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CRISPR selection

Jinzhong Zhang, Jing He, Jennifer L Johnson, Farhana Rahman, Evripidis Gavathiotis, Ana Maria Cuervo, Sergio D Catz
Cystinosis is a lysosomal storage disorder caused by defects in CTNS , the gene that encodes the lysosomal cystine transporter cystinosin. Patients with nephropathic cystinosis are characterized by endocrine defects, defective proximal tubule cell (PTC) function, the development of Fanconi syndrome and, eventually, end-stage renal disease. Kidney disease is developed despite the use of cysteamine, a drug that decreases lysosomal cystine overload but fails to correct overload-independent defects. Chaperone-mediated autophagy (CMA), a selective form of autophagy, is defective in cystinotic mouse fibroblasts, and treatment with cysteamine is unable to correct CMA defects in vivo , but whether the vesicular trafficking mechanisms that lead to defective CMA in cystinosis are manifested in human PTCs is not currently known and whether PTC-specific mechanisms are corrected upon CMA upregulation remains to be elucidated...
2019: Frontiers in Endocrinology
Shannon Kitchen, Ramesh Ratnappan, Suhao Han, Caitlyn Leasure, Emilia Grill, Zahra Iqbal, Olivia Granger, Damien M O'Halloran, John M Hawdon
Soil-transmitted nematodes (STNs) infect over a billion people and place several billion more at risk of infection. Hookworm disease is the most significant of these STNs, with over 500 million people infected. Hookworm infection can result in debilitating and sometimes fatal iron-deficiency anemia, which is particularly devastating in children and pregnant women. Currently, hookworms and other soil-transmitted helminths (STHs) are controlled by administration of a single dose of a benzimidazole to targeted populations in endemic areas...
February 13, 2019: International Journal for Parasitology
Ismail Ben Soussia, Sonia El Mouridi, Dawon Kang, Alice Leclercq-Blondel, Lamyaa Khoubza, Philippe Tardy, Nora Zariohi, Marie Gendrel, Florian Lesage, Eun-Jin Kim, Delphine Bichet, Olga Andrini, Thomas Boulin
Mutations that modulate the activity of ion channels are essential tools to understand the biophysical determinants that control their gating. Here, we reveal the conserved role played by a single amino acid position (TM2.6) located in the second transmembrane domain of two-pore domain potassium (K2P) channels. Mutations of TM2.6 to aspartate or asparagine increase channel activity for all vertebrate K2P channels. Using two-electrode voltage-clamp and single-channel recording techniques, we find that mutation of TM2...
February 15, 2019: Nature Communications
Zhixin Wu, Zhongqiu Chen, Xinyue Gao, Jing Li, Guangdong Shang
Pseudomonas putida KT2440 is a Gram-negative, biosafety strain that plays important roles in environmental and biotechnological applications. Highly efficient genome editing strategy is essential to the elucidation of gene function and construction of metabolic engineered strains. Building on our previously established recombineering-mediated markerless and scarless P. putida KT2440 chromosomal gene deletion methods, herein we combined single-stranded DNA (ssDNA) recombineering and CRISPR-Cas9 technologies for P...
February 14, 2019: Applied Microbiology and Biotechnology
Stevephen Hung, Alina Saiakhova, Zachary J Faber, Cynthia F Bartels, Devin Neu, Ian Bayles, Evelyn Ojo, Ellen S Hong, W Dean Pontius, Andrew R Morton, Ruifu Liu, Matthew F Kalady, David N Wald, Sanford Markowitz, Peter C Scacheri
Commonly-mutated genes have been found for many cancers, but less is known about mutations in cis-regulatory elements. We leverage gains in tumor-specific enhancer activity, coupled with allele-biased mutation detection from H3K27ac ChIP-seq data, to pinpoint potential enhancer-activating mutations in colorectal cancer (CRC). Analysis of a genetically-diverse cohort of CRC specimens revealed that microsatellite instable (MSI) samples have a high indel rate within active enhancers. Enhancers with indels show evidence of positive selection, increased target gene expression, and a subset is highly recurrent...
February 13, 2019: ELife
Nadia Bouhamdani, Dominique Comeau, Kevin Cormier, Sandra Turcotte
Autophagy is a highly conserved, homeostatic process by which cytosolic components reach lysosomes for degradation. The roles that play different autophagic processes in cancer are complex and remain cancer-type and -stage dependent. Renal cell carcinoma (RCC) is the most common subtype of kidney cancer and is characterized by the inactivation of the VHL tumor suppressor. Our previous study identified a small compound, STF-62247 as an autophagy-modulating molecule causing selective cytotoxicity for VHL-inactivated cells...
