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https://www.readbyqxmd.com/read/28108186/editing-the-trypanosoma-cruzi-genome-with-zinc-finger-nucleases
#1
Gabriela Assis Burle-Caldas, Viviane Grazielle-Silva, Melissa Soares-Simões, Gabriela Schumann Burkard, Isabel Roditi, Wanderson Duarte DaRocha, Santuza M Teixeira
Gene function studies in Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, have been hindered by the lack of efficient genetic manipulation protocols. In most organisms, insertion and deletion of DNA fragments in the genome are dependent on the generation of double-stranded DNA break (DSB) and repair. By inducing a site-specific DSB, zinc finger nucleases (ZFNs) have proven to be useful to enhance gene editing in many cell types. Using a pair of ZFNs targeted to the T. cruzi gp72 gene, we were able to generate gp72 knockout parasites with improved efficiency compared to the conventional gene knockout protocol...
January 17, 2017: Molecular and Biochemical Parasitology
https://www.readbyqxmd.com/read/28107987/generation-of-biostable-l-aptamers-against-achiral-targets-by-chiral-inversion-of-existing-d-aptamers
#2
Huapei Chen, Sitao Xie, Hao Liang, Cuichen Wu, Liang Cui, Shuang-Yan Huan, Xiaobing Zhang
In this paper, based on reciprocal chiral substrate specificity, taking achiral molecules, ethanolamine (EA) and malachite green (MG) as two model targets, biostable L- DNA aptamers and L-RNA aptamers were generated respectively by chiral inversion of existing D-aptamers. In the detection of EA with L-DNA aptamer-based sensors, the feasibility of our strategy was confirmed, while in the detection of MG with L-RNA aptamers, linear calibration curves were obtained in the range from 0.1 to 5µm with the detection limit of 0...
March 1, 2017: Talanta
https://www.readbyqxmd.com/read/28103141/modification-of-the-genome-of-domestic-animals
#3
Samantha N Lotti, Kathryn M Polkoff, Marcello Rubessa, Matthew B Wheeler
In the past few years, new technologies have arisen that enable higher efficiency of gene editing. With the increase ease of using gene editing technologies, it is important to consider the best method for transferring new genetic material to livestock animals. Microinjection is a technique that has proven to be effective in mice but is less efficient in large livestock animals. Over the years, a variety of methods have been used for cloning as well as gene transfer including; nuclear transfer, sperm mediated gene transfer (SMGT), and liposome-mediated DNA transfer...
January 19, 2017: Animal Biotechnology
https://www.readbyqxmd.com/read/28103009/fully-automated-one-step-synthesis-of-single-transcript-talen-pairs-using-a-biological-foundry
#4
Ran Chao, Jing Liang, Ipek Tasan, Tong Si, Linyang Ju, Huimin Zhao
Transcription activator-like effector nuclease (TALEN) is a programmable genome editing tool with wide applications. Since TALENs perform cleavage of DNA as heterodimers, a pair of TALENs must be synthesized for each target genome locus. Conventionally, TALEN pairs are either expressed on separate vectors or synthesized separately and then subcloned to the same vector. Neither approach allows high-throughput construction of TALEN libraries for large-scale applications. Here we present an assembly scheme to synthesize and express a pair of TALENs in a single transcript format with the help of a P2A self-cleavage sequence...
January 19, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28101376/atpase-activity-tightly-regulates-reca-nucleofilaments-to-promote-homologous-recombination
#5
Bailin Zhao, Dapeng Zhang, Chengmin Li, Zheng Yuan, Fangzhi Yu, Shangwei Zhong, Guibin Jiang, Yun-Gui Yang, X Chris Le, Michael Weinfeld, Ping Zhu, Hailin Wang
Homologous recombination (HR), catalyzed in an evolutionarily conserved manner by active RecA/Rad51 nucleofilaments, maintains genomic integrity and promotes biological evolution and diversity. The structures of RecA/Rad51 nucleofilaments provide information critical for the entire HR process. By exploiting a unique capillary electrophoresis-laser-induced fluorescence polarization assay, we have discovered an active form of RecA nucleofilament, stimulated by ATP hydrolysis, that contains mainly unbound nucleotide sites...
