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Svetlana N Tamkovich, Natalia A Kirushina, Vladimir E Voytsitskiy, Vsevolod A Tkachuk, Pavel P Laktionov
Size and termini of cell-free DNA molecules circulating in blood plasma and being bound with blood cell surface of healthy females and untreated breast cancer patients were investigated. The size and concentration of circulating blood DNA were analyzed by Agilent 2100 Bioanalyser (TM) and TaqMan PCR. The termini of circulating DNA were examined by ligation using biotinylated double-stranded oligonucleotide adapters with random 1-3 b overhangs of both chains and subsequent quantification by PCR. Short (180 bp) and longer (>8 kbp) DNA fragments were found in cell free DNA from both groups, but short were less represented in primary breast cancer patient plasma...
2016: Advances in Experimental Medicine and Biology
Bingjie Shi, Juan Li, Xuanling Shi, Wenxu Jia, Yi Wen, Xiongbing Hu, Fengfeng Zhuang, Jianzhong Xi, Linqi Zhang
Transcription activator-like effector nuclease (TALEN) represents a valuable tool for genomic engineering due to its single-nucleotide precision, high nuclease activity and low cytotoxicity. We report here systematic design and characterization of twenty eight novel TALENs targeting multiple regions of CCR5 gene (CCR5-TALEN) which encodes the co-receptor critical for entry of human immunodeficiency virus type I (HIV-1). By systemic characterization of these CCR5-TALENs, we have identified one (CCR5-TALEN-515) with higher nuclease activity, specificity and lower cytotoxicity compared to zinc-finger nuclease (CCR5-ZFN) currently undergoing clinical trials...
October 3, 2016: Journal of Acquired Immune Deficiency Syndromes: JAIDS
Liezhen Fu, Luan Wen, Nga Luu, Yun-Bo Shi
Genome editing with designer nucleases such as TALEN and CRISPR/Cas enzymes has broad applications. Delivery of these designer nucleases into organisms induces various genetic mutations including deletions, insertions and nucleotide substitutions. Characterizing those mutations is critical for evaluating the efficacy and specificity of targeted genome editing. While a number of methods have been developed to identify the mutations, none other than sequencing allows the identification of the most desired mutations, i...
October 17, 2016: Scientific Reports
Bastian Minkenberg, Kabin Xie, Yinong Yang
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system depends on a guide RNA (gRNA) to specify its target. By efficiently co-expressing multiple gRNAs that target different genomic sites, the polycistronic tRNA-gRNA gene (PTG) strategy enables multiplex gene editing in the family of closely related rice mitogen-activated protein kinase (MPK) genes. In this study, we identified MPK1 and MPK6 (Arabidopsis AtMPK6 and AtMPK4 orthologues, respectively) as essential genes for rice development by finding the preservation of MPK functional alleles and normal phenotypes in CRISPR-edited mutants...
October 16, 2016: Plant Journal: for Cell and Molecular Biology
Tetsuya Ishii
Genome editing based on site-directed nucleases facilitated efficient and versatile genetic modifications in human cells. However, recent reports, demonstrating CRISPR/Cas9-mediated genome editing in human embryos have raised profound concerns worldwide. This commentary explores the clinical justification and feasibility of reproductive medicine using germline genome editing. Despite the perceived utility of reproductive medicine for treating intractable infertility, it is difficult to justify germline genome editing from the perspective of the prospective child...
October 5, 2016: Reproductive Biomedicine Online
Yinyin Li, Yi Shen, Philipp Hohensinner, Jihang Ju, Zhenke Wen, Stuart B Goodman, Hui Zhang, Jörg J Goronzy, Cornelia M Weyand
Immune aging manifests with a combination of failing adaptive immunity and insufficiently restrained inflammation. In patients with rheumatoid arthritis (RA), T cell aging occurs prematurely, but the mechanisms involved and their contribution to tissue-destructive inflammation remain unclear. We found that RA CD4(+) T cells showed signs of aging during their primary immune responses and differentiated into tissue-invasive, proinflammatory effector cells. RA T cells had low expression of the double-strand-break repair nuclease MRE11A, leading to telomeric damage, juxtacentromeric heterochromatin unraveling, and senescence marker upregulation...
