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https://www.readbyqxmd.com/read/27932481/modeling-monogenic-human-nephrotic-syndrome-in-the-drosophila-garland-cell-nephrocyte
#1
Tobias Hermle, Daniela A Braun, Martin Helmstädter, Tobias B Huber, Friedhelm Hildebrandt
Steroid-resistant nephrotic syndrome is characterized by podocyte dysfunction. Drosophila garland cell nephrocytes are podocyte-like cells and thus provide a potential in vivo model in which to study the pathogenesis of nephrotic syndrome. However, relevant pathomechanisms of nephrotic syndrome have not been studied in nephrocytes. Here, we discovered that two Drosophila slit diaphragm proteins, orthologs of the human genes encoding nephrin and nephrin-like protein 1, colocalize within a fingerprint-like staining pattern that correlates with ultrastructural morphology...
December 8, 2016: Journal of the American Society of Nephrology: JASN
https://www.readbyqxmd.com/read/27924039/flyrnai-org-the-database-of-the-drosophila-rnai-screening-center-and-transgenic-rnai-project-2017-update
#2
Yanhui Hu, Aram Comjean, Charles Roesel, Arunachalam Vinayagam, Ian Flockhart, Jonathan Zirin, Lizabeth Perkins, Norbert Perrimon, Stephanie E Mohr
The FlyRNAi database of the Drosophila RNAi Screening Center (DRSC) and Transgenic RNAi Project (TRiP) at Harvard Medical School and associated DRSC/TRiP Functional Genomics Resources website (http://fgr.hms.harvard.edu) serve as a reagent production tracking system, screen data repository, and portal to the community. Through this portal, we make available protocols, online tools, and other resources useful to researchers at all stages of high-throughput functional genomics screening, from assay design and reagent identification to data analysis and interpretation...
October 23, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27918485/somatic-cell-nuclear-transfer-followed-by-cripsr-cas9-microinjection-results-in-highly-efficient-genome-editing-in-cloned-pigs
#3
Timothy P Sheets, Chi-Hun Park, Ki-Eun Park, Anne Powell, David M Donovan, Bhanu P Telugu
The domestic pig is an ideal "dual purpose" animal model for agricultural and biomedical research. With the availability of genome editing tools such as clustered regularly interspaced short palindromic repeat (CRISPR) and associated nuclease Cas9 (CRISPR/Cas9), it is now possible to perform site-specific alterations with relative ease, and will likely help realize the potential of this valuable model. In this article, we investigated for the first time a combination of somatic cell nuclear transfer (SCNT) and direct injection of CRISPR/Cas ribonucleoprotein complex targeting GRB10 into the reconstituted oocytes to generate GRB10 ablated Ossabaw fetuses...
December 3, 2016: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/27911376/rapid-and-efficient-generation-of-recombinant-human-pluripotent-stem-cells-by-recombinase-mediated-cassette-exchange-in-the-aavs1-locus
#4
Laura Ordovás, Ruben Boon, Mariaelena Pistoni, Yemiao Chen, Rangarajan Sambathkumar, Nicky Helsen, Jolien Vanhove, Pieter Berckmans, Qing Cai, Kim Vanuytsel, Susanna Raitano, Catherine M Verfaillie
Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells (hPSCs) is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects...
November 20, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/27898061/crispr-cas9-aid-base-editor-is-a-powerful-gain-of-function-screening-tool
#5
Cem Kuscu, Mazhar Adli
No abstract text is available yet for this article.
November 29, 2016: Nature Methods
https://www.readbyqxmd.com/read/27890641/specific-point-mutations-in-key-redox-enzymes-are-associated-with-chemoresistance-in-epithelial-ovarian-cancer
#6
Nicole M Fletcher, Jimmy Belotte, Mohammed G Saed, Ira Memaj, Michael P Diamond, Robert T Morris, Ghassan M Saed
Oxidative stress plays an important role in the pathophysiology of ovarian cancer. Resistance to chemotherapy presents a significant challenge for ovarian cancer treatment. Specific single nucleotide polymorphisms (SNPs) in key redox enzymes have been associated with ovarian cancer survival and progression. The objective of this study was to determine whether chemotherapy induces point mutations in key redox enzymes that lead to the acquisition of chemoresistance in epithelial ovarian cancer (EOC). Human EOC cell lines and their chemoresistant counterpart were utilized for this study...
