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clustered regularly interspaced short palindromic repeats

Romain Da Costa, Carsten Röger, Jasmin Segelken, Maya Barben, Christian Grimm, John Neidhardt
Purpose: Gene therapies to treat eye disorders have been extensively studied in the past 20 years. Frequently, adeno-associated viruses were applied to the subretinal or intravitreal space of the eye to transduce retinal cells with nucleotide sequences of therapeutic potential. In this study we describe a novel intravitreal injection procedure that leads to a reproducible adeno-associated virus (AAV)2/8-mediated transduction of more than 70% of the retina. Methods: Prior to a single intravitreal injection of a enhanced green fluorescent protien (GFP)-expressing viral suspension, we performed an aspiration of vitreous tissue from wild-type C57Bl/6J mice...
October 1, 2016: Investigative Ophthalmology & Visual Science
Jihye Chung, Shunsuke Aburaya, Wataru Aoki, Mitsuyoshi Ueda
In very early stages of cancer development, one or a few cells expressing cancer-associated genes appear among a much larger number of surrounding normal cells. To analyze the molecular changes induced by this co-existence, we artificially prepared transformed cells with complete loss of tumor suppressor gene, SCRIB, among normal human embryonic kidney (HEK293T) cells. A cell strain with SCRIB-knockout was successfully constructed by using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nuclease system and co-cultured with normal cells...
October 21, 2016: Journal of Bioscience and Bioengineering
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
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
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
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
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
Yunqing Ma, Jiayuan Zhang, Weijie Yin, Zhenchao Zhang, Yan Song, Xing Chang
A large number of genetic variants have been associated with human diseases. However, the lack of a genetic diversification approach has impeded our ability to interrogate functions of genetic variants in mammalian cells. Current screening methods can only be used to disrupt a gene or alter its expression. Here we report the fusion of activation-induced cytidine deaminase (AID) with nuclease-inactive clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCas9) for efficient genetic diversification, which enabled high-throughput screening of functional variants...
October 10, 2016: Nature Methods
Xiangna Zhao, Mikael Skurnik
Bacteriophage play many varied roles in microbial ecology and evolution. This chapter collates a vast body of knowledge and expertise on Yersinia pestis phages, including the history of their isolation and classical methods for their isolation and identification. The genomic diversity of Y. pestis phage and bacteriophage islands in the Y. pestis genome are also discussed because all phage research represents a branch of genetics. In addition, our knowledge of the receptors that are recognized by Y. pestis phage, advances in phage therapy for Y...
2016: Advances in Experimental Medicine and Biology
Zhizhen Qi, Yujun Cui, Qingwen Zhang, Ruifu Yang
This chapter summarized the taxonomy and typing works of Yersinia pestis since it's firstly identified in Hong Kong in 1894. Phenotyping methods that based on phenotypic characteristics, including biotyping, serotyping, antibiogram analysis, bacteriocin typing, phage typing, and plasmid typing, were firstly applied in classification of Y. pestis in subspecies level. And then, with the advancement of molecular biological technology, the methods based on outer membrane protein profiles, fatty acid composition, and bacterial mass fingerprinting were also used to identify the populations within Y...
2016: Advances in Experimental Medicine and Biology
Fangkun Liu, Jing Huang, Bo Ning, Zhixiong Liu, Shen Chen, Wei Zhao
Patient-derived cell lines and animal models have proven invaluable for the understanding of human intestinal diseases and for drug development although both inherently comprise disadvantages and caveats. Many genetically determined intestinal diseases occur in specific tissue microenvironments that are not adequately modeled by monolayer cell culture. Likewise, animal models incompletely recapitulate the complex pathologies of intestinal diseases of humans and fall short in predicting the effects of candidate drugs...
2016: Frontiers in Pharmacology
Bang Shen, Kevin Brown, Shaojun Long, L David Sibley
Efficient and site-specific alteration of the genome is key to decoding and altering the genomic information of an organism. Over the last couple of years, the RNA-guided Cas9 nucleases derived from the prokaryotic type 2 CRISPR (clustered regularly interspaced short palindromic repeats) systems have drastically improved our ability to engineer the genomes of a variety of organisms including Toxoplasma gondii. In this chapter, we describe detailed protocols for using the CRISPR/Cas9 system adapted from Streptococcus pyogenes to perform efficient genetic manipulations in T...
2017: Methods in Molecular Biology
Rongfang Xu, Pengcheng Wei, Jianbo Yang
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system is a newly emerging mutagenesis (gene-editing) tool in genetic engineering. Among the agriculturally important crops, several genes have been successfully mutated by the system, and some agronomic important traits have been rapidly generated, which indicates the potential applications in both scientific research and plant breeding. In this chapter, we describe a standard gene-editing procedure to effectively target rice genes and to make specific rice mutants using the CRISPR/Cas9 system mediated by Agrobacterium transformation...
2017: Methods in Molecular Biology
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