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https://www.readbyqxmd.com/read/29467311/epstein-barr-virus-nuclear-antigen-leader-protein-co-activates-ep300
#1
Chong Wang, Hufeng Zhou, Yong Xue, Jun Liang, Yohei Narita, Catherine Gerdt, Amy Y Zheng, Runsheng Jiang, Stephen Trudeau, Chih-Wen Peng, Benjamin Gewurz, Bo Zhao
Epstein-Barr virus nuclear antigen (EBNA) leader protein (EBNALP) is one of the first viral genes expressed upon B-cell infection. EBNALP is essential for EBV-mediated B cell immortalization. EBNALP is thought to function primarily by co-activaing EBNA2-mediated transcription. Chromatin immune precipitation followed by deep-sequencing (ChIP-seq) studies highlight that EBNALP frequently co-occupies DNA sites with host cell transcription factors (TFs), in particular EP300, implicating a broader role in transcription regulation...
February 21, 2018: Journal of Virology
https://www.readbyqxmd.com/read/29446747/crispr-cas9-genome-editing-a-promising-tool-for-therapeutic-applications-of-induced-pluripotent-stem-cells
#2
Yanli Zhang, Danuta Sastre, Feng Wang
Induced pluripotent stem cells hold tremendous potential for biological and therapeutic applications. The development of efficient technologies for the targeted genome alteration of stem cells in disease models is a prerequisite for utilizing stem cells to their full potential. The revolutionary technology for genome editing known as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) system is recently recognized as a powerful tool for editing DNA at specific loci...
February 14, 2018: Current Stem Cell Research & Therapy
https://www.readbyqxmd.com/read/29444854/bet-bromodomain-proteins-regulate-enhancer-function-during-adipogenesis
#3
Jonathan D Brown, Zachary B Feldman, Sean P Doherty, Jaime M Reyes, Peter B Rahl, Charles Y Lin, Quanhu Sheng, Qiong Duan, Alexander J Federation, Andrew L Kung, Saptarsi M Haldar, Richard A Young, Jorge Plutzky, James E Bradner
Developmental transitions are guided by master regulatory transcription factors. During adipogenesis, a transcriptional cascade culminates in the expression of PPARγ and C/EBPα, which orchestrate activation of the adipocyte gene expression program. However, the coactivators controlling PPARγ and C/EBPα expression are less well characterized. Here, we show the bromodomain-containing protein, BRD4, regulates transcription of PPARγ and C/EBPα. Analysis of BRD4 chromatin occupancy reveals that induction of adipogenesis in 3T3L1 fibroblasts provokes dynamic redistribution of BRD4 to de novo super-enhancers proximal to genes controlling adipocyte differentiation...
February 14, 2018: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29403034/crispr-interference-based-specific-and-efficient-gene-inactivation-in-the-brain
#4
Yi Zheng, Wei Shen, Jian Zhang, Bo Yang, Yao-Nan Liu, Huihui Qi, Xia Yu, Si-Yao Lu, Yun Chen, Yu-Zhou Xu, Yun Li, Fred H Gage, Shuangli Mi, Jun Yao
CRISPR-Cas9 has been demonstrated to delete genes in postmitotic neurons. Compared to the establishment of proliferative cell lines or animal strains, it is more challenging to acquire a highly homogeneous consequence of gene editing in a stable neural network. Here we show that dCas9-based CRISPR interference (CRISPRi) can efficiently silence genes in neurons. Using a pseudotarget fishing strategy, we demonstrate that CRISPRi shows superior targeting specificity without detectable off-target activity. Furthermore, CRISPRi can achieve multiplex inactivation of genes fundamental for neurotransmitter release with high efficiency...
February 5, 2018: Nature Neuroscience
https://www.readbyqxmd.com/read/29385696/crispr-cas9-genetic-analysis-of-virus-host-interactions
#5
REVIEW
Makda Gebre, Jason L Nomburg, Benjamin E Gewurz
Clustered regularly interspaced short palindromic repeats (CRISPR) has greatly expanded the ability to genetically probe virus-host interactions. CRISPR systems enable focused or systematic, genomewide studies of nearly all aspects of a virus lifecycle. Combined with its relative ease of use and high reproducibility, CRISPR is becoming an essential tool in studies of the host factors important for viral pathogenesis. Here, we review the use of CRISPR-Cas9 for the loss-of-function analysis of host dependency factors...
