To identify genes for radioresistance in lung cancer
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE155758 | GEO | 2022/08/03
REPOSITORIES: GEO
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Project description:Cellular phenotypes arise from the degrees to which different genes are expressed, yet the ability to monitor these phenotypes is limited by a lack of tools to precisely control gene expression. We describe the development of allelic series of systematically compromised sgRNAs to titrate expression of human genes with CRISPR interference. Using large-scale measurements of compromised sgRNA activities, we both identify empirically validated intermediate-activity sgRNAs and derive the factors governing sgRNA activity using deep learning, enabling construction of a compact sgRNA library to titrate expression of ~2,400 essential genes and a genome-wide in silico library. Staging cells along a continuum of essential gene expression levels using sgRNA series combined with rich single-cell RNA-seq readout reveals expression threshold-specific responses and gene-specific expression-to-phenotype relationships. Our work provides a general tool to control gene expression with applications ranging from tuning of biochemical pathways to identification of suppressors for diseases of dysregulated gene expression.
2019-11-18 | GSE132080 | GEO
Project description:tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is an RNA-mediated gene expression control technology that has therapeutic potential. This technology is based on the properties of tRNase ZL that it can cleave any target RNA at any desired site under the direction of an appropriate artificial small guide RNA (sgRNA) and that cytosolic tRNase ZL can modulate gene expression by cleaving mRNA under the direction of cellular 5M-bM-^@M-2-half-tRNA or microRNA as sgRNA. In order to estimate a number of potential therapeutic heptamer-type sgRNAs for hematological malignancies, we constructed an sgRNA library composed of 156 heptamer-type sgRNAs, and examined how the sgRNAs affect viability of leukemia and myeloma cells. And we found that 20 of the 156 sgRNAs can efficiently induce apoptosis in at least one of the cancer cell lines. Furthermore, we demonstrated that 4 of the 20 effective sgRNAs can reduce growth rates of HL60 cells in mouse xenograft models. DNA microarray analysis for changes in an mRNA profile by these four heptamer-type sgRNAs suggested at least one candidate target mRNA that contains a potential tRNase ZL target site for each sgRNA. Changes in gene expression in HL60 cells were measured after 18-hour incubation in the absence or presence of one of five different heptamer-type sgRNAs. *Heptamer sequences requested but not provided by submitter
2014-06-30 | E-GEOD-49578 | biostudies-arrayexpress
Project description:West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen using CRISPR/Cas9. HEK 293FT cells were infected with lentivirus expressing sgRNAs and then transfected with a Cas9 expressing construct. WNV infection killed most cells during a 12d selection. Survivor cells were harvested, from which DNA was isolated. The sgRNAs integrated in genome of survivor cells were amplified with PCR. The PCR product was sequenced with Illumina MiSeq to profile the sgRNA population in the survivor cells. Three replicates were conducted. Similarly, a second round of screen was conducted. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J2, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the endoplasmic reticulum-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death. Examination of sgRNA populations in survival 293FT cells
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Project description:sgRNA whole genome library sequencing in OCI-AML5-Cas9 EKO library cells overexpressing HMGA-YFP or control YFP vectors. The goal of this experiment is to identify synthetic lethal and synthetic rescue sgRNAs with regard to HMGA2 overexpression in AML.
2022-07-11 | GSE171061 | GEO
Project description:tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is an RNA-mediated gene expression control technology that has therapeutic potential. This technology is based on the properties of tRNase ZL that it can cleave any target RNA at any desired site under the direction of an appropriate artificial small guide RNA (sgRNA) and that cytosolic tRNase ZL can modulate gene expression by cleaving mRNA under the direction of cellular 5′-half-tRNA or microRNA as sgRNA. In order to estimate a number of potential therapeutic heptamer-type sgRNAs for hematological malignancies, we constructed an sgRNA library composed of 156 heptamer-type sgRNAs, and examined how the sgRNAs affect viability of leukemia and myeloma cells. And we found that 20 of the 156 sgRNAs can efficiently induce apoptosis in at least one of the cancer cell lines. Furthermore, we demonstrated that 4 of the 20 effective sgRNAs can reduce growth rates of HL60 cells in mouse xenograft models. DNA microarray analysis for changes in an mRNA profile by these four heptamer-type sgRNAs suggested at least one candidate target mRNA that contains a potential tRNase ZL target site for each sgRNA.
