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https://www.readbyqxmd.com/read/28212417/splitax-a-novel-method-to-assess-the-function-of-engineered-nucleases
#1
Richard A Axton, Sharmin S Haideri, Martha Lopez-Yrigoyen, Helen A Taylor, Lesley M Forrester
Engineered nucleases have been used to generate knockout or reporter cell lines and a range of animal models for human disease. These new technologies also hold great promise for therapeutic genome editing. Current methods to evaluate the activity of these nucleases are time consuming, require extensive optimization and are hampered by readouts with low signals and high background. We have developed a simple and easy to perform method (SplitAx) that largely addresses these issues and provides a readout of nuclease activity...
2017: PloS One
https://www.readbyqxmd.com/read/28207001/genome-editing-using-facs-enrichment-of-nuclease-expressing-cells-and-indel-detection-by-amplicon-analysis
#2
Lindsey A Lonowski, Yoshiki Narimatsu, Anjum Riaz, Catherine E Delay, Zhang Yang, Francesco Niola, Katarzyna Duda, Elke A Ober, Henrik Clausen, Hans H Wandall, Steen H Hansen, Eric P Bennett, Morten Frödin
This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28205594/genome-editing-reveals-dmrt1-as-an-essential-male-sex-determining-gene-in-chinese-tongue-sole-cynoglossus-semilaevis
#3
Zhongkai Cui, Yun Liu, Wenwen Wang, Qian Wang, Ning Zhang, Fan Lin, Na Wang, Changwei Shao, Zhongdian Dong, Yangzhen Li, Yingming Yang, Mengzhu Hu, Hailong Li, Fengtao Gao, Zhanfei Wei, Liang Meng, Yang Liu, Min Wei, Ying Zhu, Hua Guo, Christopher H K Cheng, Manfred Schartl, Songlin Chen
Chinese tongue sole is a marine fish with ZW sex determination. Genome sequencing suggested that the Z-linked dmrt1 is a putative male determination gene, but direct genetic evidence is still lacking. Here we show that TALEN of dmrt1 efficiently induced mutations of this gene. The ZZ dmrt1 mutant fish developed ovary-like testis, and the spermatogenesis was disrupted. The female-related genes foxl2 and cyp19a1a were significantly increased in the gonad of the ZZ dmrt1 mutant. Conversely, the male-related genes Sox9a and Amh were significantly decreased...
February 16, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28202780/erratum-for-the-report-molecular-remission-of-infant-b-all-after-infusion-of-universal-talen-gene-edited-car-t-cells-by-w-qasim-h-zhan-s-samarasinghe-s-adams-p-amrolia-s-stafford-k-butler-c-rivat-g-wright-k-somana-s-ghorashian-d-pinner-g-ahsan-k-gilmour-g-lucchini
#4
https://www.readbyqxmd.com/read/28192479/comparison-of-zebrafish-tmem88a-mutant-and-morpholino-knockdown-phenotypes
#5
Alexander M J Eve, Elsie S Place, James C Smith
Tmem88a is a transmembrane protein that is thought to be a negative regulator of the Wnt signalling pathway. Several groups have used antisense morpholino oligonucleotides in an effort to characterise the role of tmem88a in zebrafish cardiovascular development, but they have not obtained consistent results. Here, we generate an 8 bp deletion in the coding region of the tmem88a locus using TALENs, and we have gone on to establish a viable homozygous tmem88aΔ8 mutant line. Although tmem88aΔ8 mutants have reduced expression of some key haematopoietic genes, differentiation of erythrocytes and neutrophils is unaffected, contradicting our previous study using antisense morpholino oligonucleotides...