January 30, 2018: Viruses
https://www.readbyqxmd.com/read/29377933/tailor-made-gene-silencing-of-staphylococcus-aureus-clinical-isolates-by-crispr-interference
#6
Yusuke Sato'o, Junzo Hisatsune, Liansheng Yu, Tetsushi Sakuma, Takashi Yamamoto, Motoyuki Sugai
Preparing the genetically modified organisms have required much time and labor, making it the rate-limiting step but CRISPR/Cas9 technology appearance has changed this difficulty. Although reports on CRISPR/Cas9 technology such as genome editing and CRISPR interference (CRISPRi) in eukaryotes increased, those in prokaryotes especially in Staphylococci were limited. Thus, its potential in the bacteriology remains unexplored. This is attributed to ecological difference between eukaryotes and prokaryotes. Here, we constructed a novel CRISPRi plasmid vector, pBACi for Staphylococcus aureus...
2018: PloS One
https://www.readbyqxmd.com/read/29366319/molecular-toolkit-for-gene-expression-control-and-genome-modification-in-rhodococcus-opacus-pd630
#7
Drew M DeLorenzo, Austin G Rottinghaus, William R Henson, Tae Seok Moon
Rhodococcus opacus PD630 is a non-model Gram-positive bacterium that possesses desirable traits for lignocellulosic biomass conversion. In particular, it has a relatively rapid growth rate, exhibits genetic tractability, produces high quantities of lipids, and can tolerate and consume toxic lignin-derived aromatic compounds. Despite these unique, industrially relevant characteristics, R. opacus has been underutilized because of a lack of reliable genetic parts and engineering tools. In this work, we developed a molecular toolbox for reliable gene expression control and genome modification in R...
February 16, 2018: ACS Synthetic Biology
https://www.readbyqxmd.com/read/29359686/targeting-ras-driven-human-cancer-cells-with-antibodies-to-upregulated-and-essential-cell-surface-proteins
#8
Alexander John Martinko, Charles Truillet, Olivier Julien, Juan Diaz, Max A Horlbeck, Gordon Whiteley, Josip Blonder, Jonathan S Weissman, Sourav Bandyopadhyay, Michael Evans, James A Wells
While there have been tremendous efforts to target oncogenic RAS signaling from inside the cell, little effort has focused on the cell-surface. Here, we used quantitative surface proteomics to reveal a signature of proteins that are upregulated on cells transformed with KRASG12V, and driven by MAPK pathway signaling. We next generated a toolkit of recombinant antibodies to seven of these RAS induced proteins. We found that five of these proteins are broadly distributed on cancer cell lines harboring RAS mutations...
January 23, 2018: ELife
https://www.readbyqxmd.com/read/29344811/metabolic-evolution-and-a-comparative-omics-analysis-of-corynebacterium-glutamicum-for-putrescine-production
#9
Zhen Li, Yu-Ping Shen, Xuan-Long Jiang, Li-Shen Feng, Jian-Zhong Liu
Putrescine is widely used in the industrial production of bioplastics, pharmaceuticals, agrochemicals, and surfactants. Because the highest titer of putrescine is much lower than that of its precursor L-ornithine reported in microorganisms to date, further work is needed to increase putrescine production in Corynebacterium glutamicum. We first compared 7 ornithine decarboxylase genes and found that the Enterobacter cloacae ornithine decarboxylase gene speC1 was most suitable for putrescine production in C. glutamicum...
January 17, 2018: Journal of Industrial Microbiology & Biotechnology
https://www.readbyqxmd.com/read/29343791/single-plasmid-systems-for-inducible-dual-protein-expression-and-for-crispr-cas9-crispri-gene-regulation-in-lactic-acid-bacterium-lactococcus-lactis
#10
Aleš Berlec, Katja Škrlec, Janja Kocjan, Maria Olenic, Borut Štrukelj
Lactococcus lactis is a food-grade lactic acid bacterium that is used in the dairy industry as a cell factory and as a host for recombinant protein expression. The nisin-controlled inducible expression (NICE) system is frequently applied in L. lactis; however new tools for its genetic modification are highly desirable. In this work NICE was adapted for dual protein expression. Plasmid pNZDual, that contains two nisin promoters and multiple cloning sites (MCSs), and pNZPolycist, that contains a single nisin promoter and two MCSs separated by the ribosome binding site, were constructed...
