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https://www.readbyqxmd.com/read/29154869/evaluating-different-dna-binding-domains-to-modulate-l1-orf2p-driven-site-specific-retrotransposition-events-in-human-cells
#1
Catherine M Ade, Rebecca S Derbes, Bradley J Wagstaff, Sara B Linker, Travis B White, Dawn Deharo, Victoria P Belancio, Zoltán Ivics, Astrid M Roy-Engel
DNA binding domains (DBDs) have been used with great success to impart targeting capabilities to a variety of proteins creating highly useful genomic tools. We evaluated the ability of five types of DBDs and strategies (AAV Rep proteins, Cre, TAL effectors, zinc finger proteins, and Cas9/gRNA system) to target the L1 ORF2 protein to drive retrotransposition of Alu inserts to specific sequences in the human genome. First, we find that the L1 ORF2 protein tolerates the addition of protein domains both at the amino- and carboxy-terminus...
November 14, 2017: Gene
https://www.readbyqxmd.com/read/29150011/optimization-of-crispr-cas9-genome-editing-for-loss-of-function-in-the-early-chick-embryo
#2
Shashank Gandhi, Michael L Piacentino, Felipe M Vieceli, Marianne E Bronner
The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions...
December 1, 2017: Developmental Biology
https://www.readbyqxmd.com/read/29150004/dynamics-of-indel-profiles-induced-by-various-crispr-cas9-delivery-methods
#3
Michael Kosicki, Sandeep S Rajan, Flaminia C Lorenzetti, Hans H Wandall, Yoshiki Narimatsu, Emmanouil Metzakopian, Eric P Bennett
The introduction of CRISPR/Cas9 gene editing in mammalian cells is a scientific breakthrough, which has greatly affected basic research and gene therapy. The simplicity and general access to CRISPR/Cas9 reagents has in an unprecedented manner "democratized" gene targeting in biomedical research, enabling genetic engineering of any gene in any cell, tissue, organ, and organism. The ability for fast, precise, and efficient profiling of the double-stranded break induced insertions and deletions (indels), mediated by any of the available programmable nucleases, is paramount to any given gene targeting approach...
2017: Progress in Molecular Biology and Translational Science
https://www.readbyqxmd.com/read/29145843/development-of-a-crispr-cas9-genome-editing-toolbox-for-corynebacterium-glutamicum
#4
Jiao Liu, Yu Wang, Yujiao Lu, Ping Zheng, Jibin Sun, Yanhe Ma
BACKGROUND: Corynebacterium glutamicum is an important industrial workhorse and advanced genetic engineering tools are urgently demanded. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) have revolutionized the field of genome engineering. The CRISPR/Cas9 system that utilizes NGG as protospacer adjacent motif (PAM) and has good targeting specificity can be developed into a powerful tool for efficient and precise genome editing of C...
November 16, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/29143989/nanos2-promotes-differentiation-of-chicken-gallus-gallus-embryonic-stem-cells-to-male-germ-cells
#5
Zhang Wenhui, Bi Yulin, Wang Yingjie, Li Dong, He Nana, Wang Man, Jin Jing, Zuo Qisheng, Zhang Yani, Li Bichun
Nanos2 is an evolutionarily conserved RNA-binding protein containing 2 CCHC-type zinc finger motives. Here, we report that Nanos2 is strongly expressed in the testis compared to other tissues in chicken (Gallus gallus). Overexpression and knockout plasmid vectors were constructed, and in-vitro Cas9/gRNA digestion and T7 endonuclease I (T7E1) assay indicated that Nanos2-g1 possessed the highest knockout activity. In vitro and in vivo, Nanos2 overexpression accelerated the production of embryoid bodies (EBs) and SSC-like cells and promoted cvh, c-kit and integrin α6 expression...
