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https://www.readbyqxmd.com/read/29773976/facing-the-challenges-of-neuropeptide-gene-knockouts-why-do-they-not-inhibit-reproduction-in-adult-teleost-fish
#1
REVIEW
Vance L Trudeau
Genetic manipulation of teleost endocrine systems started with transgenic overexpression of pituitary growth hormone. Such strategies enhance growth and reduce fertility, but the fish still breed. Genome editing using transcription activator-like effector nuclease in zebrafish and medaka has established the role of follicle stimulating hormone for gonadal development and luteinizing hormone for ovulation. Attempts to genetically manipulate the hypophysiotropic neuropeptidergic systems have been less successful...
2018: Frontiers in Neuroscience
https://www.readbyqxmd.com/read/29773895/crispr-cas9-system-targeting-regulatory-genes-of-hiv-1-inhibits-viral-replication-in-infected-t-cell-cultures
#2
Youdiil Ophinni, Mari Inoue, Tomohiro Kotaki, Masanori Kameoka
The CRISPR/Cas9 system provides a novel and promising tool for editing the HIV-1 proviral genome. We designed RNA-guided CRISPR/Cas9 targeting the HIV-1 regulatory genes tat and rev with guide RNAs (gRNA) selected from each gene based on CRISPR specificity and sequence conservation across six major HIV-1 subtypes. Each gRNA was cloned into lentiCRISPRv2 before co-transfection to create a lentiviral vector and transduction into target cells. CRISPR/Cas9 transduction into 293 T and HeLa cells stably expressing Tat and Rev proteins successfully abolished the expression of each protein relative to that in non-transduced and gRNA-absent vector-transduced cells...
May 17, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29771955/crispr-cas9-mediated-high-efficiency-knockout-of-the-eye-color-gene-vermillion-in-helicoverpa-zea-boddie
#3
Omaththage P Perera, Nathan S Little, Calvin A Pierce
Among various genome editing tools available for functional genomic studies, reagents based on clustered regularly interspersed palindromic repeats (CRISPR) have gained popularity due to ease and versatility. CRISPR reagents consist of ribonucleoprotein (RNP) complexes formed by combining guide RNA (gRNA) that target specific genomics regions and a CRISPR associated nuclease (Cas). The gRNA targeting specific gene sequences may be delivered as a plasmid construct that needs to be transcribed or as a synthetic RNA...
2018: PloS One
https://www.readbyqxmd.com/read/29771329/regulation-of-the-neuropathy-associated-pmp22-gene-by-a-distal-super-enhancer
#4
Harrison Pantera, John J Moran, Holly A Hung, Evgenia Pak, Amalia Dutra, John Svaren
Peripheral nerve myelination is adversely affected in the most common form of the hereditary peripheral neuropathy called Charcot-Marie-Tooth Disease. This form, classified as CMT1A, is caused by a 1.4 Mb duplication on chromosome 17, which includes the abundantly expressed Schwann cell myelin gene, Peripheral Myelin Protein 22 (PMP22). This is one of the most common copy number variants causing neurological disease. Overexpression of Pmp22 in rodent models recapitulates several aspects of neuropathy, and reduction of Pmp22 in such models results in amelioration of the neuropathy phenotype...
May 16, 2018: Human Molecular Genetics
https://www.readbyqxmd.com/read/29770349/single-step-precision-genome-editing-in-yeast-using-crispr-cas9
#5
Azat Akhmetov, Jon M Laurent, Jimmy Gollihar, Elizabeth C Gardner, Riddhiman K Garge, Andrew D Ellington, Aashiq H Kachroo, Edward M Marcotte
Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended modification. Here, we describe a CRISPR-Cas9 mediated genome editing protocol for modifying any yeast gene of interest (either essential or nonessential) in a single-step transformation without any selectable marker...