2017: Cell Discovery
https://www.readbyqxmd.com/read/28100693/viral-genome-packaging-terminase-cleaves-dna-using-the-canonical-ruvc-like-two-metal-catalysis-mechanism
#6
Rui-Gang Xu, Huw T Jenkins, Maria Chechik, Elena V Blagova, Anna Lopatina, Evgeny Klimuk, Leonid Minakhin, Konstantin Severinov, Sandra J Greive, Alfred A Antson
Bacteriophages and large dsDNA viruses encode sophisticated machinery to translocate their DNA into a preformed empty capsid. An essential part of this machine, the large terminase protein, processes viral DNA into constituent units utilizing its nuclease activity. Crystal structures of the large terminase nuclease from the thermophilic bacteriophage G20c show that it is most similar to the RuvC family of the RNase H-like endonucleases. Like RuvC proteins, the nuclease requires either Mn(2+), Mg(2+) or Co(2+) ions for activity, but is inactive with Zn(2+) and Ca(2+) High resolution crystal structures of complexes with different metals reveal that in the absence of DNA, only one catalytic metal ion is accommodated in the active site...
January 18, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28096446/the-rpn-yhga-like-proteins-of-escherichia-coli-k-12-and-their-contribution-to-reca-independent-horizontal-transfer
#7
Anthony W Kingston, Christine Ponkratz, Elisabeth A Raleigh
: Bacteria use a variety of DNA mobilizing enzymes to facilitate environmental niche adaptation via horizontal gene transfer. This has led to real world problems, like the spread of antibiotic resistance, yet many mobilization proteins remain undefined. Here, we investigate the uncharacterized family of YhgA-like "transposase_31" proteins (PF04754). Our primary focus was the genetic and biochemical properties of the five E. coli K-12 members of this family, which we designate RpnA-RpnE for recombination promoting nuclease...
January 17, 2017: Journal of Bacteriology
https://www.readbyqxmd.com/read/28096179/dbf4-dependent-kinase-and-the-rtt107-scaffold-promote-mus81-mms4-resolvase-activation-during-mitosis
#8
Lissa N Princz, Philipp Wild, Julia Bittmann, F Javier Aguado, Miguel G Blanco, Joao Matos, Boris Pfander
DNA repair by homologous recombination is under stringent cell cycle control. This includes the last step of the reaction, disentanglement of DNA joint molecules (JMs). Previous work has established that JM resolving nucleases are activated specifically at the onset of mitosis. In case of budding yeast Mus81-Mms4, this cell cycle stage-specific activation is known to depend on phosphorylation by CDK and Cdc5 kinases. Here, we show that a third cell cycle kinase, Cdc7-Dbf4 (DDK), targets Mus81-Mms4 in conjunction with Cdc5-both kinases bind to as well as phosphorylate Mus81-Mms4 in an interdependent manner...
January 17, 2017: EMBO Journal
https://www.readbyqxmd.com/read/28095622/talens-and-crispr-cas9-fuel-genetically-engineered-clinically-relevant-xenopus-tropicalis-tumor-models
#9
REVIEW
Thomas Naert, Tom van Nieuwenhuysen, Kris Vleminckx
The targeted nuclease revolution (TALENs, CRISPR/Cas9) now allows Xenopus researchers to rapidly generate custom on-demand genetic knockout models. These novel methods to perform reverse genetics are unprecedented and are fueling a wide array of human disease models within the aquatic diploid model organism Xenopus tropicalis (X. tropicalis). This emerging technology review focuses on the tools to rapidly generate genetically engineered X. tropicalis models (GEXM), with a focus on establishment of genuine genetic and clinically relevant cancer models...
January 17, 2017: Genesis: the Journal of Genetics and Development
https://www.readbyqxmd.com/read/28095454/non-homologous-end-joining-and-homology-directed-dna-repair-frequency-of-double-stranded-breaks-introduced-by-genome-editing-reagents
#10
Michail Zaboikin, Tatiana Zaboikina, Carl Freter, Narasimhachar Srinivasakumar
Genome editing using transcription-activator like effector nucleases or RNA guided nucleases allows one to precisely engineer desired changes within a given target sequence. The genome editing reagents introduce double stranded breaks (DSBs) at the target site which can then undergo DNA repair by non-homologous end joining (NHEJ) or homology directed recombination (HDR) when a template DNA molecule is available. NHEJ repair results in indel mutations at the target site. As PCR amplified products from mutant target regions are likely to exhibit different melting profiles than PCR products amplified from wild type target region, we designed a high resolution melting analysis (HRMA) for rapid identification of efficient genome editing reagents...
