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https://www.readbyqxmd.com/read/28179526/a-sequence-independent-unstructured-ires-is-responsible-for-internal-expression-of-the-coat-protein-of-turnip-crinkle-virus
#1
Jared May, Philip Johnson, Huma Saleem, Anne E Simon
To maximize the coding potential of viral genomes, internal ribosome entry sites (IRES) can be used to bypass the traditional requirement of a 5' cap and some/all of the associated translation initiation factors. Although viral IRES typically contain higher order RNA structure, an unstructured sequence of about 84-nt immediately upstream of the Turnip crinkle virus (TCV) coat protein (CP) ORF has been found to promote internal expression of the CP from the genomic (g)RNA both in vitro and in vivo Absence of extensive RNA structure was predicted using RNA folding algorithms and confirmed by SHAPE structure probing...
February 8, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28177825/enhancement-of-single-guide-rna-transcription-for-efficient-crispr-cas-based-genomic-engineering
#2
Kumiko Ui-Tei, Shohei Maruyama, Yuko Nakano
Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein...
January 26, 2017: Genome Génome / Conseil National de Recherches Canada
https://www.readbyqxmd.com/read/28176813/a-combinational-crispr-cas9-gene-editing-approach-can-halt-hiv-replication-and-prevent-viral-escape
#3
Robert Jan Lebbink, Dorien C M de Jong, Femke Wolters, Elisabeth M Kruse, Petra M van Ham, Emmanuel J H J Wiertz, Monique Nijhuis
HIV presents one of the highest evolutionary rates ever detected and combination antiretroviral therapy is needed to overcome the plasticity of the virus population and control viral replication. Conventional treatments lack the ability to clear the latent reservoir, which remains the major obstacle towards a cure. Novel strategies, such as CRISPR/Cas9 gRNA-based genome-editing, can permanently disrupt the HIV genome. However, HIV genome-editing may accelerate viral escape, questioning the feasibility of the approach...
February 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28124028/optimized-crispr-cas9-genome-editing-for-leishmania-and-its-use-to-target-a-multigene-family-induce-chromosomal-translocation-and-study-dna-break-repair-mechanisms
#4
Wen-Wei Zhang, Patrick Lypaczewski, Greg Matlashewski
CRISPR-Cas9-mediated genome editing has recently been adapted for Leishmania spp. parasites, the causative agents of human leishmaniasis. We have optimized this genome-editing tool by selecting for cells with CRISPR-Cas9 activity through cotargeting the miltefosine transporter gene; mutation of this gene leads to miltefosine resistance. This cotargeting strategy integrated into a triple guide RNA (gRNA) expression vector was used to delete all 11 copies of the A2 multigene family; this was not previously possible with the traditional gene-targeting method...
January 2017: MSphere
https://www.readbyqxmd.com/read/28123598/lentiviral-crispr-cas9-vector-mediated-mir-21-gene-editing-inhibits-the-epithelial-to-mesenchymal-transition-in-ovarian-cancer-cells
#5
Wenying Huo, Guannan Zhao, Jinggang Yin, Xuan Ouyang, Yinan Wang, Chuanhe Yang, Baojing Wang, Peixin Dong, Zhixiang Wang, Hidemichi Watari, Edward Chaum, Lawrence M Pfeffer, Junming Yue
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) mediated genome editing is a powerful approach for loss of function studies. Here we report that lentiviral CRISPR/Cas9 vectors are highly efficient in introducing mutations in the precursor miRNA sequence, thus leading to the loss of miRNA expression and function. We constructed four different lentiviral CRISPR/Cas9 vectors that target different regions of the precursor miR-21 sequence and found that these lentiviral CRISPR/Cas9 miR-21 gRNA vectors induced mutations in the precursor sequences as shown by DNA surveyor mutation assay and Sanger sequencing...
2017: Journal of Cancer
https://www.readbyqxmd.com/read/28119062/baculovirus-based-genome-editing-in-primary-cells
#6
Maysam Mansouri, Zahra Ehsaei, Verdon Taylor, Philipp Berger
Genome editing in eukaryotes became easier in the last years with the development of nucleases that induce double strand breaks in DNA at user-defined sites. CRISPR/Cas9-based genome editing is currently one of the most powerful strategies. In the easiest case, a nuclease (e.g. Cas9) and a target defining guide RNA (gRNA) are transferred into a target cell. Non-homologous end joining (NHEJ) repair of the DNA break following Cas9 cleavage can lead to inactivation of the target gene. Specific repair or insertion of DNA with Homology Directed Repair (HDR) needs the simultaneous delivery of a repair template...
January 22, 2017: Plasmid
https://www.readbyqxmd.com/read/28116670/an-inducible-crispr-on-system-for-controllable-gene-activation-in-human-pluripotent-stem-cells
#7
Jianying Guo, Dacheng Ma, Rujin Huang, Jia Ming, Min Ye, Kehkooi Kee, Zhen Xie, Jie Na
Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line...