March 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29770269/ten-years-of-progress-and-promise-of-induced-pluripotent-stem-cells-historical-origins-characteristics-mechanisms-limitations-and-potential-applications
#6
Adekunle Ebenezer Omole, Adegbenro Omotuyi John Fakoya
The discovery of induced pluripotent stem cells (iPSCs) by Shinya Yamanaka in 2006 was heralded as a major breakthrough of the decade in stem cell research. The ability to reprogram human somatic cells to a pluripotent embryonic stem cell-like state through the ectopic expression of a combination of embryonic transcription factors was greeted with great excitement by scientists and bioethicists. The reprogramming technology offers the opportunity to generate patient-specific stem cells for modeling human diseases, drug development and screening, and individualized regenerative cell therapy...
2018: PeerJ
https://www.readbyqxmd.com/read/29769737/next-generation-biocontainment-systems-for-engineered-organisms
#7
REVIEW
Jeong Wook Lee, Clement T Y Chan, Shimyn Slomovic, James J Collins
The increasing use of engineered organisms for industrial, clinical, and environmental applications poses a growing risk of spreading hazardous biological entities into the environment. To address this biosafety issue, significant effort has been invested in creating ways to confine these organisms and transgenic materials. Emerging technologies in synthetic biology involving genetic circuit engineering, genome editing, and gene expression regulation have led to the development of novel biocontainment systems...
May 16, 2018: Nature Chemical Biology
https://www.readbyqxmd.com/read/29769725/mxra8-is-a-receptor-for-multiple-arthritogenic-alphaviruses
#8
Rong Zhang, Arthur S Kim, Julie M Fox, Sharmila Nair, Katherine Basore, William B Klimstra, Rebecca Rimkunas, Rachel H Fong, Hueylie Lin, Subhajit Poddar, James E Crowe, Benjamin J Doranz, Daved H Fremont, Michael S Diamond
Arthritogenic alphaviruses comprise a group of enveloped RNA viruses that are transmitted to humans by mosquitoes and cause debilitating acute and chronic musculoskeletal disease 1 . The host factors required for alphavirus entry remain poorly characterized 2 . Here we use a genome-wide CRISPR-Cas9-based screen to identify the cell adhesion molecule Mxra8 as an entry mediator for multiple emerging arthritogenic alphaviruses, including chikungunya, Ross River, Mayaro and O'nyong nyong viruses. Gene editing of mouse Mxra8 or human MXRA8 resulted in reduced levels of viral infection of cells and, reciprocally, ectopic expression of these genes resulted in increased infection...
May 16, 2018: Nature
https://www.readbyqxmd.com/read/29769555/cattle-with-a-precise-zygote-mediated-deletion-safely-eliminate-the-major-milk-allergen-beta-lactoglobulin
#9
Jingwei Wei, Stefan Wagner, Paul Maclean, Brigid Brophy, Sally Cole, Grant Smolenski, Dan F Carlson, Scott C Fahrenkrug, David N Wells, Götz Laible
We applied precise  zygote-mediated genome editing to eliminate beta-lactoglobulin (BLG), a major allergen in cows' milk. To efficiently generate LGB knockout cows, biopsied embryos were screened to transfer only appropriately modified embryos. Transfer of 13 pre-selected embryos into surrogate cows resulted in the birth of three calves, one dying shortly after birth. Deep sequencing results confirmed conversion of the genotype from wild type to the edited nine bp deletion by more than 97% in the two male calves...
May 16, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29766028/a-non-integrating-lentiviral-approach-overcomes-cas9-induced-immune-rejection-to-establish-an-immunocompetent-metastatic-renal-cancer-model
#10
Junhui Hu, Shiruyeh Schokrpur, Maani Archang, Kip Hermann, Allison C Sharrow, Prateek Khanna, Jesse Novak, Sabina Signoretti, Rupal S Bhatt, Beatrice S Knudsen, Hua Xu, Lily Wu
The CRISPR-based technology has revolutionized genome editing in recent years. This technique allows for gene knockout and evaluation of function in cell lines in a manner that is far easier and more accessible than anything previously available. Unfortunately, the ability to extend these studies to in vivo syngeneic murine cell line implantation is limited by an immune response against cells transduced to stably express Cas9. In this study, we demonstrate that a non-integrating lentiviral vector approach can overcome this immune rejection and allow for the growth of transduced cells in an immunocompetent host...
