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CRISPR/Cas9

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https://www.readbyqxmd.com/read/29679176/a-versatile-vector-toolkit-for-functional-analysis-of-rice-genes
#1
Feng He, Fan Zhang, Wenxian Sun, Yuese Ning, Guo-Liang Wang
BACKGROUND: Rice (Oryza sativa) is the main food for half of the world's population, and is considered the model for molecular biology studies of monocotyledon species. Although the rice genome was completely sequenced about 15 years ago, the function of most rice genes is still unknown. RESULTS: In this study, we developed a vector toolkit that contains 42 vectors for transient expression studies in rice protoplasts and stable expression analysis in transgenic rice...
April 20, 2018: Rice
https://www.readbyqxmd.com/read/29678421/growth-hormone-receptor-deficient-pigs-resemble-the-pathophysiology-of-human-laron-syndrome-and-reveal-altered-activation-of-signaling-cascades-in-the-liver
#2
Arne Hinrichs, Barbara Kessler, Mayuko Kurome, Andreas Blutke, Elisabeth Kemter, Maren Bernau, Armin M Scholz, Birgit Rathkolb, Simone Renner, Sebastian Bultmann, Heinrich Leonhardt, Martin Hrabĕ de Angelis, Hiroshi Nagashima, Andreas Hoeflich, Werner F Blum, Martin Bidlingmaier, Rüdiger Wanke, Maik Dahlhoff, Eckhard Wolf
OBJECTIVE: Laron syndrome (LS) is a rare, autosomal recessive disorder in humans caused by loss-of-function mutations of the growth hormone receptor (GHR) gene. To establish a large animal model for LS, pigs with GHR knockout (KO) mutations were generated and characterized. METHODS: CRISPR/Cas9 technology was applied to mutate exon 3 of the GHR gene in porcine zygotes. Two heterozygous founder sows with a 1-bp or 7-bp insertion in GHR exon 3 were obtained, and their heterozygous F1 offspring were intercrossed to produce GHR-KO, heterozygous GHR mutant, and wild-type pigs...
March 15, 2018: Molecular Metabolism
https://www.readbyqxmd.com/read/29676930/high-value-bioproducts-from-microalgae-strategies-and-progress
#3
Ming-Hua Liang, Jianhua Zhu, Jian-Guo Jiang
Microalgae have been considered as alternative sustainable resources for high-value bioproducts such as lipids (especially triacylglycerides [TAGs]), polyunsaturated fatty acids (PUFAs), and carotenoids, due to their relatively high photosynthetic efficiency, no arable land requirement, and ease of scale-up. It is of great significance to exploit microalgae for the production of high-value bioproducts. How to improve the content or productivity of specific bioproducts has become one of the most urgent challenges...
April 20, 2018: Critical Reviews in Food Science and Nutrition
https://www.readbyqxmd.com/read/29676720/mutagenic-repair-of-double-stranded-dna-breaks-in-vaccinia-virus-genomes-requires-cellular-dna-ligase-iv-activity-in-the-cytosol
#4
Rutger David Luteijn, Ingo Drexler, Geoffrey L Smith, Robert Jan Lebbink, Emmanuel J H J Wiertz
Poxviruses comprise a group of large dsDNA viruses that include members relevant to human and animal health, such as variola virus, monkeypox virus, cowpox virus and vaccinia virus (VACV). Poxviruses are remarkable for their unique replication cycle, which is restricted to the cytoplasm of infected cells. The independence from the host nucleus requires poxviruses to encode most of the enzymes involved in DNA replication, transcription and processing. Here, we use the CRISPR/Cas9 genome engineering system to induce DNA damage to VACV (strain Western Reserve) genomes...
April 20, 2018: Journal of General Virology
https://www.readbyqxmd.com/read/29676032/use-of-two-grnas-for-crispr-cas9-improves-bi-allelic-homologous-recombination-efficiency-in-mouse-embryonic-stem-cells
#5
Sandra Acosta, Luciano Fiore, Isabel Anna Carota, Guillermo Oliver
Targeted genome editing in mouse embryonic stem cells (ESCs) is a powerful resource to functionally characterize genes and regulatory elements. The use of the CRISPR/Cas9 genome editing approach has remarkably improved the time and efficiency of targeted recombination. However, the efficiency of this protocol is still far from ideal when aiming for bi-allelic homologous recombination, requiring at least two independent targeting recombination events. Here we describe an improved protocol that uses two gRNAs flanking the selected targeted region, leading to highly efficient homologous recombination in mouse ESCs...