November 16, 2017: Journal of Cellular Biochemistry
https://www.readbyqxmd.com/read/29140168/revisiting-the-roles-of-tobamovirus-replicase-complex-proteins-in-viral-replication-and-silencing-suppression
#6
Nachelli Malpica-López, Rajendran Rajeswaran, Daria Beknazariants, Jonathan Seguin, Victor Golyaev, Laurent Farinelli, Mikhail M Pooggin
Tobamoviral replicase possesses an RNA-dependent RNA polymerase (RDR) domain and is translated from genomic (g)RNA via a stop codon readthrough mechanism at a one-to-ten ratio relative to a shorter protein lacking the RDR domain. The two proteins share methyltransferase and helicase domains and form a heterodimer implicated in gRNA replication. The shorter protein is also implicated in suppressing RNA silencing-based antiviral defenses. Using a stop codon mutant of Oilseed rape mosaic tobamovirus (ORMV), we demonstrate that the readthrough replicase (p182) is sufficient for gRNA replication and for subgenomic RNA transcription during systemic infection in Nicotiana benthamiana and Arabidopsis thaliana...
November 15, 2017: Molecular Plant-microbe Interactions: MPMI
https://www.readbyqxmd.com/read/29133816/disruption-of-diphthamide-synthesis-genes-and-resulting-toxin-resistance-as-a-robust-technology-for-quantifying-and-optimizing-crispr-cas9-mediated-gene-editing
#7
Tobias Killian, Steffen Dickopf, Alexander K Haas, Claudia Kirstenpfad, Klaus Mayer, Ulrich Brinkmann
We have devised an effective and robust method for the characterization of gene-editing events. The efficacy of editing-mediated mono- and bi-allelic gene inactivation and integration events is quantified based on colony counts. The combination of diphtheria toxin (DT) and puromycin (PM) selection enables analyses of 10,000-100,000 individual cells, assessing hundreds of clones with inactivated genes per experiment. Mono- and bi-allelic gene inactivation is differentiated by DT resistance, which occurs only upon bi-allelic inactivation...
November 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29121951/increasing-the-cpg-dinucleotide-abundance-in-the-hiv-1-genomic-rna-inhibits-viral-replication
#8
Irati Antzin-Anduetza, Charlotte Mahiet, Luke A Granger, Charlotte Odendall, Chad M Swanson
BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) structural protein Gag is necessary and sufficient to form viral particles. In addition to encoding the amino acid sequence for Gag, the underlying RNA sequence could encode cis-acting elements or nucleotide biases that are necessary for viral replication. Furthermore, RNA sequences that inhibit viral replication could be suppressed in gag. However, the functional relevance of RNA elements and nucleotide biases that promote or repress HIV-1 replication remain poorly understood...
November 9, 2017: Retrovirology
https://www.readbyqxmd.com/read/29118738/monitoring-of-dual-crispr-cas9-mediated-steroidogenic-acute-regulatory-protein-gene-deletion-and-cholesterol-accumulation-using-high-resolution-fluorescence-in-situ-hybridization-in-a-single-cell
#9
Jinwoo Lee, Colin Jefcoate
Recent advances in fluorescence microscopy, coupled with CRISPR/Cas9 gene editing technology, provide opportunities for understanding gene regulation at the single-cell level. The application of direct imaging shown here provides an in situ side-by-side comparison of CRISPR/Cas9-edited cells and adjacent unedited cells. We apply this methodology to the steroidogenic acute regulatory protein (StAR) gene in Y-1 adrenal cells and MA-10 testis cells. StAR is a gatekeeper protein that controls the access of cholesterol from the cytoplasm to the inner mitochondria...
2017: Frontiers in Endocrinology
https://www.readbyqxmd.com/read/29091307/expanding-the-crispr-cas9-toolkit-for-pichia-pastoris-with-efficient-donor-integration-and-alternative-resistance-markers
#10
Astrid Weninger, Jasmin Fischer, Hana Raschmanová, Claudia Kniely, Thomas Vogl, Anton Glieder
Komagataella phaffii (syn. Pichia pastoris) is one of the most commonly used host systems for recombinant protein expression. Achieving targeted genetic modifications had been hindered by low frequencies of homologous recombination (HR). Recently, a CRISPR/Cas9 genome editing system has been implemented for P. pastoris enabling gene knockouts based on indels (insertion, deletions) via non-homologous end joining (NHEJ) at near 100% efficiency. However, specifically integrating homologous donor cassettes via HR for replacement studies had proven difficult resulting at most in ∼20% correct integration using CRISPR/Cas9...
