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Crispr-cas9

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https://www.readbyqxmd.com/read/28732214/crispr-cas9-technology-based-xenograft-tumors-as-candidate-reference-materials-for-multiple-eml4-alk-rearrangements-testing
#1
Rongxue Peng, Rui Zhang, Guigao Lin, Xin Yang, Ziyang Li, Kuo Zhang, Jiawei Zhang, Jinming Li
The echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (EML4-ALK) rearrangement is an important biomarker that plays a pivotal role in therapeutic decision making for non-small-cell lung cancer (NSCLC) patients. Ensuring accuracy and reproducibility of EML4-ALK testing by fluorescence in situ hybridization, immunohistochemistry, RT-PCR, and next-generation sequencing requires reliable reference materials for monitoring assay sensitivity and specificity...
July 18, 2017: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/28732207/a-crispr-resource-for-individual-combinatorial-or-multiplexed-gene-knockout
#2
Nicolas Erard, Simon R V Knott, Gregory J Hannon
We have combined a machine-learning approach with other strategies to optimize knockout efficiency with the CRISPR/Cas9 system. In addition, we have developed a multiplexed sgRNA expression strategy that promotes the functional ablation of single genes and allows for combinatorial targeting. These strategies have been combined to design and construct a genome-wide, sequence-verified, arrayed CRISPR library. This resource allows single-target or combinatorial genetic screens to be carried out at scale in a multiplexed or arrayed format...
July 20, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28730433/computational-prediction-of-the-immunomodulatory-potential-of-rna-sequences
#3
Gandharva Nagpal, Kumardeep Chaudhary, Sandeep Kumar Dhanda, Gajendra Pal Singh Raghava
Advances in the knowledge of various roles played by non-coding RNAs have stimulated the application of RNA molecules as therapeutics. Among these molecules, miRNA, siRNA, and CRISPR-Cas9 associated gRNA have been identified as the most potent RNA molecule classes with diverse therapeutic applications. One of the major limitations of RNA-based therapeutics is immunotoxicity of RNA molecules as it may induce the innate immune system. In contrast, RNA molecules that are potent immunostimulators are strong candidates for use in vaccine adjuvants...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28729840/cardiac-subtype-specific-modeling-of-kv1-5-ion-channel-deficiency-using-human-pluripotent-stem-cells
#4
Maike Marczenke, Ilaria Piccini, Isabella Mengarelli, Jakob Fell, Albrecht Röpke, Guiscard Seebohm, Arie O Verkerk, Boris Greber
The ultrarapid delayed rectifier K(+) current (IKur), mediated by Kv1.5 channels, constitutes a key component of the atrial action potential. Functional mutations in the underlying KCNA5 gene have been shown to cause hereditary forms of atrial fibrillation (AF). Here, we combine targeted genetic engineering with cardiac subtype-specific differentiation of human induced pluripotent stem cells (hiPSCs) to explore the role of Kv1.5 in atrial hiPSC-cardiomyocytes. CRISPR/Cas9-mediated mutagenesis of integration-free hiPSCs was employed to generate a functional KCNA5 knockout...
2017: Frontiers in Physiology
https://www.readbyqxmd.com/read/28729655/a-crispr-cas9-guidance-rna-screen-platform-for-hiv-provirus-disruption-and-hiv-aids-gene-therapy-in-astrocytes
#5
Zaohua Huang, Madahavan Nair
HIV/AIDS remains a major health threat despite significant advances in the prevention and treatment of HIV infection. The major reason is the inability of existing treatments to eradicate the multiple HIV reservoirs in the human body, including astrocytes in the human brain. CRISPR/Cas9 system is an emerging gene-editing technique with the potential to eliminate or disrupt HIV provirus in HIV reservoir cells, which may lead to a complete cure of HIV/AIDS. The key components of CRISPR/Cas9 are guide RNAs (gRNAs) which determine specific sequence targeting of DNAs...