June 15, 2018: Molecular Therapy. Methods & Clinical Development
https://www.readbyqxmd.com/read/29765551/functional-significance-of-co-occurring-mutations-in-pik3ca-and-map3k1-in-breast-cancer
#11
Alvaro Avivar-Valderas, Robert McEwen, Amir Taheri-Ghahfarokhi, Larissa S Carnevalli, Elizabeth L Hardaker, Marcello Maresca, Kevin Hudson, Elizabeth A Harrington, Francisco Cruzalegui
The PI3Kα signaling pathway is frequently hyper-activated in breast cancer (BrCa), as a result of mutations/amplifications in oncogenes (e.g. HER2 ), decreased function in tumor suppressors (e.g. PTEN ) or activating mutations in key components of the pathway. In particular, activating mutations of PIK3CA (~45%) are frequently found in luminal A BrCa samples. Genomic studies have uncovered inactivating mutations in MAP3K1 (13-20%) and MAP2K4 (~8%), two upstream kinases of the JNK apoptotic pathway in luminal A BrCa samples...
April 20, 2018: Oncotarget
https://www.readbyqxmd.com/read/29765029/the-crispr-tool-kit-for-genome-editing-and-beyond
#12
REVIEW
Mazhar Adli
CRISPR is becoming an indispensable tool in biological research. Once known as the bacterial immune system against invading viruses, the programmable capacity of the Cas9 enzyme is now revolutionizing diverse fields of medical research, biotechnology, and agriculture. CRISPR-Cas9 is no longer just a gene-editing tool; the application areas of catalytically impaired inactive Cas9, including gene regulation, epigenetic editing, chromatin engineering, and imaging, now exceed the gene-editing functionality of WT Cas9...
May 15, 2018: Nature Communications
https://www.readbyqxmd.com/read/29764955/the-new-normal-of-structure-function-studies-in-the-era-of-crispr-cas9
#13
EDITORIAL
Glennis A Logsdon, Ben E Black
Major advances in gene-editing technologies have enabled the rapid dissection of proteins in complex biological systems, facilitating biological experiments to complement biochemical studies with purified components. In this editorial, we highlight CRISPR/Cas9-based strategies to rapidly manipulate endogenous genes - strategies that have already transformed functional studies of proteins in metazoan systems. We further describe emerging tools using a catalytically dead version of Cas9 (dCas9) that do not cleave DNA, but can alter gene expression and/or local chromatin states, edit single nucleotide bases, and permit the visualization of specific genomic loci...
May 15, 2018: Biochemical Journal
https://www.readbyqxmd.com/read/29764585/-construction-of-ezh2-knockout-animal-model-by-crispr-cas9-technology
#14
Fanrong Meng, Dan Zhao, Qinghua Zhou, Zhe Liu
BACKGROUND: It has been proven that CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9) system was the modern gene-editing technology through the constitutive expression of nucleases Cas9 in the mammalian, which binds to the specific site in the genome mediated by single-guide RNA (sgRNA) at desired genomic loci. The aim of this study is that the animal model of EZH2 gene knockout was constructed using CRISPR/Cas9 technology. METHODS: In this study, we designed two single-guide RNAs targeting the Exon3 and Exon4 of EZH2 gene...
May 20, 2018: Zhongguo Fei Ai za Zhi, Chinese Journal of Lung Cancer
https://www.readbyqxmd.com/read/29764251/future-of-human-mitochondrial-dna-editing-technologies
#15
N Verechshagina, N Nikitchina, Y Yamada, Н Harashima, M Tanaka, K Orishchenko, I Mazunin
ATP and other metabolites, which are necessary for the development, maintenance, and functioning of bodily cells are all synthesized in the mitochondria. Multiple copies of the genome, present within the mitochondria, together with its maternal inheritance, determine the clinical manifestation and spreading of mutations in mitochondrial DNA (mtDNA). The main obstacle in the way of thorough understanding of mitochondrial biology and the development of gene therapy methods for mitochondrial diseases is the absence of systems that allow to directly change mtDNA sequence...
