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https://www.readbyqxmd.com/read/29229870/what-history-tells-us-xliv-the-construction-of-the-zinc-finger-nucleases
#1
Michel Morange
No abstract text is available yet for this article.
December 2017: Journal of Biosciences
https://www.readbyqxmd.com/read/29229813/human-genetic-variation-alters-crispr-cas9-on-and-off-targeting-specificity-at-therapeutically-implicated-loci
#2
Samuel Lessard, Laurent Francioli, Jessica Alfoldi, Jean-Claude Tardif, Patrick T Ellinor, Daniel G MacArthur, Guillaume Lettre, Stuart H Orkin, Matthew C Canver
The CRISPR-Cas9 nuclease system holds enormous potential for therapeutic genome editing of a wide spectrum of diseases. Large efforts have been made to further understanding of on- and off-target activity to assist the design of CRISPR-based therapies with optimized efficacy and safety. However, current efforts have largely focused on the reference genome or the genome of cell lines to evaluate guide RNA (gRNA) efficiency, safety, and toxicity. Here, we examine the effect of human genetic variation on both on- and off-target specificity...
December 11, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29228730/wisp2-disruption-represses-cxcr4-expression-and-inhibits-bmscs-homing-to-injured-liver
#3
Dan Qin, Yi Yan, Bian Hu, Wanpo Zhang, Hanmin Li, Xiaodong Li, Shenghui Liu, Depeng Dai, Xiongji Hu, Xingxu Huang, Lisheng Zhang
Liver regeneration/repair is a compensatory regrowth following acute liver failure, and bone marrow-derived mesenchyme stem cell (BMSC) transplantation is an effective therapy that promotes liver regeneration/repair. Wnt1 inducible signaling pathway protein 2 (Wisp2) is highly expressed in BMSCs, however, its function remains unclear. In this work, we used clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein -9 nuclease (CRISPR/Cas9) genome editing technology to knockdown Wisp2 in BMSCs, and these modified cells were then transplanted into rats which were induced by the 2-AAF/PH...
November 17, 2017: Oncotarget
https://www.readbyqxmd.com/read/29228332/prespacer-processing-and-specific-integration-in-a-type-i-a-crispr-system
#4
Clare Rollie, Shirley Graham, Christophe Rouillon, Malcolm F White
The CRISPR-Cas system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. Adaptation is dependent on the Cas1 and Cas2 proteins along with varying accessory proteins. Here we analyse the process in Sulfolobus solfataricus, showing that while Cas1 and Cas2 catalyze spacer integration in vitro, host factors are required for specificity. Specific integration also requires at least 400 bp of the leader sequence, and is dependent on the presence of hydrolysable ATP, suggestive of an active process that may involve DNA remodelling...
December 8, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/29228103/fertility-impairment-with-defective-spermatogenesis-and-steroidogenesis-in-male-zebrafish-lacking-androgen-receptor
#5
Haipei Tang, Yu Chen, Le Wang, Yike Yin, Gaofei Li, Yin Guo, Yun Liu, Haoran Lin, Christopher H K Cheng, Xiaochun Liu
The pivotal role of androgen receptor (AR) in regulating male fertility has attracted much research attention in the past two decades. Previous studies have shown that total androgen receptor knockout would lead to incomplete spermatogenesis and a lowered serum testosterone levels in mice, resulting in azoospermia and infertility. However, the precise physiological role of ar in controlling fertility of male fish is still poorly understood. In this study, we have established an ar knockout zebrafish line by transcription activator-like effectors nucleases (TALENs)...
December 6, 2017: Biology of Reproduction
https://www.readbyqxmd.com/read/29227966/pot1-inhibits-the-efficiency-but-promotes-the-fidelity-of-nonhomologous-end-joining-at-non-telomeric-dna-regions
#6
Yang Yu, Rong Tan, Qian Ren, Boya Gao, Zhejin Sheng, Juanlian Zhang, Xiaoqing Zheng, Ying Jiang, Li Lan, Zhiyong Mao
Robust DNA double strand break (DSB) repair and stabilized telomeres help maintain genome integrity, preventing the onset of aging or tumorigenesis. POT1 is one of the six factors in the shelterin complex, which protects telomeres from being recognized as DNA damages. TRF1 and TRF2, two other shelterin proteins, have been shown to participate in DNA DSB repair at non-telomeric regions, but whether POT1, which binds to single strand telomeric DNA at chromosomal ends, is involved in DNA DSB repair has not been assessed...
