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https://www.readbyqxmd.com/read/28072927/artificial-induction-of-native-aquaporin-1-expression-in-human-salivary-cells
#1
Z Wang, S Pradhan-Bhatt, M C Farach-Carson, M J Passineau
Gene therapy for dry mouth disorders has transitioned in recent years from theoretical to clinical proof of principle with the publication of a first-in-man phase I/II dose escalation clinical trial in patients with radiation-induced xerostomia. This trial used a prototype adenoviral vector to express aquaporin-1 (AQP1), presumably in the ductal cell layer and/or in surviving acinar cells, to drive transcellular flux of interstitial fluid into the labyrinth of the salivary duct. As the development of this promising gene therapy continues, safety considerations are a high priority, particularly those that remove nonhuman agents (i...
January 1, 2017: Journal of Dental Research
https://www.readbyqxmd.com/read/28069948/genomic-targeting-of-epigenetic-probes-using-a-chemically-tailored-cas9-system
#2
Glen P Liszczak, Zachary Z Brown, Samuel H Kim, Rob C Oslund, Yael David, Tom W Muir
Recent advances in the field of programmable DNA-binding proteins have led to the development of facile methods for genomic localization of genetically encodable entities. Despite the extensive utility of these tools, locus-specific delivery of synthetic molecules remains limited by a lack of adequate technologies. Here we combine the flexibility of chemical synthesis with the specificity of a programmable DNA-binding protein by using protein trans-splicing to ligate synthetic elements to a nuclease-deficient Cas9 (dCas9) in vitro and subsequently deliver the dCas9 cargo to live cells...
January 9, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28046023/a-model-to-investigate-single-strand-dna-responses-in-g1-human-cells-via-a-telomere-targeted-nuclease-deficient-crispr-cas9-system
#3
Remco P Crefcoeur, Omar Zgheib, Thanos D Halazonetis
DNA replication stress has the potential to compromise genomic stability and, therefore, cells have developed elaborate mechanisms to detect and resolve problems that may arise during DNA replication. The presence of single-stranded DNA (ssDNA) is often associated with DNA replication stress and serves as a signal for both checkpoint and repair responses. In this study, we exploited a CRISPR-Cas9 system to induce regions of ssDNA in the genome. Specifically, single-guide RNAs bearing sequence complementarity to human telomeric repeats, were used to target nuclease-deficient Cas9 (dCas9) to telomeres...
2017: PloS One
https://www.readbyqxmd.com/read/27983975/current-status-of-potential-applications-of-repurposed-cas9-for-structural-and-functional-genomics-of-plants
#4
Kunal Seth, Harish
Redesigned Cas9 has emerged as a tool with various applications like gene editing, gene regulation, epigenetic modification and chromosomal imaging. Target specific single guide RNA (sgRNA) can be used with Cas9 for precise gene editing with high efficiency than previously known methods. Further, nuclease-deactivated Cas9 (dCas9) can be fused with activator or repressor for activation (CRISPRa) and repression (CRISPRi) of gene expression, respectively. dCas9 fused with epigenetic modifier like methylase or acetylase further expand the scope of this technique...
October 28, 2016: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/27965712/targeted-inhibition-of-the-mir-199a-214-cluster-by-crispr-interference-augments-the-tumor-tropism-of-human-induced-pluripotent-stem-cell-derived-neural-stem-cells-under-hypoxic-condition
#5
Yumei Luo, Xuehu Xu, Xiuli An, Xiaofang Sun, Shu Wang, Detu Zhu
The human induced pluripotent stem cell (hiPSC) provides a breakthrough approach that helps overcoming ethical and allergenic challenges posed in application of neural stem cells (NSCs) in targeted cancer gene therapy. However, the tumor-tropic capacity of hiPSC-derived NSCs (hiPS-NSCs) still has much room to improve. Here we attempted to promote the tumor tropism of hiPS-NSCs by manipulating the activity of endogenous miR-199a/214 cluster that is involved in regulation of hypoxia-stimulated cell migration...
2016: Stem Cells International
https://www.readbyqxmd.com/read/27960483/single-molecule-localization-and-discrimination-of-dna-protein-complexes-by-controlled-translocation-through-nanocapillaries
#6
Roman D Bulushev, Sanjin Marion, Ekaterina Petrova, Sebastian J Davis, Sebastian J Maerkl, Aleksandra Radenovic
Through the use of optical tweezers we performed controlled translocations of DNA-protein complexes through nanocapillaries. We used RNA polymerase (RNAP) with two binding sites on a 7.2 kbp DNA fragment and a dCas9 protein tailored to have five binding sites on λ-DNA (48.5 kbp). Measured localization of binding sites showed a shift from the expected positions on the DNA that we explained using both analytical fitting and a stochastic model. From the measured force versus stage curves we extracted the nonequilibrium work done during the translocation of a DNA-protein complex and used it to obtain an estimate of the effective charge of the complex...
