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https://www.readbyqxmd.com/read/28213525/conformational-profiling-of-the-at1-angiotensin-ii-receptor-reflects-biased-agonism-g-protein-coupling-and-cellular-context
#1
Dominic Devost, Rory Sleno, Darlaine Petrin, Alice Zhang, Yuji Shinjo, Rakan Okde, Junken Aoki, Asuka Inoue, Terence E Hebert
Here, we report the design and use of GPCR-based biosensors to monitor ligand-mediated conformational changes in receptors in intact cells. These biosensors use Bioluminescence Resonance Energy Transfer (BRET) with Renilla luciferase (RlucII) as an energy donor, placed at the distal end of the receptor C-tail and the small fluorescent molecule FlAsH, as an energy acceptor, its binding site inserted at different positions throughout the intracellular loops and carboxy-terminal tail of the angiotensin II type I receptor (AT1R)...
February 17, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28212815/systematic-testing-of-enzyme-perturbation-sensitivities-via-graded-dcas9-modulation-in-saccharomyces-cerevisiae
#2
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/28212662/marked-central-nervous-system-pathology-in-cd59-knockout-rats-following-passive-transfer-of-neuromyelitis-optica-immunoglobulin-g
#3
Xiaoming Yao, Alan S Verkman
Neuromyelitis optica spectrum disorders (herein called NMO) is an inflammatory demyelinating disease of the central nervous system in which pathogenesis involves complement-dependent cytotoxicity (CDC) produced by immunoglobulin G autoantibodies targeting aquaporin-4 (AQP4-IgG) on astrocytes. We reported evidence previously, using CD59(-/-) mice, that the membrane-associated complement inhibitor CD59 modulates CDC in NMO (Zhang and Verkman, J. Autoimmun. 53:67-77, 2014). Motivated by the observation that rats, unlike mice, have human-like complement activity, here we generated CD59(-/-) rats to investigate the role of CD59 in NMO and to create NMO pathology by passive transfer of AQP4-IgG under conditions in which minimal pathology is produced in normal rats...
February 17, 2017: Acta Neuropathologica Communications
https://www.readbyqxmd.com/read/28212528/the-crispr-cas9%C3%A2-system-efficiently-reverts-the-tumorigenic-ability-of-bcr-abl-in-vitro-and-in-a-xenograft-model-of-chronic-myeloid-leukemia
#4
Ignacio García-Tuñón, María Hernández-Sánchez, José Luis Ordoñez, Veronica Alonso-Pérez, Miguel Álamo-Quijada, Rocio Benito, Carmen Guerrero, Jesús María Hernández-Rivas, Manuel Sánchez-Martín
CRISPR/Cas9 technology was used to abrogate p210 oncoprotein expression in the Boff-p210 cell line, a pro-B line derived from interlukin-3-dependent Baf/3, that shows IL-3-independence arising from the constitutive expression of BCR-ABL p210. Using this approach, pools of Boff-p210-edited cells and single edited cell-derived clones were obtained and functionally studied in vitro. The loss of p210 expression in Boff-p210 cells resulted in the loss of ability to grow in the absence of IL-3, as the Baf/3 parental line, showing significantly increased apoptosis levels...
February 9, 2017: Oncotarget
https://www.readbyqxmd.com/read/28211892/high-throughput-in-situ-cell-electroporation-microsystem-for-parallel-delivery-of-single-guide-rnas-into-mammalian-cells
#5
Shengtai Bian, Yicen Zhou, Yawei Hu, Jing Cheng, Xiaofang Chen, Youchun Xu, Peng Liu
Arrayed genetic screens mediated by the CRISPR/Cas9 technology with single guide RNA (sgRNA) libraries demand a high-throughput platform capable of transfecting diverse cell types at a high efficiency in a genome-wide scale for detection and analysis of sophisticated cellular phenotypes. Here we developed a high-throughput in situ cell electroporation (HiCEP) microsystem which leveraged the superhydrophobic feature of the microwell array to achieve individually controlled conditions in each microwell and coupled an interdigital electrode array chip with the microwells in a modular-based scheme for highly efficient delivery of exogenous molecules into cells...