December 8, 2017: Aging
https://www.readbyqxmd.com/read/29227487/hairpin-loop-enhanced-fluorescent-copper-nanoclusters-and-application-in-s1-nuclease-detection
#7
Xian-Sui Peng, Si-Yu Chen, Li-Juan Ou, Feng-Wu Luo, Si-Wen Qin, Ai-Ming Sun
Novel highly fluorescent copper nanoclusters (CuNCs) were prepared by using 24 adenine-thymine pair dsDNA (AT24) with six-base (X6) loops (AT24-X6-hairpin DNA) as an effective template. The AT24 double strand stem serves as a template for CuNC formation, and the six-base sequence loop acts as specific regions to enhance the fluorescence intensity of CuNCs. Relative to the AT24-CuNCs, AT24-X6-hairpin CuNCs have greater fluorescence (5 times enhancement). What's more, the influence of the hairpin loop with different base types and base numbers on the fluorescence of CuNCs was first proposed and investigated...
December 11, 2017: Analyst
https://www.readbyqxmd.com/read/29226219/systematic-optimization-and-modification-of-a-dna-aptamer-with-2-o-methyl-rna-analogues
#8
George Maio, Osita Enweronye, Hasan E Zumrut, Sana Batool, Nabeela Van, Prabodhika Mallikaratchy
Nucleic acid aptamers (NAAs) are short synthetic DNA or RNA molecules that specifically fold into distinct three-dimensional structures able to specifically recognize a target. While NAAs show unprecedented promise in a variety of applications, including sensing, therapeutics and diagnostics, one major limitation involves the lack of stability towards omnipresent nucleases. Therefore, we herein report a systematic truncation and incorporation of 2'-O-methyl bases to a DNA aptamer, which results in increased stability without affecting affinity...
March 1, 2017: ChemistrySelect
https://www.readbyqxmd.com/read/29225226/a-novel-biosensor-based-on-terminal-protection-and-fluorescent-copper-nanoparticles-for-detecting-potassium-ion
#9
WenJing Deng, Chang Liu, YanLei Hu, RuQin Yu, TingTing Chen, Xia Chu
A novel biosensor for sensitively detecting potassium ion (K+) has been developed based on fluorescent copper nanoparticles (Cu NPs). In our design, we employ a label-free single-strand DNA (ssDNA) that contains two parts. One is 3'-terminus structure-switching aptamers (SSAs) that can fold into G-quadruplex after binding with its target K+. The other is 5'-terminus poly thymine (polyT) which works as a template to construct fluorescent Cu NPs. After incubating with K+, the part SSAs go through target-induced conformational changes...
2017: Analytical Sciences: the International Journal of the Japan Society for Analytical Chemistry
https://www.readbyqxmd.com/read/29225080/second-shell-basic-residues-expand-the-two-metal-ion-architecture-of-dna-and-rna-processing-enzymes
#10
Vito Genna, Matteo Colombo, Marco De Vivo, Marco Marcia
Synthesis and scission of phosphodiester bonds in DNA and RNA regulate vital processes within the cell. Enzymes that catalyze these reactions operate mostly via the recognized two-metal-ion mechanism. Our analysis reveals that basic amino acids and monovalent cations occupy structurally conserved positions nearby the active site of many two-metal-ion enzymes for which high-resolution (<3 Å) structures are known, including DNA and RNA polymerases, nucleases such as Cas9, and splicing ribozymes. Integrating multiple-sequence and structural alignments with molecular dynamics simulations, electrostatic potential maps, and mutational data, we found that these elements always interact with the substrates, suggesting that they may play an active role for catalysis, in addition to their electrostatic contribution...
