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https://www.readbyqxmd.com/read/29779948/the-eukaryotic-proteome-is-shaped-by-e3-ubiquitin-ligases-targeting-c-terminal-degrons
#1
Itay Koren, Richard T Timms, Tomasz Kula, Qikai Xu, Mamie Z Li, Stephen J Elledge
Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small, and a facile technology to characterize them is lacking. Using a strategy combining global protein stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Employing CRISPR screening, we establish that the stability of many proteins is regulated through degrons located at their C terminus...
May 11, 2018: Cell
https://www.readbyqxmd.com/read/29778836/interrogation-of-mammalian-protein-complex-structure-function-and-membership-using-genome-scale-fitness-screens
#2
Joshua Pan, Robin M Meyers, Brittany C Michel, Nazar Mashtalir, Ann E Sizemore, Jonathan N Wells, Seth H Cassel, Francisca Vazquez, Barbara A Weir, William C Hahn, Joseph A Marsh, Aviad Tsherniak, Cigall Kadoch
Protein complexes are assemblies of subunits that have co-evolved to execute one or many coordinated functions in the cellular environment. Functional annotation of mammalian protein complexes is critical to understanding biological processes, as well as disease mechanisms. Here, we used genetic co-essentiality derived from genome-scale RNAi- and CRISPR-Cas9-based fitness screens performed across hundreds of human cancer cell lines to assign measures of functional similarity. From these measures, we systematically built and characterized functional similarity networks that recapitulate known structural and functional features of well-studied protein complexes and resolve novel functional modules within complexes lacking structural resolution, such as the mammalian SWI/SNF complex...
May 14, 2018: Cell Systems
https://www.readbyqxmd.com/read/29777802/derivation-and-characterization-of-a-ucp1-reporter-human-es-cell-line
#3
Suranjit Mukherjee, Tuo Zhang, Lauretta A Lacko, Lei Tan, Jenny Zhaoying Xiang, Jason M Butler, Shuibing Chen
Interest in human brown fat as a novel therapeutic target to tackle the growing obesity and diabetes epidemic has increased dramatically in recent years. While much insight into brown fat biology has been gained from murine cell lines and models, few resources are available to study human brown fat in vitro, which makes the need for new ways to derive and study human brown adipocytes imperative. Human ES cell based reporter systems present an excellent tool to identify, mark, and purify cell populations of choice...
April 22, 2018: Stem Cell Research
https://www.readbyqxmd.com/read/29776993/tumor-immune-evasion-arises-through-loss-of-tnf-sensitivity
#4
Conor J Kearney, Stephin J Vervoort, Simon J Hogg, Kelly M Ramsbottom, Andrew J Freeman, Najoua Lalaoui, Lizzy Pijpers, Jessica Michie, Kristin K Brown, Deborah A Knight, Vivien Sutton, Paul A Beavis, Ilia Voskoboinik, Phil K Darcy, John Silke, Joseph A Trapani, Ricky W Johnstone, Jane Oliaro
Immunotherapy has revolutionized outcomes for cancer patients, but the mechanisms of resistance remain poorly defined. We used a series of whole-genome clustered regularly interspaced short palindromic repeat (CRISPR)-based screens performed in vitro and in vivo to identify mechanisms of tumor immune evasion from cytotoxic lymphocytes [CD8+ T cells and natural killer (NK) cells]. Deletion of key genes within the tumor necrosis factor (TNF) signaling, interferon-γ (IFN-γ) signaling, and antigen presentation pathways provided protection of tumor cells from CD8+ T cell-mediated killing and blunted antitumor immune responses in vivo...
May 18, 2018: Science Immunology
https://www.readbyqxmd.com/read/29769725/mxra8-is-a-receptor-for-multiple-arthritogenic-alphaviruses
#5
Rong Zhang, Arthur S Kim, Julie M Fox, Sharmila Nair, Katherine Basore, William B Klimstra, Rebecca Rimkunas, Rachel H Fong, Hueylie Lin, Subhajit Poddar, James E Crowe, Benjamin J Doranz, Daved H Fremont, Michael S Diamond
Arthritogenic alphaviruses comprise a group of enveloped RNA viruses that are transmitted to humans by mosquitoes and cause debilitating acute and chronic musculoskeletal disease 1 . The host factors required for alphavirus entry remain poorly characterized 2 . Here we use a genome-wide CRISPR-Cas9-based screen to identify the cell adhesion molecule Mxra8 as an entry mediator for multiple emerging arthritogenic alphaviruses, including chikungunya, Ross River, Mayaro and O'nyong nyong viruses. Gene editing of mouse Mxra8 or human MXRA8 resulted in reduced levels of viral infection of cells and, reciprocally, ectopic expression of these genes resulted in increased infection...