October 10, 2016: Immunity
Yan Sun, Ping Ji, Tao Chen, Xinhui Zhou, Da Yang, Yuhong Guo, Yuexin Liu, Limei Hu, Dianren Xia, Yanxue Liu, Asha S Multani, Ilya Shmulevich, Raju Kucherlapati, Scott Kopetz, Anil K Sood, Stanley R Hamilton, Baocun Sun, Wei Zhang
The gene encoding Migration and Invasion Inhibitory Protein (MIIP), located on 1p36.22, is a potential tumour suppressor gene in glioma. In this study, we aimed to explore the role and mechanism of action of MIIP in colorectal cancer (CRC). MIIP protein expression gradually decreased along the colorectal adenoma-carcinoma sequence and was negatively correlated with lymph node and distant metastasis in 526 colorectal tissue samples (P<0.05 for all). Analysis of The Cancer Genome Atlas (TCGA) data showed that decreased MIIP expression was significantly associated with MIIP hemizygous deletion (P=0...
October 14, 2016: Journal of Pathology
Ayman Eid, Magdy M Mahfouz
Molecular scissors engineered for site-specific modification of the genome hold great promise for effective functional analyses of genes, genomes and epigenomes and could improve our understanding of the molecular underpinnings of disease states and facilitate novel therapeutic applications. Several platforms for molecular scissors that enable targeted genome engineering have been developed, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and, most recently, clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated-9 (Cas9)...
October 14, 2016: Experimental & Molecular Medicine
Guillermo S Romano Ibarra, Biswajit Paul, Blythe D Sather, Patrick M Younan, Karen Sommer, John P Kowalski, Malika Hale, Barry Stoddard, Jordan Jarjour, Alexander Astrakhan, Hans-Peter Kiem, David J Rawlings
A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4(+) T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4(+) T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis...
August 23, 2016: Molecular Therapy. Nucleic Acids
Jason L J Lin, Chyuan-Chuan Wu, Wei-Zen Yang, Hanna S Yuan
Endonuclease G (EndoG) is an evolutionarily conserved mitochondrial protein in eukaryotes that digests nucleus chromosomal DNA during apoptosis and paternal mitochondrial DNA during embryogenesis. Under oxidative stress, homodimeric EndoG becomes oxidized and converts to monomers with diminished nuclease activity. However, it remains unclear why EndoG has to function as a homodimer in DNA degradation. Here, we report the crystal structure of the Caenorhabditis elegans EndoG homologue, CPS-6, in complex with single-stranded DNA at a resolution of 2...
October 13, 2016: Nucleic Acids Research
(no author information available yet)
Howard Jacob is best known for pioneering genomic sequencing of a patient to solve a mysterious pediatric case in 2010. With roots in pharmacology and cardiovascular disease, however, his career has largely been dedicated to dissecting the physiology and genetics of the rat to help understand complex human diseases. Howard was Director of the Human and Molecular Genetics Center at the Medical College of Wisconsin for 16 years, during which time he applied a combination of approaches, including quantitative genetics, integrative physiology and next-generation sequencing, in rat models to shed light on cardiovascular, metabolic and renal disorders...
October 1, 2016: Disease Models & Mechanisms
Chance M Nowak, Seth Lawson, Megan Zerez, Leonidas Bleris
The Clustered Regularly Interspaced Short Palindromic Repeats system allows a single guide RNA (sgRNA) to direct a protein with combined helicase and nuclease activity to the DNA. Streptococcus pyogenes Cas9 (SpCas9), a CRISPR-associated protein, has revolutionized our ability to probe and edit the human genome in vitro and in vivo Arguably, the true modularity of the Cas9 platform is conferred through the ease of sgRNA programmability as well as the degree of modifications the sgRNA can tolerate without compromising its association with SpCas9 and function...
October 12, 2016: Nucleic Acids Research
Zachary L Demorest, Andrew Coffman, Nicholas J Baltes, Thomas J Stoddard, Benjamin M Clasen, Song Luo, Adam Retterath, Ann Yabandith, Maria Elena Gamo, Jeff Bissen, Luc Mathis, Daniel F Voytas, Feng Zhang
BACKGROUND: The ability to modulate levels of individual fatty acids within soybean oil has potential to increase shelf-life and frying stability and to improve nutritional characteristics. Commodity soybean oil contains high levels of polyunsaturated linoleic and linolenic acid, which contribute to oxidative instability - a problem that has been addressed through partial hydrogenation. However, partial hydrogenation increases levels of trans-fatty acids, which have been associated with cardiovascular disease...
October 13, 2016: BMC Plant Biology
Fillip Port, Simon L Bullock
Reverse genetics-the creation of mutations in preselected target genes-has until recently been a bottleneck in many Drosophila projects. The advent of clustered, regularly interspaced, short palindromic repeat (CRISPR) genome engineering systems has transformed this situation. A short time after the in vitro demonstration of target site cleavage by the RNA-guided endonuclease CRISPR-associated nuclease 9 (Cas9) (Jinek et al., Science 337:816-821, 2012), hundreds of fly researchers are using CRISPR technology to generate loss-of-function mutant alleles in specific genes, as well as to create specific point mutations or tagged protein products...