November 25, 2016: Free Radical Biology & Medicine
https://www.readbyqxmd.com/read/27883055/gapmer-cellular-internalization-by-macropinocytosis-induces-sequence-specific-gene-silencing-in-human-primary-t-cells
#7
Mobashar Hussain Urf Turabe Fazil, Seow Theng Ong, Madhavi Latha Somaraju Chalasani, Jian Hui Low, Atish Kizhakeyil, Akshay Mamidi, Carey Fang Hui Lim, Graham D Wright, Rajamani Lakshminarayanan, Dermot Kelleher, Navin Kumar Verma
Post-transcriptional gene silencing holds great promise in discovery research for addressing intricate biological questions and as therapeutics. While various gene silencing approaches, such as siRNA and CRISPR-Cas9 techniques, are available, these cannot be effectively applied to "hard-to-transfect" primary T-lymphocytes. The locked nucleic acid-conjugated chimeric antisense oligonucleotide, called "GapmeR", is an emerging new class of gene silencing molecule. Here, we show that GapmeR internalizes into human primary T-cells through macropinocytosis...
November 24, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27875524/microrna-137-promotes-apoptosis-in-ovarian-cancer-cells-via-the-regulation-of-xiap
#8
Xiaodi Li, Wei Chen, Wenshu Zeng, Chunling Wan, Shiwei Duan, Songshan Jiang
BACKGROUND: microRNAs (miRNAs) have regulatory roles in various cellular processes, including apoptosis. Recently, X-linked inhibitor of apoptosis protein (XIAP) has been reported to be dysregulated in epithelial ovarian cancer (EOC). However, the mechanism underlying this dysregulation is largely unknown. METHODS: Using bioinformatics and a literature analysis, a panel of miRNAs dysregulated in EOC was chosen for further experimental confirmation from hundreds of miRNAs that were predicted to interact with the XIAP 3'UTR...
November 22, 2016: British Journal of Cancer
https://www.readbyqxmd.com/read/27869803/genome-wide-crispr-screens-reveal-a-wnt-fzd5-signaling-circuit-as-a-druggable-vulnerability-of-rnf43-mutant-pancreatic-tumors
#9
Zachary Steinhart, Zvezdan Pavlovic, Megha Chandrashekhar, Traver Hart, Xiaowei Wang, Xiaoyu Zhang, Mélanie Robitaille, Kevin R Brown, Sridevi Jaksani, René Overmeer, Sylvia F Boj, Jarrett Adams, James Pan, Hans Clevers, Sachdev Sidhu, Jason Moffat, Stéphane Angers
Forward genetic screens with CRISPR-Cas9 genome editing enable high-resolution detection of genetic vulnerabilities in cancer cells. We conducted genome-wide CRISPR-Cas9 screens in RNF43-mutant pancreatic ductal adenocarcinoma (PDAC) cells, which rely on Wnt signaling for proliferation. Through these screens, we discovered a unique requirement for a Wnt signaling circuit: engaging FZD5, one of the ten Frizzled receptors encoded in the human genome. Our results uncover an underappreciated level of context-dependent specificity at the Wnt receptor level...
November 21, 2016: Nature Medicine
https://www.readbyqxmd.com/read/27866084/engineering-cell-signaling-modulators-from-native-protein-protein-interactions
#10
REVIEW
Wei Zhang, Moshe Ben-David, Sachdev S Sidhu
Recent studies on genome sequencing and genetic screens with RNAi and CRISPR technology have revolutionized our understanding of aberrant signaling networks in human diseases. A strategy combining both genetic and protein-based technologies should be at the heart of modern drug-development efforts, particularly in the era of precision medicine. Thus, there is an urgent need for efficient platforms to develop probes that can modulate protein function in cells to validate drug targets and to develop therapeutic leads...
