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CRISPR/Cas9

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https://www.readbyqxmd.com/read/28102837/precise-and-efficient-scarless-genome-editing-in-stem-cells-using-correct
#1
Dylan Kwart, Dominik Paquet, Shaun Teo, Marc Tessier-Lavigne
CRISPR/Cas9 is a promising tool for genome-editing DNA in cells with single-base-pair precision, which allows novel in vitro models of human disease to be generated-e.g., in pluripotent stem cells. However, the accuracy of intended sequence changes can be severely diminished by CRISPR/Cas9's propensity to re-edit previously modified loci, causing unwanted mutations (indels) alongside intended changes. Here we describe a genome-editing framework termed consecutive re-guide or re-Cas steps to erase CRISPR/Cas-blocked targets (CORRECT), which, by exploiting the use of highly efficacious CRISPR/Cas-blocking mutations in two rounds of genome editing, enables accurate, efficient and scarless introduction of specific base changes-for example, in human induced pluripotent (iPS) stem cells...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28102301/corrigendum-crispr-cas9-mediated-efficient-pd-1-disruption-on-human-primary-t-cells-from-cancer-patients
#2
Shu Su, Bian Hu, Jie Shao, Bin Shen, Juan Du, Yinan Du, Jiankui Zhou, Lixia Yu, Lianru Zhang, Fangjun Chen, Huizi Sha, Lei Cheng, Fanyan Meng, Zhengyun Zou, Xingxu Huang, Baorui Liu
No abstract text is available yet for this article.
January 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28101903/development-and-characterization-of-anti-glycopeptide-monoclonal-antibodies-against-human-podoplanin-using-glycan-deficient-cell-lines-generated-by-crispr-cas9-and-talen
#3
Mika K Kaneko, Takuro Nakamura, Ryusuke Honma, Satoshi Ogasawara, Yuki Fujii, Shinji Abe, Michiaki Takagi, Hiroyuki Harada, Hiroyoshi Suzuki, Yasuhiko Nishioka, Yukinari Kato
Human podoplanin (hPDPN), which binds to C-type lectin-like receptor-2 (CLEC-2), is involved in platelet aggregation and cancer metastasis. The expression of hPDPN in cancer cells or cancer-associated fibroblasts indicates poor prognosis. Human lymphatic endothelial cells, lung-type I alveolar cells, and renal glomerular epithelial cells express hPDPN. Although numerous monoclonal antibodies (mAbs) against hPDPN are available, they recognize peptide epitopes of hPDPN. Here, we generated a novel anti-hPDPN mAb, LpMab-21...
January 19, 2017: Cancer Medicine
https://www.readbyqxmd.com/read/28099519/crispr-cas9-mediated-nox4-knockout-inhibits-cell-proliferation-and-invasion-in-hela-cells
#4
Naser Jafari, Hyunju Kim, Rackhyun Park, Liqing Li, Minsu Jang, Andrew J Morris, Junsoo Park, Cai Huang
Increased expression of NOX4 protein is associated with cancer progression and metastasis but the role of NOX4 in cell proliferation and invasion is not fully understood. We generated NOX4 knockout HeLa cell lines using the CRISPR-Cas9 gene editing system to explore the cellular functions of NOX4. After transfection of CRISPR-Cas9 construct, we performed T7 endonuclease 1 assays and DNA sequencing to generate and identify insertion and deletion of the NOX4 locus. We confirmed the knockout of NOX4 by Western blotting...
2017: PloS One
https://www.readbyqxmd.com/read/28098238/expression-of-the-novel-maternal-centrosome-assembly-factor-wdr8-is-required-for-vertebrate-embryonic-mitoses
#5
Daigo Inoue, Manuel Stemmer, Thomas Thumberger, Thomas Ruppert, Felix Bärenz, Joachim Wittbrodt, Oliver J Gruss
The assembly of the first centrosome occurs upon fertilisation when male centrioles recruit pericentriolar material (PCM) from the egg cytoplasm. The mechanisms underlying the proper assembly of centrosomes during early embryogenesis remain obscure. We identify Wdr8 as a novel maternally essential protein that is required for centrosome assembly during embryonic mitoses of medaka (Oryzias latipes). By CRISPR-Cas9-mediated knockout, maternal/zygotic Wdr8-null (m/zWdr8(-/-)) blastomeres exhibit severe defects in centrosome structure that lead to asymmetric division, multipolar mitotic spindles and chromosome alignment errors...