2016: Methods in Molecular Biology
Huimin Zhang, Yu Hua, Rui Li, Daochun Kong
Double-stranded DNA breaks (DSBs) are highly detrimental DNA lesions, which may be repaired by the homologous recombination-mediated repair pathway. The 5 prime to 3 prime direction of long-range end resection on one DNA strand, in which 3 prime-single-strand DNA overhangs are created from broken DNA ends, is an essential step in this pathway. Dna2 has been demonstrated as an essential nuclease in this event, but the molecular mechanism how Dna2 is recruited to DNA break sites in vivo is not elucidated. In this study, a novel recombination factor called Cdc24 was identified in fission yeast...
October 11, 2016: Journal of Biological Chemistry
František Liška, Renata Peterková, Miroslav Peterka, Vladimír Landa, Václav Zídek, Petr Mlejnek, Jan Šilhavý, Miroslava Šimáková, Vladimír Křen, Colby G Starker, Daniel F Voytas, Zsuzsanna Izsvák, Michal Pravenec
Recently, it has been found that spontaneous mutation Lx (polydactyly-luxate syndrome) in the rat is determined by deletion of a conserved intronic sequence of the Plzf (Promyelocytic leukemia zinc finger protein) gene. In addition, Plzf is a prominent candidate gene for quantitative trait loci (QTLs) associated with cardiac hypertrophy and fibrosis in the spontaneously hypertensive rat (SHR). In the current study, we tested the effects of Plzf gene targeting in the SHR using TALENs (transcription activator-like effector nucleases)...
2016: PloS One
Donna J Palmer, Nathan C Grove, Jordan Ing, Ana M Crane, Koen Venken, Brian R Davis, Philip Ng
Helper-dependent adenoviral vectors mediate high efficiency gene editing in induced pluripotent stem cells without needing a designer nuclease thereby avoiding off-target cleavage. Because of their large cloning capacity of 37 kb, helper-dependent adenoviral vectors with long homology arms are used for gene editing. However, this makes vector construction and recombinant analysis difficult. Conversely, insufficient homology may compromise targeting efficiency. Thus, we investigated the effect of homology length on helper-dependent adenoviral vector targeting efficiency at the cystic fibrosis transmembrane conductance regulator locus in induced pluripotent stem cells and found a positive correlation...
October 11, 2016: Molecular Therapy. Nucleic Acids
Julia Hilscher, Hermann Bürstmayr, Eva Stoger
The development of gene targeting and gene editing techniques based on programmable site-directed nucleases (SDNs) has increased the precision of genome modification and made the outcomes more predictable and controllable. These approaches have achieved rapid advances in plant biotechnology, particularly the development of improved crop varieties. Here, we review the range of alterations which have already been implemented in plant genomes, and summarize the reported efficiencies of precise genome modification...
October 11, 2016: Biotechnology Journal
Yang Yang, Jian-Ge Qiu, Yong Li, Jin-Ming Di, Wen-Ji Zhang, Qi-Wei Jiang, Di-Wei Zheng, Yao Chen, Meng-Ning Wei, Jia-Rong Huang, Kun Wang, Zhi Shi
The RNA-guided clustered regularly interspaced short palindromic (CRISPR) in combination with a CRISPR-associated nuclease 9 (Cas9) nuclease system is a new rapid and precise technology for genome editing. In the present study, we applied the CRISPR/Cas9 system to target ABCB1 (also named MDR1) gene which encodes a 170 kDa transmembrane glycoprotein (P-glycoprotein/P-gp) transporting multiple types of chemotherapeutic drugs including taxanes, epipodophyllotoxins, vinca alkaloids and anthracyclines out of cells to contribute multidrug resistance (MDR) in cancer cells...
2016: American Journal of Translational Research
Satoshi Ota, Kiyohito Taimatsu, Kanoko Yanagi, Tomohiro Namiki, Rie Ohga, Shin-Ichi Higashijima, Atsuo Kawahara
The CRISPR/Cas9 complex, which is composed of a guide RNA (gRNA) and the Cas9 nuclease, is useful for carrying out genome modifications in various organisms. Recently, the CRISPR/Cas9-mediated locus-specific integration of a reporter, which contains the Mbait sequence targeted using Mbait-gRNA, the hsp70 promoter and the eGFP gene, has allowed the visualization of the target gene expression. However, it has not been ascertained whether the reporter integrations at both targeted alleles cause loss-of-function phenotypes in zebrafish...
October 11, 2016: Scientific Reports
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