November 17, 2016: Current Opinion in Structural Biology
https://www.readbyqxmd.com/read/27865852/delivery-methods-for-site-specific-nucleases-achieving-the-full-potential-of-therapeutic-gene-editing
#11
REVIEW
Jia Liu, Sai-Lan Shui
The advent of site-specific nucleases, particularly CRISPR/Cas9, provides researchers with the unprecedented ability to manipulate genomic sequences. These nucleases are used to create model cell lines, engineer metabolic pathways, produce transgenic animals and plants, perform genome-wide functional screen and, most importantly, treat human diseases that are difficult to tackle by traditional medications. Considerable efforts have been devoted to improving the efficiency and specificity of nucleases for clinical applications...
November 16, 2016: Journal of Controlled Release: Official Journal of the Controlled Release Society
https://www.readbyqxmd.com/read/27862876/tomato-facultative-parthenocarpy-results-from-slagamous-like-6-loss-of-function
#12
Chen Klap, Ester Yeshayahou, Anthony M Bolger, Tzahi Arazi, Suresh K Gupta, Sara Shabtai, Björn Usadel, Yehiam Salts, Rivka Barg
The extreme sensitivity of the microsporogenesis process to moderately high or low temperatures is a major hindrance for tomato (Solanum lycopersicum) sexual reproduction and hence year-round cropping. Consequently, breeding for parthenocarpy, i.e. fertilization-independent fruit set, is considered a valuable goal especially for maintaining sustainable agriculture in the face of global warming. A mutant capable of setting high quality seedless (parthenocarpic) fruit was found following a screen of EMS mutagenized tomato population for yielding under heat stress...
November 11, 2016: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/27860197/crispr-cas9-the-ultimate-weapon-to-battle-infectious-diseases
#13
REVIEW
M Doerflinger, W Forsyth, G Ebert, M Pellegrini, M J Herold
Infectious diseases are a leading cause of death worldwide. Novel therapeutics are urgently required to treat multidrug-resistant organisms such as Mycobacterium tuberculosis and to mitigate morbidity and mortality caused by acute infections such as malaria and dengue fever virus as well as chronic infections such as human immunodeficiency virus-1 and hepatitis B virus. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, which has revolutionized biomedical research, holds great promise for the identification and validation of novel drug targets...
November 16, 2016: Cellular Microbiology
https://www.readbyqxmd.com/read/27845624/targeting-mutant-ras-in-patient-derived-colorectal-cancer-organoids-by-combinatorial-drug-screening
#14
Carla S Verissimo, René M Overmeer, Bas Ponsioen, Jarno Drost, Sander Mertens, Ingrid Verlaan-Klink, Bastiaan van Gerwen, Marieke van der Ven, Marc van de Wetering, David A Egan, René Bernards, Hans Clevers, Johannes L Bos, Hugo J Snippert
Colorectal cancer (CRC) organoids can be derived from almost all CRC patients and therefore capture the genetic diversity of this disease. We assembled a panel of CRC organoids carrying either wild-type or mutant RAS, as well as normal organoids and tumor organoids with a CRISPR-introduced oncogenic KRAS mutation. Using this panel, we evaluated RAS pathway inhibitors and drug combinations that are currently in clinical trial for RAS mutant cancers. Presence of mutant RAS correlated strongly with resistance to these targeted therapies...
November 15, 2016: ELife
https://www.readbyqxmd.com/read/27842490/coralina-a-universal-method-for-the-generation-of-grna-libraries-for-crispr-based-screening
#15
Anna Köferle, Karolina Worf, Christopher Breunig, Valentin Baumann, Javier Herrero, Maximilian Wiesbeck, Lukas H Hutter, Magdalena Götz, Christiane Fuchs, Stephan Beck, Stefan H Stricker
BACKGROUND: The bacterial CRISPR system is fast becoming the most popular genetic and epigenetic engineering tool due to its universal applicability and adaptability. The desire to deploy CRISPR-based methods in a large variety of species and contexts has created an urgent need for the development of easy, time- and cost-effective methods enabling large-scale screening approaches. RESULTS: Here we describe CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a method for the generation of comprehensive gRNA libraries for CRISPR-based screens...
