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https://www.readbyqxmd.com/read/28079885/mirna182-regulates-percentage-of-myeloid-and-erythroid-cells-in-chronic-myeloid-leukemia
#1
Deepak Arya, Sasikala P Sachithanandan, Cecil Ross, Dasaradhi Palakodeti, Shang Li, Sudhir Krishna
The deregulation of lineage control programs is often associated with the progression of haematological malignancies. The molecular regulators of lineage choices in the context of tyrosine kinase inhibitor (TKI) resistance remain poorly understood in chronic myeloid leukemia (CML). To find a potential molecular regulator contributing to lineage distribution and TKI resistance, we undertook an RNA-sequencing approach for identifying microRNAs (miRNAs). Following an unbiased screen, elevated miRNA182-5p levels were detected in Bcr-Abl-inhibited K562 cells (CML blast crisis cell line) and in a panel of CML patients...
January 12, 2017: Cell Death & Disease
https://www.readbyqxmd.com/read/28069571/apc-c-dysfunction-limits-excessive-cancer-chromosomal-instability
#2
Laurent Sansregret, James O Patterson, Sally Dewhurst, Carlos López-García, André Koch, Nicholas McGranahan, William Chong Hang Chao, David J Barry, Andrew Rowan, Rachael Instrell, Stuart Horswell, Michael Way, Michael Howell, Martin R Singleton, René H Medema, Paul Nurse, Mark Petronczki, Charles Swanton
: Intercellular heterogeneity, exacerbated by chromosomal instability (CIN), fosters tumor heterogeneity and drug resistance. However, extreme CIN correlates with improved cancer outcome, suggesting that karyotypic diversity required to adapt to selection pressures might be balanced in tumors against the risk of excessive instability. Here, we used a functional genomics screen, genome editing, and pharmacologic approaches to identify CIN-survival factors in diploid cells. We find partial anaphase-promoting complex/cyclosome (APC/C) dysfunction lengthens mitosis, suppresses pharmacologically induced chromosome segregation errors, and reduces naturally occurring lagging chromosomes in cancer cell lines or following tetraploidization...
January 9, 2017: Cancer Discovery
https://www.readbyqxmd.com/read/28065598/cas13b-is-a-type-vi-b-crispr-associated-rna-guided-rnase-differentially-regulated-by-accessory-proteins-csx27-and-csx28
#3
Aaron A Smargon, David B T Cox, Neena K Pyzocha, Kaijie Zheng, Ian M Slaymaker, Jonathan S Gootenberg, Omar A Abudayyeh, Patrick Essletzbichler, Sergey Shmakov, Kira S Makarova, Eugene V Koonin, Feng Zhang
CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 and Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity...
January 4, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28065270/functional-study-of-the-vitamin-k-cycle-enzymes-in-live-cells
#4
J-K Tie, D W Stafford
Vitamin K-dependent carboxylation, an essential posttranslational modification catalyzed by gamma-glutamyl carboxylase, is required for the biological functions of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes. Concomitant with carboxylation, reduced vitamin K (KH2) is oxidized to vitamin K epoxide (KO). KO must be recycled back to KH2 by the enzymes vitamin K epoxide reductase and vitamin K reductase in a pathway known as the vitamin K cycle...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28062688/piggybac-mediates-efficient-in-vivo-crispr-library-screening-for-tumorigenesis-in-mice
#5
Chunlong Xu, Xiaolan Qi, Xuguang Du, Huiying Zou, Fei Gao, Tao Feng, Hengxing Lu, Shenglan Li, Xiaomeng An, Lijun Zhang, Yuanyuan Wu, Ying Liu, Ning Li, Mario R Capecchi, Sen Wu
CRISPR/Cas9 is becoming an increasingly important tool to functionally annotate genomes. However, because genome-wide CRISPR libraries are mostly constructed in lentiviral vectors, in vivo applications are severely limited as a result of difficulties in delivery. Here, we examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Although tumor induction has previously been achieved in mice by targeting cancer genes with the CRISPR/Cas9 system, in vivo genome-scale screening has not been reported...