January 23, 2017: Protein & Cell
https://www.readbyqxmd.com/read/28106276/crispr-cas9-mediated-deletion-of-c1eis-inhibits-chicken-embryonic-stem-cell-differentiation-into-male-germ-cells-gallus-gallus
#8
Qisheng Zuo, Kai Jin, Yingjie Wang, Jiuzhou Song, Yani Zhang, Bichun Li
We previously found that C1EIS is preferentially expressed in Chicken spermatogonial stem cells (SSCs) by RNA sequencing (RNA-seq), so our current study focused on C1EIS's role in Chicken embryonic stem cells (ESCs) differentiation into male germ cells. We constructed a CRISPR/Cas9 vector targeting C1EIS. T7 endonuclease I (T7EI) digestion method and sequencing of TA cloning were used to detect the knock-out efficiency of the Single guide RNA (sgRNA) after the cas9/gRNA vector transfected into D fibroblasts 1(DF-1), ESCs and Chicken embryos...
January 20, 2017: Journal of Cellular Biochemistry
https://www.readbyqxmd.com/read/28062688/piggybac-mediates-efficient-in-vivo-crispr-library-screening-for-tumorigenesis-in-mice
#9
Chunlong Xu, Xiaolan Qi, Xuguang Du, Huiying Zou, Fei Gao, Tao Feng, Hengxing Lu, Shenglan Li, Xiaomeng An, Lijun Zhang, Yuanyuan Wu, Ying Liu, Ning Li, Mario R Capecchi, Sen Wu
CRISPR/Cas9 is becoming an increasingly important tool to functionally annotate genomes. However, because genome-wide CRISPR libraries are mostly constructed in lentiviral vectors, in vivo applications are severely limited as a result of difficulties in delivery. Here, we examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Although tumor induction has previously been achieved in mice by targeting cancer genes with the CRISPR/Cas9 system, in vivo genome-scale screening has not been reported...
January 24, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28053097/subcellular-localization-of-hiv-1-gag-pol-mrnas-regulates-sites-of-virion-assembly
#10
Jordan T Becker, Nathan M Sherer
: HIV-1 full-length, unspliced RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells...
January 4, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28034301/mechanistic-differences-between-hiv-1-and-siv-nucleocapsid-proteins-and-cross-species-hiv-1-genomic-rna-recognition
#11
Klara Post, Erik D Olson, M Nabuan Naufer, Robert J Gorelick, Ioulia Rouzina, Mark C Williams, Karin Musier-Forsyth, Judith G Levin
BACKGROUND: The nucleocapsid (NC) domain of HIV-1 Gag is responsible for specific recognition and packaging of genomic RNA (gRNA) into new viral particles. This occurs through specific interactions between the Gag NC domain and the Psi packaging signal in gRNA. In addition to this critical function, NC proteins are also nucleic acid (NA) chaperone proteins that facilitate NA rearrangements during reverse transcription. Although the interaction with Psi and chaperone activity of HIV-1 NC have been well characterized in vitro, little is known about simian immunodeficiency virus (SIV) NC...
December 29, 2016: Retrovirology
https://www.readbyqxmd.com/read/28030843/targeted-delivery-of-crispr-cas9-to-prostate-cancer-by-modified-grna-using-a-flexible-aptamer-cationic-liposome
#12
Shuai Zhen, Y Takahashi, S Narita, Yi-Chen Yang, Xu Li
The potent ability of CRISPR/Cas9 system to inhibit the expression of targeted gene is being exploited as a new class of therapeutics for a variety of diseases. However, the efficient and safe delivery of CRISPR/Cas9 into specific cell populations is still the principal challenge in the clinical development of CRISPR/Cas9 therapeutics. In this study, a flexible aptamer-liposome-CRISPR/Cas9 chimera was designed to combine efficient delivery and increased flexibility. Our chimera incorporated an RNA aptamer that specifically binds prostate cancer cells expressing the prostate-specific membrane antigen as a ligand...
December 21, 2016: Oncotarget
https://www.readbyqxmd.com/read/28011935/ttll12-inhibits-the-activation-of-cellular-antiviral-signaling-through-interaction-with-visa-mavs
#13
Lin-Gao Ju, Yuan Zhu, Pin-Ji Lei, Dong Yan, Kun Zhu, Xiang Wang, Qing-Lan Li, Xue-Jing Li, Jian-Wen Chen, Lian-Yun Li, Min Wu
Upon virus infection, host cells use retinoic-acid-inducible geneI I (RIG-I)-like receptors to recognize viral RNA and activate type I IFN expression. To investigate the role of protein methylation in the antiviral signaling pathway, we screened all the SET domain-containing proteins and identified TTLL12 as a negative regulator of RIG-I signaling. TTLL12 contains SET and TTL domains, which are predicted to have lysine methyltransferase and tubulin tyrosine ligase activities, respectively. Exogenous expression of TTLL12 represses IFN-β expression induced by Sendai virus...
