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Gene edition

Deepthi Alapati, Edward E Morrisey
While our understanding of the genetics and pathology of congenital lung diseases such as surfactant protein deficiency, cystic fibrosis and alpha 1 antitrypsin deficiency is extensive, treatment options are lacking. Since the lung is a barrier organ in direct communication with the external environment, targeted delivery of gene corrective technologies to the respiratory system via intra-tracheal or intranasal routes is an attractive option for therapy. CRISPR/Cas9 gene editing technology is a promising approach to repair or inactivate disease causing mutations...
October 25, 2016: American Journal of Respiratory Cell and Molecular Biology
Shilpa Nagaraju, Naomi Kathleen Davies, David Jeffrey Fraser Walker, Michael Köpke, Séan Dennis Simpson
BACKGROUND: Impactful greenhouse gas emissions abatement can now be achieved through gas fermentation using acetogenic microbes for the production of low-carbon fuels and chemicals. However, compared to traditional hosts like Escherichia coli or yeast, only basic genetic tools exist for gas-fermenting acetogens. To advance the process, a robust genetic engineering platform for acetogens is essential. RESULTS: In this study, we report scarless genome editing of an industrially used model acetogen, Clostridium autoethanogenum, using the CRISPR/Cas9 system...
2016: Biotechnology for Biofuels
Miri Danan-Gotthold, Clotilde Guyon, Matthieu Giraud, Erez Y Levanon, Jakub Abramson
BACKGROUND: In order to become functionally competent but harmless mediators of the immune system, T cells undergo a strict educational program in the thymus, where they learn to discriminate between self and non-self. This educational program is, to a large extent, mediated by medullary thymic epithelial cells that have a unique capacity to express, and subsequently present, a large fraction of body antigens. While the scope of promiscuously expressed genes by medullary thymic epithelial cells is well-established, relatively little is known about the expression of variants that are generated by co-transcriptional and post-transcriptional processes...
October 24, 2016: Genome Biology
Wenshu Luo, Hidenobu Mizuno, Ryohei Iwata, Shingo Nakazawa, Kosuke Yasuda, Shigeyoshi Itohara, Takuji Iwasato
Here we describe "Supernova" series of vector systems that enable single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV)-mediated gene delivery. In Supernova, sparse labeling relies on low TRE leakage. In a small population of cells with over-threshold leakage, initial tTA-independent weak expression is enhanced by tTA/TRE-positive feedback along with a site-specific recombination system (e.g., Cre/loxP, Flpe/FRT). Sparse and bright labeling by Supernova with little background enables the visualization of the morphological details of individual neurons in densely packed brain areas such as the cortex and hippocampus, both during development and in adulthood...
October 24, 2016: Scientific Reports
Fang Chen, Weifeng Zhang, Junli Zhao, Peiyan Yang, Rui Ma, Haibin Xia
Objective To prepare Rev-erbβ knockout HEK293 cells using clustered regularly interspaced short palindromic repeats/Cas 9 nuclease (CRISPR/Cas9) gene editing technology. Methods The knock-in or knockout of Rev-erbβ gene could be realized by single-guide RNA (sgRNA)-mediated Cas9 cutting of target DNA, and followed by DNA homologous recombination or non-homologous end joining-mediated DNA repair. Firstly, four sgRNAs were designed for Rev-erbβ gene. The sgRNA1 and sgRNA2 with the higher activity were respectively used to construct pCMV-hCas9-U6-Rev-erbβ sgRNA1 and pCMV-hCas9-U6-Rev-erbβ sgRNA2...
November 2016: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
John D Chan, Dan Zhang, Xiaolong Liu, Magdalena Z Zarowiecki, Matthew Berriman, Jonathan S Marchant
The robust regenerative capacity of planarian flatworms depends on the orchestration of signaling events from early wounding responses through the stem cell enacted differentiative outcomes that restore appropriate tissue types. Acute signaling events in excitable cells play an important role in determining regenerative polarity, rationalized by the discovery that sub-epidermal muscle cells express critical patterning genes known to control regenerative outcomes. These data imply a dual conductive (neuromuscular signaling) and instructive (anterior-posterior patterning) role for Ca(2+) signaling in planarian regeneration...
