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https://www.readbyqxmd.com/read/28418635/enhancing-protein-production-yield-from-cho-cells-by-crispr-interference-crispri
#1
Chih-Che Shen, Li-Yu Sung, Shih-Yeh Lin, Mei-Wei Lin, Yu-Chen Hu
CHO cell is an important host for biopharmaceutical production. Generation of stable CHO cell typically requires co-integration of dhfr and foreign gene into chromosomes and subsequent methotrexate (MTX) selection for co-amplification of dhfr and foreign gene. CRISPR interference (CRISPRi) is an emerging system that effectively suppresses gene transcription through the coordination of dCas9 protein and guide RNA (gRNA). However, CRISPRi has yet to be exploited in CHO cells. Here we constructed vectors expressing the functional CRISPRi system and proved effective CRISPRi-mediated suppression of dhfr transcription in CHO cells...
April 18, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28416141/multiplexed-engineering-and-analysis-of-combinatorial-enhancer-activity-in-single-cells
#2
Shiqi Xie, Jialei Duan, Boxun Li, Pei Zhou, Gary C Hon
The study of enhancers has been hampered by the scarcity of methods to systematically quantify their endogenous activity. We develop Mosaic-seq to systematically perturb enhancers and measure their endogenous activities at single-cell resolution. Mosaic-seq uses a CRISPR barcoding system to jointly measure a cell's transcriptome and its sgRNA modulators, thus quantifying the effects of dCas9-KRAB-mediated enhancer repression in single cells. Applying Mosaic-seq to 71 constituent enhancers from 15 super-enhancers, our analysis of 51,448 sgRNA-induced transcriptomes finds that only a small number of constituents are major effectors of target gene expression...
April 20, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28412663/a-facile-rapid-and-sensitive-detection-of-mrsa-using-a-crispr-mediated-dna-fish-method-antibody-like-dcas9-sgrna-complex
#3
Kyeonghye Guk, Joo Oak Keem, Seul Gee Hwang, Hyeran Kim, Taejoon Kang, Eun-Kyung Lim, Juyeon Jung
Rapid and reliable diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is crucial for guiding effective patient treatment and preventing the spread of MRSA infections. Nonetheless, further simplification of MRSA detection procedures to shorten detection time and reduce labor relative to that of conventional methods remains a challenge. Here, we have demonstrated a Clustered regularly interspaced palindromic repeats (CRISPR)-mediated DNA-FISH method for the simple, rapid and highly sensitive detection of MRSA; this method uses CRISPR associated protein 9/single-guide RNA (dCas9/sgRNA) complex as a targeting material and SYBR Green I (SG I) as a fluorescent probe...
April 13, 2017: Biosensors & Bioelectronics
https://www.readbyqxmd.com/read/28411216/construction-of-a-dcas9-based-gene-knockdown-system-in-staphylococcus-aureus
#4
Changlong Zhao, Xueqin Shu, Baolin Sun
There has been constant absence of an efficient gene knockdown method in the important human pathogen Staphylococcus aureus like RNA interference in eukaryotes. The early developed antisense RNA technology is mainly applied for forward genetic screen, but is rather limiting in specific gene knockdown for the lack of rational antisense RNA design strategies. Here we report an efficient and specific gene knockdown system in S. aureus based on type II clustered regularly interspaced short palindromic repeats (CRISPR) system from Streptococcus pyogenes...
April 14, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28369033/crispr-cas9-epigenome-editing-enables-high-throughput-screening-for-functional-regulatory-elements-in-the-human-genome
#5
Tyler S Klann, Joshua B Black, Malathi Chellappan, Alexias Safi, Lingyun Song, Isaac B Hilton, Gregory E Crawford, Timothy E Reddy, Charles A Gersbach
Large genome-mapping consortia and thousands of genome-wide association studies have identified non-protein-coding elements in the genome as having a central role in various biological processes. However, decoding the functions of the millions of putative regulatory elements discovered in these studies remains challenging. CRISPR-Cas9-based epigenome editing technologies have enabled precise perturbation of the activity of specific regulatory elements. Here we describe CRISPR-Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context...
April 3, 2017: Nature Biotechnology
https://www.readbyqxmd.com/read/28325301/increased-expression-of-laminin-subunit-alpha-1-chain-by-dcas9-vp160
#6
Arnaud Perrin, Joël Rousseau, Jacques P Tremblay
Laminin-111 protein complex links the extracellular matrix to integrin α7β1 in sarcolemma, thus replacing in dystrophic muscles links normally insured by the dystrophin complex. Laminin-111 injection in mdx mouse stabilized sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscles from exercised-induced damages. These results suggested that increased laminin-111 is a potential therapy for DMD. Laminin subunit beta 1 and laminin subunit gamma 1 are expressed in adult human muscle, but laminin subunit alpha 1 (LAMA1) gene is expressed only during embryogenesis...
