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Crispr screen

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https://www.readbyqxmd.com/read/29352288/phospholipid-scramblase-1-interacts-with-influenza-a-virus-np-impairing-its-nuclear-import-and-thereby-suppressing-virus-replication
#1
Weiyu Luo, Jie Zhang, Libin Liang, Guangwen Wang, Qibing Li, Pengyang Zhu, Yuan Zhou, Junping Li, Yuhui Zhao, Nan Sun, Shanyu Huang, Chenchen Zhou, Yu Chang, Pengfei Cui, Pucheng Chen, Yongping Jiang, Guohua Deng, Zhigao Bu, Chengjun Li, Li Jiang, Hualan Chen
Transcription and replication of the influenza A virus (IAV) genome occur in the nucleus of infected cells and are carried out by the viral ribonucleoprotein complex (vRNP). As a major component of the vRNP complex, the viral nucleoprotein (NP) mediates the nuclear import of the vRNP complex via its nuclear localization signals (NLSs). Clearly, an effective way for the host to antagonize IAV infection would be by targeting vRNP nuclear import. Here, we identified phospholipid scramblase 1 (PLSCR1) as a binding partner of NP by using a yeast two-hybrid (Y2H) screen...
January 19, 2018: PLoS Pathogens
https://www.readbyqxmd.com/read/29348144/a-crispr-tagging-based-screen-reveals-localized-players-in-wnt-directed-asymmetric-cell-division
#2
Jennifer K Heppert, Ariel M Pani, Allyson M Roberts, Daniel J Dickinson, Bob Goldstein
Oriented cell divisions are critical to establish and maintain cell fates and tissue organization. Diverse extracellular and intracellular cues have been shown to provide spatial information for mitotic spindle positioning, however, the molecular mechanisms by which extracellular signals communicate with cells to direct mitotic spindle positioning are largely unknown. In animal cells, oriented cell divisions are often achieved by the localization of force-generating, motor protein complexes to discrete cortical domains...
January 18, 2018: Genetics
https://www.readbyqxmd.com/read/29344886/identifying-genetic-dependencies-in-cancer-by-analyzing-sirna-screens-in-tumor-cell-line-panels
#3
James Campbell, Colm J Ryan, Christopher J Lord
Loss-of-function screening using RNA interference or CRISPR approaches can be used to identify genes that specific tumor cell lines depend upon for survival. By integrating the results from screens in multiple cell lines with molecular profiling data, it is possible to associate the dependence upon specific genes with particular molecular features (e.g., the mutation of a cancer driver gene, or transcriptional or proteomic signature). Here, using a panel of kinome-wide siRNA screens in osteosarcoma cell lines as an example, we describe a computational protocol for analyzing loss-of-function screens to identify genetic dependencies associated with particular molecular features...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29337376/crispr-cas9-mediated-genome-editing-in-epstein-barr-virus-transformed-lymphoblastoid-b-cell-lines
#4
Sizun Jiang, Liang Wei Wang, Michael J Walsh, Stephen J Trudeau, Catherine Gerdt, Bo Zhao, Benjamin E Gewurz
Epstein-Barr virus (EBV) efficiently transforms primary human B cells into immortalized lymphoblastoid cell lines (LCLs), which are extensively used in human genetic, immunological and virological studies. LCLs provide unlimited sources of DNA for genetic investigation, but can be difficult to manipulate, for instance because low retroviral or lentiviral transduction frequencies hinder experiments that require co-expression of multiple components. This unit details Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 engineering for robust LCL genome editing...
January 16, 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29337374/pooled-lentiviral-delivery-genetic-screens
#5
Federica Piccioni, Scott T Younger, David E Root
Pooled cell-based screens of mammalian genetic perturbations enable systematic large-scale, even genome-scale, evaluation of gene function. Pooled screens introduce genetic perturbations into a cell population through viral transduction such that each cell integrates into its DNA a single or small number of library perturbations with barcodes identifying the perturbations. One then selects and physically isolates the subset of cells that exhibit the phenotype of interest. Sequencing the barcodes in the hit cells reveals which genes favored or inhibited the hit phenotype...