2017: PloS One
https://www.readbyqxmd.com/read/28182397/comeback-of-the-rat-in-biomedical-research
#6
Judith R Homberg, Markus Wöhr, Natalia Alenina
Rats were the first mammalian species domesticated for scientific purposes, and they soon became the most widely used animal model in biomedical sciences, including cardiovascular research and behavioral neuroscience. Yet, after the development of technologies to manipulate genes, researchers largely shifted to the use of mice. However, as we lay out with examples from drug addiction, social behavior, and cardiovascular research, rats have experimental advantages over mice. With the introduction of zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) methodologies, and, specifically, the clustered regularly interspaced short palindromic repeats (CRISPR) associated system, gene targeting is no longer limited to mice...
February 9, 2017: ACS Chemical Neuroscience
https://www.readbyqxmd.com/read/28178187/may-i-cut-in-gene-editing-approaches-in-human-induced-pluripotent-stem-cells
#7
REVIEW
Nicholas Brookhouser, Sreedevi Raman, Christopher Potts, David A Brafman
In the decade since Yamanaka and colleagues described methods to reprogram somatic cells into a pluripotent state, human induced pluripotent stem cells (hiPSCs) have demonstrated tremendous promise in numerous disease modeling, drug discovery, and regenerative medicine applications. More recently, the development and refinement of advanced gene transduction and editing technologies have further accelerated the potential of hiPSCs. In this review, we discuss the various gene editing technologies that are being implemented with hiPSCs...
February 6, 2017: Cells
https://www.readbyqxmd.com/read/28174243/efficient-talen-mediated-gene-targeting-of-chicken-primordial-germ-cells
#8
Lorna Taylor, Daniel F Carlson, Sunil Nandi, Adrian Sherman, Scott C Fahrenkrug, Michael J McGrew
In this work we use TALE nucleases (TALENs) to target a reporter construct to the DDX4 (vasa) locus in chicken primordial germ cells. Vasa is a key germ cell determinant in many animal species and is posited to control avian germ cell formation. We show that TALENs mediate homology directed repair of the DDX4 locus on the Z sex chromosome at high (8.1%) efficiencies. Large genetic deletions of 30kb encompassing the entire DDX4 locus were also created using a single TALEN pair. The targeted PGCs were germ line competent and were used to produce DDX4 null offspring...
February 7, 2017: Development
https://www.readbyqxmd.com/read/28167953/biofortification-in-millets-a-sustainable-approach-for-nutritional-security
#9
REVIEW
A Vinoth, R Ravindhran
Nutritional insecurity is a major threat to the world's population that is highly dependent on cereals-based diet, deficient in micronutrients. Next to cereals, millets are the primary sources of energy in the semi-arid tropics and drought-prone regions of Asia and Africa. Millets are nutritionally superior as their grains contain high amount of proteins, essential amino acids, minerals, and vitamins. Biofortification of staple crops is proved to be an economically feasible approach to combat micronutrient malnutrition...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/28166285/p38%C3%AE-regulates-actin-cytoskeleton-and-cytokinesis-in-hepatocytes-during-development-and-aging
#10
Ana M Tormos, Sergio Rius-Pérez, María Jorques, Patricia Rada, Lorena Ramirez, Ángela M Valverde, Ángel R Nebreda, Juan Sastre, Raquel Taléns-Visconti
BACKGROUND: Hepatocyte poliploidization is an age-dependent process, being cytokinesis failure the main mechanism of polyploid hepatocyte formation. Our aim was to study the role of p38α MAPK in the regulation of actin cytoskeleton and cytokinesis in hepatocytes during development and aging. METHODS: Wild type and p38α liver-specific knock out mice at different ages (after weaning, adults and old) were used. RESULTS: We show that p38α MAPK deficiency induces actin disassembly upon aging and also cytokinesis failure leading to enhanced binucleation...
