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https://www.readbyqxmd.com/read/28718799/zebrafish-as-a-model-organism-for-the-development-of-drugs-for-skin-cancer
#1
REVIEW
Fatemeh Bootorabi, Hamed Manouchehri, Reza Changizi, Harlan Barker, Elisabetta Palazzo, Annalisa Saltari, Mataleena Parikka, Carlo Pincelli, Ashok Aspatwar
Skin cancer, which includes melanoma and squamous cell carcinoma, represents the most common type of cutaneous malignancy worldwide, and its incidence is expected to rise in the near future. This condition derives from acquired genetic dysregulation of signaling pathways involved in the proliferation and apoptosis of skin cells. The development of animal models has allowed a better understanding of these pathomechanisms, with the possibility of carrying out toxicological screening and drug development. In particular, the zebrafish (Danio rerio) has been established as one of the most important model organisms for cancer research...
July 18, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28718764/crispr-mediated-genetic-interaction-profiling-identifies-rna-binding-proteins-controlling-metazoan-fitness
#2
Adam D Norris, Xicotencatl Gracida, John Calarco
<span style="font-size: 12pt; font-family: 'Times';">Genetic interaction screens have aided our understanding of complex genetic traits,</span><span style="font-size: 11.000000pt; font-family: 'Calibri';"> </span><span style="font-size: 12pt; font-family: 'Times';">diseases, and biological pathways. However, approaches for synthetic genetic analysis with</span><span style="font-size: 11.000000pt; font-family: 'Calibri';"> </span><span style="font-size: 12pt; font-family: 'Times';">null-alleles in metazoans have not been feasible...
July 18, 2017: ELife
https://www.readbyqxmd.com/read/28712454/crispr-cas9-mediated-scanning-for-regulatory-elements-required-for-hprt1-expression-via-thousands-of-large-programmed-genomic-deletions
#3
Molly Gasperini, Gregory M Findlay, Aaron McKenna, Jennifer H Milbank, Choli Lee, Melissa D Zhang, Darren A Cusanovich, Jay Shendure
The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion ("ScanDel"). We applied ScanDel to HPRT1, the housekeeping gene underlying Lesch-Nyhan syndrome, an X-linked recessive disorder...
July 1, 2017: American Journal of Human Genetics
https://www.readbyqxmd.com/read/28711281/cancergd-a-resource-for-identifying-and-interpreting-genetic-dependencies-in-cancer
#4
Stephen Bridgett, James Campbell, Christopher J Lord, Colm J Ryan
Genes whose function is selectively essential in the presence of cancer-associated genetic aberrations represent promising targets for the development of precision therapeutics. Here, we present CancerGD, a resource that integrates genotypic profiling with large-scale loss-of-function genetic screens in tumor cell lines to identify such genetic dependencies. CancerGD provides tools for searching, visualizing, and interpreting these genetic dependencies through the integration of functional interaction networks...
July 11, 2017: Cell Systems
https://www.readbyqxmd.com/read/28710008/identifying-synthetic-lethal-targets-using-crispr-cas9-system
#5
Jaspreet Kaur Dhanjal, Navaneethan Radhakrishnan, Durai Sundar
Synthetic lethality occurs when co-occurrence of two genetic events is unfavorable for the survival of the cell or organism. The conventional approach of high throughput screening of synthetic lethal targets using chemical compounds has been replaced by RNAi technology. CRISPR/Cas9, an RNA guided endonuclease system is the most recent technology for this work. Here, we have discussed the major considerations involved in designing a CRISPR/Cas9 based screening experiment for identification of synthetic lethal targets...
July 11, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28700943/a-role-for-mitochondrial-translation-in-promotion-of-viability-in-k-ras-mutant-cells
#6
Timothy D Martin, Danielle R Cook, Mei Yuk Choi, Mamie Z Li, Kevin M Haigis, Stephen J Elledge
Activating mutations in the KRAS oncogene are highly prevalent in tumors, especially those of the colon, lung, and pancreas. To better understand the genetic dependencies that K-Ras mutant cells rely upon for their growth, we employed whole-genome CRISPR loss-of-function screens in two isogenic pairs of cell lines. Since loss of essential genes is uniformly toxic in CRISPR-based screens, we also developed a small hairpin RNA (shRNA) library targeting essential genes. These approaches uncovered a large set of proteins whose loss results in the selective reduction of K-Ras mutant cell growth...