February 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28209983/generation-and-quantitative-proteomics-analysis-of-ck2%C3%AE-%C3%AE-cells
#6
Christian Borgo, Cinzia Franchin, Stefano Scalco, Valentina Bosello-Travain, Arianna Donella-Deana, Giorgio Arrigoni, Mauro Salvi, Lorenzo A Pinna
CK2 is a ubiquitous, constitutively active, highly pleiotropic, acidophilic Ser/Thr protein kinase whose holoenzyme is composed of two catalytic (α and/or α') subunits and a dimer of a non-catalytic β subunit. Abnormally high CK2 level/activity is often associated with malignancy and a variety of cancer cells have been shown to rely on it to escape apoptosis. To gain information about the actual "druggability" of CK2 and to dissect CK2 dependent cellular processes that are instrumental to the establishment and progression of neoplasia we have exploited the CRISPR/Cas9 genome editing technology to generate viable clones of C2C12 myoblasts devoid of either both the CK2 catalytic subunits or its regulatory β-subunit...
February 17, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28209967/optimization-of-the-production-of-knock-in-alleles-by-crispr-cas9-microinjection-into-the-mouse-zygote
#7
Aurélien Raveux, Sandrine Vandormael-Pournin, Michel Cohen-Tannoudji
Microinjection of the CRISPR/Cas9 system in zygotes is an efficient and comparatively fast method to generate genetically modified mice. So far, only few knock-in mice have been generated using this approach, and because no systematic study has been performed, parameters controlling the efficacy of CRISPR/Cas9-mediated targeted insertion are not fully established. Here, we evaluated the effect of several parameters on knock-in efficiency changing only one variable at a time. We found that knock-in efficiency was dependent on injected Cas9 mRNA and single-guide RNA concentrations and that cytoplasmic injection resulted in more genotypic complexity compared to pronuclear injection...
February 17, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28209587/genome-surgery-using-cas9-ribonucleoproteins-for-the-treatment-of-age-related-macular-degeneration
#8
Kyoungmi Kim, Sung Wook Park, Jin Hyoung Kim, Seung Hwan Lee, Daesik Kim, Taeyoung Koo, Kwang-Eun Kim, Jeong Hun Kim, Jin-Soo Kim
RNA-guided genome surgery using CRISPR-Cas9 nucleases has shown promise for the treatment of diverse genetic diseases. Yet, the potential of such nucleases for therapeutic applications in nongenetic diseases is largely unexplored. Here, we focus on age-related macular degeneration (AMD), a leading cause of blindness in adults, which is associated with retinal overexpression of, rather than mutations in, the VEGFA gene. Subretinal injection of preassembled, Vegfa gene-specific Cas9 ribonucleoproteins (RNPs) into the adult mouse eye gave rise to mutagenesis at the target site in the retinal pigment epithelium...
February 16, 2017: Genome Research
https://www.readbyqxmd.com/read/28207183/combining-the-auxin-inducible-degradation-system-with-crispr-cas9-based-genome-editing-for-the-conditional-depletion-of-endogenous-drosophila-melanogaster-proteins
#9
Melinda Bence, Ferenc Jankovics, Tamás Lukácsovich, Miklós Erdélyi
Inducible protein degradation techniques have considerable advantages over classical genetic approaches, which generate loss-of-function phenotypes at the gene or mRNA level. The plant-derived auxin-inducible degradation system (AID) is a promising technique which enables the degradation of target proteins tagged with the AID motif in non-plant cells. Here, we present a detailed characterization of this method employed during the adult oogenesis of Drosophila. Furthermore, with the help of CRISPR/Cas9-based genome editing, we improve the utility of the AID system in the conditional elimination of endogenously expressed proteins...