January 16, 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29337372/crispr-cas9-edited-site-sequencing-cres-seq-an-efficient-and-high-throughput-method-for-the-selection-of-crispr-cas9-edited-clones
#6
Yaligara Veeranagouda, Delphine Debono-Lagneaux, Hamida Fournet, Gilbert Thill, Michel Didier
The emergence of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) gene editing systems has enabled the creation of specific mutants at low cost, in a short time and with high efficiency, in eukaryotic cells. Since a CRISPR-Cas9 system typically creates an array of mutations in targeted sites, a successful gene editing project requires careful selection of edited clones. This process can be very challenging, especially when working with multiallelic genes and/or polyploid cells (such as cancer and plants cells)...
January 16, 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29335603/in-vivo-simultaneous-transcriptional-activation-of-multiple-genes-in-the-brain-using-crispr-dcas9-activator-transgenic-mice
#7
Haibo Zhou, Junlai Liu, Changyang Zhou, Ni Gao, Zhiping Rao, He Li, Xinde Hu, Changlin Li, Xuan Yao, Xiaowen Shen, Yidi Sun, Yu Wei, Fei Liu, Wenqin Ying, Junming Zhang, Cheng Tang, Xu Zhang, Huatai Xu, Linyu Shi, Leping Cheng, Pengyu Huang, Hui Yang
Despite rapid progresses in the genome-editing field, in vivo simultaneous overexpression of multiple genes remains challenging. We generated a transgenic mouse using an improved dCas9 system that enables simultaneous and precise in vivo transcriptional activation of multiple genes and long noncoding RNAs in the nervous system. As proof of concept, we were able to use targeted activation of endogenous neurogenic genes in these transgenic mice to directly and efficiently convert astrocytes into functional neurons in vivo...
January 15, 2018: Nature Neuroscience
https://www.readbyqxmd.com/read/29335437/deubiquitinase-usp13-dictates-mcl1-stability-and-sensitivity-to-bh3-mimetic-inhibitors
#8
Shengzhe Zhang, Meiying Zhang, Ying Jing, Xia Yin, Pengfei Ma, Zhenfeng Zhang, Xiaojie Wang, Wen Di, Guanglei Zhuang
MCL1 is a pivot member of the anti-apoptotic BCL-2 family proteins. While a distinctive feature of MCL1 resides in its efficient ubiquitination and destruction, the deubiquitinase USP9X has been implicated in the preservation of MCL1 expression by removing the polyubiquitin chains. Here we perform an unbiased siRNA screen and identify that the second deubiquitinase, USP13, regulates MCL1 stability in lung and ovarian cancer cells. Mechanistically, USP13 interacts with and stabilizes MCL1 via deubiquitination...
January 15, 2018: Nature Communications
https://www.readbyqxmd.com/read/29332168/efficient-crispr-cas9-based-genome-editing-in-carrot-cells
#9
Magdalena Klimek-Chodacka, Tomasz Oleszkiewicz, Levi G Lowder, Yiping Qi, Rafal Baranski
The first report presenting successful and efficient carrot genome editing using CRISPR/Cas9 system. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) is a powerful genome editing tool that has been widely adopted in model organisms recently, but has not been used in carrot-a model species for in vitro culture studies and an important health-promoting crop grown worldwide. In this study, for the first time, we report application of the CRISPR/Cas9 system for efficient targeted mutagenesis of the carrot genome...
January 13, 2018: Plant Cell Reports
https://www.readbyqxmd.com/read/29327641/generation-of-an-arrayed-crispr-cas9-library-targeting-epigenetic-regulators-from-high-content-screens-to-in-vivo-assays
#10
Tristan Henser-Brownhill, Josep Monserrat, Paola Scaffidi
The CRISPR-Cas9 system has revolutionized genome engineering, allowing precise modification of DNA in various organisms. The most popular method for conducting CRISPR-based functional screens involves the use of pooled lentiviral libraries in selection screens coupled with next-generation sequencing. Screens employing genome-scale pooled small guide RNA (sgRNA) libraries are demanding, particularly when complex assays are used. Furthermore, pooled libraries are not suitable for microscopy-based high-content screens or for systematic interrogation of protein function...