January 17, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29337370/modulating-gene-expression-in-epstein-barr-virus-ebv-positive-b-cell-lines-with-crispra-and-crispri
#11
Liang Wei Wang, Stephen J Trudeau, Chong Wang, Catherine Gerdt, Sizun Jiang, Bo Zhao, Benjamin E Gewurz
Epstein-Barr virus (EBV) transforms small resting primary B cells into large lymphoblastoid cells which are able to grow and survive in vitro indefinitely. These cells represent a model for oncogenesis. In this unit, variants of conventional clustered regularly interspaced short palindromic repeats (CRISPR), namely the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) methods, are discussed in the context of gene regulation at genomic DNA promoter and enhancer elements. Lymphoblastoid B cell lines (LCLs) stably expressing nuclease-deficient Cas9 (dCas9)-VP64 (Cas9 associated with CRISPRa) or dCas9-KRAB (Cas9 associated with CRISPRi) are transduced with lentivirus that encodes a single guide RNA (sgRNA) that targets a specific gene locus...
January 16, 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29316926/rna-guided-single-double-gene-repressions-in-corynebacterium-glutamicum-using-an-efficient-crispr-interference-and-its-application-to-industrial-strain
#12
Jaehyun Park, Hyojung Shin, Sun-Mi Lee, Youngsoon Um, Han Min Woo
BACKGROUND: The construction of microbial cell factories requires cost-effective and rapid strain development through metabolic engineering. Recently, RNA-guided CRISPR technologies have been developed for metabolic engineering of industrially-relevant host. RESULTS: To demonstrate the application of the CRISPR interference (CRISPRi), we developed two-plasmid CRISPRi vectors and applied the CRISPRi in Corynebacterium glutamicum to repress single target genes and double target genes simultaneously...
January 9, 2018: Microbial Cell Factories
https://www.readbyqxmd.com/read/29311279/a-robust-crispri-gene-repression-system-in-pseudomonas
#13
Sue Zanne Tan, Christopher R Reisch, Kristala L J Prather
Pseudomonas spp. are widely used model organisms in different areas of research. Despite its relevance in many applications, protein depletion tools in this host remain limited. Here, we developed the CRISPR interference system for gene repression in Pseudomonas spp. using the Streptococcus pasteurianus dCas9. We demonstrate a robust and titratable gene depletion system with up to 100-fold repression in β-galactosidase activity in P. aeruginosa and 300-fold repression in pyoverdine production in P. putida This inducible system enables the study of essential genes, as shown by ftsZ depletions in P...
January 8, 2018: Journal of Bacteriology
https://www.readbyqxmd.com/read/29259518/%C3%A2-genome-surgery-and-gene-therapy-in-retinal-disorders
#14
REVIEW
Lawrence Chan, Vinit B Mahajan, Stephen H Tsang
The emergence of genome surgery techniques like the clustered regularly interspaced short palindromic repeats (CRISPR) editing technology has given researchers a powerful tool for precisely introducing targeted changes within the genome. New modifications to the CRISPR-Cas system have been made since its recent discovery, such as high-fidelity Cas9 variants to reduce off-target effects and transcriptional activation/silencing with CRISPRa/CRISPRi. The applications of CRISPR-Cas and gene therapy in ophthalmic diseases have been necessary and fruitful, especially given the impact of blinding diseases on society and the large number of monogenic disorders of the eye...
December 2017: Yale Journal of Biology and Medicine
https://www.readbyqxmd.com/read/29247166/increasing-the-permeability-of-escherichia-coli-using-mac13243
#15
Claudio Muheim, Hansjörg Götzke, Anna U Eriksson, Stina Lindberg, Ida Lauritsen, Morten H H Nørholm, Daniel O Daley
The outer membrane of gram-negative bacteria is a permeability barrier that prevents the efficient uptake of molecules with large scaffolds. As a consequence, a number of antibiotic classes are ineffective against gram-negative strains. Herein we carried out a high throughput screen for small molecules that make the outer membrane of Escherichia coli more permeable. We identified MAC13243, an inhibitor of the periplasmic chaperone LolA that traffics lipoproteins from the inner to the outer membrane. We observed that cells were (1) more permeable to the fluorescent probe 1-N-phenylnapthylamine, and (2) more susceptible to large-scaffold antibiotics when sub-inhibitory concentrations of MAC13243 were used...
