scholarly journals Genome-wide CRISPR-based gene knockout screens reveal cellular factors and pathways essential for nasopharyngeal carcinoma

2019 ◽  
Vol 294 (25) ◽  
pp. 9734-9745 ◽  
Author(s):  
Chong Wang ◽  
Sizun Jiang ◽  
Liangru Ke ◽  
Luyao Zhang ◽  
Difei Li ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Advanced Disease ◽  
Gene Knockout ◽  
Histone Acetyl Transferase ◽  
Knock Out ◽  
Genome Wide ◽  
A Genome ◽  
Cellular Factors ◽  
New Treatments ◽  
Specific Symptoms

Early diagnosis of nasopharyngeal carcinoma (NPC) is difficult because of a lack of specific symptoms. Many patients have advanced disease at diagnosis, and these patients respond poorly to treatment. New treatments are therefore needed to improve the outcome of NPC. To better understand the molecular pathogenesis of NPC, here we used an NPC cell line in a genome-wide CRISPR-based knockout screen to identify the cellular factors and pathways essential for NPC (i.e. dependence factors). This screen identified the Moz, Ybf2/Sas3, Sas2, Tip60 histone acetyl transferase complex, NF-κB signaling, purine synthesis, and linear ubiquitination pathways; and MDM2 proto-oncogene as NPC dependence factors/pathways. Using gene knock out, complementary DNA rescue, and inhibitor assays, we found that perturbation of these pathways greatly reduces the growth of NPC cell lines but does not affect growth of SV40-immortalized normal nasopharyngeal epithelial cells. These results suggest that targeting these pathways/proteins may hold promise for achieving better treatment of patients with NPC.

BIOM-32. ENDOPLASMIC RETICULUM PROTEIN SSR3 DETERMINES AND PREDICTS RESPONSE TO PACLITAXEL IN BREAST CANCER AND GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi17-vi18
Author(s):  
Crismita Dmello ◽  
Aarón Sonabend ◽  
Víctor Arrieta ◽  
Daniel Zhang ◽  
Deepak Kanojia ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Endoplasmic Reticulum ◽  
Glioma Cells ◽  
Predictive Biomarkers ◽  
Predictive Biomarker ◽  
Precision Oncology ◽  
Patient Response ◽  
Knock Out ◽  
Genome Wide ◽  
A Genome

Abstract Paclitaxel (PTX) is one the most potent and commonly used chemotherapies for breast and pancreatic cancer. Given the potency of this drug for glioblastomas (GBM) several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood-brain barrier for this disease. In spite of the efficacy of PTX, individual tumors exhibit variable susceptibility to this drug, with response rate in the range of 30%-60%. To identify predictive biomarkers for response to PTX, we performed a genome-wide CRISPR knock-out screen using human glioma cells. The most enriched genes in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort. This led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with response to PTX in breast cancer cells, glioma cells, in multiple intracranial glioma xenografts and in GBM patient derived explant cultures. Knockout of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. In gliomas, SSR3-mediated susceptibility to PTX relates to modulation of phosphorylation of ER stress sensor IRE1α. Thus, by using genome-wide screen combined with patient response data, we discovered a biomarker that demonstrates causal and correlative relationship with response to PTX in breast cancer and GBM. Prospective validation of this biomarker is warranted for its broad implementation for precision oncology.

scholarly journals A genome-wide scan suggests a susceptibility locus on 5p13 for nasopharyngeal carcinoma

2008 ◽  
Vol 16 (3) ◽  
pp. 343-349 ◽  
Author(s):  
Li-Fu Hu ◽  
Qian-Hui Qiu ◽  
Sheng-Miao Fu ◽  
Di Sun ◽  
Kristinn Magnusson ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Susceptibility Locus ◽  
Genome Wide ◽  
A Genome ◽  
Genome Wide Scan

scholarly journals Towards a Systems Approach in the Genetic Analysis of Archaea: Accelerating Mutant Construction and Phenotypic Analysis inHaloferax volcanii