May 15, 2018: Mitochondrial DNA. Part A. DNA Mapping, Sequencing, and Analysis
https://www.readbyqxmd.com/read/29763675/efficient-genome-editing-in-fusarium-oxysporum-based-on-crispr-cas9-ribonucleoprotein-complexes
#16
Qiang Wang, Paul A Cobine, Jeffrey J Coleman
The Fusarium oxysporum species complex (FOSC) is an economically important group of pathogenic filamentous fungi that are able to infect both animals and plants. Reverse genetic techniques, including gene disruption/deletion methods, to study these fungi are available although limitations exist resulting in decreased efficiency. Herein we describe a gene editing system developed using a F. oxysporum-optimized Cas9 ribonucleoprotein (RNP) and protoplast transformation method. The Cas9 protein and sgRNA were assembled to form a stable RNP in vitro and this complex was transferred into fungal protoplasts for gene editing with PEG-mediated transformation...
May 12, 2018: Fungal Genetics and Biology: FG & B
https://www.readbyqxmd.com/read/29762716/crispor-intuitive-guide-selection-for-crispr-cas9-genome-editing-experiments-and-screens
#17
Jean-Paul Concordet, Maximilian Haeussler
CRISPOR.org is a web tool for genome editing experiments with the CRISPR-Cas9 system. It finds guide RNAs in an input sequence and ranks them according to different scores that evaluate potential off-targets in the genome of interest and predict on-target activity. The list of genomes is continuously expanded, with more 150 genomes added in the last two years. CRISPOR tries to provide a comprehensive solution from selection, cloning and expression of guide RNA as well as providing primers needed for testing guide activity and potential off-targets...
May 14, 2018: Nucleic Acids Research
https://www.readbyqxmd.com/read/29761268/towards-a-comprehensive-picture-of-c-to-u-rna-editing-sites-in-angiosperm-mitochondria
#18
Alejandro A Edera, Carolina L Gandini, M Virginia Sanchez-Puerta
Our understanding of the dynamic and evolution of RNA editing in angiosperms is in part limited by the few editing sites identified to date. This study identified 10,217 editing sites from 17 diverse angiosperms. Our analyses confirmed the universality of certain features of RNA editing, and offer new evidence behind the loss of editing sites in angiosperms. RNA editing is a post-transcriptional process that substitutes cytidines (C) for uridines (U) in organellar transcripts of angiosperms. These substitutions mostly take place in mitochondrial messenger RNAs at specific positions called editing sites...
May 14, 2018: Plant Molecular Biology
https://www.readbyqxmd.com/read/29759817/enhancer-logic-and-mechanics-in-development-and-disease
#19
REVIEW
Ryan Rickels, Ali Shilatifard
Enhancers are distally located genomic cis-regulatory elements that integrate spatiotemporal cues to coordinate gene expression in a tissue-specific manner during metazoan development. Enhancer function depends on a combination of bound transcription factors and cofactors that regulate local chromatin structure, as well as on the topological interactions that are necessary for their activity. Numerous genome-wide studies concur that the vast majority of disease-associated variations occur within non-coding genomic sequences, in other words the 'cis-regulome', and this underscores their relevance for human health...
May 11, 2018: Trends in Cell Biology
https://www.readbyqxmd.com/read/29758287/pseudomonas-putida-as-a-functional-chassis-for-industrial-biocatalysis-from-native-biochemistry-to-trans-metabolism
#20
REVIEW
Pablo I Nikel, Víctor de Lorenzo
The itinerary followed by Pseudomonas putida from being a soil-dweller and plant colonizer bacterium to become a flexible and engineer-able platform for metabolic engineering stems from its natural lifestyle, which is adapted to harsh environmental conditions and all sorts of physicochemical stresses. Over the years, these properties have been capitalized biotechnologically owing to the expanding wealth of genetic tools designed for deep-editing the P. putida genome. A suite of dedicated vectors inspired in the core tenets of synthetic biology have enabled to suppress many of the naturally-occurring undesirable traits native to this species while enhancing its many appealing properties, and also to import catalytic activities and attributes from other biological systems...
May 11, 2018: Metabolic Engineering
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