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https://www.readbyqxmd.com/read/27910942/improved-bi-allelic-modification-of-a-transcriptionally-silent-locus-in-patient-derived-ipsc-by-cas9-nickase
#1
Reto Eggenschwiler, Mohsen Moslem, Mariane Serra Fráguas, Melanie Galla, Oliver Papp, Maximilian Naujock, Ines Fonfara, Ingrid Gensch, Annabell Wähner, Abbas Beh-Pajooh, Claudio Mussolino, Marcel Tauscher, Doris Steinemann, Florian Wegner, Susanne Petri, Axel Schambach, Emmanuelle Charpentier, Toni Cathomen, Tobias Cantz
Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella...
December 2, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27908936/genome-editing-technologies-principles-and-applications
#2
REVIEW
Thomas Gaj, Shannon J Sirk, Sai-Lan Shui, Jia Liu
Targeted nucleases have provided researchers with the ability to manipulate virtually any genomic sequence, enabling the facile creation of isogenic cell lines and animal models for the study of human disease, and promoting exciting new possibilities for human gene therapy. Here we review three foundational technologies-clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs)...
December 1, 2016: Cold Spring Harbor Perspectives in Biology
https://www.readbyqxmd.com/read/27905217/to-crispr-and-beyond-the-evolution-of-genome-editing-in-stem-cells
#3
Kuang-Yui Chen, Paul S Knoepfler
The goal of editing the genomes of stem cells to generate model organisms and cell lines for genetic and biological studies has been pursued for decades. There is also exciting potential for future clinical impact in humans. While recent, rapid advances in targeted nuclease technologies have led to unprecedented accessibility and ease of gene editing, biology has benefited from past directed gene modification via homologous recombination, gene traps and other transgenic methodologies. Here we review the history of genome editing in stem cells (including via zinc finger nucleases, transcription activator-like effector nucleases and CRISPR-Cas9), discuss recent developments leading to the implementation of stem cell gene therapies in clinical trials and consider the prospects for future advances in this rapidly evolving field...
December 1, 2016: Regenerative Medicine
https://www.readbyqxmd.com/read/27903891/cloning-independent-markerless-gene-editing-in-streptococcus-sanguinis-novel-insights-in-type-iv-pilus-biology
#4
Ishwori Gurung, Jamie-Lee Berry, Alexander M J Hall, Vladimir Pelicic
Streptococcus sanguinis, a naturally competent opportunistic human pathogen, is a Gram-positive workhorse for genomics. It has recently emerged as a model for the study of type IV pili (Tfp)-exceptionally widespread and important prokaryotic filaments. To enhance genetic manipulation of Streptococcus sanguinis, we have developed a cloning-independent methodology, which uses a counterselectable marker and allows sophisticated markerless gene editing in situ We illustrate the utility of this methodology by answering several questions regarding Tfp biology by (i) deleting single or mutiple genes, (ii) altering specific bases in genes of interest, and (iii) engineering genes to encode proteins with appended affinity tags...
November 29, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27899664/integration-defective-lentiviral-vector-mediates-efficient-gene-editing-through-homology-directed-repair-in-human-embryonic-stem-cells
#5
Yebo Wang, Yingjia Wang, Tammy Chang, He Huang, Jiing-Kuan Yee
Human embryonic stem cells (hESCs) are used as platforms for disease study, drug screening and cell-based therapy. To facilitate these applications, it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However, the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09...
November 28, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27899645/efficient-targeted-dna-methylation-with-chimeric-dcas9-dnmt3a-dnmt3l-methyltransferase
#6
Peter Stepper, Goran Kungulovski, Renata Z Jurkowska, Tamir Chandra, Felix Krueger, Richard Reinhardt, Wolf Reik, Albert Jeltsch, Tomasz P Jurkowski
DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a-Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters...