March 17, 2017: Molecular Therapy. Nucleic Acids
https://www.readbyqxmd.com/read/28298224/transcriptional-reprogramming-in-yeast-using-dcas9-and-combinatorial-grna-strategies
#7
Emil D Jensen, Raphael Ferreira, Tadas Jakočiūnas, Dushica Arsovska, Jie Zhang, Ling Ding, Justin D Smith, Florian David, Jens Nielsen, Michael K Jensen, Jay D Keasling
BACKGROUND: Transcriptional reprogramming is a fundamental process of living cells in order to adapt to environmental and endogenous cues. In order to allow flexible and timely control over gene expression without the interference of native gene expression machinery, a large number of studies have focused on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene transcription start site...
March 15, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28217741/transcriptome-level-signatures-in-gene-expression-and-gene-expression-variability-during-bacterial-adaptive-evolution
#8
Keesha E Erickson, Peter B Otoupal, Anushree Chatterjee
Antibiotic-resistant bacteria are an increasingly serious public health concern, as strains emerge that demonstrate resistance to almost all available treatments. One factor that contributes to the crisis is the adaptive ability of bacteria, which exhibit remarkable phenotypic and gene expression heterogeneity in order to gain a survival advantage in damaging environments. This high degree of variability in gene expression across biological populations makes it a challenging task to identify key regulators of bacterial adaptation...
January 2017: MSphere
https://www.readbyqxmd.com/read/28212815/systematic-testing-of-enzyme-perturbation-sensitivities-via-graded-dcas9-modulation-in-saccharomyces-cerevisiae
#9
Matthew Deaner, Hal S Alper
Dissecting genotype-phenotype relationships in a high-throughput and scalable manner is still an unresolved problem facing metabolic engineers. While the RNA-guided nuclease Cas9 has been repurposed as a programmable transcription regulator, its application has typically been limited to binary on/off regulation and thus misses informative and potentially optimal intermediate levels of gene expression. In this work, we establish a rapid method for fine-tuned, graded expression of pathway enzymes via dCas9 regulation by varying sgRNA target location as the dominant parameter...
February 14, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28179021/switch-a-dynamic-crispr-tool-for-genome-engineering-and-metabolic-pathway-control-for-cell-factory-construction-in-saccharomyces-cerevisiae
#10
Katherina García Vanegas, Beata Joanna Lehka, Uffe Hasbro Mortensen
BACKGROUND: The yeast Saccharomyces cerevisiae is increasingly used as a cell factory. However, cell factory construction time is a major obstacle towards using yeast for bio-production. Hence, tools to speed up cell factory construction are desirable. RESULTS: In this study, we have developed a new Cas9/dCas9 based system, SWITCH, which allows Saccharomyces cerevisiae strains to iteratively alternate between a genetic engineering state and a pathway control state...
February 8, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28143571/single-cas9-nickase-induced-generation-of-nramp1-knockin-cattle-with-reduced-off-target-effects
#11
Yuanpeng Gao, Haibo Wu, Yongsheng Wang, Xin Liu, Linlin Chen, Qian Li, Chenchen Cui, Xu Liu, Jingcheng Zhang, Yong Zhang
BACKGROUND: The CRISPR-Cas9 system is a widely utilized platform for transgenic animal production in various species, although its off-target effects should be addressed. Several applications of this tool have been proposed in model animals but remain insufficient for transgenic livestock production. RESULTS: Here, we report the first application of single Cas9 nickase (Cas9n) to induce gene insertion at a selected locus in cattle. We identify the main binding sites of a catalytically inactive Cas9 (dCas9) protein in bovine fetal fibroblast cells (BFFs) with chromatin immunoprecipitation sequencing (ChIP-seq)...
February 1, 2017: Genome Biology
https://www.readbyqxmd.com/read/28136159/crispr-cas-type-ii-based-synthetic-biology-applications-in-eukaryotic-cells
#12
Mario Andrea Marchisio, Zhiwei Huang
The CRISPR-Cas system has rapidly reached a huge popularity as a new, powerful method for precise DNA editing and genome reengineering. In Synthetic Biology, the CRISPR-Cas type II system has inspired the construction of a novel class of RNA-based transcription factors. In their simplest form, they are made of a CRISPR RNA molecule, which targets a promoter sequence, and a deficient Cas9 (i.e. deprived of any nuclease activity) that has been fused to an activation or a repression domain. Up- and down-regulation of single genes in mammalian and yeast cells have been achieved with satisfactory results...
January 31, 2017: RNA Biology
https://www.readbyqxmd.com/read/28116670/an-inducible-crispr-on-system-for-controllable-gene-activation-in-human-pluripotent-stem-cells
#13
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/28095853/crispri-mediated-metabolic-engineering-of-e-coli-for-o-methylated-anthocyanin-production
#14
Brady F Cress, Quentin D Leitz, Daniel C Kim, Teresita D Amore, Jon Y Suzuki, Robert J Linhardt, Mattheos A G Koffas
BACKGROUND: Anthocyanins are a class of brightly colored, glycosylated flavonoid pigments that imbue their flower and fruit host tissues with hues of predominantly red, orange, purple, and blue. Although all anthocyanins exhibit pH-responsive photochemical changes, distinct structural decorations on the core anthocyanin skeleton also cause dramatic color shifts, in addition to improved stabilities and unique pharmacological properties. In this work, we report for the first time the extension of the reconstituted plant anthocyanin pathway from (+)-catechin to O-methylated anthocyanins in a microbial production system, an effort which requires simultaneous co-option of the endogenous metabolites UDP-glucose and S-adenosyl-L-methionine (SAM or AdoMet)...
