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Genome scale screen

Meng Cai, Sitao Li, Yunfei Shuai, Jie Li, Jieqiong Tan, Qiyi Zeng
Tumor necrosis factor α (TNF-α), a pivotal cytokine in sepsis, protects the host against pathogens by promoting an inflammatory response while simultaneously inducing apoptosis of the vascular endothelium. Unfortunately, inhibitors targeting certain components of the TNF-α signaling pathway to reduce cellular apoptosis have failed to translate into clinical applications, partly due to the adverse effects of excessive immunosuppression. In an attempt to discover potential targets in the TNF-α signaling pathway to modulate moderate inflammation and apoptosis during the development of sepsis, we performed a pooled genome-wide CRISPR/Cas9 knockout screen in human umbilical vein endothelial cells (HUVECs)...
October 14, 2018: Journal of Cellular Physiology
Michael Inouye, Gad Abraham, Christopher P Nelson, Angela M Wood, Michael J Sweeting, Frank Dudbridge, Florence Y Lai, Stephen Kaptoge, Marta Brozynska, Tingting Wang, Shu Ye, Thomas R Webb, Martin K Rutter, Ioanna Tzoulaki, Riyaz S Patel, Ruth J F Loos, Bernard Keavney, Harry Hemingway, John Thompson, Hugh Watkins, Panos Deloukas, Emanuele Di Angelantonio, Adam S Butterworth, John Danesh, Nilesh J Samani
BACKGROUND: Coronary artery disease (CAD) has substantial heritability and a polygenic architecture. However, the potential of genomic risk scores to help predict CAD outcomes has not been evaluated comprehensively, because available studies have involved limited genomic scope and limited sample sizes. OBJECTIVES: This study sought to construct a genomic risk score for CAD and to estimate its potential as a screening tool for primary prevention. METHODS: Using a meta-analytic approach to combine large-scale, genome-wide, and targeted genetic association data, we developed a new genomic risk score for CAD (metaGRS) consisting of 1...
October 16, 2018: Journal of the American College of Cardiology
Brian Gibson, Virve Vidgren, Gopal Peddinti, Kristoffer Krogerus
Diacetyl contributes to the flavor profile of many fermented products. Its typical buttery flavor is considered as an off flavor in lager-style beers, and its removal has a major impact on time and energy expenditure in breweries. Here, we investigated the possibility of lowering beer diacetyl levels through evolutionary engineering of lager yeast for altered synthesis of α-acetolactate, the precursor of diacetyl. Cells were exposed repeatedly to a sub-lethal level of chlorsulfuron, which inhibits the acetohydroxy acid synthase responsible for α-acetolactate production...
October 10, 2018: Journal of Industrial Microbiology & Biotechnology
Violaine Dubois, Albertus Viljoen, Laura Laencina, Vincent Le Moigne, Audrey Bernut, Faustine Dubar, Mickaël Blaise, Jean-Louis Gaillard, Yann Guérardel, Laurent Kremer, Jean-Louis Herrmann, Fabienne Girard-Misguich
Mycobacterium abscessus is a peculiar rapid-growing Mycobacterium (RGM) capable of surviving within eukaryotic cells thanks to an arsenal of virulence genes also found in slow-growing mycobacteria (SGM), such as Mycobacterium tuberculosis A screen based on the intracellular survival in amoebae and macrophages (MΦ) of an M. abscessus transposon mutant library revealed the important role of MAB_0855, a yet uncharacterized Mycobacterial membrane protein Large (MmpL). Large-scale comparisons with SGM and RGM genomes uncovered MmpL12 proteins as putative orthologs of MAB_0855 and a locus-scale synteny between the MAB_0855 and Mycobacterium chelonae mmpL8 loci...
October 9, 2018: Proceedings of the National Academy of Sciences of the United States of America
Pamela Y Ting, Albert E Parker, J Scott Lee, Chris Trussell, Orzala Sharif, Fabio Luna, Glenn Federe, S Whitney Barnes, John R Walker, Julie Vance, Mu-Yun Gao, Heath E Klock, Scott Clarkson, Carsten Russ, Loren J Miraglia, Michael P Cooke, Anthony E Boitano, Peter McNamara, John Lamb, Christian Schmedt, Jennifer L Snead
CRISPR-Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells-an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR-Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA...
October 8, 2018: Nature Methods
Timothy P Daley, Zhixiang Lin, Xueqiu Lin, Yanxia Liu, Wing Hung Wong, Lei S Qi
Pooled CRISPR screens allow researchers to interrogate genetic causes of complex phenotypes at the genome-wide scale and promise higher specificity and sensitivity compared to competing technologies. Unfortunately, two problems exist, particularly for CRISPRi/a screens: variability in guide efficiency and large rare off-target effects. We present a method, CRISPhieRmix, that resolves these issues by using a hierarchical mixture model with a broad-tailed null distribution. We show that CRISPhieRmix allows for more accurate and powerful inferences in large-scale pooled CRISPRi/a screens...