Archaea ◽  
2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Ian K. Blaby ◽  
Gabriela Phillips ◽  
Crysten E. Blaby-Haas ◽  
Kevin S. Gulig ◽  
Basma El Yacoubi ◽  
...  
Keyword(s):  
High Throughput ◽  
Gene Deletion ◽  
Systems Approach ◽  
Essential Gene ◽  
Gene Knockout ◽  
Deletion Mutants ◽  
Mutant Library ◽  
Phenotypic Analysis ◽  
Genome Wide ◽  
A Genome

With the availability of a genome sequence and increasingly sophisticated genetic tools,Haloferax volcaniiis becoming a model for both Archaea and halophiles. In order forH. volcaniito reach a status equivalent toEscherichia coli, Bacillus subtilis, orSaccharomyces cerevisiae, a gene knockout collection needs to be constructed in order to identify the archaeal essential gene set and enable systematic phenotype screens. A streamlined gene-deletion protocol adapted for potential automation was implemented and used to generate 22H. volcaniideletion strains and identify several potentially essential genes. These gene deletion mutants, generated in this and previous studies, were then analyzed in a high-throughput fashion to measure growth rates in different media and temperature conditions. We conclude that these high-throughput methods are suitable for a rapid investigation of anH. volcaniimutant library and suggest that they should form the basis of a larger genome-wide experiment.

scholarly journals A genome-wide CRISPR/Cas9 screen to identify phagocytosis modulators in monocytic THP-1 cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Benjamin Lindner ◽  
Eva Martin ◽  
Monika Steininger ◽  
Aleksandra Bundalo ◽  
Martin Lenter ◽  
...  
Keyword(s):  
Translational Control ◽  
Molecular Mechanisms ◽  
Gene Knockout ◽  
Single Gene ◽  
Microbial Pathogens ◽  
Protein Accumulation ◽  
Cellular Mechanisms ◽  
Developmental Defects ◽  
Genome Wide ◽  
A Genome

AbstractPhagocytosis of microbial pathogens, dying or dead cells, and cell debris is essential to maintain tissue homeostasis. Impairment of these processes is associated with autoimmunity, developmental defects and toxic protein accumulation. However, the underlying molecular mechanisms of phagocytosis remain incompletely understood. Here, we performed a genome-wide CRISPR knockout screen to systematically identify regulators involved in phagocytosis of Staphylococcus (S.) aureus by human monocytic THP-1 cells. The screen identified 75 hits including known regulators of phagocytosis, e.g. members of the actin cytoskeleton regulation Arp2/3 and WAVE complexes, as well as genes previously not associated with phagocytosis. These novel genes are involved in translational control (EIF5A and DHPS) and the UDP glycosylation pathway (SLC35A2, SLC35A3, UGCG and UXS1) and were further validated by single gene knockout experiments. Whereas the knockout of EIF5A and DHPS impaired phagocytosis, knocking out SLC35A2, SLC35A3, UGCG and UXS1 resulted in increased phagocytosis. In addition to S. aureus phagocytosis, the above described genes also modulate phagocytosis of Escherichia coli and yeast-derived zymosan A. In summary, we identified both known and unknown genetic regulators of phagocytosis, the latter providing a valuable resource for future studies dissecting the underlying molecular and cellular mechanisms and their role in human disease.

scholarly journals A genome-wide CRISPR/Cas9 gene knockout screen identifies immunoglobulin superfamily DCC subclass member 4 as a key host factor that promotes influenza virus endocytosis

2021 ◽  
Vol 17 (12) ◽  
pp. e1010141
Author(s):  
Yangming Song ◽  
Haixiang Huang ◽  
Yuzhen Hu ◽  
Jiwen Zhang ◽  
Fang Li ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Infection ◽  
H5n1 Virus ◽  
Gene Knockout ◽  
A549 Cells ◽  
Influenza Virus Infection ◽  
Immunoglobulin Superfamily ◽  
Wild Type ◽  
Genome Wide ◽  
A Genome