2017: PloS One
https://www.readbyqxmd.com/read/28093313/knockdown-of-nuclease-activity-in-the-gut-enhances-rnai-efficiency-in-the-colorado-potato-beetle-leptinotarsa-decemlineata-but-not-in-the-desert-locust-schistocerca-gregaria
#11
Jornt Spit, Annelies Philips, Niels Wynant, Dulce Santos, Geert Plaetinck, Jozef Vanden Broeck
The responsiveness towards orally delivered dsRNA and the potency of a subsequent environmental RNA interference (RNAi) response strongly differs between different insect species. While some species are very sensitive to dsRNA delivery through the diet, others are not. The underlying reasons for this may vary, but degradation of dsRNA by nucleases in the gut lumen is believed to play a crucial role. The Colorado potato beetle, Leptinotarsa decemlineata, is a voracious defoliator of potato crops worldwide, and is currently under investigation for novel control methods based on dsRNA treatments...
January 13, 2017: Insect Biochemistry and Molecular Biology
https://www.readbyqxmd.com/read/28088460/ultrasound-mediated-delivery-of-rna-to-colonic-mucosa-of-live-mice
#12
Carl M Schoellhammer, Gregory Y Lauwers, Jeremy A Goettel, Matthias A Oberli, Cody Cleveland, June Y Park, Daniel Minahan, Yiyun Chen, Daniel G Anderson, Ana Jaklenec, Scott B Snapper, Robert Langer, Giovanni Traverso
BACKGROUND & AIMS: It is a challenge to deliver nucleic acids to gastrointestinal (GI) tissues due to their size and need for intracellular delivery. They are also extremely susceptible to degradation by nucleases, which are ubiquitous in the GI tract. We investigated whether ultrasound, which can permeabilize tissue through a phenomenon known as transient cavitation, can be used to deliver RNA to the colonic mucosa of living mice. METHODS: We investigated delivery of fluorescently labeled permeants to colon tissues of Yorkshire pigs ex vivo and mice in vivo...
January 11, 2017: Gastroenterology
https://www.readbyqxmd.com/read/28081439/knotty-zika-virus-blocks-exonuclease-to-produce-subgenomic-flaviviral-rnas
#13
Nandan S Gokhale, Stacy M Horner
In a recent issue of Science, Akiyama et al. (2016) prove the existence of a pseudoknot that stabilizes a nuclease-resistant RNA structure in the 3' untranslated region of Zika virus. This reinforced structure blocks the 5'→3' exonuclease Xrn1 for the production of pathogenic subgenomic flaviviral RNAs.
January 11, 2017: Cell Host & Microbe
https://www.readbyqxmd.com/read/28081254/maternal-supply-of-cas9-to-zygotes-facilitates-the-efficient-generation-of-site-specific-mutant-mouse-models
#14
Alberto Cebrian-Serrano, Shijun Zha, Lars Hanssen, Daniel Biggs, Christopher Preece, Benjamin Davies
Genome manipulation in the mouse via microinjection of CRISPR/Cas9 site-specific nucleases has allowed the production time for genetically modified mouse models to be significantly reduced. Successful genome manipulation in the mouse has already been reported using Cas9 supplied by microinjection of a DNA construct, in vitro transcribed mRNA and recombinant protein. Recently the use of transgenic strains of mice overexpressing Cas9 has been shown to facilitate site-specific mutagenesis via maternal supply to zygotes and this route may provide an alternative to exogenous supply...
2017: PloS One
https://www.readbyqxmd.com/read/28081204/deletion-of-ssna-attenuates-the-pathogenicity-of-streptococcus-suis-and-confers-protection-against-serovar-2-strain-challenge
#15
Miao Li, Ru-Jian Cai, Chun-Ling Li, Shuai Song, Yan Li, Zhi-Yong Jiang, Dong-Xia Yang
Streptococcus suis serotype 2 (SS2) is a major porcine and human pathogen which causes arthritis, meningitis, and septicemia. Streptococcus suis nuclease A (SsnA) is a recently discovered deoxyribonuclease (DNase), which has been demonstrated to contribute to escape killing in neutrophil extracellular traps (NETs). To further determine the effects of ssnA on virulence, the ssnA deletion mutant (ΔssnA) and its complemented strain (C-ΔssnA) were constructed. The ability of ΔssnA mutant to interact with human laryngeal epithelial cell (Hep-2) was evaluated and it exhibited dramatically decreased ability to adhere to and invade Hep-2 cells...