November 29, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27899449/deficiency-of-a-retinal-dystrophy-protein-acbd5-impairs-peroxisomal-%C3%AE-oxidation-of-very-long-chain-fatty-acids
#7
Yuichi Yagita, Kyoko Shinohara, Yuichi Abe, Keiko Nakagawa, Mohammed Al-Owain, Fowzan S Alkuraya, Yukio Fujiki
Acyl-CoA binding domain containing 5 (ACBD5) is a peroxisomal protein that carries an acyl-CoA binding domain (ACBD) at its N-terminal region. The recent identification of a mutation in the ACBD5 gene in patients with a syndromic form of retinal dystrophy highlights the physiological importance of ACBD5 in humans. However, the underlying pathogenic mechanisms and the precise function of ACBD5 remain unclear. We herein report that ACBD5 is a peroxisomal tail-anchored membrane protein exposing its ACBD to the cytosol...
November 29, 2016: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/27898094/crispr-cas9-aav-mediated-knock-in-at-nrl-locus-in-human-embryonic-stem-cells
#8
Xianglian Ge, Haitao Xi, Fayu Yang, Xiao Zhi, Yanghua Fu, Ding Chen, Ren-He Xu, Ge Lin, Jia Qu, Junzhao Zhao, Feng Gu
Clustered interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low...
November 29, 2016: Molecular Therapy. Nucleic Acids
https://www.readbyqxmd.com/read/27892925/a-mouse-model-for-mers-coronavirus-induced-acute-respiratory-distress-syndrome
#9
Adam S Cockrell, Boyd L Yount, Trevor Scobey, Kara Jensen, Madeline Douglas, Anne Beall, Xian-Chun Tang, Wayne A Marasco, Mark T Heise, Ralph S Baric
Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel virus that emerged in 2012, causing acute respiratory distress syndrome (ARDS), severe pneumonia-like symptoms and multi-organ failure, with a case fatality rate of ∼36%. Limited clinical studies indicate that humans infected with MERS-CoV exhibit pathology consistent with the late stages of ARDS, which is reminiscent of the disease observed in patients infected with severe acute respiratory syndrome coronavirus. Models of MERS-CoV-induced severe respiratory disease have been difficult to achieve, and small-animal models traditionally used to investigate viral pathogenesis (mouse, hamster, guinea-pig and ferret) are naturally resistant to MERS-CoV...
November 28, 2016: Nature Microbiology
https://www.readbyqxmd.com/read/27890672/epigenetics-of-cell-fate-reprogramming-and-its-implications-for-neurological-disorders-modelling
#10
REVIEW
Maciej Grzybek, Aleksandra Golonko, Marta Walczak, Pawel Lisowski
The reprogramming of human induced pluripotent stem cells (hiPSCs) proceeds in a stepwise manner with reprogramming factors binding and epigenetic composition changes during transition to maintain the epigenetic landscape, important for pluripotency. There arises a question as to whether the aberrant epigenetic state after reprogramming leads to epigenetic defects in induced stem cells causing unpredictable long term effects in differentiated cells. In this review, we present a comprehensive view of epigenetic alterations accompanying reprogramming, cell maintenance and differentiation as factors that influence applications of hiPSCs in stem cell based technologies...
November 23, 2016: Neurobiology of Disease
https://www.readbyqxmd.com/read/27890617/polycistronic-trna-and-crispr-guide-rna-enables-highly-efficient-multiplexed-genome-engineering-in-human-cells
#11
Fengping Dong, Kabin Xie, Yueying Chen, Yinong Yang, Yingwei Mao
CRISPR/Cas9 has been widely used for genomic editing in many organisms. Many human diseases are caused by multiple mutations. The CRISPR/Cas9 system provides a potential tool to introduce multiple mutations in a genome. To mimic complicated genomic events in human diseases, such as multiple gene deletions or mutations, two or more small guide RNAs (sgRNA) must be introduced all together. This can be achieved by separate Pol III promoters in a construct. However, limited enzyme sites and the increased insertion size lower the efficiency to make a construct...