November 1, 2017: Journal of Cellular Biochemistry
https://www.readbyqxmd.com/read/29091290/combinatorial-genetics-in-liver-repopulation-and-carcinogenesis-with-a-novel-in-vivo-crispr-activation-platform
#11
Kirk J Wangensteen, Yue J Wang, Zhixun Dou, Amber W Wang, Elham Mosleh-Shirazi, Max A Horlbeck, Luke A Gilbert, Jonathan S Weissman, Shelley L Berger, Klaus H Kaestner
CRISPR/Cas9 activation (CRISPRa) systems have enabled genetic screens in cultured cells lines to discover and characterize drivers and inhibitors of cancer cell growth. We adapted this system for use in vivo to assess whether modulating endogenous gene expression levels can result in functional outcomes in the native environment of the liver. We engineered the dCas9(+) mouse, a Cre-inducible CRISPRa system for cell type-specific gene activation in vivo. We tested the capacity for genetic screening in live animals by applying CRISPRa in a clinically relevant model of liver injury and repopulation...
November 1, 2017: Hepatology: Official Journal of the American Association for the Study of Liver Diseases
https://www.readbyqxmd.com/read/29089503/designing-broad-spectrum-anti-hiv-1-grnas-to-target-patient-derived-variants
#12
Will Dampier, Neil T Sullivan, Cheng-Han Chung, Joshua Chang Mell, Michael R Nonnemacher, Brian Wigdahl
Clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated protein 9 (Cas9), including specific guide RNAs (gRNAs), can excise integrated human immunodeficiency virus type 1 (HIV-1) provirus from host chromosomes. To date, anti-HIV-1 gRNAs have been designed to account for off-target activity, however, they seldom account for genetic variation in the HIV-1 genome within and between patients, which will be crucial for therapeutic application of this technology. This analysis tests the ability of published anti-HIV-1 gRNAs to cleave publicly available patient-derived HIV-1 sequences to inform gRNA design and provides basic computational tools to researchers in the field...
October 31, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29078304/structures-of-q%C3%AE-virions-virus-like-particles-and-the-q%C3%AE-mura-complex-reveal-internal-coat-proteins-and-the-mechanism-of-host-lysis
#13
Zhicheng Cui, Karl V Gorzelnik, Jeng-Yih Chang, Carrie Langlais, Joanita Jakana, Ry Young, Junjie Zhang
In single-stranded RNA bacteriophages (ssRNA phages) a single copy of the maturation protein binds the genomic RNA (gRNA) and is required for attachment of the phage to the host pilus. For the canonical Allolevivirus Qβ the maturation protein, A2, has an additional role as the lysis protein, by its ability to bind and inhibit MurA, which is involved in peptidoglycan biosynthesis. Here, we determined structures of Qβ virions, virus-like particles, and the Qβ-MurA complex using single-particle cryoelectron microscopy, at 4...
October 31, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29075312/a-highly-efficient-ligation-independent-cloning-system-for-crispr-cas9-based-genome-editing-in-plants
#14
Aftab A Khan, Ashraf El-Sayed, Asma Akbar, Arianna Mangravita-Novo, Shaheen Bibi, Zunaira Afzal, David J Norman, Gul Shad Ali
BACKGROUND: Most current methods for constructing guide RNAs (gRNA) for the CRISPR/Cas9 genome editing system, depend on traditional cloning using specific type IIS restriction enzymes and DNA ligation. These methods consist of multiple steps of cloning, and are time consuming, resource intensive and not flexible. These issues are particularly exacerbated when multiple guide RNAs need to be assembled in one plasmid such as for multiplexing or for the paired nickases approach. Furthermore, identification of functional gRNA clones usually requires expensive in vitro screening...