November 14, 2016: BMC Genomics
https://www.readbyqxmd.com/read/27829120/crispr-guide-rna-validation-in-vitro
#16
Stephanie Grainger, Brianna Lonquich, Chet Huan Oon, Nicole Nguyen, Karl Willert, David Traver
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has been applied to edit genomes in a wide variety of model systems. Although this process can be quite efficient, editing at precise locations in the genome remains difficult without a suitable single guide RNA (sgRNA). We have developed a method for screening sgRNA function in vitro, using reagents that most zebrafish laboratories are already using. The results from our in vitro assay correlate with function in vivo in every sgRNA that we have examined so far...
November 9, 2016: Zebrafish
https://www.readbyqxmd.com/read/27825983/accelerating-glioblastoma-drug-discovery-convergence-of-patient-derived-models-genome-editing-and-phenotypic-screening
#17
Eoghan O'Duibhir, Neil Carragher, Steven M Pollard
Patients diagnosed with glioblastoma (GBM) continue to face a bleak prognosis. It is critical that new effective therapeutic strategies are developed. GBM stem cells have molecular hallmarks of neural stem and progenitor cells and it is possible to propagate both non-transformed normal neural stem cells and GBM stem cells, in defined, feeder-free, adherent culture. These primary stem cell lines provide an experimental model that is ideally suited to cell-based drug discovery or genetic screens in order to identify tumour-specific vulnerabilities...
November 4, 2016: Molecular and Cellular Neurosciences
https://www.readbyqxmd.com/read/27811178/crispr-cas9-the-new-kid-on-the-block-of-fungal-molecular-biology
#18
Sven Krappmann
Research on fungal pathogens with the aim to identify virulence determinants strictly relies on the generation of defined, recombinant strains, a task that is executed by means of a sophisticated molecular biology toolbox. Recent developments in fungal genome engineering have opened a new frontier by implementing the CRISPR-Cas9 technology, based on expression of the Cas9 endonuclease that is loaded by a single guiding RNA (sgRNA) molecule to target a defined site in the recipient genome. This novel approach has been adapted successfully to engineer fungal genomes, among them the one of the human-pathogenic mould Aspergillus fumigatus Implementation of the required components was achieved by various means that differ with respect to expression of the Cas9 enzyme and sgRNA delivery...
November 2, 2016: Medical Mycology: Official Publication of the International Society for Human and Animal Mycology
https://www.readbyqxmd.com/read/27809318/a-high-throughput-functional-genomics-workflow-based-on-crispr-cas9-mediated-targeted-mutagenesis-in-zebrafish
#19
Gaurav K Varshney, Blake Carrington, Wuhong Pei, Kevin Bishop, Zelin Chen, Chunxin Fan, Lisha Xu, Marypat Jones, Matthew C LaFave, Johan Ledin, Raman Sood, Shawn M Burgess
The zebrafish is a popular model organism for studying development and disease, and genetically modified zebrafish provide an essential tool for functional genomic studies. Numerous publications have demonstrated the efficacy of gene targeting in zebrafish using CRISPR/Cas9, and they have included descriptions of a variety of tools and methods for guide RNA synthesis and mutant identification. However, most of the published techniques are not readily scalable to increase throughput. We recently described a CRISPR/Cas9-based high-throughput mutagenesis and phenotyping pipeline in zebrafish...
December 2016: Nature Protocols
https://www.readbyqxmd.com/read/27809196/making-better-crispr-libraries
#20
Shiyou Zhu, Wensheng Wei
A new algorithm improves the performance of CRISPR-based genetic screens in mammals.
November 3, 2016: ELife
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