July 20, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28729543/evaluation-of-atm-heterozygous-mutations-underlying-individual-differences-in-radiosensitivity-using-genome-editing-in-human-cultured-cells
#6
Ekaterina Royba, Tatsuo Miyamoto, Silvia Natsuko Akutsu, Kosuke Hosoba, Hiroshi Tauchi, Yoshiki Kudo, Satoshi Tashiro, Takashi Yamamoto, Shinya Matsuura
Ionizing radiation (IR) induces DNA double-strand breaks (DSBs), which are an initial step towards chromosomal aberrations and cell death. It has been suggested that there are individual differences in radiosensitivity within human populations, and that the variations in DNA repair genes might determine this heterogeneity. However, it is difficult to quantify the effect of genetic variants on the individual differences in radiosensitivity, since confounding factors such as smoking and the diverse genetic backgrounds within human populations affect radiosensitivity...
July 20, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28729249/deletion-of-transcription-factor-binding-motifs-using-the-crispr-spcas9-system-in-the-%C3%AE-globin-lcr
#7
Yea Woon Kim, AeRi Kim
Transcription factors play roles in gene transcription through direct binding to their motifs in genome, and inhibiting this binding provides an effective strategy for studying their roles. Here we applied the CRISPR/spCas9 system to mutate the binding motifs of transcription factors. Binding motifs for erythroid specific transcription factors were mutated in the locus control region hypersensitive sites of the human β-globin locus. Guide RNAs targeting binding motifs were cloned into lentiviral CRISPR vector containing the spCas9 gene, and transduced into MEL/ch11 cells carrying a human chromosome 11...
July 20, 2017: Bioscience Reports
https://www.readbyqxmd.com/read/28728294/-depletion-of-gp73-inhibits-invasion-and-metastasis-of-hepatocellular-carcinoma-cells
#8
L P Liu, J K Chen, Y M Liu, D H Zhang, J Zhang, X L Yang
Objective: To explore the in vitro and in vivo effect of GP73 on the proliferation, invasion and metastasis in hepatocellular carcinoma. Methods: GP73 gene was knocked out using CRISPR/Cas9 gene editing system in H22 and HepG2 cells, and stable knock out strains were constructed. The knockout efficiency was measured by western blot. Colony formation assay was used to detect the effect of GP73 on long-term survival ability. Cells were then highly synchronized in G(1) phase upon treatment with cell synchronization reagents (mimosine), and the percentage of cells in G(2)/M phase at different time points was detected by flow cytometry...
July 23, 2017: Zhonghua Zhong Liu za Zhi [Chinese Journal of Oncology]
https://www.readbyqxmd.com/read/28727785/novel-crispr-cas9-gene-drive-constructs-reveal-insights-into-mechanisms-of-resistance-allele-formation-and-drive-efficiency-in-genetically-diverse-populations
#9
Jackson Champer, Riona Reeves, Suh Yeon Oh, Chen Liu, Jingxian Liu, Andrew G Clark, Philipp W Messer
A functioning gene drive system could fundamentally change our strategies for the control of vector-borne diseases by facilitating rapid dissemination of transgenes that prevent pathogen transmission or reduce vector capacity. CRISPR/Cas9 gene drive promises such a mechanism, which works by converting cells that are heterozygous for the drive construct into homozygotes, thereby enabling super-Mendelian inheritance. Although CRISPR gene drive activity has already been demonstrated, a key obstacle for current systems is their propensity to generate resistance alleles, which cannot be converted to drive alleles...
July 2017: PLoS Genetics
https://www.readbyqxmd.com/read/28727005/rapid-communication-generation-of-fgf5-knockout-sheep-via-the-crispr-cas9-system
#10
R Hu, Z Y Fan, B Y Wang, S L Deng, X S Zhang, J L Zhang, H B Han, Z X Lian
Sheep are an important source of fiber production. Fibroblast growth factor 5 (FGF5) is a dominant inhibitor of length of the anagen phase of the hair cycle. Knockout or silencing of the gene results in a wooly coat in mice, donkeys, dogs, and rabbits. In sheep breeding, wool length is one of the most important wool quality traits. However, traditional breeding cannot accurately and efficiently mediate an advanced genotype into the sheep genome. In this study, we generated 3 knockout sheep via the 1-step clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system...