January 17, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28072927/artificial-induction-of-native-aquaporin-1-expression-in-human-salivary-cells
#15
Z Wang, S Pradhan-Bhatt, M C Farach-Carson, M J Passineau
Gene therapy for dry mouth disorders has transitioned in recent years from theoretical to clinical proof of principle with the publication of a first-in-man phase I/II dose escalation clinical trial in patients with radiation-induced xerostomia. This trial used a prototype adenoviral vector to express aquaporin-1 (AQP1), presumably in the ductal cell layer and/or in surviving acinar cells, to drive transcellular flux of interstitial fluid into the labyrinth of the salivary duct. As the development of this promising gene therapy continues, safety considerations are a high priority, particularly those that remove nonhuman agents (i...
April 2017: Journal of Dental Research
https://www.readbyqxmd.com/read/28069948/genomic-targeting-of-epigenetic-probes-using-a-chemically-tailored-cas9-system
#16
Glen P Liszczak, Zachary Z Brown, Samuel H Kim, Rob C Oslund, Yael David, Tom W Muir
Recent advances in the field of programmable DNA-binding proteins have led to the development of facile methods for genomic localization of genetically encodable entities. Despite the extensive utility of these tools, locus-specific delivery of synthetic molecules remains limited by a lack of adequate technologies. Here we combine the flexibility of chemical synthesis with the specificity of a programmable DNA-binding protein by using protein trans-splicing to ligate synthetic elements to a nuclease-deficient Cas9 (dCas9) in vitro and subsequently deliver the dCas9 cargo to live cells...
January 24, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28046023/a-model-to-investigate-single-strand-dna-responses-in-g1-human-cells-via-a-telomere-targeted-nuclease-deficient-crispr-cas9-system
#17
Remco P Crefcoeur, Omar Zgheib, Thanos D Halazonetis
DNA replication stress has the potential to compromise genomic stability and, therefore, cells have developed elaborate mechanisms to detect and resolve problems that may arise during DNA replication. The presence of single-stranded DNA (ssDNA) is often associated with DNA replication stress and serves as a signal for both checkpoint and repair responses. In this study, we exploited a CRISPR-Cas9 system to induce regions of ssDNA in the genome. Specifically, single-guide RNAs bearing sequence complementarity to human telomeric repeats, were used to target nuclease-deficient Cas9 (dCas9) to telomeres...
2017: PloS One
https://www.readbyqxmd.com/read/27983975/current-status-of-potential-applications-of-repurposed-cas9-for-structural-and-functional-genomics-of-plants
#18
Kunal Seth, Harish
Redesigned Cas9 has emerged as a tool with various applications like gene editing, gene regulation, epigenetic modification and chromosomal imaging. Target specific single guide RNA (sgRNA) can be used with Cas9 for precise gene editing with high efficiency than previously known methods. Further, nuclease-deactivated Cas9 (dCas9) can be fused with activator or repressor for activation (CRISPRa) and repression (CRISPRi) of gene expression, respectively. dCas9 fused with epigenetic modifier like methylase or acetylase further expand the scope of this technique...
October 28, 2016: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/27965712/targeted-inhibition-of-the-mir-199a-214-cluster-by-crispr-interference-augments-the-tumor-tropism-of-human-induced-pluripotent-stem-cell-derived-neural-stem-cells-under-hypoxic-condition
#19
Yumei Luo, Xuehu Xu, Xiuli An, Xiaofang Sun, Shu Wang, Detu Zhu
The human induced pluripotent stem cell (hiPSC) provides a breakthrough approach that helps overcoming ethical and allergenic challenges posed in application of neural stem cells (NSCs) in targeted cancer gene therapy. However, the tumor-tropic capacity of hiPSC-derived NSCs (hiPS-NSCs) still has much room to improve. Here we attempted to promote the tumor tropism of hiPS-NSCs by manipulating the activity of endogenous miR-199a/214 cluster that is involved in regulation of hypoxia-stimulated cell migration...
2016: Stem Cells International
https://www.readbyqxmd.com/read/27960483/single-molecule-localization-and-discrimination-of-dna-protein-complexes-by-controlled-translocation-through-nanocapillaries
#20
Roman D Bulushev, Sanjin Marion, Ekaterina Petrova, Sebastian J Davis, Sebastian J Maerkl, Aleksandra Radenovic
Through the use of optical tweezers we performed controlled translocations of DNA-protein complexes through nanocapillaries. We used RNA polymerase (RNAP) with two binding sites on a 7.2 kbp DNA fragment and a dCas9 protein tailored to have five binding sites on λ-DNA (48.5 kbp). Measured localization of binding sites showed a shift from the expected positions on the DNA that we explained using both analytical fitting and a stochastic model. From the measured force versus stage curves we extracted the nonequilibrium work done during the translocation of a DNA-protein complex and used it to obtain an estimate of the effective charge of the complex...
December 14, 2016: Nano Letters
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