May 16, 2018: Nature
https://www.readbyqxmd.com/read/29768208/the-cst-complex-mediates-end-protection-at-double-strand-breaks-and-promotes-parp-inhibitor-sensitivity-in-brca1-deficient-cells
#6
Marco Barazas, Stefano Annunziato, Stephen J Pettitt, Inge de Krijger, Hind Ghezraoui, Stefan J Roobol, Catrin Lutz, Jessica Frankum, Fei Fei Song, Rachel Brough, Bastiaan Evers, Ewa Gogola, Jinhyuk Bhin, Marieke van de Ven, Dik C van Gent, Jacqueline J L Jacobs, Ross Chapman, Christopher J Lord, Jos Jonkers, Sven Rottenberg
Selective elimination of BRCA1-deficient cells by inhibitors of poly(ADP-ribose) polymerase (PARP) is a prime example of the concept of synthetic lethality in cancer therapy. This interaction is counteracted by the restoration of BRCA1-independent homologous recombination through loss of factors such as 53BP1, RIF1, and REV7/MAD2L2, which inhibit end resection of DNA double-strand breaks (DSBs). To identify additional factors involved in this process, we performed CRISPR/SpCas9-based loss-of-function screens and selected for factors that confer PARP inhibitor (PARPi) resistance in BRCA1-deficient cells...
May 15, 2018: Cell Reports
https://www.readbyqxmd.com/read/29766738/the-role-of-gene-editing-in-neurodegenerative-diseases
#7
Hueng-Chuen Fan, Ching-Shiang Chi, Yih-Jing Lee, Jeng-Dau Tsai, Shinn-Zong Lin, Horng-Jyh Harn
Neurodegenerative diseases (NDs), at least including Alzheimer's, Huntington's, and Parkinson's diseases, have become the most dreaded maladies because there are no precise diagnostic tools or definite treatments for these debilitating diseases. The increased prevalence and a substantial impact on the social-economic and medical care of NDs propel governments to develop policies to counteract the impact. Although the etiologies of NDs are still unknown, growing evidence suggests that genetic, cellular, and circuit alternations may cause the generation of abnormal misfolded proteins, which uncontrolledly accumulate to damage and eventually overwhelm the protein-disposal mechanisms of these neurons, leading to a common pathological feature of NDs...
January 1, 2018: Cell Transplantation
https://www.readbyqxmd.com/read/29765036/a-crispri-screen-in-e-coli-reveals-sequence-specific-toxicity-of-dcas9
#8
Lun Cui, Antoine Vigouroux, François Rousset, Hugo Varet, Varun Khanna, David Bikard
High-throughput CRISPR-Cas9 screens have recently emerged as powerful tools to decipher gene functions and genetic interactions. Here we use a genome-wide library of guide RNAs to direct the catalytically dead Cas9 (dCas9) to block gene transcription in Escherichia coli. Using a machine-learning approach, we reveal that guide RNAs sharing specific 5-nucleotide seed sequences can produce strong fitness defects or even kill E. coli regardless of the other 15 nucleotides of guide sequence. This effect occurs at high dCas9 concentrations and can be alleviated by tuning the expression of dCas9 while maintaining strong on-target repression...
May 15, 2018: Nature Communications
https://www.readbyqxmd.com/read/29762716/crispor-intuitive-guide-selection-for-crispr-cas9-genome-editing-experiments-and-screens
#9
Jean-Paul Concordet, Maximilian Haeussler
CRISPOR.org is a web tool for genome editing experiments with the CRISPR-Cas9 system. It finds guide RNAs in an input sequence and ranks them according to different scores that evaluate potential off-targets in the genome of interest and predict on-target activity. The list of genomes is continuously expanded, with more 150 genomes added in the last two years. CRISPOR tries to provide a comprehensive solution from selection, cloning and expression of guide RNA as well as providing primers needed for testing guide activity and potential off-targets...
