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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
#1
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
#2
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 events in human diseases, such as multiple gene deletions or mutations, two or more small guide RNAs (sgRNA) must be introduced all together. This can be achieved by separate Pol III promoters in a construct. However, limited enzyme sites and the increased insertion size lower the efficiency to make a construct...
November 24, 2016: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/27866656/an-analysis-of-possible-off-target-effects-following-cas9-crispr-targeted-deletions-of-neuropeptide-gene-enhancers-from-the-mouse-genome
#3
Elizabeth Anne Hay, Abdulla Razak Khalaf, Pietro Marini, Andrew Brown, Karyn Heath, Darrin Sheppard, Alasdair MacKenzie
We have successfully used comparative genomics to identify putative regulatory elements within the human genome that contribute to the tissue specific expression of neuropeptides such as galanin and receptors such as CB1. However, a previous inability to rapidly delete these elements from the mouse genome has prevented optimal assessment of their function in-vivo. This has been solved using CAS9/CRISPR genome editing technology which uses a bacterial endonuclease called CAS9 that, in combination with specifically designed guide RNA (gRNA) molecules, cuts specific regions of the mouse genome...
November 4, 2016: Neuropeptides
https://www.readbyqxmd.com/read/27852925/anticodon-like-binding-of-the-hiv-1-trna-like-element-to-human-lysyl-trna-synthetase
#4
Sheng Liu, Roopa Comandur, Christopher P Jones, Pearl Tsang, Karin Musier-Forsyth
A critical step in the HIV-1 lifecycle involves reverse transcription of the viral genomic RNA (gRNA). Human tRNA(Lys3) serves as a primer for transcription initiation and is selectively enriched in virus particles. Human lysyl-tRNA synthetase (hLysRS) is also packaged into virions. Recently, a tRNA-like element (TLE) within the HIV-1 gRNA was shown to mimic the global tRNA fold and bind competitively to hLysRS, suggesting a mechanism of tRNA targeting to the primer binding site (PBS) and release from the synthetase...
December 2016: RNA
https://www.readbyqxmd.com/read/27848933/genome-editing-in-maize-directed-by-crispr-cas9-ribonucleoprotein-complexes
#5
Sergei Svitashev, Christine Schwartz, Brian Lenderts, Joshua K Young, A Mark Cigan
Targeted DNA double-strand breaks have been shown to significantly increase the frequency and precision of genome editing. In the past two decades, several double-strand break technologies have been developed. CRISPR-Cas9 has quickly become the technology of choice for genome editing due to its simplicity, efficiency and versatility. Currently, genome editing in plants primarily relies on delivering double-strand break reagents in the form of DNA vectors. Here we report biolistic delivery of pre-assembled Cas9-gRNA ribonucleoproteins into maize embryo cells and regeneration of plants with both mutated and edited alleles...
November 16, 2016: Nature Communications
https://www.readbyqxmd.com/read/27845387/adenoviral-vectors-encoding-crispr-cas9-multiplexes-rescue-dystrophin-synthesis-in-unselected-populations-of-dmd-muscle-cells
#6
Ignazio Maggio, Jin Liu, Josephine M Janssen, Xiaoyu Chen, Manuel A F V Gonçalves
Mutations disrupting the reading frame of the ~2.4 Mb dystrophin-encoding DMD gene cause a fatal X-linked muscle-wasting disorder called Duchenne muscular dystrophy (DMD). Genome editing based on paired RNA-guided nucleases (RGNs) from CRISPR/Cas9 systems has been proposed for permanently repairing faulty DMD loci. However, such multiplexing strategies require the development and testing of delivery systems capable of introducing the various gene editing tools into target cells. Here, we investigated the suitability of adenoviral vectors (AdVs) for multiplexed DMD editing by packaging in single vector particles expression units encoding the Streptococcus pyogenes Cas9 nuclease and sequence-specific gRNA pairs...
November 15, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27845164/enhanced-crispr-cas9-mediated-precise-genome-editing-by-improved-design-and-delivery-of-grna-cas9-nuclease-and-donor-dna
#7
Xiquan Liang, Jason Potter, Shantanu Kumar, Namritha Ravinder, Jonathan D Chesnut
While CRISPR-based gene knock out in mammalian cells has proven to be very efficient, precise insertion of genetic elements via the cellular homology directed repair (HDR) pathway remains a rate-limiting step to seamless genome editing. Under the conditions described here, we achieved up to 56% targeted integration efficiency with up to a six-nucleotide insertion in HEK293 cells. In induced pluripotent stem cells (iPSCs), we achieved precise genome editing rates of up to 45% by co-delivering the Cas9 RNP and donor DNA...