2017: PloS One
https://www.readbyqxmd.com/read/28079956/an-exonuclease-iii-powered-on-particle-stochastic-dna-walker
#16
Xiangmeng Qu, Dan Zhu, Guangbao Yao, Shao Su, Jie Chao, Huajie Liu, Xiaolei Zuo, Lihua Wang, Jiye Shi, Lianhui Wang, Wei Huang, Hao Pei, Chunhai Fan
DNA-based machines have attracted rapidly growing interest owing to their potential in drug delivery, biocomputing, and diagnostic applications. Herein, we report a type of exonuclease III (Exo III)-powered stochastic DNA walker that can autonomously move on a spherical nucleic acid (SNA)-based 3D track. The motion is propelled by unidirectional Exo III digestion of hybridized DNA tracks in a burnt-bridge mechanism. The operation of this Exo III-propelled DNA walker was monitored in real time and at the single-particle resolution using total internal reflection fluorescence microscopy (TIRF)...
January 12, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28079255/moonlighting-at-replication-forks-a-new-life-for-homologous-recombination-proteins-brca1-brca2-and-rad51
#17
REVIEW
Arun Mouli Kolinjivadi, Vincenzo Sannino, Anna de Antoni, Hervé Técher, Giorgio Baldi, Vincenzo Costanzo
Coordination between DNA replication and DNA repair ensures maintenance of genome integrity, which is lost in cancer cells. Emerging evidence has linked Homologous Recombination (HR) proteins RAD51, BRCA1 and BRCA2 to the stability of nascent DNA. This function appears to be distinct from Double Strand Break (DSB) repair and is in part due to the prevention of MRE11-mediated degradation of nascent DNA at stalled forks. The role of RAD51 in fork protection resembles the activity described for its prokaryotic ortholog RecA, which prevents nuclease-mediated degradation of DNA and promotes replication fork restart in cells challenged by DNA damaging agents...
January 12, 2017: FEBS Letters
https://www.readbyqxmd.com/read/28079019/an-efficient-targeted-nuclease-strategy-for-high-resolution-mapping-of-dna-binding-sites
#18
Peter J Skene, Steven Henikoff
We describe Cleavage Under Targets and Release Using Nuclease (CUT&RUN), a chromatin profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes total chromatin, CUT&RUN is performed in situ, allowing for both quantitative high-resolution chromatin mapping and probing of the local chromatin environment. When applied to yeast and human nuclei, CUT&RUN yielded precise transcription factor profiles while avoiding cross-linking and solubilization issues...
January 12, 2017: ELife
https://www.readbyqxmd.com/read/28073246/a-new-era-of-genome-integration-simply-cut-and-paste
#19
Zihe Liu, Youyun Liang, Ee-Lui Ang, Huimin Zhao
Genome integration is a powerful tool in both basic and applied biological research. However, traditional genome integration, which is typically mediated by homologous recombination, has been constrained by low efficiencies and limited host range. In recent years, the emergence of homing endonucleases and programmable nucleases has greatly enhanced integration efficiencies and allowed alternative integration mechanisms such as non-homologous end joining and microhomology-mediated end joining, enabling integration in hosts deficient in homologous recombination...
January 10, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28070592/therapeutic-genome-editing-with-engineered-nucleases
#20
Simone A Haas, Viviane Dettmer, Toni Cathomen
Targeted genome editing with designer nucleases, such as zinc finger nucleases, TALE nucleases, and CRISPR-Cas nucleases, has heralded a new era in gene therapy. Genetic disorders, which have not been amenable to conventional gene-addition-type gene therapy approaches, such as disorders with dominant inheritance or diseases caused by mutations in tightly regulated genes, can now be treated by precise genome surgery. Moreover, engineered nucleases enable novel genetic interventions to fight infectious diseases or to improve cancer immunotherapies...
January 10, 2017: Hämostaseologie
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