scholarly journals A genome-wide CRISPR screen identifies the glycosylation enzyme DPM1 as a modifier of DPAGT1 deficiency and ER stress

2021 ◽  
Author(s):  
Hans M. Dalton ◽  
Raghuvir Viswanatha ◽  
Ricky Brathwaite ◽  
Jae Sophia Zuno ◽  
Stephanie E. Mohr ◽  
...  
Keyword(s):  
Er Stress ◽  
Therapeutic Targets ◽  
Therapeutic Benefit ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Fructose Metabolism ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

AbstractPartial loss-of-function mutations in glycosylation pathways underlie a set of rare diseases called Congenital Disorders of Glycosylation (CDGs). In particular, DPAGT1-CDG is caused by mutations in the gene encoding the first step in N-glycosylation, DPAGT1, and this disorder currently lacks effective therapies. To identify potential therapeutic targets for DPAGT1-CDG, we performed CRISPR knockout screens in Drosophila cells for genes associated with better survival and glycoprotein levels under DPAGT1 inhibition. We identified hundreds of candidate genes that may be of therapeutic benefit. Intriguingly, inhibition of the mannosyltransferase Dpm1, or its downstream glycosylation pathways, could rescue two in vivo models of DPAGT1 inhibition and ER stress, even though impairment of these pathways alone usually cause CDGs. While both in vivo models ostensibly cause ER stress (through DPAGT1 inhibition or a misfolded protein), we found a novel difference in fructose metabolism that may indicate glycolysis as a modulator of DPAGT1-CDG. Our results provide new therapeutic targets for DPAGT1-CDG, include the unique finding of Dpm1-related pathways rescuing DPAGT1 inhibition, and reveal a novel interaction between fructose metabolism and ER stress.

scholarly journals In vivo CRISPR/Cas9 knockout screen: TCEAL1 silencing enhances docetaxel efficacy in prostate cancer

2020 ◽  
Vol 3 (12) ◽  
pp. e202000770 ◽  
Author(s):  
Linda K Rushworth ◽  
Victoria Harle ◽  
Peter Repiscak ◽  
William Clark ◽  
Robin Shaw ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Elongation Factor ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Genome Wide ◽  
A Genome ◽  
Docetaxel Treatment ◽  
Crispr Screen ◽  
Altered Cell

Docetaxel chemotherapy in metastatic prostate cancer offers only a modest survival benefit because of emerging resistance. To identify candidate therapeutic gene targets, we applied a murine prostate cancer orthograft model that recapitulates clinical invasive prostate cancer in a genome-wide CRISPR/Cas9 screen under docetaxel treatment pressure. We identified 17 candidate genes whose suppression may enhance the efficacy of docetaxel, with transcription elongation factor A–like 1 (Tceal1) as the top candidate. TCEAL1 function is not fully characterised; it may modulate transcription in a promoter dependent fashion. Suppressed TCEAL1 expression in multiple human prostate cancer cell lines enhanced therapeutic response to docetaxel. Based on gene set enrichment analysis from transcriptomic data and flow cytometry, we confirmed that loss of TCEAL1 in combination with docetaxel leads to an altered cell cycle profile compared with docetaxel alone, with increased subG1 cell death and increased polyploidy. Here, we report the first in vivo genome-wide treatment sensitisation CRISPR screen in prostate cancer, and present proof of concept data on TCEAL1 as a candidate for a combinational strategy with the use of docetaxel.

scholarly journals Genome-wide synthetic lethal CRISPR screen identifies FIS1 as a genetic interactor of ALS-linked C9ORF72

2019 ◽  
Author(s):  
Noori Chai ◽  
Michael S. Haney ◽  
Julien Couthouis ◽  
David W. Morgens ◽  
Alyssa Benjamin ◽  
...  
Keyword(s):  
Genetic Interaction ◽  
Mitochondrial Fission ◽  
Loss Of Function ◽  
Synthetic Lethal ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen ◽  
Insight Into ◽  
Mitochondrial Membrane Protein ◽  
Lateral Sclerosis

AbstractMutations in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis (ALS). Both toxic gain of function and loss of function pathogenic mechanisms have been proposed. Accruing evidence from mouse knockout studies point to a role for C9ORF72 as a regulator of immune function. To provide further insight into its cellular function, we performed a genome-wide synthetic lethal CRISPR screen in human myeloid cells lacking C9ORF72. We discovered a strong synthetic lethal genetic interaction between C9ORF72 and FIS1, which encodes a mitochondrial membrane protein involved in mitochondrial fission and mitophagy. Mass spectrometry experiments revealed that in C9ORF72 knockout cells, FIS1 strongly bound to a class of immune regulators that activate the receptor for advanced glycation end (RAGE) products and trigger inflammatory cascades. These findings present a novel genetic interactor for C9ORF72 and suggest a compensatory role for FIS1 in suppressing inflammatory signaling in the absence of C9ORF72.