November 11, 2016: Journal of Biotechnology
https://www.readbyqxmd.com/read/27842490/coralina-a-universal-method-for-the-generation-of-grna-libraries-for-crispr-based-screening
#8
Anna Köferle, Karolina Worf, Christopher Breunig, Valentin Baumann, Javier Herrero, Maximilian Wiesbeck, Lukas H Hutter, Magdalena Götz, Christiane Fuchs, Stephan Beck, Stefan H Stricker
BACKGROUND: The bacterial CRISPR system is fast becoming the most popular genetic and epigenetic engineering tool due to its universal applicability and adaptability. The desire to deploy CRISPR-based methods in a large variety of species and contexts has created an urgent need for the development of easy, time- and cost-effective methods enabling large-scale screening approaches. RESULTS: Here we describe CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a method for the generation of comprehensive gRNA libraries for CRISPR-based screens...
November 14, 2016: BMC Genomics
https://www.readbyqxmd.com/read/27834588/generation-of-ggta1-mutant-pigs-by-direct-pronuclear-microinjection-of-crispr-cas9-plasmid-vectors
#9
Chin-Kai Chuang, Chien-Hong Chen, Chung-Ling Huang, Yu-Hsiu Su, Shu-Hui Peng, Tai-Yun Lin, Hao-Chih Tai, Tien-Shuh Yang, Ching-Fu Tu
This study was conducted to confirm that 1-site and 4-site ppU6-GGTA1-gRNA CRISPR vectors together with the pCX-Flag2-NLS1-Cas9-NLS2 plasmid can both generate KO pigs by direct pronuclear microinjection. In total, 41 and 53 fertilized eggs were microinjected on 1-site and 4-site strategies, respectively. The 1-site construction generated a litter of 8 piglets, and 2 were mono-allelic mutant (mMt). The injection of 4-site constructions resulted in one biallelic mutant (bMt) and one mMt piglet in a litter of 7...
November 11, 2016: Animal Biotechnology
https://www.readbyqxmd.com/read/27832146/ribozyme-mediated-grna-generation-for-in-vitro-and-in-vivo-crispr-cas9-mutagenesis
#10
Raymond Teck Ho Lee, Ashley Shu Mei Ng, Philip W Ingham
CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites available for mutagenesis without compromising mutagenesis efficiency. We have tested this by comparing the efficiency of mutagenesis of gRNA constructs with and without ribozymes and also generated a transgenic zebrafish expressing gRNA using a heat shock promoter (RNA polymerase II-dependent promoter) that was able to induce mutagenesis of its target...
2016: PloS One
https://www.readbyqxmd.com/read/27808171/using-genome-wide-crispr-library-screening-with-library-resistant-dck-to-find-new-sources-of-ara-c-drug-resistance-in-aml
#11
Morito Kurata, Susan K Rathe, Natashay J Bailey, Natalie K Aumann, Justine M Jones, G Willemijn Veldhuijzen, Branden S Moriarity, David A Largaespada
Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance...
November 3, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27804953/structural-roles-of-guide-rnas-in-the-nuclease-activity-of-cas9-endonuclease
#12
Youngbin Lim, So Young Bak, Keewon Sung, Euihwan Jeong, Seung Hwan Lee, Jin-Soo Kim, Sangsu Bae, Seong Keun Kim
The type II CRISPR-associated protein Cas9 recognizes and cleaves target DNA with the help of two guide RNAs (gRNAs; tracrRNA and crRNA). However, the detailed mechanisms and kinetics of these gRNAs in the Cas9 nuclease activity are unclear. Here, we investigate the structural roles of gRNAs in the CRISPR-Cas9 system by single-molecule spectroscopy and reveal a new conformation of inactive Cas9 that is thermodynamically more preferable than active apo-Cas9. We find that tracrRNA prevents Cas9 from changing into the inactive form and leads to the Cas9:gRNA complex...
November 2, 2016: Nature Communications
https://www.readbyqxmd.com/read/27768202/genomic-disruption-of-vegf-a-expression-in-human-retinal-pigment-epithelial-cells-using-crispr-cas9-endonuclease
#13
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
https://www.readbyqxmd.com/read/27754889/the-zebrafish-homologs-of-set-i2pp2a-oncoprotein-expression-patterns-and-insights-into-their-physiological-roles-during-development
#14
Iliana Serifi, Eleni Tzima, Katerina Soupsana, Zoe Karetsou, Dimitris Beis, Thomais Papamarcaki
The oncoprotein SET/I2PP2A participates in various cellular mechanisms such as transcription, cell cycle regulation and cell migration. SET is also an inhibitor of the serine/threonine phosphatase PP2A, which is involved in the regulation of cell homeostasis. In zebrafish there are two paralogous set genes that encode Seta (269 aa) and Setb (275 aa) proteins which share 94% identity. We show here that seta and set b are similarly expressed in the eye, the otic vesicle, the brain and the lateral line system, as indicated by in situ hybridization labeling...