July 11, 2017: Cell Reports
https://www.readbyqxmd.com/read/28698801/tak1-inhibition-attenuates-both-inflammation-and-fibrosis-in-experimental-pneumoconiosis
#7
Jie Li, Chao Liang, Zong-Kang Zhang, Xiaohua Pan, Songlin Peng, Wing-Sze Lee, Aiping Lu, Zhixiu Lin, Ge Zhang, Wing-Nang Leung, Bao-Ting Zhang
Pneumoconiosis, caused by inhalation of mineral dusts, is a major occupational disease worldwide. Currently, there are no effective drugs owing to a lack of potential therapeutic targets during either the inflammation or fibrosis molecular events in pneumoconiosis. Here, we performed microarrays to identify aberrantly expressed genes in the above molecular events in vitro and found a hub gene transforming growth factor-β-activated kinase 1 (TAK1), which was highly expressed and activated in pneumoconiosis patients as well as silica-exposed rats with experimental pneumoconiosis...
2017: Cell Discovery
https://www.readbyqxmd.com/read/28679166/a-convenient-method-to-pre-screen-candidate-guide-rnas-for-crispr-cas9-gene-editing-by-nhej-mediated-integration-of-a-self-cleaving-gfp-expression-plasmid
#8
András Tálas, Péter István Kulcsár, Nóra Weinhardt, Adrienn Borsy, Eszter Tóth, Kornélia Szebényi, Sarah Laura Krausz, Krisztina Huszár, István Vida, Ádám Sturm, Bianka Gordos, Orsolya Ivett Hoffmann, Petra Bencsura, Antal Nyeste, Zoltán Ligeti, Elfrieda Fodor, Ervin Welker
The efficacies of guide RNAs (gRNAs), the short RNA molecules that bind to and determine the sequence specificity of the Streptococcus pyogenes Cas9 nuclease, to mediate DNA cleavage vary dramatically. Thus, the selection of appropriate target sites, and hence spacer sequence, is critical for most applications. Here, we describe a simple, unparalleled method for experimentally pre-testing the efficiencies of various gRNAs targeting a gene. The method explores NHEJ-cloning, genomic integration of a GFP-expressing plasmid without homologous arms and linearized in-cell...
June 30, 2017: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes
https://www.readbyqxmd.com/read/28678442/crispr-cas9-directed-gene-editing-for-the-generation-of-loss-of-function-mutants-in-high-throughput-zebrafish-f0-screens
#9
Sunita S Shankaran, Timothy J Dahlem, Brent W Bisgrove, H Joseph Yost, Martin Tristani-Firouzi
The ability to perform reverse genetics in the zebrafish model organism has been greatly advanced with the advent of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated) system. The high level of efficiency in generating mutations when using the CRISPR/Cas9 system combined with the rapid generation time of the zebrafish model organism has made the possibility of performing F0 screens in this organism a reality. This unit describes a detailed protocol for performing an F0 screen using the CRISPR/Cas9 system in zebrafish starting with the design and production of custom CRISPR/Cas9 reagents for injection...
July 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28677529/generation-and-characterization-of-a-human-ipsc-cell-line-expressing-inducible-cas9-in-the-safe-harbor-aavs1-locus
#10
Julio Castaño, Clara Bueno, Senda Jiménez-Delgado, Heleia Roca-Ho, Mario F Fraga, Agustín F Fernandez, Mahito Nakanishi, Raúl Torres-Ruiz, Sandra Rodríguez-Perales, Pablo Menéndez
We report the generation-characterization of a fetal liver (FL) B-cell progenitor (BCP)-derived human induced pluripotent stem cell (hiPSC) line CRISPR/Cas9-edited to carry/express a single copy of doxycycline-inducible Cas9 gene in the "safe locus" AAVS1 (iCas9-FL-BCP-hiPSC). Gene-edited iPSCs remained pluripotent after CRISPR/Cas9 genome-edition. Correct genomic integration of a unique copy of Cas9 was confirmed by PCR and Southern blot. Cas9 was robustly and specifically expressed on doxycycline exposure...