scholarly journals Genome-wide CRISPR Screen to Identify Genes that Suppress Transformation in the Presence of Endogenous KrasG12D

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jianguo Huang ◽  
Mark Chen ◽  
Eric S. Xu ◽  
Lixia Luo ◽  
Yan Ma ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Gene Mutations ◽  
Soft Agar ◽  
Reading Frame ◽  
Immunodeficient Mice ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

AbstractCooperating gene mutations are typically required to transform normal cells enabling growth in soft agar or in immunodeficient mice. For example, mutations in Kras and transformation-related protein 53 (Trp53) are known to transform a variety of mesenchymal and epithelial cells in vitro and in vivo. Identifying other genes that can cooperate with oncogenic Kras and substitute for Trp53 mutation has the potential to lead to new insights into mechanisms of carcinogenesis. Here, we applied a genome-wide CRISPR/Cas9 knockout screen in KrasG12D immortalized mouse embryonic fibroblasts (MEFs) to search for genes that when mutated cooperate with oncogenic Kras to induce transformation. We also tested if mutation of the identified candidate genes could cooperate with KrasG12D to generate primary sarcomas in mice. In addition to identifying the well-known tumor suppressor cyclin dependent kinase inhibitor 2A (Cdkn2a), whose alternative reading frame product p19 activates Trp53, we also identified other putative tumor suppressors, such as F-box/WD repeat-containing protein 7 (Fbxw7) and solute carrier family 9 member 3 (Slc9a3). Remarkably, the TCGA database indicates that both FBXW7 and SLC9A3 are commonly co-mutated with KRAS in human cancers. However, we found that only mutation of Trp53 or Cdkn2a, but not Fbxw7 or Slc9a3 can cooperate with KrasG12D to generate primary sarcomas in mice. These results show that mutations in oncogenic Kras and either Fbxw7 or Slc9a3 are sufficient for transformation in vitro, but not for in vivo sarcomagenesis.

scholarly journals Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection

2020 ◽  
Author(s):  
Jin Wei ◽  
Mia Madel Alfajaro ◽  
Ruth E. Hanna ◽  
Peter C. DeWeirdt ◽  
Madison S. Strine ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Cathepsin L ◽  
Therapeutic Targets ◽  
Chromatin Remodeling Complex ◽  
Genome Wide ◽  
A Genome ◽  
Viral Genes ◽  
Crispr Screen ◽  
Chaperone Complex ◽  
Host Genes

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-β signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis.

scholarly journals Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation

2018 ◽  
Vol 217 (11) ◽  
pp. 3817-3828 ◽  
Author(s):  
Keigo Morita ◽  
Yutaro Hama ◽  
Tamaki Izume ◽  
Norito Tamura ◽  
Toshihide Ueno ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Degradation Process ◽  
Autophagic Flux ◽  
Autophagosome Formation ◽  
Early Step ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

Macroautophagy is an intracellular degradation process that requires multiple autophagy-related (ATG) genes. In this study, we performed a genome-wide screen using the autophagic flux reporter GFP-LC3-RFP and identified TMEM41B as a novel ATG gene. TMEM41B is a multispanning membrane protein localized in the endoplasmic reticulum (ER). It has a conserved domain also found in vacuole membrane protein 1 (VMP1), another ER multispanning membrane protein essential for autophagy, yeast Tvp38, and the bacterial DedA family of putative half-transporters. Deletion of TMEM41B blocked the formation of autophagosomes at an early step, causing accumulation of ATG proteins and small vesicles but not elongating autophagosome-like structures. Furthermore, lipid droplets accumulated in TMEM41B-knockout (KO) cells. The phenotype of TMEM41B-KO cells resembled those of VMP1-KO cells. Indeed, TMEM41B and VMP1 formed a complex in vivo and in vitro, and overexpression of VMP1 restored autophagic flux in TMEM41B-KO cells. These results suggest that TMEM41B and VMP1 function together at an early step of autophagosome formation.