January 12, 2018: Epigenetics: Official Journal of the DNA Methylation Society
https://www.readbyqxmd.com/read/29326299/targeting-mutant-kras-with-crispr-cas9-controls-tumor-growth
#11
Hyongbum Henry Kim, Wonjoo Kim, Sangeun Lee, Han Sang Kim, Minjung Song, Yong Hoon Cha, Young-Hoon Kim, Jeonghong Shin, Eun-Seo Lee, Yeonsoo Joo, Jae J Song, Eun Ju Choi, Jae W Choi, Jinu Lee, Moonkyung Kang, Jong In Yook, Min Goo Lee, Yeon-Soo Kim, Soonmyung Paik
KRAS is the most frequently mutated oncogene in human tumors and its activating mutations represents important therapeutic targets. The combination of Cas9 and guide RNA from the CRISPR-Cas system recognizes a specific DNA sequence and makes a double-strand break, which enables editing of the relevant genes. Here we harnessed CRISPR to specifically target mutant KRAS alleles in cancer cells. We screened guide RNAs using a reporter system and validated them in cancer cells after lentiviral delivery of Cas9 and guide RNA...
January 11, 2018: Genome Research
https://www.readbyqxmd.com/read/29326075/use-of-crispr-cas9-gene-editing-tools-for-developing-models-in-drug-discovery
#12
REVIEW
Gulzar Ahmad, Mansoor Amiji
Clustered regularly interspaced short palindromic repeat/CRISPR-associated 9 (CRISPR/Cas9) enables targeted genome engineering. The simplicity of this system, its facile engineering, and amenability to multiplex genes make it the system of choice for many applications. This system has revolutionized our ability to carry out gene editing, transcription regulation, genome imaging, and epigenetic modification. In this review, we discuss the discovery of CRISPR/Cas9, its mechanism of action, its application in medicine and animal model development, and its delivery...
January 8, 2018: Drug Discovery Today
https://www.readbyqxmd.com/read/29323662/crispr-cas9-based-genome-wide-screening-of-toxoplasma-gondii
#13
Saima M Sidik, Diego Huet, Sebastian Lourido
Apicomplexan parasites, such as Toxoplasma gondii, cause extensive morbidity and mortality in humans and livestock, highlighting the need for a deeper understanding of their molecular biology. Although techniques for the generation of targeted gene disruptions have long been available for apicomplexans, such methods are not readily scalable to the entire genome. We recently used CRISPR-Cas9 to disrupt all nuclear protein-coding genes in T. gondii using a pooled format. The method relies on transfection of a guide RNA library into parasites constitutively expressing Cas9...
January 2018: Nature Protocols
https://www.readbyqxmd.com/read/29322420/a-comprehensive-protocol-resource-for-performing-pooled-shrna-and-crispr-screens
#14
Leonie A Cluse, Iva Nikolic, Deborah Knight, Piyush B Madhamshettiwar, Jennii Luu, Karla J Cowley, Timothy Semple, Gisela Mir Arnau, Jake Shortt, Ricky W Johnstone, Kaylene J Simpson
This chapter details a compendium of protocols that collectively enable the reader to perform a pooled shRNA and/or CRISPR screen-with methods to identify and validate positive controls and subsequent hits; establish a viral titer in the cell line of choice; create and screen libraries, sequence strategies, and bioinformatics resources to analyze outcomes. Collectively, this provides an overarching resource from the start to finish of a screening project, making this technology possible in all laboratories.
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29304331/rapid-and-scalable-characterization-of-crispr-technologies-using-an-e-%C3%A2-coli-cell-free-transcription-translation-system
#15
Ryan Marshall, Colin S Maxwell, Scott P Collins, Thomas Jacobsen, Michelle L Luo, Matthew B Begemann, Benjamin N Gray, Emma January, Anna Singer, Yonghua He, Chase L Beisel, Vincent Noireaux
CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression-all without protein purification or live cells...