May 14, 2018: Nucleic Acids Research
https://www.readbyqxmd.com/read/29761864/high-throughput-detection-and-screening-of-plantsmodified-by-geneediting-using-quantitative-real-time-pcr
#10
Cheng Peng, Hua Wang, Xiaoli Xu, Xiaofu Wang, Xiaoyun Chen, Wei Wei, Yongmin Lai, Guoquan Liu, Godwin Ian, Jieqin Li, Ling Zhang, Junfeng Xu
Gene editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been developed to detect gene-edited organisms, these techniques are time- and labour-intensive. Meanwhile, few studies have investigated high-throughput detection and screening strategies for plants modified by gene editing. In this study, we developed a simple, sensitive and high-throughput quantitative real-time(qPCR)-based method. The qPCR-based methodexploits two differently labelled probes that are placed within one amplicon at the gene editing target site to simultaneously detect the wild type and a gene-edited mutant...
May 15, 2018: Plant Journal: for Cell and Molecular Biology
https://www.readbyqxmd.com/read/29760161/ctcf-boundary-remodels-chromatin-domain-and-drives-aberrant-hox-gene-transcription-in-acute-myeloid-leukemia
#11
Huacheng Luo, Fei Wang, Jie Zha, Haoli Li, Bowen Yan, Qinghua Du, Fengchun Yang, Amin Sobh, Christopher Vulpe, Leylah Drusbosky, Christopher Cogle, Iouri Chepelev, Bing Xu, Stephen D Nimer, Jonathan Licht, Yi Qiu, Baoan Chen, Mingjiang Xu, Suming Huang
HOX gene dysregulation is a common feature of acute myeloid leukemia (AML). The molecular mechanisms underlying aberrant HOX gene expression and associated AML pathogenesis remain unclear. The nuclear protein CCCTC-binding factor (CTCF), when bound to insulator sequences, constrains temporal HOX gene expression patterns within confined chromatin domains for normal development. Here, we employed targeted pooled CRISPR-Cas9 knockout library screening to interrogate the function of CTCF boundaries in the HOX gene loci...
May 14, 2018: Blood
https://www.readbyqxmd.com/read/29759937/identification-of-epigenetic-regulators-of-dux4-fl-for-targeted-therapy-of-facioscapulohumeral-muscular-dystrophy
#12
Charis L Himeda, Takako I Jones, Ching-Man Virbasius, Lihua Julie Zhu, Michael R Green, Peter L Jones
Facioscapulohumeral muscular dystrophy (FSHD) is caused by epigenetic de-repression of the disease locus, leading to pathogenic misexpression of the DUX4 gene in skeletal muscle. While the factors and pathways involved in normal repression of the FSHD locus in healthy cells have been well characterized, very little is known about those responsible for the aberrant activation of DUX4-fl in FSHD myocytes. Reasoning that DUX4-fl activators might represent useful targets for small molecule inhibition, we performed a highly targeted, candidate-based screen of epigenetic regulators in primary FSHD myocytes...
April 26, 2018: Molecular Therapy: the Journal of the American Society of Gene Therapy
https://www.readbyqxmd.com/read/29757293/a-rapid-and-facile-pipeline-for-generating-genomic-point-mutants-in-c-elegans-using-crispr-cas9-ribonucleoproteins
#13
Harriet Prior, Lauren MacConnachie, Jose L Martinez, Georgina C B Nicholl, Asim A Beg
The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals...
April 30, 2018: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29756770/versatile-high-throughput-fluorescence-assay-for-monitoring-cas9-activity
#14
Kyle Seamon, Yooli K Light, Edwin Saada, Joseph S Schoeniger, Brooke Harmon
The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of CRISPR systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, under-scoring the need for therapeutically-effective inhibitors of Cas9...