2014-06-30 | GSE49578 | GEO
Project description:We conducted a two-vector CRISPR/Cas13d proliferation screening experiment. The screening library contains 10,830 sgRNAs targeting 192 protein-coding genes and 234 lncRNAs, and the screening experiment was performed using a melanoma cell line A375. It provides a unique dataset to model Cas13d sgRNA efficiency and specificity. We designed a deep learning model, named DeepCas13, to predict the sgRNA on-target activity with high accuracy from sgRNA sequences and RNA secondary structures.
2023-01-16 | GSE183256 | GEO
Project description:Purpose: To identify critical epigenetic regulators of acute myeloid leukemia cell surival (AML), we performed a CRISPR screen with an sgRNA library containing 6121 sgRNAs against the epigenome and controls. We collected samples at day 7 (reference) and day 21 to look for sgRNAs that dropped out of the population and hence, may be critical for for AML cell growth or survival. Conclusions: Our study reveals a novel epigenetic regulator of innate immunity in AML. This gene is critical for AML surivival and loss of this gene induces a type I IFN response. This particular dataset show the dropout of this gene at day 21, as well as other potential genes that regulate AML cell survival or growth.
2017-09-06 | GSE103407 | GEO
Project description:Here, we introduce CRaft-ID (CRISPR-based microRaft followed by gRNA identification) to enable the screening of image-based phenotypes such as subcellular protein localization from a pool of lenti-CRISPR bulk infected cells. Clonal colonies selected from microRaft arrays are isolated after phenotyping for a targeted-sequencing library preparation to identify the infected sgRNA in each colony. sgRNA representation in the pool of cells is measured with a targeted-sequencing library preparation from the bulk sample.
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Project description:We performed tiling CRISPR activation (CRISPRa) screens in Jurkat T cells to discover enhancers controlling IL2RA and CD69 expression. For each target gene, we constructed a pooled lentiviral library of sgRNAs that targeted sites at all Cas9 PAMs throughout the window starting 100 kb upstream of the transcription start site, extending through the gene body, and 25 kb downstream. A separate library was constructed for each gene, and a separate screen was performed for each gene. For each gene, Jurkat cells stably expressing the dCas9-VP64 transcriptional activator were transduced with the lentiviral sgRNA library. Cells were stained for the protein of interested and sorted by flow cytometry into four bins of expression. Distribution of sgRNAs in each of the sorted populations was compared to the unsorted population. Regions where dCas9-VP64 recruitment was sufficient to drive target gene expression were identified as putative enhancers. The hg19 coordinates of the CD69 library window were chr12:9,880,082-10,013,497. The library contained 2,244 negative control sgRNAs taken from the CRISPRi/a libraries described in Gilbert et al., Cell 150, 647-661 (2014). The hg19 coordinates of the IL2RA library window were chr10:6,027,657- 6,204,333. The library contained 2,244 negative control sgRNAs taken from the CRISPRi/a libraries described in Gilbert et al., Cell 150, 647-661 (2014).
2017-04-26 | GSE98178 | GEO
Project description:We have combined a machine-learning approach with other strategies to optimize the efficiency of sgRNAs for CRISPR screens and have constructed a genome-wide, sequence-verified, arrayed CRISPR library. This incorporates expression strategies to facilitate multiplexed or combinatorial screening. By conducting parallel loss-of-function screens, we compare our approach to existing sgRNA design and expression strategies.
2017-07-20 | GSE97433 | GEO