May 2017: Journal of Animal Science
https://www.readbyqxmd.com/read/28726781/autophagy-inhibition-reduces-chemoresistance-and-tumorigenic-potential-of-human-ovarian-cancer-stem-cells
#11
Anna Pagotto, Giorgia Pilotto, Elena Laura Mazzoldi, Maria Ornella Nicoletto, Simona Frezzini, Anna Pastò, Alberto Amadori
Epithelial ovarian cancer (EOC) is one of the most malignant gynecological tumors with a high mortality rate owing to tumor relapse after anticancer therapies. It is widely accepted that a rare tumor cell population, known as cancer stem cells (CSC), is responsible for tumor progression and relapse; intriguingly, these cells are able to survive nutrient starvation (such as in vitro culture in the absence of glucose) and chemotherapy treatment. Recent data also indicated that chemotherapy resistance is associated with autophagy activation...
July 20, 2017: Cell Death & Disease
https://www.readbyqxmd.com/read/28726007/sharing-mutations-are-biobanks-still-required-in-the-post-crispr-cas9-era
#12
Janet Kenyon, Martin D Fray
Cryopreservation is seen as a key aspect of good colony management which supports the drive towards improvements in animal care and the implementation of the 3Rs. However, following the advent of gene editing technologies, the generation of new mouse models is quicker and cheaper than ever before. This has led some to question the future value of biobanks around the world. In the following commentary, we argue that the need to cryopreserve mouse strains and distribute them from well-funded repositories is as strong as it has ever been...
July 18, 2017: Mammalian Genome: Official Journal of the International Mammalian Genome Society
https://www.readbyqxmd.com/read/28725235/gateway-compatible-crispr-cas9-vectors-and-a-rapid-detection-by-high-resolution-melting-curve-analysis
#13
Cynthia J Denbow, Samantha Lapins, Nick Dietz, Raelynn Scherer, Zachary L Nimchuk, Sakiko Okumoto
CRISPR-Cas9 system rapidly became an indispensable tool in plant biology to perform targeted mutagenesis. A CRISPR-Cas9-mediated double strand break followed by non-homologous end joining (NHEJ) repair most frequently results in a single base pair deletion or insertions (indels), which is hard to detect using methods based on enzymes that detect heteroduplex DNA. In addition, somatic tissues of the T1 generation inevitably contain a mosaic population, in which the portion of cells carrying the mutation can be too small to be detected by the enzyme-based methods...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/28724963/midnolin-is-a-novel-regulator-of-parkin-expression-and-is-associated-with-parkinson-s-disease
#14
Yutaro Obara, Toru Imai, Hidenori Sato, Yuji Takeda, Takeo Kato, Kuniaki Ishii
Midnolin (MIDN) was first discovered in embryonic stem cells, but its physiological and pathological roles are, to date, poorly understood. In the present study, we therefore examined the role of MIDN in detail. We found that in PC12 cells, a model of neuronal cells, MIDN localized primarily to the nucleus and intracellular membranes. Nerve growth factor promoted MIDN gene expression, which was attenuated by specific inhibitors of extracellular signal-regulated kinases 1/2 and 5. MIDN-deficient PC12 cells created using CRISPR/Cas9 technology displayed significantly impaired neurite outgrowth...
July 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28724960/sgrna-expression-of-cripsr-cas9-system-based-on-mirna-polycistrons-as-a-versatile-tool-to-manipulate-multiple-and-tissue-specific-genome-editing
#15
Chen Xie, Yan-Lian Chen, Dong-Fang Wang, Yi-Lin Wang, Tian-Peng Zhang, Hui Li, Fu Liang, Yong Zhao, Guang-Ya Zhang
CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications. Typically, sgRNA is constitutively expressed relying on RNA polymerase III promoters. Polymerase II promoters initiate transcription in a flexible manner, but sgRNAs generated by RNA polymerase II promoter lost their nuclease activity. To express sgRNAs in a tissue-specific fashion and endow CRISPR with more versatile function, a novel system was established in a polycistron, where miRNAs (or shRNAs) and sgRNAs alternately emerged and co-expressed under the control of a single polymerase II promoter...