2017: PloS One
https://www.readbyqxmd.com/read/28161521/germ-cell-regeneration-mediated-enhanced-mutagenesis-in-the-ascidian-ciona-intestinalis-reveals-flexible-germ-cell-formation-from-different-somatic-cells
#11
Keita Yoshida, Akiko Hozumi, Nicholas Treen, Tetsushi Sakuma, Takashi Yamamoto, Maki Shirae-Kurabayashi, Yasunori Sasakura
The ascidian Ciona intestinalis has a high regeneration capacity that enables the regeneration of artificially removed primordial germ cells (PGCs) from somatic cells. We utilized PGC regeneration to establish efficient methods of germ line mutagenesis with transcription activator-like effector nucleases (TALENs). When PGCs were artificially removed from animals in which a TALEN pair was expressed, somatic cells harboring mutations in the target gene were converted into germ cells, this germ cell population exhibited higher mutation rates than animals not subjected to PGC removal...
February 1, 2017: Developmental Biology
https://www.readbyqxmd.com/read/28153086/targeted-repair-of-cybb-in-x-cgd-ipscs-requires-retention-of-intronic-sequences-for-expression-and-functional-correction
#12
Colin L Sweeney, Jizhong Zou, Uimook Choi, Randall K Merling, Alexander Liu, Aaron Bodansky, Sandra Burkett, Jung-Woong Kim, Suk See De Ravin, Harry L Malech
X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the CYBB gene, resulting in absent or defective gp91(phox) protein expression. To correct CYBB exon 5 mutations while retaining normal gene regulation, we utilized TALEN or Cas9 for exon 5 replacement in induced pluripotent stem cells (iPSCs) from patients, which restored gp91(phox) expression and ROS production in iPSC-derived granulocytes...
February 1, 2017: Molecular Therapy: the Journal of the American Society of Gene Therapy
https://www.readbyqxmd.com/read/28129851/small-teleost-fish-provide-new-insights-into-human-skeletal-diseases
#13
P E Witten, M P Harris, A Huysseune, C Winkler
Small teleost fish such as zebrafish and medaka are increasingly studied as models for human skeletal diseases. Efficient new genome editing tools combined with advances in the analysis of skeletal phenotypes provide new insights into fundamental processes of skeletal development. The skeleton among vertebrates is a highly conserved organ system, but teleost fish and mammals have evolved unique traits or have lost particular skeletal elements in each lineage. Several unique features of the skeleton relate to the extremely small size of early fish embryos and the small size of adult fish used as models...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28129844/studying-disorders-of-vertebrate-iron-and-heme-metabolism-using-zebrafish
#14
Lisa N van der Vorm, Barry H Paw
Iron is a crucial component of heme- and iron-sulfur clusters, involved in vital cellular functions such as oxygen transport, DNA synthesis, and respiration. Both excess and insufficient levels of iron and heme-precursors cause human disease, such as iron-deficiency anemia, hemochromatosis, and porphyrias. Hence, their levels must be tightly regulated, requiring a complex network of transporters and feedback mechanisms. The use of zebrafish to study these pathways and the underlying genetics offers many advantages, among others their optical transparency, ex-vivo development and high genetic and physiological conservations...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28129840/infectious-disease-models-in-zebrafish
#15
C Sullivan, M A Matty, D Jurczyszak, K A Gabor, P J Millard, D M Tobin, C H Kim
In recent years, the zebrafish (Danio rerio) has developed as an important alternative to mammalian models for the study of hostpathogen interactions. Because they lack a functional adaptive immune response during the first 4-6weeks of development, zebrafish rely upon innate immune responses to protect against injuries and infections. During this early period of development, it is possible to isolate and study mechanisms of infection and inflammation arising from the innate immune response without the complications presented by the adaptive immune response...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28128770/modern-genome-editing-technologies-in-huntington-s-disease-research
#16
Tuyana Bairovna Malankhanova, Anastasia Aleksandrovna Malakhova, Sergey Petrovich Medvedev, Suren Minasovich Zakian
The development of new revolutionary technologies for directed gene editing has made it possible to thoroughly model and study human diseases at the cellular and molecular levels. Gene editing tools like ZFN, TALEN, CRISPR-based systems, NgAgo and SGN can introduce different modifications in gene sequences and regulate gene expression in different types of cells including induced pluripotent stem cells (iPSCs). These tools can be successfully used for Huntington's disease (HD) modeling, for example, to generate isogenic cell lines bearing different numbers of CAG repeats or to correct the mutation causing the disease...