November 24, 2016: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/27889058/mutations-in-reep6-cause-autosomal-recessive-retinitis-pigmentosa
#12
Gavin Arno, Smriti A Agrawal, Aiden Eblimit, James Bellingham, Mingchu Xu, Feng Wang, Christina Chakarova, David A Parfitt, Amelia Lane, Thomas Burgoyne, Sarah Hull, Keren J Carss, Alessia Fiorentino, Matthew J Hayes, Peter M Munro, Ralph Nicols, Nikolas Pontikos, Graham E Holder, Chinwe Asomugha, F Lucy Raymond, Anthony T Moore, Vincent Plagnol, Michel Michaelides, Alison J Hardcastle, Yumei Li, Catherine Cukras, Andrew R Webster, Michael E Cheetham, Rui Chen
Retinitis pigmentosa (RP) is the most frequent form of inherited retinal dystrophy. RP is genetically heterogeneous and the genes identified to date encode proteins involved in a wide range of functional pathways, including photoreceptor development, phototransduction, the retinoid cycle, cilia, and outer segment development. Here we report the identification of biallelic mutations in Receptor Expression Enhancer Protein 6 (REEP6) in seven individuals with autosomal-recessive RP from five unrelated families...
December 1, 2016: American Journal of Human Genetics
https://www.readbyqxmd.com/read/27879221/generation-of-a-tle3-heterozygous-knockout-human-embryonic-stem-cell-line-using-crispr-cas9
#13
Anne M Bara, Angelica Messana, Amanda Herring, Dane Z Hazelbaker, Kevin Eggan, Lindy E Barrett
Here, we generated a monoallelic mutation in the TLE3 (Transducin Like Enhancer of Split 3) gene using CRISPR-Cas9 editing in the human embryonic stem cell (hESC) line WA01. The heterozygous knockout cell line, TLE3-447-D08-A01, displays partial loss of TLE3 protein expression while maintaining pluripotency, differentiation potential and genomic integrity.
September 2016: Stem Cell Research
https://www.readbyqxmd.com/read/27879218/generation-of-a-tle1-homozygous-knockout-human-embryonic-stem-cell-line-using-crispr-cas9
#14
Amanda Herring, Angelica Messana, Anne M Bara, Dane Z Hazelbaker, Kevin Eggan, Lindy E Barrett
Here, we generated a biallelic mutation in the TLE1 (Transducin Like Enhancer of Split 1) gene using CRISPR-Cas9 editing in the human embryonic stem cell (hESC) line WA01. The homozygous knockout cell line, TLE1-464-G04, displays loss of TLE1 protein expression while maintaining pluripotency, differentiation potential and genomic integrity.
September 2016: Stem Cell Research
https://www.readbyqxmd.com/read/27872471/recent-progress-in-xenotransplantation-with-emphasis-on-virological-safety
#15
Joachim Denner
Xenotransplantation is a new technology that may help to overcome the shortage of human tissues and organs available for the treatment of tissue and organ failure. Remarkable progress has recently been made in this field. First, understanding of the mechanisms of immunological rejection, mainly of the hyperacute rejection, allowed generating numerous genetically modified pigs to overcome rejection. Second, based on these genetically modified animals and new immunosuppression regimens, long-term survival of non-human primate recipients of heart, kidney, and islet cell cells has been reported...
November 22, 2016: Annals of Transplantation: Quarterly of the Polish Transplantation Society
https://www.readbyqxmd.com/read/27869803/genome-wide-crispr-screens-reveal-a-wnt-fzd5-signaling-circuit-as-a-druggable-vulnerability-of-rnf43-mutant-pancreatic-tumors
#16
Zachary Steinhart, Zvezdan Pavlovic, Megha Chandrashekhar, Traver Hart, Xiaowei Wang, Xiaoyu Zhang, Mélanie Robitaille, Kevin R Brown, Sridevi Jaksani, René Overmeer, Sylvia F Boj, Jarrett Adams, James Pan, Hans Clevers, Sachdev Sidhu, Jason Moffat, Stéphane Angers
Forward genetic screens with CRISPR-Cas9 genome editing enable high-resolution detection of genetic vulnerabilities in cancer cells. We conducted genome-wide CRISPR-Cas9 screens in RNF43-mutant pancreatic ductal adenocarcinoma (PDAC) cells, which rely on Wnt signaling for proliferation. Through these screens, we discovered a unique requirement for a Wnt signaling circuit: engaging FZD5, one of the ten Frizzled receptors encoded in the human genome. Our results uncover an underappreciated level of context-dependent specificity at the Wnt receptor level...