February 16, 2017: FEBS Journal
https://www.readbyqxmd.com/read/28207001/genome-editing-using-facs-enrichment-of-nuclease-expressing-cells-and-indel-detection-by-amplicon-analysis
#10
Lindsey A Lonowski, Yoshiki Narimatsu, Anjum Riaz, Catherine E Delay, Zhang Yang, Francesco Niola, Katarzyna Duda, Elke A Ober, Henrik Clausen, Hans H Wandall, Steen H Hansen, Eric P Bennett, Morten Frödin
This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28205648/identification-of-a-novel-nampt-inhibitor-by-crispr-cas9-chemogenomic-profiling-in-mammalian-cells
#11
David Estoppey, Jeffrey W Hewett, Chantale T Guy, Edmund Harrington, Jason R Thomas, Markus Schirle, Rachel Cuttat, Annick Waldt, Bertran Gerrits, Zinger Yang, Sven Schuierer, Xuewen Pan, Kevin Xie, Walter Carbone, Judith Knehr, Alicia Lindeman, Carsten Russ, Elizabeth Frias, Gregory R Hoffman, Malini Varadarajan, Nadire Ramadan, John S Reece-Hoyes, Qiong Wang, Xin Chen, Gregory McAllister, Guglielmo Roma, Tewis Bouwmeester, Dominic Hoepfner
Chemogenomic profiling is a powerful and unbiased approach to elucidate pharmacological targets and the mechanism of bioactive compounds. Until recently, genome-wide, high-resolution experiments of this nature have been limited to fungal systems due to lack of mammalian genome-wide deletion collections. With the example of a novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, we demonstrate that the CRISPR/Cas9 system enables the generation of transient homo- and heterozygous deletion libraries and allows for the identification of efficacy targets and pathways mediating hypersensitivity and resistance relevant to the compound mechanism of action...
February 16, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28205605/the-role-of-the-rna-demethylase-fto-fat-mass-and-obesity-associated-and-mrna-methylation-in-hippocampal-memory-formation
#12
Brandon J Walters, Valentina Mercaldo, Colleen J Gillon, Matthew Yip, Rachael L Neve, Frederick M Boyce, Paul W Frankland, Sheena A Josselyn
The formation of long-lasting memories requires coordinated changes in gene expression and protein synthesis (Davis and Squire, 1984; Duvarci et al, 2008; Hernandez and Abel, 2008). Although many studies implicate DNA modifications (DNA methylation, histone modifications) in memory formation, the contributions of RNA modifications remain largely unexplored. Here, we investigated the role of mRNA methylation in hippocampal-dependent memory formation in mice. RNA modifications are highly dynamic and readily reversible (Jia et al, 2013)...
February 16, 2017: Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology
https://www.readbyqxmd.com/read/28205168/crispr-cas9-mediated-generation-of-niemann-pick-c1-knockout-cell-line
#13
Ximing Du, Ivan Lukmantara, Hongyuan Yang
Generating a cholesterol storage phenotype of Niemann-Pick Type C (NPC) disease is important for investigating the mechanisms of intracellular cholesterol trafficking, as well as screening drugs for potential treatment of NPC disease. The use of the CRISPR/Cas9 technology to knockout specific genes within the genome of mammals has become routine in the past few years. Here, we describe a protocol for producing a cellular NPC cholesterol storage phenotype in HeLa cells using the CRISPR-Cas9 system to disrupt the NPC1 gene...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28203643/establishing-elements-of-a-synthetic-biology-platform-for-vaccinia-virus-production-biobrick%C3%A2-design-serum-free-virus-production-and-microcarrier-based-cultivation-of-cv-1-cells
#14
Shuchang Liu, Ludmila Ruban, Yaohe Wang, Yuhong Zhou, Darren N Nesbeth
Vaccinia virus (VACV) is an established vector for vaccination and is beginning to prove effective as an oncolytic agent. Industrial production of VACV stands to benefit in future from advances made by synthetic biology in genome engineering and standardisation. The CV-1 cell line can be used for VACV propagation and has been used extensively with the CRISPR/Cas9 system for making precise edits of the VACV genome. Here we take first steps toward establishing a scalable synthetic biology platform for VACV production with CV-1 cells featuring standardised biological tools and serum free cell cultivation...
February 2017: Heliyon
https://www.readbyqxmd.com/read/28202911/what-rheumatologists-need-to-know-about-crispr-cas9
#15
REVIEW
Gary J Gibson, Maozhou Yang
CRISPR/Cas9 genome editing technology has taken the research world by storm since its use in eukaryotes was first proposed in 2012. Publications describing advances in technology and new applications have continued at an unrelenting pace since that time. In this Review, we discuss the application of CRISPR/Cas9 for creating gene mutations - the application that initiated the current avalanche of interest - and new developments that have largely answered initial concerns about its specificity and ability to introduce new gene sequences...