October 8, 2018: Genome Biology
Yi Zhao, Guili Song, Jing Ren, Qing Li, Shan Zhong, Zongbin Cui
Saturation mutagenesis of all endogenous genes within the mouse genome remains a challenging task, although a plenty of gene-editing approaches are available for this purpose. Here, a poly(A)-trap vector was generated for insertion mutagenesis in mouse embryonic stem (mES) cells. This vector contains an expression cassette of neomycin (Neo)-resistant gene lacking a poly(A) signal and flanked by two inverted terminal repeats of the Sleeping Beauty (SB) transposon. The whole poly(A)-trap cassette can transpose into target TA dinucleotides, properly splice with endogenous genes and effectively interrupt the transcription of trapped genes in mES cells after transient induction of SB expression by doxycycline (DOX)-treatment at 1 μg/ml, leading to the formation of multiple geneticin (G418)-resistant cell clones...
October 2, 2018: Environmental and Molecular Mutagenesis
C Koriath, J Kenny, G Adamson, R Druyeh, W Taylor, J Beck, L Quinn, T H Mok, A Dimitriadis, P Norsworthy, N Bass, J Carter, Z Walker, C Kipps, E Coulthard, J M Polke, M Bernal-Quiros, N Denning, R Thomas, R Raybould, J Williams, C J Mummery, E J Wild, H Houlden, S J Tabrizi, M N Rossor, H Hummerich, J D Warren, J B Rowe, J D Rohrer, J M Schott, N C Fox, J Collinge, S Mead
Next-generation genetic sequencing (NGS) technologies facilitate the screening of multiple genes linked to neurodegenerative dementia, but there are few reports about their use in clinical practice. Which patients would most profit from testing, and information on the likelihood of discovery of a causal variant in a clinical syndrome, are conspicuously absent from the literature, mostly for a lack of large-scale studies. We applied a validated NGS dementia panel to 3241 patients with dementia and healthy aged controls; 13,152 variants were classified by likelihood of pathogenicity...
October 2, 2018: Molecular Psychiatry
R Craig Albertson, Kenta C Kawasaki, Emily R Tetrault, Kara E Powder
Elasmoid scales are the most common epithelial appendage among vertebrates, however an understanding of the genetic mechanisms that underlie variation in scale shape is lacking. Using an F2 mapping cross between morphologically distinct cichlid species, we identified >40 QTL for scale shape at different body positions. We show that while certain regions of the genome regulate variation in multiple scales, most are specific to scales at distinct positions. This suggests a degree of regional modularity in scale development...
2018: Communications biology
Maris Alver, Marili Palover, Aet Saar, Kristi Läll, Seyedeh Maryam Zekavat, Neeme Tõnisson, Liis Leitsalu, Anu Reigo, Tiit Nikopensius, Tiia Ainla, Mart Kals, Reedik Mägi, Stacey B Gabriel, Jaan Eha, Eric S Lander, Alar Irs, Anthony Philippakis, Toomas Marandi, Pradeep Natarajan, Andres Metspalu, Sekar Kathiresan, Tõnu Esko
PURPOSE: Large-scale, population-based biobanks integrating health records and genomic profiles may provide a platform to identify individuals with disease-predisposing genetic variants. Here, we recall probands carrying familial hypercholesterolemia (FH)-associated variants, perform cascade screening of family members, and describe health outcomes affected by such a strategy. METHODS: The Estonian Biobank of Estonian Genome Center, University of Tartu, comprises 52,274 individuals...
October 1, 2018: Genetics in Medicine: Official Journal of the American College of Medical Genetics
Anbu K Karuppannan, Tanja Opriessnig
Routine large-scale xenotransplantation from pigs to humans is getting closer to clinical reality owing to several state-of-the-art technologies, especially the ability to rapidly engineer genetically defined pigs. However, using pig organs in humans poses risks including unwanted cross-species transfer of viruses and adaption of these pig viruses to the human organ recipient. Recent developments in the field of virology, including the advent of metagenomic techniques to characterize entire viromes, have led to the identification of a plethora of viruses in many niches...
July 2018: Xenotransplantation
Richard Preiss, Caroline Tyrawa, Kristoffer Krogerus, Lars Marius Garshol, George van der Merwe
The widespread production of fermented food and beverages has resulted in the domestication of Saccharomyces cerevisiae yeasts specifically adapted to beer production. While there is evidence beer yeast domestication was accelerated by industrialization of beer, there also exists a farmhouse brewing culture in western Norway which has passed down yeasts referred to as kveik for generations. This practice has resulted in ale yeasts which are typically highly flocculant, phenolic off flavor negative (POF-), and exhibit a high rate of fermentation, similar to previously characterized lineages of domesticated yeast...
2018: Frontiers in Microbiology
Rachel M Guerra, Gregory H Bird, Edward P Harvey, Neekesh V Dharia, Kyle J Korshavn, Michelle S Prew, Kimberly Stegmaier, Loren D Walensky
Cancer cells overexpress a diversity of anti-apoptotic BCL-2 family proteins, such as BCL-2, MCL-1, and BFL-1/A1, to enforce cellular immortality. Thus, intensive drug development efforts have focused on targeting this class of oncogenic proteins to overcome treatment resistance. Whereas a selective BCL-2 inhibitor has been FDA approved and several small molecule inhibitors of MCL-1 have recently entered phase I clinical testing, BFL-1/A1 remains undrugged. Here, we developed a series of stapled peptide design principles to engineer a functionally selective and cell-permeable BFL-1/A1 inhibitor that is specifically cytotoxic to BFL-1/A1-dependent human cancer cells...