January 4, 2018: Molecular Cell
https://www.readbyqxmd.com/read/29301958/a-major-chromatin-regulator-determines-resistance-of-tumor-cells-to-t-cell-mediated-killing
#16
Deng Pan, Aya Kobayashi, Peng Jiang, Lucas Ferrari de Andrade, Rong En Tay, Adrienne Luoma, Daphne Tsoucas, Xintao Qiu, Klothilda Lim, Prakash Rao, Henry W Long, Guo-Cheng Yuan, John Doench, Myles Brown, Shirley Liu, Kai W Wucherpfennig
Many human cancers are resistant to immunotherapy for reasons that are poorly understood. We used a genome-scale CRISPR/Cas9 screen to identify mechanisms of tumor cell resistance to killing by cytotoxic T cells, the central effectors of anti-tumor immunity. Inactivation of >100 genes sensitized mouse B16F10 melanoma cells to killing by T cells, including Pbrm1, Arid2 and Brd7, which encode components of the PBAF form of the SWI/SNF chromatin remodeling complex. Loss of PBAF function increased tumor cell sensitivity to interferon-γ, resulting in enhanced secretion of chemokines that recruit effector T cells...
January 4, 2018: Science
https://www.readbyqxmd.com/read/29300083/identifying-novel-enhancer-elements-with-crispr-based-screens
#17
Jason C Klein, Wei Chen, Molly Gasperini, Jay Shendure
Enhancers control the spatiotemporal expression of genes and are essential for encoding differentiation and development. Since their discovery more than three decades ago, researchers have largely studied enhancers removed from their genomic context. The recent adaptation of CRISPR/Cas9 to genome editing in higher organisms now allows researchers to perturb and test these elements in their genomic context, through both mutation and epigenetic modulation. In this perspective, we discuss recent advances in scanning non-coding regions of the genome for enhancer activity using CRISPR-based tools...
January 4, 2018: ACS Chemical Biology
https://www.readbyqxmd.com/read/29291512/generation-of-fhl2-homozygous-knockout-lines-from-human-embryonic-stem-cells-by-crispr-cas9-mediated-ablation
#18
Chia-Wei Chang, Chih-Chia Chang, Kuo-Chiang Hsia, Su-Yi Tsai
Cardiovascular disease is the leading cause of morbidity and mortality in the world. Mutations in the FHL2 (Four and a half LIM domains protein 2) gene are associated with cardiomyopathy in patients. Here, we generated two homozygous knockout lines using CRISPR/Cas9-mediated ablation in a human embryonic stem cell (hESC) WA09 line. These knockout lines exhibit a normal karyotype without expressing FHL2 protein, while maintaining pluripotency and differentiation properties. These isogenic mutation lines will be provided as a disease model for cardiomyopathy studies and drug screening...
December 23, 2017: Stem Cell Research
https://www.readbyqxmd.com/read/29290584/crispr-screens-uncover-genes-that-regulate-target-cell-sensitivity-to-the-morphogen-sonic-hedgehog
#19
Ganesh V Pusapati, Jennifer H Kong, Bhaven B Patel, Arunkumar Krishnan, Andreas Sagner, Maia Kinnebrew, James Briscoe, L Aravind, Rajat Rohatgi
To uncover regulatory mechanisms in Hedgehog (Hh) signaling, we conducted genome-wide screens to identify positive and negative pathway components and validated top hits using multiple signaling and differentiation assays in two different cell types. Most positive regulators identified in our screens, including Rab34, Pdcl, and Tubd1, were involved in ciliary functions, confirming the central role for primary cilia in Hh signaling. Negative regulators identified included Megf8, Mgrn1, and an unannotated gene encoding a tetraspanin protein we named Atthog...
December 26, 2017: Developmental Cell
https://www.readbyqxmd.com/read/29286403/a-universal-protocol-for-large-scale-grna-library-production-from-any-dna-source
#20
Anna Köferle, Stefan H Stricker
The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment...
December 6, 2017: Journal of Visualized Experiments: JoVE
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