Genome-wide CRISPR/Cas9 library screening to identify PAK1 as a critical driver for lenvatinib resistance in HCC.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e16687-e16687
Author(s):  
Shanzhou Huang ◽  
Chuanzhao Zhang ◽  
Baohua Hou
Keyword(s):  
Drug Resistance ◽  
Progression Free Survival ◽  
Sphingolipid Metabolism ◽  
Library Screening ◽  
Loss Of Function ◽  
Free Survival ◽  
Genome Wide ◽  
A Genome ◽  
Hcc Cells

e16687 Background: Lenvatinib demonstrated noninferiority to sorafenib in overall survival and a statistically significant increase in progression-free survival in patients with unresectable hepatocellular carcinoma (HCC). However, the development of drug resistance of lenvatinib is becoming common. Methods: Here, we performed the first genome-wide CRISPR/Cas9-based screening on lenvatinib-treated HCC cells to identify essential genes for non-mutational mechanisms related to acquired lenvatinib resistance and/or sensitivity in HCC cells. A genome-scale CRISPR knockout (mGeCKOa) library containing 123,411 single guide RNAs (sgRNAs) was used to identify loss-of-function mutations conferring lenvatinib resistance upon HCC cells. CCK8, wound-healing, transwell and flow cytometry assays were used. Subcutaneous xenotransplantation models were used to investigate the role in vivo. Results: By using genome-wide CRISPR/Cas9 library screening, we identify PAK1, a family member of serine/threonine p21-activating kinases, as a critical driver for lenvatinib resistance and the most-effective genetic suppressor of lenvatinib activity. Analysis of the survival of HCC after hepatic resection revealed that low PAK1 expression shortened overall and tumor-free survival (P < 0.001). Further, loss of PAK1 from HCC cell increased the cytotoxicity of lenvatinib in vivo. Lenvatinib treatment activates series of metabolism, including “Glycolysis/ Gluconeogenesis、Cysteine and methionine metabolism、Selenocompound metabolism、Sphingolipid metabolism、Pyruvate metabolism、Metabolic pathways”. With RNAi knockdown and CRISPR/Cas9 knockout models, we show that loss of PAK1 activates the Glycolysis/ Gluconeogenesis and increase the production related metabolites. Concomitantly, activation of PAK1 reduces ROS level and HCC apoptosis upon Lenvatinib treatment. Lenvatinib treatment activates series of metabolism, including “Glycolysis/ Gluconeogenesis”. With RNAi knockdown and CRISPR/Cas9 knockout models, we show that loss of PAK1 activates the Glycolysis/ Gluconeogenesis and increase the production related metabolites. Concomitantly, activation of PAK1 reduces ROS level and HCC apoptosis upon Lenvatinib treatment. Conclusions: In summary, our results demonstrate that targeting PAK1 is an effective approach to overcome lenvatinib drug resistance in HCC.

scholarly journals Optimized strategy for in vivo Cas9-activation in Drosophila

2017 ◽  
Vol 114 (35) ◽  
pp. 9409-9414 ◽  
Author(s):  
Ben Ewen-Campen ◽  
Donghui Yang-Zhou ◽  
Vitória R. Fernandes ◽  
Delfina P. González ◽  
Lu-Ping Liu ◽  
...  
Keyword(s):  
Large Scale ◽  
Target Gene ◽  
In Vivo Studies ◽  
Transcriptional Activators ◽  
Loss Of Function ◽  
Guide Rnas ◽  
Genome Wide ◽  
A Genome ◽  
Endogenous Loci

While several large-scale resources are available for in vivo loss-of-function studies in Drosophila, an analogous resource for overexpressing genes from their endogenous loci does not exist. We describe a strategy for generating such a resource using Cas9 transcriptional activators (CRISPRa). First, we compare a panel of CRISPRa approaches and demonstrate that, for in vivo studies, dCas9-VPR is the most optimal activator. Next, we demonstrate that this approach is scalable and has a high success rate, as >75% of the lines tested activate their target gene. We show that CRISPRa leads to physiologically relevant levels of target gene expression capable of generating strong gain-of-function (GOF) phenotypes in multiple tissues and thus serves as a useful platform for genetic screening. Based on the success of this CRISRPa approach, we are generating a genome-wide collection of flies expressing single-guide RNAs (sgRNAs) for CRISPRa. We also present a collection of more than 30 Gal4 > UAS:dCas9-VPR lines to aid in using these sgRNA lines for GOF studies in vivo.

scholarly journals SUT1-promoted sterol uptake involves the ABC transporter Aus1 and the mannoprotein Dan1 whose synergistic action is sufficient for this process