2017: Plant Methods
https://www.readbyqxmd.com/read/29067670/from-grna-identification-to-the-restoration-of-dystrophin-expression-a-dystrophin-gene-correction-strategy-for-duchenne-muscular-dystrophy-mutations-using-the-crispr-induced-deletion-method
#15
Benjamin Duchêne, Jean-Paul Iyombe-Engembe, Joël Rousseau, Jacques P Tremblay, Dominique L Ouellet
The discovery of the CRISPR-Cas9 system raises hope for the treatment of many genetic disorders. We describe here an approach based on the use of a pair of single guide RNAs to form a hybrid exon that does not only restore the dystrophin gene reading frame but also results in the production of a dystrophin protein with an adequate structure of the central rod-domain, with a correct spectrin-like repeat. The therapeutic approach described here involved DMD patient cells having a deletion of exons 51-53 of the DMD gene...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29056323/impeding-transcription-of-expanded-microsatellite-repeats-by-deactivated-cas9
#16
Belinda S Pinto, Tanvi Saxena, Ruan Oliveira, Héctor R Méndez-Gómez, John D Cleary, Lance T Denes, Ona McConnell, Juan Arboleda, Guangbin Xia, Maurice S Swanson, Eric T Wang
Transcription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs endothelial corneal dystrophy, and C9orf72-ALS/FTD. Reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that deactivated Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences; targeting the non-template strand was more effective...
November 2, 2017: Molecular Cell
https://www.readbyqxmd.com/read/29045453/crispr-cas-mediated-knock-in-via-non-homologous-end-joining-in-the-crustacean-daphnia-magna
#17
Hitoshi Kumagai, Takashi Nakanishi, Tomoaki Matsuura, Yasuhiko Kato, Hajime Watanabe
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) is widely used for mediating the knock-in of foreign DNA into the genomes of various organisms. Here, we report a process of CRISPR/Cas-mediated knock-in via non-homologous end joining by the direct injection of Cas9/gRNA ribonucleoproteins (RNPs) in the crustacean Daphnia magna, which is a model organism for studies on toxicology, ecology, and evolution. First, we confirmed the cleavage activity of Cas9 RNPs comprising purified Cas9 proteins and gRNAs in D...
2017: PloS One
https://www.readbyqxmd.com/read/29040704/in-planta-processing-of-the-spcas9-grna-complex
#18
Masafumi Mikami, Seiichi Toki, Masaki Endo
In CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9)-mediated genome editing in plants, Streptococcus pyogenes Cas9 (SpCas9) protein and the required guide RNA (gRNA) are, in most cases, expressed from a stably integrated transgene. Generally, SpCas9 protein is expressed from an RNA polymerase (pol) II promoter, while gRNA is expressed from a pol III promoter. However, pol III promoters have not been much characterized other than in model plants, making it difficult to select appropriate promoters for specific applications, while pol II transcripts have to be processed to generate functional gRNAs...
November 1, 2017: Plant & Cell Physiology
https://www.readbyqxmd.com/read/29019352/hot-news-gene-therapy-with-crispr-cas9-coming-to-age-for-hiv-cure
#19
Vicente Soriano
The huge success of current antiretroviral therapy is mediated by a triple effect: (i) Halting progression to AIDS in infected persons; (ii) reducing the risk of transmission to contacts (treatment as prevention); and (iii) minimizing the risk of HIV acquisition treating uninfected persons at risk (pre-exposure prophylaxis). However, UNAIDS has estimated that only 70% of infected people globally are diagnosed, only 53% are treated, and overall 44% have undetectable viral load, which is the necessary request for ensuring any antiretroviral benefit...
October 2017: AIDS Reviews
https://www.readbyqxmd.com/read/28992181/requirement-of-glycosylation-machinery-in-toll-like-receptor-responses-revealed-by-crispr-cas9-screening
#20
Ryota Sato, Takuma Shibata, Yu Tanaka, Chiharu Kato, Kiyoshi Yamaguchi, Yoichi Furukawa, Eigo Shimizu, Rui Yamaguchi, Seiya Imoto, Satoru Miyano, Kensuke Miyake
The Toll family of receptors sense microbial products and activate a defense response. The molecular machinery required for the Toll-like receptor (TLR) response is not yet fully understood. In the present study, we used a CRISPR/CAS9 screening system to study TLR responses. We employed a cell line expressing TLR with an NF-κB-driven GFP reporter. The cell line was transduced with a guide RNA library and stimulated with TLR ligands. The cells impaired in GFP induction were sorted, and gRNAs were sequenced...
July 28, 2017: International Immunology
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