April 20, 2018: Genesis: the Journal of Genetics and Development
https://www.readbyqxmd.com/read/29675805/enhanced-extracellular-pullulanase-production-in-bacillus-subtilis-using-protease-deficient-strains-and-optimal-feeding
#6
Kang Zhang, Lingqia Su, Jing Wu
To study the effect of proteases on pullulanase production, six protease-encoding genes (nprB, bpr, mpr, epr, vpr, and wprA) in the genome of Bacillus subtilis strain WS5, which already lacks the protease-encoding genes nprE and aprE, were sequentially disrupted using a CRISPR/Cas9 system. This created strains WS6-WS11, respectively. The strains WS3 (none) and WS4 (ΔnprE) were constructed earlier. After addition of expression plasmid pHYPULd4 into the strains WS3-WS11, the pullulanase production levels of the resulting strains (WS3PUL-WS11PUL, respectively) were investigated in shake-flask cultivations and recombinant strain WS5PUL produced the highest pullulanase activity (148...
April 20, 2018: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/29675267/genetic-variation-may-confound-analysis-of-crispr-cas9-off-target-mutations
#7
Guanqun Wang, Meijie Du, Jianbin Wang, Ting F Zhu
No abstract text is available yet for this article.
2018: Cell Discovery
https://www.readbyqxmd.com/read/29675030/homology-directed-repair-of-a-defective-glabrous-gene-in-arabidopsis-with-cas9-based-gene-targeting
#8
Florian Hahn, Marion Eisenhut, Otho Mantegazza, Andreas P M Weber
The CRISPR/Cas9 system has emerged as a powerful tool for targeted genome editing in plants and beyond. Double-strand breaks induced by the Cas9 enzyme are repaired by the cell's own repair machinery either by the non-homologous end joining pathway or by homologous recombination (HR). While the first repair mechanism results in random mutations at the double-strand break site, HR uses the genetic information from a highly homologous repair template as blueprint for repair of the break. By offering an artificial repair template, this pathway can be exploited to introduce specific changes at a site of choice in the genome...
2018: Frontiers in Plant Science
https://www.readbyqxmd.com/read/29673923/crispr-cas9-for-cancer-research-and-therapy
#9
REVIEW
Tianzuo Zhan, Niklas Rindtorff, Johannes Betge, Matthias P Ebert, Michael Boutros
CRISPR/Cas9 has become a powerful method for making changes to the genome of many organisms. First discovered in bacteria as part of an adaptive immune system, CRISPR/Cas9 and modified versions have found a widespread use to engineer genomes and to activate or to repress the expression of genes. As such, CRISPR/Cas9 promises to accelerate cancer research by providing an efficient technology to dissect mechanisms of tumorigenesis, identify targets for drug development, and possibly arm cells for cell-based therapies...
April 16, 2018: Seminars in Cancer Biology
https://www.readbyqxmd.com/read/29673772/widespread-enhancer-activity-from-core-promoters
#10
REVIEW
Alejandra Medina-Rivera, David Santiago-Algarra, Denis Puthier, Salvatore Spicuglia
Gene expression in higher eukaryotes is precisely regulated in time and space through the interplay between promoters and gene-distal regulatory regions, known as enhancers. The original definition of enhancers implies the ability to activate gene expression remotely, while promoters entail the capability to locally induce gene expression. Despite the conventional distinction between them, promoters and enhancers share many genomic and epigenomic features. One intriguing finding in the gene regulation field comes from the observation that many core promoter regions display enhancer activity...
April 16, 2018: Trends in Biochemical Sciences
https://www.readbyqxmd.com/read/29673078/crispr-cas9-mediated-gfp-knock-in-at-the-map1lc3b-locus-in-293ft-cells-is-better-for-bona-fide-monitoring-cellular-autophagy
#11
Zhiqiang Wu, Jinlin Zhao, Minghan Qiu, Zeyun Mi, Maobin Meng, Yu Guo, Hui Wang, Zhiyong Yuan
Accurately identifying and quantifying cellular autophagy is very important as the significance of autophagy in physiological and pathological processes becomes increasingly evident. Ectopically expressed fluorescent-tagged microtubule-associated protein light chain 3B (MAP1LC3B, LC3) is the most widely used reporter for monitoring autophagy activity thus far. However, this approach ignored the influence of constitutively overexpressed LC3 on autophagy itself and autophagy-related processes and its accuracy in indicating autophagy is questionable...