February 13, 2019: American Journal of Physiology. Cell Physiology
Fabrizio Alberti, Daniel J Leng, Ina Wilkening, Lijiang Song, Manuela Tosin, Christophe Corre
In this study, we report the rapid characterisation of a novel microbial natural product resulting from the rational derepression of a silent gene cluster. A conserved set of five regulatory genes was used as a query to search genomic databases and identify atypical biosynthetic gene clusters (BGCs). A 20-kb BGC from the genetically intractable Streptomyces sclerotialus bacterial strain was captured using yeast-based homologous recombination and introduced into validated heterologous hosts. CRISPR/Cas9-mediated genome editing was then employed to rationally inactivate the key transcriptional repressor and trigger production of an unprecedented class of hybrid natural products exemplified by (2-(benzoyloxy)acetyl)-l-proline, named scleric acid...
January 14, 2019: Chemical Science
Tarik Exner, Inés Romero-Brey, Eden Yifrach, Jhon Rivera-Monroy, Bianca Schrul, Christos C Zouboulis, Wolfgang Stremmel, Masanori Honsho, Ralf Bartenschlager, Einat Zalckvar, Margarete Poppelreuther, Joachim Füllekrug
Fatty acyl-CoA reductase 1 (Far1) is an ubiquitously expressed peroxisomal membrane protein generating fatty alcohols required for the biosynthesis of ether lipids.Lipid droplet localization of exogenously expressed and endogenous human Far1 was observed by fluorescence microscopy under conditions of increased triglyceride synthesis in tissue culture cells. This unexpected finding was supported further by correlative light electron microscopy and subcellular fractionation. Selective permeabilization, protease sensitivity and N-glycosylation tagging suggested that Far1 is able to assume two different membrane topologies, differing in the orientation of the short hydrophilic C-terminus towards the lumen or the cytosol, respectively...
February 11, 2019: Journal of Cell Science
Sandeep Soni, Donald B Kohn
Gene modification of hematopoietic stem cells is increasingly becoming popular as a therapeutic approach, given the recent approvals and the number of new applications for clinical trials targeting monogenetic and immunodeficiency disorders. Technological advances in stem cell selection, culture, transduction and gene editing now allow for efficient ex vivo genetic manipulation of stem cells. Gene-addition techniques using viral vectors (mainly retrovirus- and lentivirus-based) and gene editing using various targeted nuclease platforms (e...
February 8, 2019: Cytotherapy
Elaine M Morazzani, Jaimee R Compton, Dagmar H Leary, Angela V Berry, Xin Hu, Juan Marugan, Pamela J Glass, Patricia M Legler
The alphaviral nonstructural protein 2 (nsP2) cysteine proteases (EC 3.4.22.-) are essential for the proteolytic processing of the nonstructural (ns) polyprotein and are validated drug targets. A common secondary role of these proteases is to antagonize the effects of interferon (IFN). After delineating the cleavage site motif of the Venezuelan equine encephalitis virus (VEEV) nsP2 cysteine protease, we searched the human genome to identify host protein substrates. Here we identify a new host substrate of the VEEV nsP2 protease, human TRIM14, a component of the mitochondrial antiviral-signaling protein (MAVS) signalosome...
February 8, 2019: Antiviral Research
Rebecca E McKenzie, Cristóbal Almendros, Jochem N A Vink, Stan J J Brouns
CRISPR-Cas systems are able to acquire immunological memories (spacers) from bacteriophages and plasmids in order to survive infection; however, this often occurs at low frequency within a population, which can make it difficult to detect. Here we describe CAPTURE (CRISPR adaptation PCR technique using reamplification and electrophoresis), a versatile and adaptable protocol to detect spacer-acquisition events by electrophoresis imaging with high-enough sensitivity to identify spacer acquisition in 1 in 105 cells...
February 11, 2019: Nature Protocols
Matthias Höllerhage, Marc Bickle, Günter U Höglinger
PURPOSE OF REVIEW: We provide an overview about unbiased screens to identify modifiers of alpha-synuclein (αSyn)-induced toxicity, present the models and the libraries that have been used for screening, and describe how hits from primary screens were selected and validated. RECENT FINDINGS: Screens can be classified as either genetic or chemical compound modifier screens, but a few screens do not fit this classification. Most screens addressing αSyn-induced toxicity, including genome-wide overexpressing and deletion, were performed in yeast...
February 9, 2019: Current Neurology and Neuroscience Reports
Thomas Gassler, Lina Heistinger, Diethard Mattanovich, Brigitte Gasser, Roland Prielhofer
State-of-the-art strain engineering techniques for the methylotrophic yeast Pichia pastoris (syn. Komagataella spp.) include overexpression of endogenous and heterologous genes and deletion of host genes. For efficient gene deletion, methods such as the split-marker technique have been established. However, synthetic biology trends move toward building up large and complex reaction networks, which often require endogenous gene knockouts and simultaneous overexpression of individual genes or whole pathways...
2019: Methods in Molecular Biology
Cara E Moravec, Francisco J Pelegri
The ability to create targeted mutations in specific genes, and therefore a loss-of-function condition, provides essential information about their endogenous functions during development and homeostasis. The discovery that CRISPR-Cas9 can target specific sequences according to base-pair complementarity and readily create knockouts in a desired gene has elevated the implementation of genetic analysis in numerous organisms. As CRISPR-Cas9 has become a powerful tool in a number of species, multiple methods for designing, creating, and screening editing efficiencies have been published, each of which has unique benefits...