November 21, 2016: Nature Medicine
https://www.readbyqxmd.com/read/27866656/an-analysis-of-possible-off-target-effects-following-cas9-crispr-targeted-deletions-of-neuropeptide-gene-enhancers-from-the-mouse-genome
#17
Elizabeth Anne Hay, Abdulla Razak Khalaf, Pietro Marini, Andrew Brown, Karyn Heath, Darrin Sheppard, Alasdair MacKenzie
We have successfully used comparative genomics to identify putative regulatory elements within the human genome that contribute to the tissue specific expression of neuropeptides such as galanin and receptors such as CB1. However, a previous inability to rapidly delete these elements from the mouse genome has prevented optimal assessment of their function in-vivo. This has been solved using CAS9/CRISPR genome editing technology which uses a bacterial endonuclease called CAS9 that, in combination with specifically designed guide RNA (gRNA) molecules, cuts specific regions of the mouse genome...
November 4, 2016: Neuropeptides
https://www.readbyqxmd.com/read/27865852/delivery-methods-for-site-specific-nucleases-achieving-the-full-potential-of-therapeutic-gene-editing
#18
REVIEW
Jia Liu, Sai-Lan Shui
The advent of site-specific nucleases, particularly CRISPR/Cas9, provides researchers with the unprecedented ability to manipulate genomic sequences. These nucleases are used to create model cell lines, engineer metabolic pathways, produce transgenic animals and plants, perform genome-wide functional screen and, most importantly, treat human diseases that are difficult to tackle by traditional medications. Considerable efforts have been devoted to improving the efficiency and specificity of nucleases for clinical applications...
November 16, 2016: Journal of Controlled Release: Official Journal of the Controlled Release Society
https://www.readbyqxmd.com/read/27865053/neural-progenitor-cells-and-their-role-in-the-development-and-evolutionary-expansion-of-the-neocortex
#19
REVIEW
Takashi Namba, Wieland B Huttner
The evolutionary expansion of the mammalian brain, notably the neocortex, provides a platform for the higher cognitive abilities that characterize humans. Cortical expansion is accompanied by increased folding of the pial surface, which gives rise to a gyrencephalic (folded) rather than lissencephalic (unfolded) neocortex. This expansion reflects the prolonged and increased proliferation of neural stem and progenitor cells (NPCs). Distinct classes of NPCs can be distinguished based on either cell biological criteria (apical progenitors [APs], basal progenitors [BPs]) or lineage (primary progenitors and secondary progenitors)...
November 16, 2016: Wiley Interdisciplinary Reviews. Developmental Biology
https://www.readbyqxmd.com/read/27860235/a-functional-pseudogene-nmral2p-is-regulated-by-nrf2-and-serves-as-a-co-activator-of-nqo1-in-sulforaphane-treated-colon-cancer-cells
#20
Gavin S Johnson, Jia Li, Laura M Beaver, W Mohaiza Dashwood, Deqiang Sun, Praveen Rajendran, David E Williams, Emily Ho, Roderick H Dashwood
SCOPE: The anticancer agent sulforaphane (SFN) acts via multiple mechanisms to modulate gene expression, including the induction of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent signaling and the inhibition of histone deacetylase activity. Transcriptomics studies were performed in SFN-treated human colon cancer cells and in non-transformed colonic epithelial cells in order to pursue new mechanistic leads. METHODS AND RESULTS: RNA-sequencing corroborated the expected changes in cancer-related pathways after SFN treatment...
November 18, 2016: Molecular Nutrition & Food Research
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