December 1, 2017: Structure
https://www.readbyqxmd.com/read/29225036/transcription-and-remodeling-produce-asymmetrically-unwrapped-nucleosomal-intermediates
#11
Srinivas Ramachandran, Kami Ahmad, Steven Henikoff
Nucleosomes are disrupted during transcription and other active processes, but the structural intermediates during nucleosome disruption in vivo are unknown. To identify intermediates, we mapped subnucleosomal protections in Drosophila cells using Micrococcal Nuclease followed by sequencing. At the first nucleosome position downstream of the transcription start site, we identified unwrapped intermediates, including hexasomes that lack either proximal or distal contacts. Inhibiting topoisomerases or depleting histone chaperones increased unwrapping, whereas inhibiting release of paused RNAPII or reducing RNAPII elongation decreased unwrapping...
December 6, 2017: Molecular Cell
https://www.readbyqxmd.com/read/29222969/copper-ii-complexes-of-n3o-tripodal-ligands-appended-with-pyrene-and-polyamine-groups-anti-proliferative-and-nuclease-activities
#12
Doti Serre, Sule Erbek, Nathalie Berthet, Xavier Ronot, Véronique Martel-Frachet, Fabrice Thomas
A series of tripodal ligands based on the 2-tert-butyl-4-R-6-phenol was synthesized, where R=aldehyde (HL1), R=putrescine-pyrene (HL2) and R=putrescine (HL3). A dinucleating ligand wherein a putrescine group connects two tripodal moieties was also prepared (H2L4). The corresponding copper complexes (1, 2, 3, and 4, respectively) were prepared and characterized. We determined the phenol's pKas in the range 2.47-3.93. The DNA binding constants were determined at 6×106, 5.5×105 and 2.7×106 for 2, 3 and 4, respectively...
November 11, 2017: Journal of Inorganic Biochemistry
https://www.readbyqxmd.com/read/29222528/structure-and-dynamics-of-cas9-hnh-domain-catalytic-state
#13
Zhicheng Zuo, Jin Liu
The bacterial CRISPR-Cas9 immune system has been harnessed as a powerful and versatile genome-editing tool and holds immense promise for future therapeutic applications. Despite recent advances in understanding Cas9 structures and its functional mechanism, little is known about the catalytic state of the Cas9 HNH nuclease domain, and identifying how the divalent metal ions affect the HNH domain conformational transition remains elusive. A deeper understanding of Cas9 activation and its cleavage mechanism can enable further optimization of Cas9-based genome-editing specificity and efficiency...
December 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29221806/characterization-of-gene-alterations-following-editing-of-the-%C3%AE-globin-gene-locus-in-hematopoietic-stem-progenitor-cells
#14
Joseph Long, Megan D Hoban, Aaron R Cooper, Michael L Kaufman, Caroline Y Kuo, Beatriz Campo-Fernandez, Dianne Lumaquin, Roger P Hollis, Xiaoyan Wang, Donald B Kohn, Zulema Romero
The use of engineered nucleases combined with a homologous DNA donor template can result in targeted gene correction of the sickle cell disease mutation in hematopoietic stem and progenitor cells. However, because of the high homology between the adjacent human β- and δ-globin genes, off-target cleavage is observed at δ-globin when using some endonucleases targeted to the sickle mutation in β-globin. Introduction of multiple double-stranded breaks by endonucleases has the potential to induce intergenic alterations...
November 9, 2017: Molecular Therapy: the Journal of the American Society of Gene Therapy
https://www.readbyqxmd.com/read/29220655/aid-and-reactive-oxygen-species-can-induce-dna-breaks-within-human-chromosomal-translocation-fragile-zones
#15
Nicholas R Pannunzio, Michael R Lieber
DNA double-strand breaks (DSBs) occurring within fragile zones of less than 200 base pairs account for the formation of the most common human chromosomal translocations in lymphoid malignancies, yet the mechanism of how breaks occur remains unknown. Here, we have transferred human fragile zones into S. cerevisiae in the context of a genetic assay to understand the mechanism leading to DSBs at these sites. Our findings indicate that a combination of factors is required to sensitize these regions. Foremost, DNA strand separation by transcription or increased torsional stress can expose these DNA regions to damage from either the expression of human AID or increased oxidative stress...
