negatively regulates autophagy

Project description:Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood. To identify potential negative regulators of autophagy, we performed a genome-wide CRISPR screen using the quantitative autophagic flux reporter GFP-LC3-RFP. We identified phosphoribosylformylglycinamidine synthase (PFAS), a component of the de novo purine synthesis pathway, as one such negative regulator of autophagy. Autophagy was activated in cells lacking PFAS or phosphoribosyl pyrophosphate amidotransferase (PPAT), another de novo purine synthesis enzyme, or treated with methotrexate when exogenous levels of purines were insufficient. Purine starvation-induced autophagy activation was concomitant with mTORC1 suppression, and was profoundly suppressed in cells deficient for TSC2, which negatively regulates mTORC1 through inhibition of RHEB, suggesting that purines regulate autophagy through the TSC-RHEB-mTORC1 signaling axis. Moreover, depletion of the pyrimidine synthesis enzymes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) and dihydroorotate dehydrogenase (DHODH) activated autophagy as well, although mTORC1 activity was not altered by pyrimidine shortage. These results suggest a different mechanism of autophagy induction between purine and pyrimidine starvation. These findings provide novel insights into the regulation of autophagy by nucleotides and possibly the role of autophagy in nucleotide metabolism, leading to further developing anticancer strategies involving nucleotide synthesis and autophagy.

2021-05-11 | GSE164778 | GEO

Transcriptomic analysis reveals autophagy flux variation within mouse pancreatic cancer organoids

Project description:Murine pancreatic cancer cells (HY15549 cells) established from genetically engineered mouse models (GEMM) of PDAC (p48-Cre+, KrasLSL-G12D/+, Trp53lox/+; KPC mice) were transfected with the GFP-LC3-RFP autophagy flux reporter from Mizushima lab, wherein reduction in the GFP/RFP ratio indicates increase in autophagy flux. Organoids derived from these cells were dissociated into single cells and sorted into autophagy-high (AThi) and autophagy-low (ATlo) populations according to GFP/RFP signal ratio. To validate this reporter, RNA was extracted from these two populations and expression of autophagy/lysosome related genes was examined.

2020-04-16 | GSE145766 | GEO

CRISPR Screens in Synthetic Lethality and Combinatorial Therapies for Cancer.

Project description: Not available

| S-EPMC8037779 | biostudies-literature