2019: Methods in Molecular Biology
Ruben V Uribe, Eric van der Helm, Maria-Anna Misiakou, Sang-Woo Lee, Stefan Kol, Morten O A Sommer
CRISPR-Cas systems in bacteria and archaea provide immunity against bacteriophages and plasmids. To overcome CRISPR immunity, phages have acquired anti-CRISPR genes that reduce CRISPR-Cas activity. Using a synthetic genetic circuit, we developed a high-throughput approach to discover anti-CRISPR genes from metagenomic libraries based on their functional activity rather than sequence homology or genetic context. We identified 11 DNA fragments from soil, animal, and human metagenomes that circumvent Streptococcus pyogenes Cas9 activity in our selection strain...
January 31, 2019: Cell Host & Microbe
Julia A Guzova, Michael J Primiano, Aiping Jiao, Jeffrey Stock, Chiachin Lee, Aaron R Winkler, J Perry Hall
The NLRP3 inflammasome is a complex multimeric signaling apparatus that regulates production of the pro-inflammatory cytokine IL-1β. To overcome both the variability among primary immune cells and the limitations of genetic manipulation of differentiated human or murine macrophages, we developed a simplified, reliable and relevant cell-based model for studying the NLRP3 inflammasome using the undifferentiated human myelomonocytic cell line THP1. Undifferentiated THP1 cells constitutively express NLRP3, and NLRP3 inflammasome activation occurred in response to canonical NLRP3 activation stimuli including nigericin, ATP, and urea crystals, culminating in pro-IL-1β cleavage, extracellular release of mature IL-1β, and pyroptosis...
February 5, 2019: Journal of Immunological Methods
Laura B Duvall, Lavoisier Ramos-Espiritu, Kyrollos E Barsoum, J Fraser Glickman, Leslie B Vosshall
Female Aedes aegypti mosquitoes bite humans to obtain blood to develop their eggs. Remarkably, their strong attraction to humans is suppressed for days after the blood meal by an unknown mechanism. We investigated a role for neuropeptide Y (NPY)-related signaling in long-term behavioral suppression and discovered that drugs targeting human NPY receptors modulate mosquito host-seeking. In a screen of all 49 predicted Ae. aegypti peptide receptors, we identified NPY-like receptor 7 (NPYLR7) as the sole target of these drugs...
February 7, 2019: Cell
Yukio Koizumi, Jun Fukushima, Yayoi Kobayashi, Ayumi Kadowaki, Miyuki Natsui, Tomokazu Yamaguchi, Yumiko Imai, Toshihiro Sugiyama, Keiji Kuba
Malformin A1 (MA1) is a fungus-produced cyclic pentapeptide. MA1 exhibits teratogenicity to plants, fibrinolysis-enhancing activity, and cytotoxicity to mammalian cells. To clarify the cytotoxic mechanism of MA1, we screened for the genes involved in the cytotoxicity of MA1 in monocytoid U937 cells by using CRISPR/Cas9-based genome-wide knockout library. Screening was performed by positive selection for the cells resistant to MA1 treatment, and sgRNAs integrated into MA1-resistant cells were analyzed with high-throughput sequencing...
February 8, 2019: Chembiochem: a European Journal of Chemical Biology
Felice Ho-Ching Tsang, Cheuk-Ting Law, Chloe Tsz-Ching Tang, Carol Lai-Hung Cheng, Don Wai-Ching Chin, Vincy Wing-Sum Tam, Lai Wei, Carmen Chak-Lui Wong, Irene Oi-Lin Ng, Chun-Ming Wong
Hepatocellular carcinoma (HCC) cells exploit an aberrant transcriptional program to sustain their infinite growth and progression. Emerging evidence indicates that the continuous and robust transcription of oncogenes in cancer cells is often driven by super-enhancers (SEs). In this study, we systematically compared the SE-landscapes between normal liver and HCC cells and revealed that the cis-acting SE-landscape was extensively reprogrammed during liver carcinogenesis. HCC cells acquired SEs at multiple prominent oncogenes to drive their vigorous expression...
February 5, 2019: Hepatology: Official Journal of the American Association for the Study of Liver Diseases
Scott A Alpizar, Arielle L Baker, Allan T Gulledge, Michael B Hoppa
The axon initial segment (AIS) is a specialized region within the proximal portion of the axon that initiates action potentials thanks in large part to an enrichment of sodium channels. The scaffolding protein ankyrinG (AnkG) is essential for the recruitment of sodium channels as well as several other intracellular and extracellular proteins to the AIS. In the present study, we explore the role of the cell adhesion molecule (CAM) neurofascin-186 (NF-186) in arranging the individual molecular components of the AIS in cultured rat hippocampal neurons...
2019: Frontiers in Cellular Neuroscience
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