April 19, 2018: Biotechnology Journal
https://www.readbyqxmd.com/read/29672884/oxidatively-modified-pdia3-promotes-dkc1-mediated-malignancy-and-survival-of-hepatocellular-carcinoma-cells
#12
Eunkyong Ko, Jong-Seo Kim, Soomi Ju, Hyun-Wook Seo, Yeonji Chang, Jung-Ah Kang, Sung-Gyoo Park, Guhung Jung
DKC1 is a conserved gene encoding the RNA-binding protein, dyskerin pseudouridine synthase 1, which is an essential component of the telomerase holoenzyme. DKC1 upregulation is frequently observed in many different human cancers including hepatocellular carcinoma (HCC); however, its regulatory mechanisms still remain unclear. Thus, we investigated the regulatory mechanism of DKC1 in HCC progression. We found that protein-disulfide isomerase-associated 3 (PDIA3) interacted with the DKC1 regulatory DNA in HCC cells, but not in HCC cells with elevated reactive oxygen species (ROS) levels, using liquid chromatography tandem-mass spectrometry analysis after isolating the DKC1 regulatory region binding proteins...
April 19, 2018: Hepatology: Official Journal of the American Association for the Study of Liver Diseases
https://www.readbyqxmd.com/read/29672770/clustered-regularly-interspaced-short-palindromic-repeats-crispr-crispr-associated-protein-9-with-improved-proof-reading-enhances-homology-directed-repair
#13
Tomoko Kato-Inui, Gou Takahashi, Szuyin Hsu, Yuichiro Miyaoka
Genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) predominantly induces non-homologous end joining (NHEJ), which generates random insertions or deletions, whereas homology-directed repair (HDR), which generates precise recombination products, is useful for wider applications. However, the factors that determine the ratio of HDR to NHEJ products after CRISPR/Cas9 editing remain unclear, and methods by which the proportion of HDR products can be increased have not yet been fully established...
April 17, 2018: Nucleic Acids Research
https://www.readbyqxmd.com/read/29672669/crispr-cas9-cleavage-efficiency-regression-through-boosting-algorithms-and-markov-sequence-profiling
#14
Hui Peng, Yi Zheng, Michael Blumenstein, Dacheng Tao, Jinyan Li
Motivation: CRISPR/Cas9 system is a widely used genome editing tool. A prediction problem of great interests for this system is: how to select optimal single guide RNAs (sgRNAs) such that its cleavage efficiency is high meanwhile the off-target effect is low. Results: This work proposed a two-step averaging method (TSAM) for the regression of cleavage efficiencies of a set of sgRNAs by averaging the predicted efficiency scores of a boosting algorithm and those by a support vector machine (SVM)...
April 16, 2018: Bioinformatics
https://www.readbyqxmd.com/read/29672586/selection-on-the-regulation-of-sympathetic-nervous-activity-in-humans-and-chimpanzees
#15
Kang Seon Lee, Paramita Chatterjee, Eun-Young Choi, Min Kyung Sung, Jaeho Oh, Hyejung Won, Seong-Min Park, Youn-Jae Kim, Soojin V Yi, Jung Kyoon Choi
Adrenergic α2C receptor (ADRA2C) is an inhibitory modulator of the sympathetic nervous system. Knockout mice for this gene show physiological and behavioural alterations that are associated with the fight-or-flight response. There is evidence of positive selection on the regulation of this gene during chicken domestication. Here, we find that the neuronal expression of ADRA2C is lower in human and chimpanzee than in other primates. On the basis of three-dimensional chromatin structure, we identified a cis-regulatory region whose DNA sequences have been significantly accelerated in human and chimpanzee...