October 19, 2016: Biochimica et Biophysica Acta
Wojciech Rosikiewicz, Izabela Makałowska
Natural antisense transcripts (NATs) are RNA molecules that originate from opposite DNA strands of the same genomic locus (cis-NAT) or unlinked genomic loci (trans-NAT). NATs may play various regulatory functions at the transcriptional level via transcriptional interference. NATs may also regulate gene expression levels post-transcriptionally via induction of epigenetic changes or double-stranded RNA formation, which may lead to endogenous RNA interference, RNA editing or RNA masking. The true biological significance of the natural antisense transcripts remains controversial despite many years of research...
October 21, 2016: Acta Biochimica Polonica
Nina Xie, He Gong, Joshua A Suhl, Pankaj Chopra, Tao Wang, Stephen T Warren
Fragile X syndrome (FXS) is a common cause of intellectual disability that is most often due to a CGG-repeat expansion mutation in the FMR1 gene that triggers epigenetic gene silencing. Epigenetic modifying drugs can only transiently and modestly induce FMR1 reactivation in the presence of the elongated CGG repeat. As a proof-of-principle, we excised the expanded CGG-repeat in both somatic cell hybrids containing the human fragile X chromosome and human FXS iPS cells using the CRISPR/Cas9 genome editing. We observed transcriptional reactivation in approximately 67% of the CRISPR cut hybrid colonies and in 20% of isolated human FXS iPSC colonies...
2016: PloS One
Glenn Yiu, Eric Tieu, Anthony T Nguyen, Brittany Wong, Zeljka Smit-McBride
Purpose: To employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells. Methods: CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcuspyogenes Cas9 endonuclease (SpCas9) gene...
October 1, 2016: Investigative Ophthalmology & Visual Science
Ji Luo, Qingyang Liu, Kunihiko Morihiro, Alexander Deiters
Using small molecules to control the function of proteins in live cells with complete specificity is highly desirable, but challenging. Here we report a small-molecule switch that can be used to control protein activity. The approach uses a phosphine-mediated Staudinger reduction to activate protein function. Genetic encoding of an ortho-azidobenzyloxycarbonyl amino acid using a pyrrolysyl transfer RNA synthetase/tRNACUA pair in mammalian cells enables the site-specific introduction of a small-molecule-removable protecting group into the protein of interest...
November 2016: Nature Chemistry
Yanina S Bogliotti, Marcela Vilarino, Pablo J Ross
Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo...
October 5, 2016: Journal of Visualized Experiments: JoVE
Elizabeth M Dlugosz, Scott C Lenaghan, C Neal Stewart
Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a 'Bright Yellow' 2 (BY-2) tobacco suspension culture using a robotic platform...
September 27, 2016: Journal of Visualized Experiments: JoVE
In K Cho, Silun Wang, Hui Mao, Anthony Ws Chan
Recent advances in stem cell-based regenerative medicine, cell replacement therapy, and genome editing technologies (i.e. CRISPR-Cas 9) have sparked great interest in in vivo cell monitoring. Molecular imaging promises a unique approach to noninvasively monitor cellular and molecular phenomena, including cell survival, migration, proliferation, and even differentiation at the whole organismal level. Several imaging modalities and strategies have been explored for monitoring cell grafts in vivo. We begin this review with an introduction describing the progress in stem cell technology, with a perspective toward cell replacement therapy...
2016: American Journal of Nuclear Medicine and Molecular Imaging
Szabolcs Makai, László Tamás, Angéla Juhász
Wheat has been cultivated for 10000 years and ever since the origin of hexaploid wheat it has been exempt from natural selection. Instead, it was under the constant selective pressure of human agriculture from harvest to sowing during every year, producing a vast array of varieties. Wheat has been adopted globally, accumulating variation for genes involved in yield traits, environmental adaptation and resistance. However, one small but important part of the wheat genome has hardly changed: the regulatory regions of both the x- and y-type high molecular weight glutenin subunit (HMW-GS) genes, which are alone responsible for approximately 12% of the grain protein content...