October 17, 2016: Biochemical Journal
https://www.readbyqxmd.com/read/27747971/discovery-of-rice-essential-genes-by-characterizing-crispr-edited-mutation-of-closely-related-rice-map-kinase-genes
#15
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
https://www.readbyqxmd.com/read/27744351/rna-editing-tutase-1-structural-foundation-of-substrate-recognition-complex-interactions-and-drug-targeting
#16
Lional Rajappa-Titu, Takuma Suematsu, Paola Munoz-Tello, Marius Long, Özlem Demir, Kevin J Cheng, Jason R Stagno, Hartmut Luecke, Rommie E Amaro, Inna Aphasizheva, Ruslan Aphasizhev, Stéphane Thore
Terminal uridyltransferases (TUTases) execute 3' RNA uridylation across protists, fungi, metazoan and plant species. Uridylation plays a particularly prominent role in RNA processing pathways of kinetoplastid protists typified by the causative agent of African sleeping sickness, Trypanosoma brucei In mitochondria of this pathogen, most mRNAs are internally modified by U-insertion/deletion editing while guide RNAs and rRNAs are U-tailed. The founding member of TUTase family, RNA editing TUTase 1 (RET1), functions as a subunit of the 3' processome in uridylation of gRNA precursors and mature guide RNAs...
October 15, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27744049/crispr-cas9-in-locusts-successful-establishment-of-an-olfactory-deficiency-line-by-targeting-the-mutagenesis-of-an-odorant-receptor-co-receptor-orco
#17
Yan Li, Jie Zhang, Dafeng Chen, Pengcheng Yang, Feng Jiang, Xianhui Wang, Le Kang
Locusts are important agricultural pests worldwide and regarded as study models for entomology. However, the absence of targeted gene manipulation systems for locusts has restricted their applications for research. Herein, we report the successful use of the CRISPR/Cas9 system to induce a targeted heritable mutagenesis of the migratory locust, Locusta migratoria. The target sequence of gRNA was designed to disrupt the gene encoding the odorant receptor co-receptor (Orco) and examine the roles of the odorant receptor pathway in the locust...
October 12, 2016: Insect Biochemistry and Molecular Biology
https://www.readbyqxmd.com/read/27739525/crispr-cas9-mediated-knockout-of-rb1-and-rbl1-leads-to-rapid-and-penetrant-retinoblastoma-development-in-xenopus-tropicalis
#18
Thomas Naert, Robin Colpaert, Tom Van Nieuwenhuysen, Dionysia Dimitrakopoulou, Jannick Leoen, Jurgen Haustraete, Annekatrien Boel, Wouter Steyaert, Trees Lepez, Dieter Deforce, Andy Willaert, David Creytens, Kris Vleminckx
Retinoblastoma is a pediatric eye tumor in which bi-allelic inactivation of the Retinoblastoma 1 (RB1) gene is the initiating genetic lesion. Although recently curative rates of retinoblastoma have increased, there are at this time no molecular targeted therapies available. This is, in part, due to the lack of highly penetrant and rapid retinoblastoma animal models that facilitate rapid identification of targets that allow therapeutic intervention. Different mouse models are available, all based on genetic deactivation of both Rb1 and Retinoblastoma-like 1 (Rbl1), and each showing different kinetics of retinoblastoma development...
October 14, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27727192/cross-and-co-packaging-of-retroviral-rnas-and-their-consequences
#19
Lizna M Ali, Tahir A Rizvi, Farah Mustafa
Retroviruses belong to the family Retroviridae and are ribonucleoprotein (RNP) particles that contain a dimeric RNA genome. Retroviral particle assembly is a complex process, and how the virus is able to recognize and specifically capture the genomic RNA (gRNA) among millions of other cellular and spliced retroviral RNAs has been the subject of extensive investigation over the last two decades. The specificity towards RNA packaging requires higher order interactions of the retroviral gRNA with the structural Gag proteins...
October 11, 2016: Viruses
https://www.readbyqxmd.com/read/27725766/functional-visualization-and-disruption-of-targeted-genes-using-crispr-cas9-mediated-egfp-reporter-integration-in-zebrafish
#20
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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