May 2017: Stem Cell Research
https://www.readbyqxmd.com/read/28663331/multiplexed-gene-editing-and-protein-over-expression-using-a-tobacco-mosaic-virus-viral-vector
#11
Will Cody, Herman B Scholthof, T Erik Mirkov
The CRISPR/Cas9 gene editing platform has been adapted as a transient screening device for most biological model systems, although a comparable transient system has yet to be regularly implemented in plant biology. Here, we use the Tobacco mosaic virus-derived viral vector, TRBO, to evaluate the ability of three single guide RNA (sgRNA) delivery constructs in Nicotiana benthamiana by measuring the presence of genomic indels (inserts and deletions) when co-delivered through agroinfiltration with a Cas9 nuclease expressing construct...
June 29, 2017: Plant Physiology
https://www.readbyqxmd.com/read/28662369/generation-of-genomic-deletions-of-rig-i-gene-in-goat-primary-cell-culture-using-crispr-cas9-method
#12
Shivani Malpotra, Ashutosh Vats, Sushil Kumar, Devika Gautam, Sachinandan De
CRISPR/Cas9 system is a natural immune system in prokaryotes protecting them from infectious viral or plasmid DNA invading the cells. This RNA-guided system can act as powerful tool for introducing genomic alterations in eukaryotic cells with high efficiency. In the present study, Rig-Igene is taken as model gene to study the efficiency of CRISPR/Cas9 system induced gene deletion in primary fibroblast cell culture. Rig-I(retinoic acid-inducible gene-1) is involved in regulating immune response in mammals. In this study, we optimized the CRISPR/Cas9 method for knocking out Rig-Igene in Goat primary fibroblasts by using a NHEJ pathway...
June 29, 2017: Animal Biotechnology
https://www.readbyqxmd.com/read/28655737/evaluation-and-design-of-genome-wide-crispr-spcas9-knockout-screens
#13
Traver Hart, Amy Hin Yan Tong, Katie Chan, Jolanda Van Leeuwen, Ashwin Seetharaman, Michael Aregger, Megha Chandrashekhar, Nicole Hustedt, Sahil Seth, Avery Noonan, Andrea Habsid, Olga Sizova, Lyudmilla Nedyalkova, Ryan Climie, Leanne Tworzyanski, Keith Lawson, Maria Augusta Sartori, Sabriyeh Alibai, David Tieu, Sanna Masud, Patricia Mero, Alexander Weiss, Kevin R Brown, Matej Ušaj, Maximilian Billmann, Mahfuzur Rahman, Michael Costanzo, Chad L Myers, Brenda J Andrews, Charles Boone, Daniel Durocher, Jason Moffat
The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols...
June 27, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/28654078/selection-dependent-and-independent-generation-of-crispr-cas9-mediated-gene-knockouts-in-mammalian-cells
#14
Erin L Sternburg, Kristen C Dias, Fedor V Karginov
The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening...
June 16, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28653435/rice-crispr-rapidly-increased-cut-ends-by-an-exonuclease-cas9-fusion-in-zebrafish
#15
Thomas P Clements, Bhavna Tandon, Hendrik A Lintel, Joseph H McCarty, Daniel S Wagner
Application of CRISPR-Cas9 technology in diverse organisms has resulted in an explosion of genome modification efforts. To expand the toolbox of applications, we have created an E. coli Exonuclease I (sbcB) - Cas9 fusion that has altered enzymatic activity in zebrafish embryos. This Cas9 variant has increased mutation efficiency and favors longer deletions relative to wild type Cas9. We anticipate that this variant will allow for more efficient screening for F0 phenotypes and mutation of a larger spectrum of genomic targets including deletion of regulatory regions and creating loss of function mutations in transcription units with poor sequence conservation such as lncRNAs where larger deletions may be required for loss of function...