2004 ◽  
Vol 381 (1) ◽  
pp. 195-202 ◽  
Author(s):  
Parissa ALIMARDANI ◽  
Matthieu RÉGNACQ ◽  
Carole MOREAU-VAUZELLE ◽  
Thierry FERREIRA ◽  
Tristan ROSSIGNOL ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Constitutive Expression ◽  
Open Reading Frames ◽  
Wild Type ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sterol Uptake ◽  
Genome Wide ◽  
A Genome

Efficient sterol influx in the yeast Saccharomyces cerevisiae is restricted to anaerobiosis or to haem deficiency resulting from mutations. Constitutive expression of SUT1, an hypoxic gene encoding a transcriptional regulator, induces sterol uptake in aerobiosis. A genome-wide approach using DNA microarray was used to identify the mediators of SUT1 effects on aerobic sterol uptake. A total of 121 ORFs (open reading frames) were significantly and differentially expressed after SUT1 overexpression, 61 down-regulated and 60 up-regulated. Among these genes, the role of the putative ABC transporter (ATP-binding-cassette transporter) Aus1, and of the cell-wall mannoprotein Dan1, was characterized better. These two genes play an essential role in aerobic sterol uptake, since their deletion compromised the SUT1 effects, but individual overexpression of either of these genes in a wild-type background was not sufficient for this process. However, constitutive co-expression of AUS1 and DAN1 in a wild-type background resulted in sterol influx in aerobiosis. These results suggest that the corresponding proteins may act synergistically in vivo to promote sterol uptake.

scholarly journals Genome-wide CRISPR screen identifies protein pathways modulating tau protein levels in neurons

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Carlos G. Sanchez ◽  
Christopher M. Acker ◽  
Audrey Gray ◽  
Malini Varadarajan ◽  
Cheng Song ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Tau Protein ◽  
Screening Strategy ◽  
Mtor Pathway ◽  
Protein Levels ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen ◽  
Hit Validation

AbstractAggregates of hyperphosphorylated tau protein are a pathological hallmark of more than 20 distinct neurodegenerative diseases, including Alzheimer’s disease, progressive supranuclear palsy, and frontotemporal dementia. While the exact mechanism of tau aggregation is unknown, the accumulation of aggregates correlates with disease progression. Here we report a genome-wide CRISPR screen to identify modulators of endogenous tau protein for the first time. Primary screens performed in SH-SY5Y cells, identified positive and negative regulators of tau protein levels. Hit validation of the top 43 candidate genes was performed using Ngn2-induced human cortical excitatory neurons. Using this approach, genes and pathways involved in modulation of endogenous tau levels were identified, including chromatin modifying enzymes, neddylation and ubiquitin pathway members, and components of the mTOR pathway. TSC1, a critical component of the mTOR pathway, was further validated in vivo, demonstrating the relevance of this screening strategy. These findings may have implications for treating neurodegenerative diseases in the future.

scholarly journals Functional proteogenomics reveals biomarkers and therapeutic targets in lymphomas

2017 ◽  
Vol 114 (25) ◽  
pp. 6581-6586 ◽  
Author(s):  
Delphine C. M. Rolland ◽  
Venkatesha Basrur ◽  
Yoon-Kyung Jeon ◽  
Carla McNeil-Schwalm ◽  
Damian Fermin ◽  
...  
Keyword(s):  
Cell Lines ◽  
Therapeutic Targets ◽  
Cancer Biomarkers ◽  
Large Cell ◽  
Primary Lymphoma ◽  
Anaplastic Lymphoma ◽  
Genome Wide ◽  
A Genome

Identification of biomarkers and therapeutic targets is a critical goal of precision medicine. N-glycoproteins are a particularly attractive class of proteins that constitute potential cancer biomarkers and therapeutic targets for small molecules, antibodies, and cellular therapies. Using mass spectrometry (MS), we generated a compendium of 1,091 N-glycoproteins (from 40 human primary lymphomas and cell lines). Hierarchical clustering revealed distinct subtype signatures that included several subtype-specific biomarkers. Orthogonal immunological studies in 671 primary lymphoma tissue biopsies and 32 lymphoma-derived cell lines corroborated MS data. In anaplastic lymphoma kinase-positive (ALK+) anaplastic large cell lymphoma (ALCL), integration of N-glycoproteomics and transcriptome sequencing revealed an ALK-regulated cytokine/receptor signaling network, including vulnerabilities corroborated by a genome-wide clustered regularly interspaced short palindromic screen. Functional targeting of IL-31 receptor β, an ALCL-enriched and ALK-regulated N-glycoprotein in this network, abrogated ALK+ALCL growth in vitro and in vivo. Our results highlight the utility of functional proteogenomic approaches for discovery of cancer biomarkers and therapeutic targets.

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