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Araceli Oropeza-Aburto, Alfredo Cruz-Ramírez, Javier Mora-Macías, Luis Herrera-Estrella
Phosphate (Pi) deficient soils are a major limitant factor for crop production in many regions in the world. Despite that plants have innovated several developmental and biochemical strategies to deal with this stress, there are still massive extensions of land which combine several abiotic stresses, including phosphate starvation, that limit their use for plant growth and food production. In several plant species a genetic program underlies the biochemical and developmental responses of the organism to cope with low phosphate (Pi) availability...
October 24, 2016: Plant Biotechnology Journal
Fang Chen, Weifeng Zhang, Junli Zhao, Peiyan Yang, Rui Ma, Haibin Xia
Objective To prepare Rev-erbβ knockout HEK293 cells using clustered regularly interspaced short palindromic repeats/Cas 9 nuclease (CRISPR/Cas9) gene editing technology. Methods The knock-in or knockout of Rev-erbβ gene could be realized by single-guide RNA (sgRNA)-mediated Cas9 cutting of target DNA, and followed by DNA homologous recombination or non-homologous end joining-mediated DNA repair. Firstly, four sgRNAs were designed for Rev-erbβ gene. The sgRNA1 and sgRNA2 with the higher activity were respectively used to construct pCMV-hCas9-U6-Rev-erbβ sgRNA1 and pCMV-hCas9-U6-Rev-erbβ sgRNA2...
November 2016: Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology
Julieta Bonacina, Nadia Suárez, Ricardo Hormigo, Silvina Fadda, Marcus Lechner, Lucila Saavedra
The study of enterococcal genomes has grown considerably in recent years. While special attention is paid to comparative genomic analysis among clinical relevant isolates, in this study we performed an exhaustive comparative analysis of enterococcal genomes of food origin and/or with potential to be used as probiotics. Beyond common genetic features, we especially aimed to identify those that are specific to enterococcal strains isolated from a certain food-related source as well as features present in a species-specific manner...
October 23, 2016: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes
Conny Turni, Denise Dayao, Gorka Aduriz, Nekane Cortabarria, Carolina Tejero, Jose C Ibabe, Reema Singh, Pat Blackall
Pasteurella multocida isolates from dairy cattle on a farm in Spain were associated with pneumonia of calves (six isolates) and mastitis of heifers (five isolates). The objective was to determine if the P. multocida isolates retrieved from both disease scenarios were the same strain or whether more than one strain was present. The isolates were identified by a species-specific polymerase chain (PCR) assay, serotyped by the Heddleston scheme and then typed by a number of molecular genotyping assays including multi-locus sequence typing (MLST)...
November 15, 2016: Veterinary Microbiology
Aura Falco, Yusibeska Ramos, Esther Franco, Alegría Guzmán, Howard Takiff
BACKGROUND: Klebsiella pneumoniae is a bacterial pathogen that has developed resistance to multiple antibiotics and is a major cause of nosocomial infections worldwide. Carbapenemase-producing Klebsiella pneumoniae have been isolated in many hospitals in Venezuela, but they have not been well-studied. The aim of this study was to characterize carbapenem-resistant Klebsiella pneumoniae isolates from the pediatric service of a hospital located in Anzoategui State, in the eastern part of Venezuela...
October 22, 2016: BMC Infectious Diseases
Johan Kreuger, Paul O'Callaghan
Here we report on a technical difficulty we encountered while optimizing genotyping strategies to identify mice derived from Exoc3l2tm1a(KOMP)Wtsi embryonic stem cells obtained from the Knockout Mouse Project Repository. The Exoc3l2tm1a(KOMP)Wtsi construct encodes a "knockout-first" design with loxP sites that confer conditional potential (KO1st). We designed primers that targeted wild-type sequences flanking the most downstream element of the construct, an 80 base pair synthetic loxP region, which BLAST alignment analysis reveals is an element common to over 10,000 conditional gene-targeting mouse models...
2016: PloS One
Glenn Yiu, Eric Tieu, Anthony T Nguyen, Brittany Wong, Zeljka Smit-McBride
Purpose: To employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells. Methods: CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcuspyogenes Cas9 endonuclease (SpCas9) gene...
October 1, 2016: Investigative Ophthalmology & Visual Science
Walter H Moos, Carl A Pinkert, Michael H Irwin, Douglas V Faller, Krishna Kodukula, Ioannis P Glavas, Kosta Steliou
Preclinical Research Approximately 2,500 years ago, Hippocrates used the word herpes as a medical term to describe lesions that appeared to creep or crawl on the skin, advocating heat as a possible treatment. During the last 50 years, pharmaceutical research has made great strides, and therapeutic options have expanded to include small molecule antiviral agents, protease inhibitors, preventive vaccines for a handful of the papillomaviruses, and even cures for hepatitis C virus infections. However, effective treatments for persistent and recurrent viral infections, particularly the highly prevalent herpesviruses, continue to represent a significant unmet medical need, affecting the majority of the world's population...