January 6, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28060762/eukaryotic-elongation-factor-2-is-a-prognostic-marker-and-its-kinase-a-potential-therapeutic-target-in-hcc
#6
Leona L Pott, Sascha Hagemann, Henning Reis, Kristina Lorenz, Thilo Bracht, Thomas Herold, Boris V Skryabin, Dominik A Megger, Julia Kälsch, Frank Weber, Barbara Sitek, Hideo A Baba
: Hepatocellular carcinoma is a cancer with increasing incidence and largely refractory to current anticancer drugs. Since Sorafenib, a multikinase inhibitor has shown modest efficacy in advanced hepatocellular carcinoma additional treatments are highly needed. Protein phosphorylation via kinases is an important post-translational modification to regulate cell homeostasis including proliferation and apoptosis. Therefore kinases are valuable targets in cancer therapy. To this end we performed 2D differential gel electrophoresis and mass spectrometry analysis of phosphoprotein-enriched lysates of tumor and corresponding non-tumorous liver samples to detect differentially abundant phosphoproteins to screen for novel kinases as potential drug targets...
January 2, 2017: Oncotarget
https://www.readbyqxmd.com/read/28030641/validation-of-synthetic-crispr-reagents-as-a-tool-for-arrayed-functional-genomic-screening
#7
Jenille Tan, Scott E Martin
To date, lentiviral-based CRISPR-Cas9 screens have largely been conducted in pooled format. However, numerous assays are not amenable to pooled approaches, and lentiviral screening in arrayed format presents many challenges. We sought to examine synthetic CRISPR reagents in the context of arrayed screening. Experiments were performed using aberrant DNA replication as an assay. Using synthetic CRISPR RNAs targeting the known control gene GMNN in HCT-116 cells stably expressing Cas9, we observed statistically significant phenotype among the majority of transfected cells within 72 hours...
2016: PloS One
https://www.readbyqxmd.com/read/28017590/fluorescence-amplification-method-for-forward-genetic-discovery-of-factors-in-human-mrna-degradation
#8
Andrei Alexandrov, Mei-Di Shu, Joan A Steitz
Nonsense-mediated decay (NMD) degrades mRNAs containing a premature termination codon (PTC). PTCs are a frequent cause of human genetic diseases, and the NMD pathway is known to modulate disease severity. Since partial NMD attenuation can potentially enhance nonsense suppression therapies, better definition of human-specific NMD is required. However, the majority of NMD factors were first discovered in model organisms and then subsequently identified by homology in human. Sensitivity and throughput limitations of existing approaches have hindered systematic forward genetic screening for NMD factors in human cells...
January 5, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28011935/ttll12-inhibits-the-activation-of-cellular-antiviral-signaling-through-interaction-with-visa-mavs
#9
Lin-Gao Ju, Yuan Zhu, Pin-Ji Lei, Dong Yan, Kun Zhu, Xiang Wang, Qing-Lan Li, Xue-Jing Li, Jian-Wen Chen, Lian-Yun Li, Min Wu
Upon virus infection, host cells use retinoic-acid-inducible geneI I (RIG-I)-like receptors to recognize viral RNA and activate type I IFN expression. To investigate the role of protein methylation in the antiviral signaling pathway, we screened all the SET domain-containing proteins and identified TTLL12 as a negative regulator of RIG-I signaling. TTLL12 contains SET and TTL domains, which are predicted to have lysine methyltransferase and tubulin tyrosine ligase activities, respectively. Exogenous expression of TTLL12 represses IFN-β expression induced by Sendai virus...
December 23, 2016: Journal of Immunology: Official Journal of the American Association of Immunologists
https://www.readbyqxmd.com/read/28008168/cornerstones-of-crispr-cas-in-drug-discovery-and-therapy
#10
Christof Fellmann, Benjamin G Gowen, Pei-Chun Lin, Jennifer A Doudna, Jacob E Corn
The recent development of CRISPR-Cas systems as easily accessible and programmable tools for genome editing and regulation is spurring a revolution in biology. Paired with the rapid expansion of reference and personalized genomic sequence information, technologies based on CRISPR-Cas are enabling nearly unlimited genetic manipulation, even in previously difficult contexts, including human cells. Although much attention has focused on the potential of CRISPR-Cas to cure Mendelian diseases, the technology also holds promise to transform the development of therapies to treat complex heritable and somatic disorders...