December 23, 2016: Journal of Immunology: Official Journal of the American Association of Immunologists
https://www.readbyqxmd.com/read/28009832/nmr-studies-of-the-structure-and-function-of-the-hiv-1-5-leader
#14
REVIEW
Sarah C Keane, Michael F Summers
The 5'-leader of the human immunodeficiency virus type 1 (HIV-1) genome plays several critical roles during viral replication, including differentially establishing mRNA versus genomic RNA (gRNA) fates. As observed for proteins, the function of the RNA is tightly regulated by its structure, and a common paradigm has been that genome function is temporally modulated by structural changes in the 5'-leader. Over the past 30 years, combinations of nucleotide reactivity mapping experiments with biochemistry, mutagenesis, and phylogenetic studies have provided clues regarding the secondary structures of stretches of residues within the leader that adopt functionally discrete domains...
December 21, 2016: Viruses
https://www.readbyqxmd.com/read/27983963/simultaneous-paralogue-knockout-using-a-crispr-concatemer-in-mouse-small-intestinal-organoids
#15
Amanda Andersson-Rolf, Alessandra Merenda, Roxana C Mustata, Taibo Li, Sabine Dietmann, Bon-Kyoung Koo
Approaches based on genetic modification have been invaluable for investigating a wide array of biological processes, with gain- and loss-of-function approaches frequently used to investigate gene function. However, the presence of paralogues, and hence possible genetic compensation, for many genes necessitates the knockout (KO) of all paralogous genes in order to observe clear phenotypic change. CRISPR technology, the most recently described tool for gene editing, can generate KOs with unprecedented ease and speed and has been used in adult stem cell-derived organoids for single gene knockout, gene knock-in and gene correction...
October 27, 2016: Developmental Biology
https://www.readbyqxmd.com/read/27974196/a-combinatorial-crispr-cas9-attack-on-hiv-1-dna-extinguishes-all-infectious-provirus-in-infected-t-cell-cultures
#16
Gang Wang, Na Zhao, Ben Berkhout, Atze T Das
Current drug therapies effectively suppress HIV-1 replication but do not inactivate the provirus that persists in latent reservoirs. Recent studies have found that the guide RNA (gRNA)-directed CRISPR/Cas9 system can be used for sequence-specific attack on this proviral DNA. Although potent inhibition of virus replication was reported, HIV-1 can escape from a single antiviral gRNA by mutation of the target sequence. Here, we demonstrate that combinations of two antiviral gRNAs delay viral escape, and identify two gRNA combinations that durably block virus replication...
December 13, 2016: Cell Reports
https://www.readbyqxmd.com/read/27936040/a-high-throughput-strategy-for-dissecting-mammalian-genetic-interactions
#17
Victoria B Stockman, Lila Ghamsari, Gorka Lasso, Barry Honig, Sagi D Shapira, Harris H Wang
Comprehensive delineation of complex cellular networks requires high-throughput interrogation of genetic interactions. To address this challenge, we describe the development of a multiplex combinatorial strategy to assess pairwise genetic interactions using CRISPR-Cas9 genome editing and next-generation sequencing. We characterize the performance of combinatorial genome editing and analysis using different promoter and gRNA designs and identified regions of the chimeric RNA that are compatible with next-generation sequencing preparation and quantification...
2016: PloS One
https://www.readbyqxmd.com/read/27929521/efficient-footprint-free-human-ipsc-genome-editing-by-consolidation-of-cas9-crispr-and-piggybac-technologies
#18
Gang Wang, Luhan Yang, Dennis Grishin, Xavier Rios, Lillian Y Ye, Yong Hu, Kai Li, Donghui Zhang, George M Church, William T Pu
Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations...
January 2017: Nature Protocols
https://www.readbyqxmd.com/read/27905467/direct-identification-of-antibiotic-resistance-genes-on-single-plasmid-molecules-using-crispr-cas9-in-combination-with-optical-dna-mapping
#19
Vilhelm Müller, Fredrika Rajer, Karolin Frykholm, Lena K Nyberg, Saair Quaderi, Joachim Fritzsche, Erik Kristiansson, Tobias Ambjörnsson, Linus Sandegren, Fredrik Westerlund
Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene...
December 1, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27890617/polycistronic-trna-and-crispr-guide-rna-enables-highly-efficient-multiplexed-genome-engineering-in-human-cells
#20
Fengping Dong, Kabin Xie, Yueying Chen, Yinong Yang, Yingwei Mao
CRISPR/Cas9 has been widely used for genomic editing in many organisms. Many human diseases are caused by multiple mutations. The CRISPR/Cas9 system provides a potential tool to introduce multiple mutations in a genome. To mimic complicated genomic variants in human diseases, such as multiple gene deletions or mutations, two or more small guide RNAs (sgRNAs) need to be introduced all together. This can be achieved by separate Pol III promoters in a construct. However, limited enzyme sites and increased insertion size lower the efficiency to make a construct...
January 22, 2017: Biochemical and Biophysical Research Communications
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