December 14, 2016: Nano Letters
https://www.readbyqxmd.com/read/27955725/current-status-of-potential-applications-of-repurposed-cas9-for-structural-and-functional-genomics-of-plants
#7
Kunal Seth, Harish
Redesigned Cas9 has emerged as a tool with various applications like gene editing, gene regulation, epigenetic modification and chromosomal imaging. Target specific single guide RNA (sgRNA) can be used with Cas9 for precise gene editing with high efficiency than previously known methods. Further, nuclease-deactivated Cas9 (dCas9) can be fused with activator or repressor for activation (CRISPRa) and repression (CRISPRi) of gene expression, respectively. dCas9 fused with epigenetic modifier like methylase or acetylase further expand the scope of this technique...
November 25, 2016: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/27934604/generation-of-induced-pluripotent-stem-cells-ipscs-stably-expressing-crispr-based-synergistic-activation-mediator-sam
#8
Kai Xiong, Yan Zhou, Poul Hyttel, Lars Bolund, Kristine Karla Freude, Yonglun Luo
Human fibroblasts were engineered to express the CRISPR-based synergistic activation mediator (SAM) complex: dCas9-VP64 and MS2-P65-HSF1. Two induced pluripotent stem cells (iPSCs) clones expressing SAM were established by transducing these fibroblasts with lentivirus expressing OCT4, SOX2, KLF4 and C-MYC. We have validated that the reprogramming cassette is silenced in the SAM iPSC clones. Expression of pluripotency genes (OCT4, SOX2, LIN28A, NANOG, GDF3, SSEA4, and TRA-1-60), differentiation potential to all three germ layers, and normal karyotypes are validated...
November 17, 2016: Stem Cell Research
https://www.readbyqxmd.com/read/27905063/efficient-gene-targeting-in-mouse-zygotes-mediated-by-crispr-cas9-protein
#9
Chris J Jung, Junli Zhang, Elizabeth Trenchard, Kent C Lloyd, David B West, Barry Rosen, Pieter J de Jong
The CRISPR/Cas9 system has rapidly advanced targeted genome editing technologies. However, its efficiency in targeting with constructs in mouse zygotes via homology directed repair (HDR) remains low. Here, we systematically explored optimal parameters for targeting constructs in mouse zygotes via HDR using mouse embryonic stem cells as a model system. We characterized several parameters, including single guide RNA cleavage activity and the length and symmetry of homology arms in the construct, and we compared the targeting efficiency between Cas9, Cas9nickase, and dCas9-FokI...
November 30, 2016: Transgenic Research
https://www.readbyqxmd.com/read/27903907/chromatin-binding-of-gcn5-in-drosophila-is-largely-mediated-by-cp190
#10
Tamer Ali, Marcus Krüger, Sabin Bhuju, Michael Jarek, Marek Bartkuhn, Rainer Renkawitz
Centrosomal 190 kDa protein (CP190) is a promoter binding factor, mediates long-range interactions in the context of enhancer-promoter contacts and in chromosomal domain formation. All Drosophila insulator proteins bind CP190 suggesting a crucial role in insulator function. CP190 has major effects on chromatin, such as depletion of nucleosomes, high nucleosomal turnover and prevention of heterochromatin expansion. Here, we searched for enzymes, which might be involved in CP190 mediated chromatin changes. Eighty percent of the genomic binding sites of the histone acetyltransferase Gcn5 are colocalizing with CP190 binding...
November 29, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27899645/efficient-targeted-dna-methylation-with-chimeric-dcas9-dnmt3a-dnmt3l-methyltransferase
#11
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/27899566/asymmetric-positioning-of-cas1-2-complex-and-integration-host-factor-induced-dna-bending-guide-the-unidirectional-homing-of-protospacer-in-crispr-cas-type-i-e-system
#12
K N R Yoganand, R Sivathanu, Siddharth Nimkar, B Anand
CRISPR-Cas system epitomizes prokaryote-specific quintessential adaptive defense machinery that limits the genome invasion of mobile genetic elements. It confers adaptive immunity to bacteria by capturing a protospacer fragment from invading foreign DNA, which is later inserted into the leader proximal end of CRIPSR array and serves as immunological memory to recognize recurrent invasions. The universally conserved Cas1 and Cas2 form an integration complex that is known to mediate the protospacer invasion into the CRISPR array...
November 29, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27846887/crispr-interference-crispri-for-gene-regulation-and-succinate-production-in-cyanobacterium-s-elongatus-pcc-7942
#13
Chun-Hung Huang, Claire R Shen, Hung Li, Li-Yu Sung, Meng-Ying Wu, Yu-Chen Hu
BACKGROUND: Cyanobacterium Synechococcus elongatus PCC 7942 holds promise for biochemical conversion, but gene deletion in PCC 7942 is time-consuming and may be lethal to cells. CRISPR interference (CRISPRi) is an emerging technology that exploits the catalytically inactive Cas9 (dCas9) and single guide RNA (sgRNA) to repress sequence-specific genes without the need of gene knockout, and is repurposed to rewire metabolic networks in various procaryotic cells. RESULTS: To employ CRISPRi for the manipulation of gene network in PCC 7942, we integrated the cassettes expressing enhanced yellow fluorescent protein (EYFP), dCas9 and sgRNA targeting different regions on eyfp into the PCC 7942 chromosome...