July 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28724787/disrupted-apical-exocytosis-of-cargo-vesicles-causes-enteropathy-in-fhl5-patients-with-munc18-2-mutations
#16
Georg F Vogel, Jorik M van Rijn, Iris M Krainer, Andreas R Janecke, Carsten Posovzsky, Marta Cohen, Claire Searle, Prevost Jantchou, Johanna C Escher, Natalie Patey, Ernest Cutz, Thomas Müller, Sabine Middendorp, Michael W Hess, Lukas A Huber
Familial hemophagocytic lymphohistiocytosis 5 (FHL5) is an autosomal recessive disease caused by mutations in STXBP2, coding for Munc18-2, which is required for SNARE-mediated membrane fusion. FHL5 causes hematologic and gastrointestinal symptoms characterized by chronic enteropathy that is reminiscent of microvillus inclusion disease (MVID). However, the molecular pathophysiology of FHL5-associated diarrhea is poorly understood. Five FHL5 patients, including four previously unreported patients, were studied...
July 20, 2017: JCI Insight
https://www.readbyqxmd.com/read/28723893/in-vivo-crispr-screening-identifies-ptpn2-as-a-cancer-immunotherapy-target
#17
Robert T Manguso, Hans W Pope, Margaret D Zimmer, Flavian D Brown, Kathleen B Yates, Brian C Miller, Natalie B Collins, Kevin Bi, Martin W LaFleur, Vikram R Juneja, Sarah A Weiss, Jennifer Lo, David E Fisher, Diana Miao, Eliezer Van Allen, David E Root, Arlene H Sharpe, John G Doench, W Nicholas Haining
Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy...
July 19, 2017: Nature
https://www.readbyqxmd.com/read/28723575/crispr-mediated-integration-of-large-gene-cassettes-using-aav-donor-vectors
#18
Rasmus O Bak, Matthew H Porteus
The CRISPR/Cas9 system has recently been shown to facilitate high levels of precise genome editing using adeno-associated viral (AAV) vectors to serve as donor template DNA during homologous recombination (HR). However, the maximum AAV packaging capacity of ∼4.5 kb limits the donor size. Here, we overcome this constraint by showing that two co-transduced AAV vectors can serve as donors during consecutive HR events for the integration of large transgenes. Importantly, the method involves a single-step procedure applicable to primary cells with relevance to therapeutic genome editing...
July 18, 2017: Cell Reports
https://www.readbyqxmd.com/read/28721587/genetically-engineered-cell-lines-for-%C3%AE-1-antitrypsin-expression
#19
Qianqian Ji, Caiping Guo, Chen Xie, Yingdan Wu, Pei Zhang, Hui Li, Yongjun Lu
OBJECTIVES: To establish genetically modified cell lines that can produce functional α1-antitrypsin (AAT), by CRISPR/Cas9-assisted homologous recombination. RESULTS: α1-Antitrypsin deficiency (AATD) is a monogenic heritable disease that often results in lungs and liver damage. Current augmentation therapy is expensive and in short of supply. To develop a safer and more effective therapeutic strategy for AATD, we integrated the AAT gene (SERPINA1, NG_008290.1) into the AAVS1 locus of human cell line HEK293T and assessed the safety and efficacy of CRISPR/Cas9 on producing potential therapeutic cell lines...
July 18, 2017: Biotechnology Letters
https://www.readbyqxmd.com/read/28720677/synthetic-circulating-cell-free-dna-as-quality-control-materials-for-somatic-mutation-detection-in-liquid-biopsy-for-cancer
#20
Rui Zhang, Rongxue Peng, Ziyang Li, Peng Gao, Shiyu Jia, Xin Yang, Jiansheng Ding, Yanxi Han, Jiehong Xie, Jinming Li
BACKGROUND: Detection of somatic genomic alterations in tumor-derived cell-free DNA (cfDNA) in the plasma is challenging owing to the low concentrations of cfDNA, variable detection methods, and complex workflows. Moreover, no proper quality control materials are available currently. METHODS: We developed a set of synthetic cfDNA quality control materials (SCQCMs) containing spike-in cfDNA on the basis of micrococcal nuclease digestion carrying somatic mutations as simulated cfDNA and matched genomic DNA as genetic background to emulate paired tumor-normal samples in real clinical tests...
July 18, 2017: Clinical Chemistry
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