October 20, 2016: Drug Development Research
Konstantinos Tzelepis, Hiroko Koike-Yusa, Etienne De Braekeleer, Yilong Li, Emmanouil Metzakopian, Oliver M Dovey, Annalisa Mupo, Vera Grinkevich, Meng Li, Milena Mazan, Malgorzata Gozdecka, Shuhei Ohnishi, Jonathan Cooper, Miten Patel, Thomas McKerrell, Bin Chen, Ana Filipa Domingues, Paolo Gallipoli, Sarah Teichmann, Hannes Ponstingl, Ultan McDermott, Julio Saez-Rodriguez, Brian J P Huntly, Francesco Iorio, Cristina Pina, George S Vassiliou, Kosuke Yusa
Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which mainstream treatments have not changed for decades. To identify additional therapeutic targets in AML, we optimize a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening platform and use it to identify genetic vulnerabilities in AML cells. We identify 492 AML-specific cell-essential genes, including several established therapeutic targets such as DOT1L, BCL2, and MEN1, and many other genes including clinically actionable candidates...
October 18, 2016: Cell 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
Zhong Qian, Sankar Adhya
Although discovered decades ago, the molecular identification, the diversity and versatility of functions, and the evolutionary origin of repeat DNA sequences (REPs) containing palindromic units in prokaryotes are now bringing attention to a wide range of biological scientists. A brief account of the current state of the repeat DNA sequences is presented here.
October 14, 2016: Current Genetics
Tsuyoshi Momose, Jean-Paul Concordet
More and more genomes are sequenced and a great range of biological questions can be examined at the genomic level in a growing number of organisms. Testing the function of genome features, from gene networks, genome organization, conserved non-coding sequences to microRNAs, and, more generally, experimentally addressing the genotype-phenotype relationship is now possible owing to the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 revolution of genome editing. In the present review, we give a brief overview of the CRISPR/Cas9 toolbox and different strategies for genome editing currently available...
October 11, 2016: Marine Genomics
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
Hasan Awad Aljohi, Wanfei Liu, Qiang Lin, Yuhui Zhao, Jingyao Zeng, Ali Alamer, Ibrahim O Alanazi, Abdullah O Alawad, Abdullah M Al-Sadi, Songnian Hu, Jun Yu
Coconut (Cocos nucifera L.), a member of the palm family (Arecaceae), is one of the most economically important crops in tropics, serving as an important source of food, drink, fuel, medicine, and construction material. Here we report an assembly of the coconut (C. nucifera, Oman local Tall cultivar) mitochondrial (mt) genome based on next-generation sequencing data. This genome, 678,653bp in length and 45.5% in GC content, encodes 72 proteins, 9 pseudogenes, 23 tRNAs, and 3 ribosomal RNAs. Within the assembly, we find that the chloroplast (cp) derived regions account for 5...
2016: PloS One
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
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
Van Trung Chu, Robin Graf, Tristan Wirtz, Timm Weber, Jeremy Favret, Xun Li, Kerstin Petsch, Ngoc Tung Tran, Michael H Sieweke, Claudia Berek, Ralf Kühn, Klaus Rajewsky
Applying clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis to primary mouse immune cells, we used high-fidelity single guide RNAs (sgRNAs) designed with an sgRNA design tool (CrispRGold) to target genes in primary B cells, T cells, and macrophages isolated from a Cas9 transgenic mouse line. Using this system, we achieved an average knockout efficiency of 80% in B cells. On this basis, we established a robust small-scale CRISPR-mediated screen in these cells and identified genes essential for B-cell activation and plasma cell differentiation...
October 11, 2016: Proceedings of the National Academy of Sciences of the United States of America
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
Muhammad Abu Bakr Shabbir, Haihong Hao, Muhammad Zubair Shabbir, Hafiz Iftikhar Hussain, Zahid Iqbal, Saeed Ahmed, Adeel Sattar, Mujahid Iqbal, Jun Li, Zonghui Yuan
Prokaryotes have developed numerous innate immune mechanisms in order to fend off bacteriophage or plasmid attack. One of these immune systems is clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR-associated proteins play a key role in survival of prokaryotes against invaders, as these systems cleave DNA of foreign genetic elements. Beyond providing immunity, these systems have significant impact in altering the bacterial physiology in term of its virulence and pathogenicity, as well as evolution...
2016: Frontiers in Immunology
Hanchao Gao, Chengjiang Zhao, Xi Xiang, Yong Li, Yanli Zhao, Zesong Li, Dengke Pan, Yifan Dai, Hidetaka Hara, David K C Cooper, Zhiming Cai, Lisha Mou
Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs...
October 8, 2016: Journal of Reproduction and Development
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