January 18, 2017: Nature Communications
https://www.readbyqxmd.com/read/28098143/efficient-dna-free-genome-editing-of-bread-wheat-using-crispr-cas9-ribonucleoprotein-complexes
#6
Zhen Liang, Kunling Chen, Tingdong Li, Yi Zhang, Yanpeng Wang, Qian Zhao, Jinxing Liu, Huawei Zhang, Cuimin Liu, Yidong Ran, Caixia Gao
Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA...
January 18, 2017: Nature Communications
https://www.readbyqxmd.com/read/28096358/repair-shielding-of-platinum-dna-lesions-in-testicular-germ-cell-tumors-by-high-mobility-group-box-protein-4-imparts-cisplatin-hypersensitivity
#7
Samuel G Awuah, Imogen A Riddell, Stephen J Lippard
Cisplatin is the most commonly used anticancer drug for the treatment of testicular germ cell tumors (TGCTs). The hypersensitivity of TGCTs to cisplatin is a subject of widespread interest. Here, we show that high-mobility group box protein 4 (HMGB4), a protein preferentially expressed in testes, uniquely blocks excision repair of cisplatin-DNA adducts, 1,2-intrastrand cross-links, to potentiate the sensitivity of TGCTs to cisplatin therapy. We used CRISPR/Cas9-mediated gene editing to knockout the HMGB4 gene in a testicular human embryonic carcinoma and examined cellular responses...
January 17, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28096221/efficient-crispr-cas9-assisted-gene-targeting-enables-rapid-and-precise-genetic-manipulation-of-mammalian-neural-stem-cells
#8
Raul Bardini Bressan, Pooran Singh Dewari, Maria Kalantzaki, Ester Gangoso, Mantas Matjusaitis, Claudia Garcia-Diaz, Carla Blin, Vivien Grant, Harry Bulstrode, Sabine Gogolok, William C Skarnes, Steven M Pollard
Mammalian neural stem (NS) cell lines provide a tractable model for discovery across stem cell and developmental biology, regenerative medicine and neuroscience. They can be derived from foetal or adult germinal tissues and continuously propagated in vitro as adherent monolayers. NS cells are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with targeted genetic manipulations. However, gene targeting - so critical for functional studies of embryonic stem cells - has not been exploited to date in NS cells...
January 17, 2017: Development
https://www.readbyqxmd.com/read/28095751/crispr-based-epigenome-editing-of-cytokine-receptors-for-the-promotion-of-cell-survival-and-tissue-deposition-in-inflammatory-environments
#9
Niloofar Farhang, Jonathan M Brunger, Joshua D Stover, Pratiksha I Thakore, Brandon Lawrence, Farshid Guilak, Charles A Gersbach, Lori A Setton, Robert D Bowles
Musculoskeletal diseases have been associated with inflammatory cytokine action, particularly action by TNF-α and IL-1β. These inflammatory cytokines promote apoptosis and senescence of cells in diseased tissue and extracellular matrix breakdown. Stem cell-based therapies are being considered for the treatment of musculoskeletal diseases, but the presence of these inflammatory cytokines will have similar deleterious action on therapeutic cells delivered to these environments. Methods that prevent inflammatory-induced apoptosis and pro-inflammatory signaling, in cell and pathway specific manners are needed...
January 17, 2017: Tissue Engineering. Part A
https://www.readbyqxmd.com/read/28095622/talens-and-crispr-cas9-fuel-genetically-engineered-clinically-relevant-xenopus-tropicalis-tumor-models
#10
REVIEW
Thomas Naert, Tom van Nieuwenhuysen, Kris Vleminckx
The targeted nuclease revolution (TALENs, CRISPR/Cas9) now allows Xenopus researchers to rapidly generate custom on-demand genetic knockout models. These novel methods to perform reverse genetics are unprecedented and are fueling a wide array of human disease models within the aquatic diploid model organism Xenopus tropicalis (X. tropicalis). This emerging technology review focuses on the tools to rapidly generate genetically engineered X. tropicalis models (GEXM), with a focus on establishment of genuine genetic and clinically relevant cancer models...