2016: Frontiers in Plant Science
Ching-Tzu Yen, Meng-Ni Fan, Yung-Li Yang, Sheng-Chieh Chou, I-Shing Yu, Shu-Wha Lin
Hemophilia is the most well-known hereditary bleeding disorder, with an incidence of one in every 5000 to 30,000 males worldwide. The disease is treated by infusion of protein products on demand and as prophylaxis. Although these therapies have been very successful, some challenging and unresolved tasks remain, such as reducing bleeding rates, presence of target joints and/or established joint damage, eliminating the development of inhibitors, and increasing the success rate of immune-tolerance induction (ITI)...
2016: Thrombosis Journal
James West, W Warren Gill
Genome editing in large animals has tremendous practical applications, from more accurate models for medical research through improved animal welfare and production efficiency. Although genetic modification in large animals has a 30 year history, until recently technical issues limited its utility. The original methods - pronuclear injection and integrating viruses - were plagued with problems associated with low efficiency, silencing, poor regulation of gene expression, and variability associated with random integration...
June 2016: Journal of Equine Veterinary Science
Alexander N Shikov, Olga N Pozharitskaya, Valery G Makarov
PURPOSE: Aralia elata var. mandshurica (Rupr. & Maxim.) J.Wen syn. A. mandshurica Rupr. & Maxim is evaluated for its medicinal application. The aim of this study is to analyze pharmacological studies on A. elata var. mandshurica published until December 2015. METHODS: The information regarding the chemistry, safety, effectiveness, and pharmacological and clinical effects of A. elata was systematically collected from the scientific literature through library catalogs; online services such as E-library...
November 15, 2016: Phytomedicine: International Journal of Phytotherapy and Phytopharmacology
Jeong Hyo Lee, Si Won Kim, Tae Sub Park
Objective: Based on rapid advancement of genetic modification techniques, genomic editing is expected to become the most efficient tool for improvement of economic traits in livestock as well as poultry. In this study, we examined and verified the nickase of mutated CRISPR-associated protein 9 (Cas9) to modulate the specific target gene in chicken DF1 cells. Methods: Chicken myostatin which inhibits muscle cell growth and differentiation during myogenesis was targeted to be deleted and mutated by the Cas9-D10A nickase...
October 19, 2016: Asian-Australasian Journal of Animal Sciences
Saura R Silva, Yani C A Diaz, Helen Alves Penha, Daniel G Pinheiro, Camila C Fernandes, Vitor F O Miranda, Todd P Michael, Alessandro M Varani
Lentibulariaceae is the richest family of carnivorous plants spanning three genera including Pinguicula, Genlisea, and Utricularia. Utricularia is globally distributed, and, unlike Pinguicula and Genlisea, has both aquatic and terrestrial forms. In this study we present the analysis of the chloroplast (cp) genome of the terrestrial Utricularia reniformis. U. reniformis has a standard cp genome of 139,725bp, encoding a gene repertoire similar to essentially all photosynthetic organisms. However, an exclusive combination of losses and pseudogenization of the plastid NAD(P)H-dehydrogenase (ndh) gene complex were observed...
2016: PloS One
Gabriella Juhasz, Eva Csepany, Mate Magyar, Andrea Edit Edes, Nora Eszlari, Gabor Hullam, Peter Antal, Gyongyi Kokonyei, Ian Muir Anderson, John Francis William Deakin, Gyorgy Bagdy
One of the main effects of the endocannabinoid system in the brain is stress adaptation with presynaptic endocannabinoid receptor 1 (CB1 receptors) playing a major role. In the present study, we investigated whether the effect of the CB1 receptor coding CNR1 gene on migraine and its symptoms is conditional on life stress. In a cross-sectional European population (n = 2426), recruited from Manchester and Budapest, we used the ID-Migraine questionnaire for migraine screening, the Life Threatening Experiences questionnaire to measure recent negative life events (RLE), and covered the CNR1 gene with 11 SNPs...
October 19, 2016: Genes, Brain, and Behavior
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