Influenza virus infection is dependent on host cellular factors, and identification of these factors and their underlying mechanisms can provide important information for the development of strategies to inhibit viral infection. Here, we used a highly pathogenic H5N1 influenza virus to perform a genome-wide CRISPR/Cas9 gene knockout screen in human lung epithelial cells (A549 cells), and found that knockout of transmembrane protein immunoglobulin superfamily DCC subclass member 4 (IGDCC4) significantly reduced the replication of the virus in A549 cells. Further studies showed that IGDCC4 interacted with the viral hemagglutinin protein and facilitated virus internalization into host cells. Animal infection studies showed that replication of H5N1 virus in the nasal turbinates, lungs, and kidneys of IGDCC4-knockout mice was significantly lower than that in the corresponding organs of wild-type mice. Half of the IGDCC4-knockout mice survived a lethal H5N1 virus challenge, whereas all of the wild-type mice died within 11 days of infection. Our study identifies a novel host factor that promotes influenza virus infection by facilitating internalization and provides insights that will support the development of antiviral therapies.

scholarly journals A genome-wide CRISPR/Cas phenotypic screen for modulators of DUX4 cytotoxicity reveals screen complications

2020 ◽  
Author(s):  
Ator Ashoti ◽  
Francesco Limone ◽  
Melissa van Kranenburg ◽  
Anna Alemany ◽  
Mirna Baak ◽  
...  
Keyword(s):  
Treatment Options ◽  
Underlying Mechanism ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Knock Out ◽  
Genome Wide ◽  
Phenotypic Screen ◽  
A Genome ◽  
The Face ◽  
Muscle Disorder ◽  
Pioneer Transcription Factor

AbstractFacioscapulohumeral muscular dystrophy (FHSD), a fundamentally complex muscle disorder that thus far remains untreatable. As the name implies, FSHD starts in the muscles of the face and shoulder gridle. The main perturbator of the disease is the pioneer transcription factor DUX4, which is misexpressed in affected tissues due to a failure in epigenetic repressive mechanisms. In pursuit of unraveling the underlying mechanism of FSHD and finding potential therapeutic targets or treatment options, we performed an exhaustive genome-wide CRISPR/Cas9 phenotypic rescue screen to identify modulators of DUX4 cytotoxicity. We found no key effectors other than DUX4 itself, suggesting treatment efforts in FSHD should be directed towards its direct modulation.The screen did however reveal some rare and unexpected Cas9-induced genomic events, that may provide important considerations for planning future CRISPR/Cas9 knock-out screens.

scholarly journals Morc3 silences endogenous retroviruses by enabling Daxx-mediated H3.3 incorporation

2020 ◽  
Author(s):  
Sophia Groh ◽  
Anna Viktoria Milton ◽  
Lisa Marinelli ◽  
Cara V. Sickinger ◽  
Heike Bollig ◽  
...  
Keyword(s):  
Binding Sites ◽  
Atp Hydrolysis ◽  
Chromatin Accessibility ◽  
Endogenous Retroviruses ◽  
Chromatin Modifications ◽  
Interaction Domain ◽  
Knock Out ◽  
Genome Wide ◽  
A Genome ◽  
Mammalian Genomes

ABSTRACTEndogenous retroviruses (ERVs) comprise a significant portion of mammalian genomes. Although, specific ERV loci feature regulatory roles for host gene expression, most ERV integrations are transcriptionally repressed by Setdb1 mediated H3K9me3 and DNA methylation. However, the protein network which regulates deposition of these chromatin modifications is still incompletely understood. Here, we performed a genome-wide sgRNA screen for genes involved in ERV silencing and identified the GHKL ATPase protein Morc3 as top scoring hit. Morc3 knock-out cells display de-repression, reduced H3K9me3 and increased chromatin accessibility of distinct ERV classes. We found that the GHKL ATPase domain of Morc3 is critical for ERV silencing, since mutants which cannot bind ATP, or which are defective in ATP hydrolysis cannot rescue the Morc3 ko phenotype. Proteomic analysis revealed that Morc3 mutant protein which cannot bind ATP fails to interact with the H3.3 chaperone Daxx. This interaction depends on Morc3 SUMOylation, as Daxx lacking the SUMO interaction domain shows reduced association with Morc3. Notably, in Morc3 ko cells, we observed strongly reduced H3.3 on Morc3 binding sites. Thus, our data demonstrate Morc3 as critical regulator of Daxx-mediated H3.3 incorporation to ERV regions.

scholarly journals New Regulators of a High Affinity Ca2+ Influx System Revealed through a Genome-wide Screen in Yeast