June 27, 2017: Genesis: the Journal of Genetics and Development
https://www.readbyqxmd.com/read/28652865/crispr-cas9-enabled-genetic-disruptions-for-understanding-ethanol-and-ethyl-acetate-biosynthesis-in-kluyveromyces-marxianus
#16
Ann-Kathrin Löbs, Ronja Engel, Cory Schwartz, Andrew Flores, Ian Wheeldon
BACKGROUND: The thermotolerant yeast Kluyveromyces marxianus shows promise as an industrial host for the biochemical production of fuels and chemicals. Wild-type strains are known to ferment high titers of ethanol and can effectively convert a wide range of C5, C6, and C12 sugars into the volatile short-chain ester ethyl acetate. Strain engineering, however, has been limited due to a lack of advanced genome-editing tools and an incomplete understanding of ester and ethanol biosynthesis...
2017: Biotechnology for Biofuels
https://www.readbyqxmd.com/read/28649363/genetic-and-epigenetic-control-of-gene-expression-by-crispr-cas-systems
#17
REVIEW
Albert Lo, Lei Qi
The discovery and adaption of bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems has revolutionized the way researchers edit genomes. Engineering of catalytically inactivated Cas variants (nuclease-deficient or nuclease-deactivated [dCas]) combined with transcriptional repressors, activators, or epigenetic modifiers enable sequence-specific regulation of gene expression and chromatin state. These CRISPR-Cas-based technologies have contributed to the rapid development of disease models and functional genomics screening approaches, which can facilitate genetic target identification and drug discovery...
2017: F1000Research
https://www.readbyqxmd.com/read/28628606/splicing-stimulates-sirna-formation-at-drosophila-dna-double-strand-breaks
#18
Karin Merk, Marco Breinig, Romy Böttcher, Stefan Krebs, Helmut Blum, Michael Boutros, Klaus Förstemann
DNA double-strand breaks trigger the production of locus-derived siRNAs in fruit flies, human cells and plants. At least in flies, their biogenesis depends on active transcription running towards the break. Since siRNAs derive from a double-stranded RNA precursor, a major question is how broken DNA ends can generate matching sense and antisense transcripts. We performed a genome-wide RNAi-screen in cultured Drosophila cells, which revealed that in addition to DNA repair factors, many spliceosome components are required for efficient siRNA generation...
June 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28625976/a-genome-wide-crispr-screen-identifies-genes-critical-for-resistance-to-flt3-inhibitor-ac220
#19
Panpan Hou, Chao Wu, Yuchen Wang, Rui Qi, Dheeraj Bhavanasi, Zhixiang Zuo, Cedric Dos Santos, Shuliang Chen, Yu Chen, Hong Zheng, Hong Wang, Alexander E Perl, Deyin Guo, Jian Huang
Acute myeloid leukemia (AML) is a malignant hematopoietic disease and the most common type of acute leukemia in adults. The mechanisms underlying drug resistance in AML are poorly understood. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are the most common molecular abnormality in AML. Quizartinib (AC220) is a potent and selective second-generation inhibitor of FLT3. It is in clinical trials for the treatment of relapsed or refractory FLT3-ITD-positive and -negative AML patients and as maintenance therapy...
June 16, 2017: Cancer Research
https://www.readbyqxmd.com/read/28625679/phenotype-databases-for-genetic-screens-in-human-cells
#20
Benedikt Rauscher, Erica Valentini, Ulrike Hardeland, Michael Boutros
Genetic screens are powerful tools to identify components that make up biological systems. Perturbations introduced by methods such as RNA interference (RNAi) or CRISPR/Cas9-mediated genome editing lead to biological phenotypes that can be examined to understand the molecular function of genes in the cell. Over the years, many of such experiments have been conducted providing a wealth of knowledge about genotype-to-phenotype relationships. These data are a rich source of information and it is in a common interest to make them available in a simplified and integrated format...
June 15, 2017: Journal of Biotechnology
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