December 15, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29199103/diversion-of-the-long-chain-acyl-acp-pool-in-synechocystis-to-fatty-alcohols-through-crispri-repression-of-the-essential-phosphate-acyltransferase-plsx
#16
Danuta Kaczmarzyk, Ivana Cengic, Lun Yao, Elton P Hudson
Fatty alcohol production in Synechocystis sp. PCC 6803 was achieved through heterologous expression of the fatty acyl-CoA/ACP reductase Maqu2220 from the bacteria Marinobacter aquaeolei VT8 and the fatty acyl-ACP reductase DPW from the rice Oryza sativa. These platform strains became models for testing multiplex CRISPR-interference (CRISPRi) metabolic engineering strategies to both improve fatty alcohol production and to study membrane homeostasis. CRISPRi allowed partial repression of up to six genes simultaneously, each encoding enzymes of acyl-ACP-consuming pathways...
November 30, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/29181009/crispri-induced-suppression-of-fimbriae-gene-fimh-of-a-uropathogenic-escherichia-coli-an-approach-to-inhibit-microbial-biofilms
#17
Azna Zuberi, Nayeem Ahmad, Asad U Khan
Urinary tract infection (UTI) is one the common infections caused by the recalcitrant nature of biofilms, developed after the pathogen has adhered to the inner lining of the urinary tract. Although significant research has been made in recent years to control these types of infection, but as of yet, no approach has sufficiently been able to reduce the prevalence of UTIs. The main objective of this study was to prevent UTIs through targeting the fimH gene, which is the major virulent factor responsible for biofilm formation...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/29174524/engineering-cell-wall-synthesis-mechanism-for-enhanced-phb-accumulation-in-e-coli
#18
Xing-Chen Zhang, Yingying Guo, Xu Liu, Xin-Guang Chen, Qiong Wu, Guo-Qiang Chen
The rigidity of bacterial cell walls synthesized by a complicated pathway limit the cell shapes as coccus, bar or ellipse or even fibers. A less rigid bacterium could be beneficial for intracellular accumulation of poly-3-hydroxybutyrate (PHB) as granular inclusion bodies. To understand how cell rigidity affects PHB accumulation, E. coli cell wall synthesis pathway was reinforced and weakened, respectively. Cell rigidity was achieved by thickening the cell walls via insertion of a constitutive gltA (encoding citrate synthase) promoter in front of a series of cell wall synthesis genes on the chromosome of several E...
November 24, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/29170951/bacterial-genome-editing-strategy-for-control-of-transcription-and-protein-stability
#19
Ida Lauritsen, Virginia Martínez, Carlotta Ronda, Alex Toftgaard Nielsen, Morten H H Nørholm
In molecular biology and cell factory engineering, tools that enable control of protein production and stability are highly important. Here, we describe protocols for tagging genes in Escherichia coli allowing for inducible degradation and transcriptional control of any soluble protein of interest. The underlying molecular biology is based on the two cross-kingdom tools CRISPRi and the N-end rule for protein degradation. Genome editing is performed with the CRMAGE technology and randomization of the translational initiation region minimizes the polar effects of tag insertion...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29158600/crispr-cas9-library-screening-for-drug-target-discovery
#20
REVIEW
Morito Kurata, Kouhei Yamamoto, Branden S Moriarity, Masanobu Kitagawa, David A Largaespada
CRISPR/Cas9-based tools have rapidly developed in recent years. These include CRISPR-based gene activation (CRISPRa) or inhibition (CRISPRi), for which there are libraries. CRISPR libraries for loss of function have been widely used to identify new biological mechanisms, such as drug resistance and cell survival signals. CRISPRa is highly useful in screening for gain of functions, and CRISPRi is a more powerful tool than RNA interference (RNAi) libraries in screening for loss of functions. Positive selection using a CRISPR library can detect survival cells with specific conditions, such as drug treatment, and it can easily clarify drug resistance mechanisms...
November 20, 2017: Journal of Human Genetics
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