2011 ◽  
Vol 286 (12) ◽  
pp. 10744-10754 ◽  
Author(s):  
D. Christian Martin ◽  
Hyemin Kim ◽  
Nancy A. Mackin ◽  
Lymarie Maldonado-Báez ◽  
Carlos C. Evangelista ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Membrane Trafficking ◽  
Knock Out ◽  
Voltage Gated ◽  
High Affinity ◽  
Voltage Gated Calcium Channels ◽  
Genome Wide ◽  
A Genome ◽  
Target Membrane ◽  
Α Subunits

The bakers' yeast Saccharomyces cerevisiae utilizes a high affinity Ca2+ influx system (HACS) to survive assaults by mating pheromones, tunicamycin, and azole-class antifungal agents. HACS consists of two known subunits, Cch1 and Mid1, that are homologous and analogous to the catalytic α-subunits and regulatory α2δ-subunits of mammalian voltage-gated calcium channels, respectively. To search for additional subunits and regulators of HACS, a collection of gene knock-out mutants was screened for abnormal uptake of Ca2+ after exposure to mating pheromone or to tunicamycin. The screen revealed that Ecm7 is required for HACS function in most conditions. Cycloheximide chase experiments showed that Ecm7 was stabilized by Mid1, and Mid1 was stabilized by Cch1 in non-signaling conditions, suggesting they all interact. Ecm7 is a member of the PMP-22/EMP/MP20/Claudin superfamily of transmembrane proteins that includes γ-subunits of voltage-gated calcium channels. Eleven additional members of this superfamily were identified in yeast, but none was required for HACS activity in response to the stimuli. Remarkably, many dozens of genes involved in vesicle-mediated trafficking and protein secretion were required to prevent spontaneous activation of HACS. Taken together, the findings suggest that HACS and calcineurin monitor performance of the membrane trafficking system in yeasts and coordinate compensatory processes. Conservation of this quality control system in Candida glabrata suggests that many pathogenic species of fungi may utilize HACS and calcineurin to resist azoles and other compounds that target membrane biosynthesis.

Escherichia coli glycogen metabolism is controlled by the PhoP-PhoQ regulatory system at submillimolar environmental Mg2+ concentrations, and is highly interconnected with a wide variety of cellular processes

2009 ◽  
Vol 424 (1) ◽  
pp. 129-141 ◽  
Author(s):  
Manuel Montero ◽  
Gustavo Eydallin ◽  
Alejandro M. Viale ◽  
Goizeder Almagro ◽  
Francisco J. Muñoz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stress Responses ◽  
Regulatory System ◽  
Glycogen Metabolism ◽  
Metabolic Model ◽  
Knock Out ◽  
Cellular Processes ◽  
Genome Wide ◽  
A Genome ◽  
Keio Collection

Using the Keio collection of gene-disrupted mutants of Escherichia coli, we have recently carried out a genome-wide screening of the genes affecting glycogen metabolism. Among the mutants identified in the study, ΔmgtA, ΔphoP and ΔphoQ cells, all lacking genes that are induced under low extracellular Mg2+ conditions, displayed glycogen-deficient phenotypes. In this work we show that these mutants accumulated normal glycogen levels when the culture medium was supplemented with submillimolar Mg2+ concentrations. Expression analyses conducted in wild-type, ΔphoP and ΔphoQ cells showed that the glgCAP operon is under PhoP-PhoQ control in the submillimolar Mg2+ concentration range. Subsequent screening of the Keio collection under non-limiting Mg2+ allowed the identification of 183 knock-out mutants with altered glycogen levels. The stringent and general stress responses, end-turnover of tRNA, intracellular AMP levels, and metabolism of amino acids, iron, carbon and sulfur were major determinants of glycogen levels. glgC::lacZY expression analyses using mutants representing different functional categories revealed that the glgCAP operon belongs to the RelA regulon. We propose an integrated metabolic model wherein glycogen metabolism is (a) tightly controlled by the energy and nutritional status of the cell and (b) finely regulated by changes in environmental Mg2+ occurring at the submillimolar concentration range.

Sign in / Sign up

Export Citation Format

Share Document