November 15, 2016: Microbial Cell Factories
https://www.readbyqxmd.com/read/27842593/easy-regulation-of-metabolic-flux-in-escherichia-coli-using-an-endogenous-type-i-e-crispr-cas-system
#14
Yizhao Chang, Tianyuan Su, Qingsheng Qi, Quanfeng Liang
BACKGROUND: Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is a recently developed powerful tool for gene regulation. In Escherichia coli, the type I CRISPR system expressed endogenously shall be easy for internal regulation without causing metabolic burden in compared with the widely used type II system, which expressed dCas9 as an additional plasmid. RESULTS: By knocking out cas3 and activating the expression of CRISPR-associated complex for antiviral defense (Cascade), we constructed a native CRISPRi system in E...
November 15, 2016: Microbial Cell Factories
https://www.readbyqxmd.com/read/27832543/using-an-inducible-crispr-dcas9-krab-effector-system-to-dissect-transcriptional-regulation-in-human-embryonic-stem-cells
#15
Krishna Mohan Parsi, Erica Hennessy, Nicola Kearns, René Maehr
CRISPR-Cas9 effector systems have wide applications for the stem cell and regenerative medicine field. The ability to dissect the functional gene regulatory networks in pluripotency and potentially in differentiation intermediates of all three germ layers makes this a valuable tool for the stem cell community. Catalytically inactive Cas9 fused to transcriptional/chromatin effector domains allows for silencing or activation of a genomic region of interest. Here, we describe the application of an inducible, RNA-guided, nuclease-deficient (d) Cas9-KRAB system (adapted from Streptococcus pyogenes) to silence target gene expression in human embryonic stem cells, via KRAB repression at the promoter region...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27799472/empower-multiplex-cell-and-tissue-specific-crispr-mediated-gene-manipulation-with-self-cleaving-ribozymes-and-trna
#16
Li Xu, Lixia Zhao, Yandi Gao, Jing Xu, Renzhi Han
Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system has emerged in recent years as a highly efficient RNA-guided gene manipulation platform. Simultaneous editing or transcriptional activation/suppression of different genes becomes feasible with the co-delivery of multiple guide RNAs (gRNAs). Here, we report that multiple gRNAs linked with self-cleaving ribozymes and/or tRNA could be simultaneously expressed from a single U6 promoter to exert genome editing of dystrophin and myosin binding protein C3 in human and mouse cells...
October 30, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27798611/directed-evolution-using-dcas9-targeted-somatic-hypermutation-in-mammalian-cells
#17
Gaelen T Hess, Laure Frésard, Kyuho Han, Cameron H Lee, Amy Li, Karlene A Cimprich, Stephen B Montgomery, Michael C Bassik
Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously...
December 2016: Nature Methods
https://www.readbyqxmd.com/read/27788303/efficient-gene-transcription-repression-in-clostridium-using-crispr-dcas9
#18
(no author information available yet)
No abstract text is available yet for this article.
December 2016: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/27786189/crispr-rna-guided-foki-nucleases-repair-a-pah-variant-in-a-phenylketonuria-model
#19
Yi Pan, Nan Shen, Sabine Jung-Klawitter, Christian Betzen, Georg F Hoffmann, Jörg D Hoheisel, Nenad Blau
The CRISPR/Cas9 system is a recently developed genome editing technique. In this study, we used a modified CRISPR system, which employs the fusion of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9) to correct the most common variant (allele frequency 21.4%) in the phenylalanine hydroxylase (PAH) gene - c.1222C>T (p.Arg408Trp) - as an approach toward curing phenylketonuria (PKU). PKU is the most common inherited diseases in amino acid metabolism. It leads to severe neurological and neuropsychological symptoms if untreated or late diagnosed...
October 27, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27776111/complex-transcriptional-modulation-with-orthogonal-and-inducible-dcas9-regulators
#20
Yuchen Gao, Xin Xiong, Spencer Wong, Emeric J Charles, Wendell A Lim, Lei S Qi
The ability to dynamically manipulate the transcriptome is important for studying how gene networks direct cellular functions and how network perturbations cause disease. Nuclease-dead CRISPR-dCas9 transcriptional regulators, while offering an approach for controlling individual gene expression, remain incapable of dynamically coordinating complex transcriptional events. Here, we describe a flexible dCas9-based platform for chemical-inducible complex gene regulation. From a screen of chemical- and light-inducible dimerization systems, we identified two potent chemical inducers that mediate efficient gene activation and repression in mammalian cells...
October 24, 2016: Nature Methods
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