January 24, 2017: Journal of Huntington's Disease
https://www.readbyqxmd.com/read/28123068/molecular-remission-of-infant-b-all-after-infusion-of-universal-talen-gene-edited-car-t-cells
#17
Waseem Qasim, Hong Zhan, Sujith Samarasinghe, Stuart Adams, Persis Amrolia, Sian Stafford, Katie Butler, Christine Rivat, Gary Wright, Kathy Somana, Sara Ghorashian, Danielle Pinner, Gul Ahsan, Kimberly Gilmour, Giovanna Lucchini, Sarah Inglott, William Mifsud, Robert Chiesa, Karl S Peggs, Lucas Chan, Farzin Farzeneh, Adrian J Thrasher, Ajay Vora, Martin Pule, Paul Veys
Autologous T cells engineered to express chimeric antigen receptor against the B cell antigen CD19 (CAR19) are achieving marked leukemic remissions in early-phase trials but can be difficult to manufacture, especially in infants or heavily treated patients. We generated universal CAR19 (UCART19) T cells by lentiviral transduction of non-human leukocyte antigen-matched donor cells and simultaneous transcription activator-like effector nuclease (TALEN)-mediated gene editing of T cell receptor α chain and CD52 gene loci...
January 25, 2017: Science Translational Medicine
https://www.readbyqxmd.com/read/28118669/the-ciliopathy-gene-ahi1-is-required-for-zebrafish-cone-photoreceptor-outer-segment-morphogenesis-and-survival
#18
Emma M Lessieur, Joseph Fogerty, Robert J Gaivin, Ping Song, Brian D Perkins
Purpose: Joubert syndrome (JBTS) is an autosomal recessive ciliopathy with considerable phenotypic variability. In addition to central nervous system abnormalities, a subset of JBTS patients exhibit retinal dystrophy and/or kidney disease. Mutations in the AHI1 gene are causative for approximately 10% of all JBTS cases. The purpose of this study was to generate ahi1 mutant alleles in zebrafish and to characterize the retinal phenotypes. Methods: Zebrafish ahi1 mutants were generated using transcription activator-like effector nucleases (TALENs)...
January 1, 2017: Investigative Ophthalmology & Visual Science
https://www.readbyqxmd.com/read/28117500/crosslinker-induced-effects-on-the-gelation-pathway-of-a-low-molecular-weight-hydrogel
#19
Willem E M Noteborn, Damy N H Zwagerman, Victorio Saez Talens, Chandan Maity, Lars van der Mee, Jos M Poolman, Serhii Mytnyk, Jan H van Esch, Alexander Kros, Rienk Eelkema, Roxanne E Kieltyka
The use of polymeric crosslinkers is an attractive method to modify the mechanical properties of supramolecular materials, but their effects on the self-assembly of the underlying supramolecular polymer networks are poorly understood. Modulation of the gelation pathway of a reaction-coupled low molecular weight hydrogelator is demonstrated using (bio)polymeric crosslinkers of disparate physicochemical identities, providing a handle for control over materials properties.
January 24, 2017: Advanced Materials
https://www.readbyqxmd.com/read/28114701/-genome-editing-tools-and-their-application-in-experimental-ophthalmology
#20
M Yanik, W Wende, K Stieger
New genome editing tools in molecular biology are revolutionising precise genome surgery and have greatly influenced experimental ophthalmology too. Aside from the commonly used nuclease-based platforms, such as the zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), CRISPR/Cas systems, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes, perform very efficiently in site-specific DNA cleavage within living cells. DNA double strand breaks (DSB) are repaired through two different conserved repair pathways: NHEJ (non-homologous end joining) and HDR (homology directed repair)...
January 23, 2017: Klinische Monatsblätter Für Augenheilkunde
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