January 17, 2017: Genesis: the Journal of Genetics and Development
https://www.readbyqxmd.com/read/28089076/comparative-analyses-of-secreted-proteins-in-plant-pathogenic-smut-fungi-and-related-basidiomycetes
#11
Mariana Schuster, Gabriel Schweizer, Regine Kahmann
In the ten years since the genome sequence of the basidiomycete corn smut fungus Ustilago maydis was published, additional genomes of smut species infecting different hosts became available. In addition, the genomes of related Malassezia species causing skin diseases and of Pseudozyma species not known to infect plants were determined. As secreted proteins are critical virulence determinants in U. maydis we compare here the secretomes of 12 basidiomycete species to gain information about their composition and conservation...
January 6, 2017: Fungal Genetics and Biology: FG & B
https://www.readbyqxmd.com/read/28088877/the-concerted-action-of-type-2-and-type-3-deiodinases-regulates-the-cell-cycle-and-survival-of-basal-cell-carcinoma-cells
#12
Caterina Miro, Raffaele Ambrosio, Maria Angela De Stefano, Daniela Di Girolamo, Emery Di Cicco, Annunziata Gaetana Cicatiello, Giuseppina Mancino, Tommaso Porcelli, Maddalena Raia, Luigi Del Vecchio, Domenico Salvatore, Monica Dentice
BACKGROUND: Thyroid hormones (THs) mediate pleiotropic cellular processes involved in metabolism, cellular proliferation and differentiation. The intracellular hormonal environment can be tailored by the deiodinase enzymes, D2 and D3, which catalyze TH activation and inactivation, respectively. In many cellular systems, THs exert well documented stimulatory or inhibitory effects on cell proliferation, however, the molecular mechanisms by which they control rates of cell cycle progression have not yet been entirely clarified...
January 15, 2017: Thyroid: Official Journal of the American Thyroid Association
https://www.readbyqxmd.com/read/28081254/maternal-supply-of-cas9-to-zygotes-facilitates-the-efficient-generation-of-site-specific-mutant-mouse-models
#13
Alberto Cebrian-Serrano, Shijun Zha, Lars Hanssen, Daniel Biggs, Christopher Preece, Benjamin Davies
Genome manipulation in the mouse via microinjection of CRISPR/Cas9 site-specific nucleases has allowed the production time for genetically modified mouse models to be significantly reduced. Successful genome manipulation in the mouse has already been reported using Cas9 supplied by microinjection of a DNA construct, in vitro transcribed mRNA and recombinant protein. Recently the use of transgenic strains of mice overexpressing Cas9 has been shown to facilitate site-specific mutagenesis via maternal supply to zygotes and this route may provide an alternative to exogenous supply...
2017: PloS One
https://www.readbyqxmd.com/read/28081156/generation-of-a-stable-transgenic-swine-model-expressing-a-porcine-histone-2b-egfp-fusion-protein-for-cell-tracking-and-chromosome-dynamics-studies
#14
Renan B Sper, Sehwon Koh, Xia Zhang, Sean Simpson, Bruce Collins, Jeff Sommer, Robert M Petters, Ignacio Caballero, Jeff L Platt, Jorge A Piedrahita
Transgenic pigs have become an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic and physiological similarities with humans. The development of fluorescent proteins as molecular tags has allowed investigators to track cell migration and engraftment levels after transplantation. Here we describe the development of two transgenic pig models via SCNT expressing a fusion protein composed of eGFP and porcine Histone 2B (pH2B)...