September 25, 2018: Cell Reports
Zeynep Hakguder, Jiang Shu, Chunxiao Liao, Kaiyue Pan, Juan Cui
BACKGROUND: MicroRNA regulation is fundamentally responsible for fine-tuning the whole gene network in human and has been implicated in most physiological and pathological conditions. Studying regulatory impact of microRNA on various cellular and disease processes has resulted in numerous computational tools that investigate microRNA-mRNA interactions through the prediction of static binding site highly dependent on sequence pairing. However, what hindered the practical use of such target prediction is the interplay between competing and cooperative microRNA binding that complicates the whole regulatory process exceptionally...
September 24, 2018: BMC Genomics
Brian T Emmer, Geoffrey G Hesketh, Emilee Kotnik, Vi T Tang, Paul J Lascuna, Jie Xiang, Anne-Claude Gingras, Xiao-Wei Chen, David Ginsburg
PCSK9 is a secreted protein that regulates plasma cholesterol levels and cardiovascular disease risk. Prior studies suggested the presence of an ER cargo receptor that recruits PCSK9 into the secretory pathway, but its identity has remained elusive. Here, we apply a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify SURF4, a homologue of the yeast cargo receptor Erv29p, as a primary mediator of PCSK9 secretion in HEK293T cells...
September 25, 2018: ELife
Sandeep Grover, Fabiola Del Greco M, Inke R König
BACKGROUND: Mendelian randomization (MR) is fast becoming a popular method to judge causality from routinely conducted observational studies. However, stringent underlying statistical assumptions, missing biological information, and high sample size requirement might make it prone to misuse. Furthermore, rapidly updating methodologies and increasingly available datasets to researchers are making the interpretations of heterogeneous results even more complicated. In this protocol, we provide our design for a multifaceted systematic review on MR studies using neurodegenerative disease as an example outcome...
September 24, 2018: Systematic Reviews
Chundi Gao, Jing Zhuang, Chao Zhou, Ke Ma, Minzhang Zhao, Cun Liu, Lijuan Liu, Huayao Li, Fubin Feng, Changgang Sun
Currently, research on genome-scale epigenetic modifications for studying the pathogenesis of lung cancer is lacking. Aberrant DNA methylation, as the most common and important modification in epigenetics, is an important means of regulating genomic function and can be used as a biomarker for the diagnosis and prognosis of lung squamous cell carcinoma (LUSC). In this paper, methylation information and gene expression data from patients with LUSC were extracted from the TCGA database. Univariate and multivariate COX analyses were used to screen abnormally methylated genes related to the prognosis of LUSC...
September 24, 2018: Journal of Cellular Physiology
Cristina Parola, Derek M Mason, Andreas Zingg, Daniel Neumeier, Sai T Reddy
From the perspective of academic and small research laboratories, the most common and practical strategy for recombinant expression of full-length monoclonal antibodies is to perform transient plasmid transfection of mammalian cells, resulting in small-scale and limited protein production. The generation of stable antibody producing mammalian cell lines enables larger-scale and consistent expression, however this approach is rarely pursued due to the time-consuming and expensive process of single colony screening and characterization...
2018: Methods in Molecular Biology
Tianmin Wang, Jiahui Guo, Yangyang Liu, Zhenglian Xue, Chong Zhang, Xin-Hui Xing
Terpenoids are a large family of natural compounds that are important for both biotechnological applications and basic microorganism physiology. Inspired by the current literature, we hypothesized that recently deciphered phosphatase promiscuity may be an unexplored factor that negatively affects terpenoid biosynthesis by redirecting carbon flux away from the pathway via unrecognized catalytic activities on the phosphorylated intermediates. We used lycopene as a proof-of-concept to test this hypothesis. Based on an extensive bioinformatics analysis, we selected 56 phosphatase-encoding genes in Escherichia coli and constructed a knockdown library for these genes in a lycopene overproducer via CRISPR interference (CRISPRi)...
September 18, 2018: Applied Microbiology and Biotechnology
R Blake Richardson, Maikke B Ohlson, Jennifer L Eitson, Ashwani Kumar, Matthew B McDougal, Ian N Boys, Katrina B Mar, Pamela C De La Cruz-Rivera, Connor Douglas, Genevieve Konopka, Chao Xing, John W Schoggins
The endoplasmic reticulum (ER) is an architecturally diverse organelle that serves as a membrane source for the replication of multiple viruses. Flaviviruses, including yellow fever virus, West Nile virus, dengue virus and Zika virus, induce unique single-membrane ER invaginations that house the viral replication machinery1 . Whether this virus-induced ER remodelling is vulnerable to antiviral pathways is unknown. Here, we show that flavivirus replication at the ER is targeted by the interferon (IFN) response...
September 17, 2018: Nature Microbiology
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