February 9, 2017: Nature Reviews. Rheumatology
https://www.readbyqxmd.com/read/28202721/distinct-c-mannosylation-of-netrin-receptor-thrombospondin-type-1-repeats-by-mammalian-dpy19l1-and-dpy19l3
#16
Aleksandra Shcherbakova, Birgit Tiemann, Falk F R Buettner, Hans Bakker
Thrombospondin type 1 repeats (TSRs) occur in diverse proteins involved in adhesion and signaling. The two extracellular TSRs of the netrin receptor UNC5A contain WxxWxxWxxC motifs that can be C-mannosylated on all tryptophans. A single C-mannosyltransferase (dumpy-19, DPY-19), modifying the first two tryptophans, occurs in Caenorhabditis elegans, but four putative enzymes (DPY-19-like 1-4, DPY19L1-4) exist in mammals. Single and triple CRISPR-Cas9 knockouts of the three homologs that are expressed in Chinese hamster ovary cells (DPY19L1, DPY19L3, and DPY19L4) and complementation experiments with mouse homologs showed that DPY19L1 preferentially mannosylates the first two tryptophans and DPY19L3 prefers the third, whereas DPY19L4 has no function in TSR glycosylation...
February 15, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28202491/organoid-technologies-meet-genome-engineering
#17
REVIEW
Jing Nie, Eri Hashino
Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications...
February 15, 2017: EMBO Reports
https://www.readbyqxmd.com/read/28198371/method-for-dual-viral-vector-mediated-crispr-cas9-gene-disruption-in-primary-human-endothelial-cells
#18
Haixia Gong, Menglin Liu, Jeff Klomp, Bradley J Merrill, Jalees Rehman, Asrar B Malik
Human endothelial cells (ECs) are widely used to study mechanisms of angiogenesis, inflammation, and endothelial permeability. Targeted gene disruption induced by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated Protein 9 (Cas9) nuclease gene editing is potentially an important tool for definitively establishing the functional roles of individual genes in ECs. We showed that co-delivery of adenovirus encoding EGFP-tagged Cas9 and lentivirus encoding a single guide RNA (sgRNA) in primary human lung microvascular ECs (HLMVECs) disrupted the expression of the Tie2 gene and protein...
February 15, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28197365/crispr-cas9-mediated-disruption-of-pd-1-on-human-t-cells-for-adoptive-cellular-therapies-of-ebv-positive-gastric-cancer
#19
Shu Su, Zhengyun Zou, Fangjun Chen, Naiqing Ding, Juan Du, Jie Shao, Lin Li, Yao Fu, Bian Hu, Yang Yang, Huizi Sha, Fanyan Meng, Jia Wei, Xingxu Huang, Baorui Liu
The successful use of immune cell checkpoint inhibitors PD-1 and PD-L1, over the past 5 y has raised the concern of using immunotherapy to treat various cancers. Epstein-Barr virus-associated gastric cancer (EBVaGC) exhibits high infiltration of lymphocytes and high amplification of immune-related genes including PD-L1 as distinguished from Epstein-Barr virus-non-associated gastric cancer (EBVnGC). Here, we presume that this PD-1/PD-L1 pathway may hinder the efficacy of adoptive T cell therapy toward EBVaGC...
2017: Oncoimmunology
https://www.readbyqxmd.com/read/28196955/identification-of-human-junctional-adhesion-molecule-1-as-a-functional-receptor-for-the-hom-1-calicivirus-on-human-cells
#20
Stanislav V Sosnovtsev, Carlos Sandoval-Jaime, Gabriel I Parra, Christine M Tin, Ronald W Jones, Jo Soden, Donna Barnes, Jim Freeth, Alvin W Smith, Kim Y Green
The Hom-1 vesivirus was reported in 1998 following the inadvertent transmission of the animal calicivirus San Miguel sea lion virus to a human host in a laboratory. We characterized the Hom-1 strain and investigated the mechanism by which human cells could be infected. An expression library of 3,559 human plasma membrane proteins was screened for reactivity with Hom-1 virus-like particles, and a single interacting protein, human junctional adhesion molecule 1 (hJAM1), was identified. Transient expression of hJAM1 conferred susceptibility to Hom-1 infection on nonpermissive Chinese hamster ovary (CHO) cells...
February 14, 2017: MBio
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