2017: PloS One
https://www.readbyqxmd.com/read/28079885/mirna182-regulates-percentage-of-myeloid-and-erythroid-cells-in-chronic-myeloid-leukemia
#15
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/28077794/large-scale-genomic-deletions-mediated-by-crispr-cas9-system
#16
EDITORIAL
Yuning Song, Liangxue Lai, Zhanjun Li
No abstract text is available yet for this article.
January 6, 2017: Oncotarget
https://www.readbyqxmd.com/read/28077679/crispr-cas9-gene-repair-of-hematopoietic-stem-cells-from-patients-with-x-linked-chronic-granulomatous-disease
#17
Suk See De Ravin, Linhong Li, Xiaolin Wu, Uimook Choi, Cornell Allen, Sherry Koontz, Janet Lee, Narda Theobald-Whiting, Jessica Chu, Mary Garofalo, Colin Sweeney, Lela Kardava, Susan Moir, Angelia Viley, Pachai Natarajan, Ling Su, Douglas Kuhns, Kol A Zarember, Madhusudan V Peshwa, Harry L Malech
Gene repair of CD34(+) hematopoietic stem and progenitor cells (HSPCs) may avoid problems associated with gene therapy, such as vector-related mutagenesis and dysregulated transgene expression. We used CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated 9) to repair a mutation in the CYBB gene of CD34(+) HSPCs from patients with the immunodeficiency disorder X-linked chronic granulomatous disease (X-CGD). Sequence-confirmed repair of >20% of HSPCs from X-CGD patients restored the function of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and superoxide radical production in myeloid cells differentiated from these progenitor cells in vitro...
January 11, 2017: Science Translational Medicine
https://www.readbyqxmd.com/read/28077621/functional-redundancy-and-non-redundancy-between-two-troponin-c-isoforms-in-drosophila-adult-muscles
#18
Maria B Chechenova, Sara Maes, Sandy T Oas, Cloyce Nelson, Kaveh G Kiani, Anton L Bryantsev, Richard M Cripps
We investigated the functional overlap of two muscle Troponin C (TpnC) genes that are expressed in the adult fruit fly, Drosophila melanogaster: TpnC4 is predominantly expressed in the indirect flight muscles (IFM), whereas TpnC41C is the main isoform in the tergal depressor of the trochanter muscle (TDT or jump muscle). Using CRISPR/Cas9, we created a transgenic line with a homozygous deletion of TpnC41C, and compared its phenotype to a line lacking functional TpnC4 We found that the removal of either of these genes leads to expression of the other isoform in both muscle types...
January 11, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28077422/antxr2-knock-out-does-not-result-in-the-development-of-hypertension-in-rats
#19
Xiaoyan Liu, Wen Yuan, Jing Li, Lei Yang, Jun Cai
BACKGROUND: Our recent genetic study as well as robust evidences reported by previous genome-wide association studies (GWASs) have indicated that the single nucleotide polymorphism rs16998073, located near gene anthrax toxin receptor 2 (ANTXR2), was significantly associated with hypertension in Asians and Europeans. The aim of the present study was to determine whether ANTXR2 is the causal gene of hypertension at the 4q21 locus using an ANTXR2 knock-out model. METHODS: Relative expression of ANTXR2 in Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) were determined by real-time quantitative polymerase chain reaction and western blot analysis...
February 2017: American Journal of Hypertension
https://www.readbyqxmd.com/read/28077285/a-fluorophore-fusion-construct-of-human-profilin-i-with-non-compromized-poly-l-proline-binding-capacity-suitable-for-imaging
#20
Michaela Nejedla, Zhilun Li, Anna E Masser, Matteo Biancospino, Matthias Spiess, Sebastian D Mackowiak, Marc R Friedländer, Roger Karlsson
Profilin is vital for actin organization in eukaryotic cells. It controls actin filament formation by binding monomeric actin and numerous proteins involved in polarized actin assembly. Important for the latter is the interaction surface formed by the N- and C-terminal helices, which pack close to each other on one side of the molecule at distance from the actin site and mediate binding to poly-proline sequences present in many of the targeted proteins. Via these interactions profilin contributes to the spatiotemporal control of actin filament growth...
January 7, 2017: Journal of Molecular Biology
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