May 14, 2018: Analytical Chemistry
https://www.readbyqxmd.com/read/29754226/construction-of-crispr-libraries-for-functional-screening
#15
Carsten P Carstens, Katherine A Felts, Sarah E Johns
Identification of gene function has been aided by the ability to generate targeted gene knockouts or transcriptional repression using the CRISPR/CAS9 system. Using pooled libraries of guide RNA expression vectors that direct CAS9 to a specific genomic site allows identification of genes that are either enriched or depleted in response to a selection scheme, thus linking the affected gene to the chosen phenotype. The quality of the data generated by the screening is dependent on the quality of the guide RNA delivery library with regards to error rates and especially evenness of distribution of the guides...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29754224/transformation-of-an-exotic-yeast-species-into-a-platform-organism-a-case-study-for-engineering-glycolipid-production-in-the-yeast-starmerella-bombicola
#16
Sofie Lodens, Marilyn De Graeve, Sophie L K W Roelants, Sofie L De Maeseneire, Wim Soetaert
In this chapter, a step-by-step approach on how to transform non-conventional yeasts or fungi into platform organisms is described. The non-conventional glycolipid producing yeast Starmerella bombicola (and in some cases also Pseudohyphozyma bogoriensis) is used as a case study. And more specifically how to engineer it toward production of new-to-nature glycolipids like bola sophorolipids. When starting genetic engineering efforts for non-lab strains, one should start at the very basis: identifying selection markers and possibly developing auxotrophic strains...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29748565/genome-wide-and-high-density-crispr-cas9-screens-identify-point-mutations-in-parp1-causing-parp-inhibitor-resistance
#17
Stephen J Pettitt, Dragomir B Krastev, Inger Brandsma, Amy Dréan, Feifei Song, Radoslav Aleksandrov, Maria I Harrell, Malini Menon, Rachel Brough, James Campbell, Jessica Frankum, Michael Ranes, Helen N Pemberton, Rumana Rafiq, Kerry Fenwick, Amanda Swain, Sebastian Guettler, Jung-Min Lee, Elizabeth M Swisher, Stoyno Stoynov, Kosuke Yusa, Alan Ashworth, Christopher J Lord
Although PARP inhibitors (PARPi) target homologous recombination defective tumours, drug resistance frequently emerges, often via poorly understood mechanisms. Here, using genome-wide and high-density CRISPR-Cas9 "tag-mutate-enrich" mutagenesis screens, we identify close to full-length mutant forms of PARP1 that cause in vitro and in vivo PARPi resistance. Mutations both within and outside of the PARP1 DNA-binding zinc-finger domains cause PARPi resistance and alter PARP1 trapping, as does a PARP1 mutation found in a clinical case of PARPi resistance...
May 10, 2018: Nature Communications
https://www.readbyqxmd.com/read/29746416/crispr-genome-surgery-in-the-retina-in-light-of-off-targeting
#18
Galaxy Y Cho, Kellie A Schaefer, Alexander G Bassuk, Stephen H Tsang, Vinit B Mahajan
PURPOSE: Recent concerns regarding the clinical utilization of clustered regularly interspaced short palindromic repeats (CRISPR) involve uncertainties about the potential detrimental effects that many arise due to unintended genetic changes, as in off-target mutagenesis, during CRISPR genome surgery. This review gives an overview of off-targeting detection methods and CRISPR's place in the clinical setting, specifically in the field of ophthalmology. RESULTS: As CRISPR utilization in the laboratory setting has increased, knowledge regarding CRISPR mechanisms including its off-target effects has also increased...
May 7, 2018: Retina
https://www.readbyqxmd.com/read/29743638/screen-and-verification-for-transgene-integration-sites-in-pigs
#19
Linyuan Ma, Yuzhe Wang, Haitao Wang, Yiqing Hu, Jingyao Chen, Tan Tan, Man Hu, Xiaojuan Liu, Ran Zhang, Yiming Xing, Yiqiang Zhao, Xiaoxiang Hu, Ning Li
Efficient transgene expression in recipient cells constitutes the primary step in gene therapy. However, random integration in host genome comprises too many uncertainties. Our study presents a strategy combining bioinformatics and functional verification to find transgene integration sites in pig genome. Using an in silico approach, we screen out two candidate sites, namely, Pifs302 and Pifs501, located in actively transcribed intergenic regions with low nucleosome formation potential and without potential non-coding RNAs...
May 9, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29742805/novel-genetic-activation-screening-in-liver-repopulation-and-cancer-now-crispr-than-ever
#20
EDITORIAL
Morgan Preziosi, Satdarshan P Monga
No abstract text is available yet for this article.
May 9, 2018: Hepatology: Official Journal of the American Association for the Study of Liver Diseases
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