April 2018: PLoS Genetics
https://www.readbyqxmd.com/read/29672299/crispr-cas9-mediated-silencing-of-cd44-in-human-highly-metastatic-osteosarcoma-cells
#16
Tang Liu, Zuyun Yan, Yong Liu, Edwin Choy, Francis J Hornicek, Henry Mankin, Zhenfeng Duan
BACKGROUND/AIMS: Metastasis is the major cause of death in patients with osteosarcoma. There is an urgent need to identify molecular markers that promote metastasis. Cluster of differentiation 44 is a receptor for hyaluronic acid (HA) and HA-binding has been proven to participate in various biological tumor activities, including tumor progression and metastasis. METHODS: We performed a meta-analysis to investigate the relationship between CD44 expression, survival, and metastasis in patients with osteosarcoma...
April 16, 2018: Cellular Physiology and Biochemistry
https://www.readbyqxmd.com/read/29671068/intracellular-generation-of-single-strand-template-increases-the-knock-in-efficiency-by-combining-crispr-cas9-with-aav
#17
Qing Xiao, Taishan Min, Shuangping Ma, Lingna Hu, Hongyan Chen, Daru Lu
Targeted integration of transgenes facilitates functional genomic research and holds prospect for gene therapy. The established microhomology-mediated end-joining (MMEJ)-based strategy leads to the precise gene knock-in with easily constructed donor, yet the limited efficiency remains to be further improved. Here, we show that single-strand DNA (ssDNA) donor contributes to efficient increase of knock-in efficiency and establishes a method to achieve the intracellular linearization of long ssDNA donor. We identified that the CRISPR/Cas9 system is responsible for breaking double-strand DNA (dsDNA) of palindromic structure in inverted terminal repeats (ITRs) region of recombinant adeno-associated virus (AAV), leading to the inhibition of viral second-strand DNA synthesis...
April 18, 2018: Molecular Genetics and Genomics: MGG
https://www.readbyqxmd.com/read/29670283/genetic-identification-of-leptin-neural-circuits-in-energy-and-glucose-homeostases
#18
Jie Xu, Christopher L Bartolome, Cho Shing Low, Xinchi Yi, Cheng-Hao Chien, Peng Wang, Dong Kong
Leptin, a hormone produced in white adipose tissue, acts in the brain to communicate fuel status, suppress appetite following a meal, promote energy expenditure and maintain blood glucose stability1,2 . Dysregulation of leptin or its receptors (LEPR) results in severe obesity and diabetes3-5 . Although intensive studies on leptin have transformed obesity and diabetes research2,6 , clinical applications of the molecule are still limited 7 , at least in part owing to the complexity and our incomplete understanding of the underlying neural circuits...
April 18, 2018: Nature
https://www.readbyqxmd.com/read/29669988/establishment-of-protocol-for-preparation-of-gene-edited-bovine-ear-derived-fibroblasts-for-somatic-cell-nuclear-transplantation
#19
Takeshi Ishino, Mayuko Hashimoto, Misato Amagasa, Natsuko Saito, Osamu Dochi, Rikio Kirisawa, Hiroshi Kitamura
Recently, gene-editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) technique has attempted to utilize fibroblasts of livestock animals for somatic cell nuclear transfer. In this study, we establish the procedure for preparing skin fibroblast clones whose genes were edited by the CRISPR/Cas9 technique. After isolating fibroblasts from earlobes of Japanese Black cattle, subsequent collagenase-digestion and extensive wash procedures enabled us to avoid contamination of fungi...
2018: Biomedical Research
https://www.readbyqxmd.com/read/29669863/continuous-signaling-of-cd79b-and-cd19-is-required-for-the-fitness-of-burkitt-lymphoma-b-cells
#20
Xiaocui He, Kathrin Kläsener, Joseena M Iype, Martin Becker, Palash C Maity, Marco Cavallari, Peter J Nielsen, Jianying Yang, Michael Reth
Expression of the B-cell antigen receptor (BCR) is essential not only for the development but also for the maintenance of mature B cells. Similarly, many B-cell lymphomas, including Burkitt lymphoma (BL), require continuous BCR signaling for their tumor growth. This growth is driven by immunoreceptor tyrosine-based activation motif (ITAM) and PI3 kinase (PI3K) signaling. Here, we employ CRISPR/Cas9 to delete BCR and B-cell co-receptor genes in the human BL cell line Ramos. We find that Ramos B cells require the expression of the BCR signaling component Igβ (CD79b), and the co-receptor CD19, for their fitness and competitive growth in culture...
April 18, 2018: EMBO Journal
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