December 23, 2016: Nature Reviews. Drug Discovery
https://www.readbyqxmd.com/read/28006686/automated-microraft-platform-to-identify-and-collect-non-adherent-cells-successfully-gene-edited-with-crispr-cas9
#11
Peter J Attayek, Jennifer P Waugh, Sally A Hunsucker, Philip J Grayeski, Christopher E Sims, Paul M Armistead, Nancy L Allbritton
Microraft arrays have been used to screen and then isolate adherent and non-adherent cells with very high efficiency and excellent viability; however, manual screening and isolation limits the throughput and utility of the technology. In this work, novel hardware and software were developed to automate the microraft array platform. The developed analysis software identified microrafts on the array with greater than 99% sensitivity and cells on the microrafts with 100% sensitivity. The software enabled time-lapse imaging and the use of temporally varying characteristics as sort criteria...
December 10, 2016: Biosensors & Bioelectronics
https://www.readbyqxmd.com/read/27994508/crispr-cas9-from-genome-editing-to-cancer-research
#12
REVIEW
Si Chen, Heng Sun, Kai Miao, Chu-Xia Deng
Cancer development is a multistep process triggered by innate and acquired mutations, which cause the functional abnormality and determine the initiation and progression of tumorigenesis. Gene editing is a widely used engineering tool for generating mutations that enhance tumorigenesis. The recent developed clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9) system renews the genome editing approach into a more convenient and efficient way. By rapidly introducing genetic modifications in cell lines, organs and animals, CRISPR-Cas9 system extends the gene editing into whole genome screening, both in loss-of-function and gain-of-function manners...
2016: International Journal of Biological Sciences
https://www.readbyqxmd.com/read/27992415/a-genome-wide-crispr-screen-identifies-a-restricted-set-of-hiv-host-dependency-factors
#13
Ryan J Park, Tim Wang, Dylan Koundakjian, Judd F Hultquist, Pedro Lamothe-Molina, Blandine Monel, Kathrin Schumann, Haiyan Yu, Kevin M Krupzcak, Wilfredo Garcia-Beltran, Alicja Piechocka-Trocha, Nevan J Krogan, Alexander Marson, David M Sabatini, Eric S Lander, Nir Hacohen, Bruce D Walker
Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability...
December 19, 2016: Nature Genetics
https://www.readbyqxmd.com/read/27989123/standardized-markerless-gene-integration-for-pathway-engineering-in-yarrowia-lipolytica
#14
Cory Schwartz, Murtaza Shabbir-Hussain, Keith Frogue, Mark Blenner, Ian Wheeldon
The yeast Yarrowia lipolytica is a promising microbial host due to its native capacity to produce lipid-based chemicals. Engineering stable production strains requires genomic integration of modified genes, avoiding episomal expression that requires specialized media to maintain selective pressures. Here, we develop a CRISPR-Cas9-based tool for targeted, markerless gene integration into the Y. lipolytica genome. A set of genomic loci was screened to identify sites that were accepting of gene integrations without impacting cell growth...
December 22, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/27987038/efficient-genome-editing-of-differentiated-renal-epithelial-cells
#15
Alexis Hofherr, Tilman Busch, Nora Huber, Andreas Nold, Albert Bohn, Amandine Viau, Frank Bienaimé, E Wolfgang Kuehn, Sebastian J Arnold, Michael Köttgen
Recent advances in genome editing technologies have enabled the rapid and precise manipulation of genomes, including the targeted introduction, alteration, and removal of genomic sequences. However, respective methods have been described mainly in non-differentiated or haploid cell types. Genome editing of well-differentiated renal epithelial cells has been hampered by a range of technological issues, including optimal design, efficient expression of multiple genome editing constructs, attainable mutation rates, and best screening strategies...