December 7, 2017: Molecular Cell
https://www.readbyqxmd.com/read/29216270/generation-and-characterization-of-kctd15-mutations-in-zebrafish
#16
Alison Heffer, Gregory D Marquart, Allisan Aquilina-Beck, Nabil Saleem, Harold A Burgess, Igor B Dawid
Potassium channel tetramerization domain containing 15 (Kctd15) was previously found to have a role in early neural crest (NC) patterning, specifically delimiting the region where NC markers are expressed via repression of transcription factor AP-2a and inhibition of Wnt signaling. We used transcription activator-like effector nucleases (TALENs) to generate null mutations in zebrafish kctd15a and kctd15b paralogs to study the in vivo role of Kctd15. We found that while deletions producing frame-shift mutations in each paralog showed no apparent phenotype, kctd15a/b double mutant zebrafish are smaller in size and show several phenotypes including some affecting the NC, such as expansion of the early NC domain, increased pigmentation, and craniofacial defects...
2017: PloS One
https://www.readbyqxmd.com/read/29215062/protein-structure-assisted-optimization-of-4-5-dihydroxypyrimidine-6-carboxamide-inhibitors-of-influenza-virus-endonuclease
#17
Diane Beylkin, Gyanendra Kumar, Wei Zhou, Jaehyeon Park, Trushar Jeevan, Chandraiah Lagisetti, Rhodri Harfoot, Richard J Webby, Stephen W White, Thomas R Webb
Influenza is a serious hazard to human health that causes hundreds of thousands of deaths annually. Though vaccines and current therapeutics can blunt some of the perilous impact of this viral infection, new treatments are needed due to the constantly evolving nature of this virus. Recently, our growing understanding of an essential influenza viral protein, PA, has led to the development of focused libraries of new small molecules that specifically target the active site of the PA influenza endonuclease, which we report here...
December 7, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29212533/subtracting-the-sequence-bias-from-partially-digested-mnase-seq-data-reveals-a-general-contribution-of-tfiis-to-nucleosome-positioning
#18
Gabriel Gutiérrez, Gonzalo Millán-Zambrano, Daniel A Medina, Antonio Jordán-Pla, José E Pérez-Ortín, Xenia Peñate, Sebastián Chávez
BACKGROUND: TFIIS stimulates RNA cleavage by RNA polymerase II and promotes the resolution of backtracking events. TFIIS acts in the chromatin context, but its contribution to the chromatin landscape has not yet been investigated. Co-transcriptional chromatin alterations include subtle changes in nucleosome positioning, like those expected to be elicited by TFIIS, which are elusive to detect. The most popular method to map nucleosomes involves intensive chromatin digestion by micrococcal nuclease (MNase)...
December 7, 2017: Epigenetics & Chromatin
https://www.readbyqxmd.com/read/29211736/versatile-single-step-assembly-crispr-cas9-vectors-for-dual-grna-expression
#19
Fatwa Adikusuma, Chandran Pfitzner, Paul Quinton Thomas
CRISPR/Cas9 technology enables efficient, rapid and cost-effective targeted genomic modification in a wide variety of cellular contexts including cultured cells. Some applications such as generation of double knock-outs, large deletions and paired-nickase cleavage require simultaneous expression of two gRNAs. Although single plasmids that enable multiplex expression of gRNAs have been developed, these require multiple rounds of cloning and/or PCR for generation of the desired construct. Here, we describe a series of vectors that enable generation of customized dual-gRNA expression constructs via an easy one-step golden gate cloning reaction using two annealed oligonucleotide inserts with different overhangs...
2017: PloS One
https://www.readbyqxmd.com/read/29210569/modulating-dna-repair-pathways-to-improve-precision-genome-engineering
#20
Katherine S Pawelczak, Navnath S Gavande, Pamela S VanderVere-Carozza, John J Turchi
Programmable nucleases like the popular CRISPR/Cas9 system allow for precision genome engineering by inducing a site-specific DNA double strand break (DSB) within a genome. The DSB is repaired by endogenous DNA repair pathways, either non-homologous end joining (NHEJ) or homology directed repair (HDR). The predominant and error-prone NHEJ pathway often results in small nucleotide insertions or deletions that can be used to construct knockout alleles. Alternatively, HDR activity can result in precise modification incorporating exogenous DNA fragments into the cut site...
December 6, 2017: ACS Chemical Biology
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