December 16, 2016: Pflügers Archiv: European Journal of Physiology
https://www.readbyqxmd.com/read/27984746/mammalian-rad52-functions-in-break-induced-replication-repair-of-collapsed-dna-replication-forks
#16
Sotirios K Sotiriou, Irene Kamileri, Natalia Lugli, Konstantinos Evangelou, Caterina Da-Ré, Florian Huber, Laura Padayachy, Sebastien Tardy, Noemie L Nicati, Samia Barriot, Fena Ochs, Claudia Lukas, Jiri Lukas, Vassilis G Gorgoulis, Leonardo Scapozza, Thanos D Halazonetis
Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci...
December 15, 2016: Molecular Cell
https://www.readbyqxmd.com/read/27984734/dissecting-immune-circuits-by-linking-crispr-pooled-screens-with-single-cell-rna-seq
#17
Diego Adhemar Jaitin, Assaf Weiner, Ido Yofe, David Lara-Astiaso, Hadas Keren-Shaul, Eyal David, Tomer Meir Salame, Amos Tanay, Alexander van Oudenaarden, Ido Amit
In multicellular organisms, dedicated regulatory circuits control cell type diversity and responses. The crosstalk and redundancies within these circuits and substantial cellular heterogeneity pose a major research challenge. Here, we present CRISP-seq, an integrated method for massively parallel single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-pooled screens. We show that profiling the genomic perturbation and transcriptome in the same cell enables us to simultaneously elucidate the function of multiple factors and their interactions...
December 15, 2016: Cell
https://www.readbyqxmd.com/read/27984733/a-multiplexed-single-cell-crispr-screening-platform-enables-systematic-dissection-of-the-unfolded-protein-response
#18
Britt Adamson, Thomas M Norman, Marco Jost, Min Y Cho, James K Nuñez, Yuwen Chen, Jacqueline E Villalta, Luke A Gilbert, Max A Horlbeck, Marco Y Hein, Ryan A Pak, Andrew N Gray, Carol A Gross, Atray Dixit, Oren Parnas, Aviv Regev, Jonathan S Weissman
Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis...
December 15, 2016: Cell
https://www.readbyqxmd.com/read/27984732/perturb-seq-dissecting-molecular-circuits-with-scalable-single-cell-rna-profiling-of-pooled-genetic-screens
#19
Atray Dixit, Oren Parnas, Biyu Li, Jenny Chen, Charles P Fulco, Livnat Jerby-Arnon, Nemanja D Marjanovic, Danielle Dionne, Tyler Burks, Raktima Raychowdhury, Britt Adamson, Thomas M Norman, Eric S Lander, Jonathan S Weissman, Nir Friedman, Aviv Regev
Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes-such as transcriptional profiles-at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells to lipopolysaccharide (LPS)...
December 15, 2016: Cell
https://www.readbyqxmd.com/read/27983599/using-crispr-cas9-mediated-gla-gene-knockout-as-an-in-vitro-drug-screening-model-for-fabry-disease
#20
Hui-Yung Song, Huai-Chih Chiang, Wei-Lien Tseng, Ping Wu, Chian-Shiu Chien, Hsin-Bang Leu, Yi-Ping Yang, Mong-Lien Wang, Yuh-Jyh Jong, Chung-Hsuan Chen, Wen-Chung Yu, Shih-Hwa Chiou
The CRISPR/Cas9 Genome-editing system has revealed promising potential for generating gene mutation, deletion, and correction in human cells. Application of this powerful tool in Fabry disease (FD), however, still needs to be explored. Enzyme replacement therapy (ERT), a regular administration of recombinant human α Gal A (rhα-GLA), is a currently available and effective treatment to clear the accumulated Gb3 in FD patients. However, the short half-life of rhα-GLA in human body limits its application. Moreover, lack of an appropriate in vitro disease model restricted the high-throughput screening of drugs for improving ERT efficacy...
December 13, 2016: International Journal of Molecular Sciences
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