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Inducible crispr

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https://www.readbyqxmd.com/read/29778644/a-trichostatin-a-tsa-sp1-mediated-mechanism-for-the-regulation-of-sall2-tumor-suppressor-in-jurkat-t-cells
#1
Matías I Hepp, David Escobar, Carlos Farkas, Viviana Hermosilla, Claudia Álvarez, Roberto Amigo, José L Gutiérrez, Ariel F Castro, Roxana Pincheira
SALL2 is a transcription factor involved in development and disease. Deregulation of SALL2 has been associated with cancer, suggesting that it plays a role in the disease. However, how SALL2 is regulated and why is deregulated in cancer remain poorly understood. We previously showed that the p53 tumor suppressor represses SALL2 under acute genotoxic stress. Here, we investigated the effect of Histone Deacetylase Inhibitor (HDACi) Trichostatin A (TSA), and involvement of Sp1 on expression and function of SALL2 in Jurkat T cells...
May 17, 2018: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/29778643/inheritance-of-co-edited-genes-by-crispr-based-targeted-nucleotide-substitutions-in-rice
#2
Zenpei Shimatani, Ushio Fujikura, Hisaki Ishii, Yusuke Matsui, Minoru Suzuki, Yuki Ueke, Ken-Ichiro Taoka, Rie Terada, Keiji Nishida, Akihiko Kondo
The CRISPR/Cas9 system is a revolutionary genome-editing tool for directed gene editing in various organisms. Cas9 variants can be applied as molecular homing devices when combined with various functional effectors such as transcriptional activators or DNA modification enzymes. Target-AID is a synthetic complex of nuclease deficient Cas9 fused to an activation-induced cytidine deaminase (AID) that enables targeted nucleotide substitution (C to T or G to A). We previously demonstrated that the introduction of desired point mutations into target genes by Target-AID confers herbicide tolerance to rice callus...
April 25, 2018: Plant Physiology and Biochemistry: PPB
https://www.readbyqxmd.com/read/29775409/transactivation-domain-of-p53-regulates-dna-repair-and-integrity-in-human-ips-cells
#3
Ramaswamy Kannappan, Saidulu Mattapally, Pooja A Wagle, Jianyi Zhang
The role of p53 transactivation domain (p53-TAD), a multifunctional and dynamic domain, on DNA repair and retaining DNA integrity in human iPS cells has never been studied. p53-TAD was knocked out in iPS cells using CRISPR/Cas9 and was confirmed by DNA sequencing. p53-TAD KO cells were characterized by: accelerated proliferation, decreased population doubling time, and unaltered Bcl2, BBC3, IGF1R, Bax and altered Mdm2, p21, and PIDD transcripts expression. In p53-TAD KO cells p53 regulated DNA repair proteins XPA, DNA polH and DDB2 expression were found to be reduced compared to p53-WT cells...
May 18, 2018: American Journal of Physiology. Heart and Circulatory Physiology
https://www.readbyqxmd.com/read/29774125/the-critical-role-that-stat3-plays-in-glioma-initiating-cells-stat3-addiction-in-glioma
#4
Debolina Ganguly, Meiyun Fan, Chuan He Yang, Blazej Zbytek, David Finkelstein, Martine F Roussel, Lawrence M Pfeffer
Glioma-Initiating Cells (GICs) are thought to be responsible for tumor initiation, progression and recurrence in glioblastoma (GBM). In previous studies, we reported the constitutive phosphorylation of the STAT3 transcription factor in GICs derived from GBM patient-derived xenografts, and that STAT3 played a critical role in GBM tumorigenesis. In this study, we show that CRISPR/Cas9-mediated deletion of STAT3 in an established GBM cell line markedly inhibited tumorigenesis by intracranial injection but had little effect on cell proliferation in vitro ...
April 24, 2018: Oncotarget
https://www.readbyqxmd.com/read/29769662/novel-internal-regulators-and-candidate-mirnas-within-mir-379-mir-656-mirna-cluster-can-alter-cellular-phenotype-of-human-glioblastoma
#5
Subhashree Nayak, Meghali Aich, Anupam Kumar, Suman Sengupta, Prajakta Bajad, Parashar Dhapola, Deepanjan Paul, Kiran Narta, Suvendu Purkrait, Bharati Mehani, Ashish Suri, Debojyoti Chakraborty, Arijit Mukhopadhyay, Chitra Sarkar
Clustered miRNAs can affect functioning of downstream pathways due to possible coordinated function. We observed 78-88% of the miR-379/miR-656 cluster (C14MC) miRNAs were downregulated in three sub-types of diffuse gliomas, which was also corroborated with analysis from The Cancer Genome Atlas (TCGA) datasets. The miRNA expression levels decreased with increasing tumor grade, indicating this downregulation as an early event in gliomagenesis. Higher expression of the C14MC miRNAs significantly improved glioblastioma prognosis (Pearson's r = 0...
May 16, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29769640/hypoxia-induced-microrna-191-contributes-to-hepatic-ischemia-reperfusion-injury-through-the-zonab-cyclin-d1-axis
#6
Wenming Pan, Lin Wang, Xiao-Fei Zhang, Hongji Zhang, Jinxiang Zhang, Guoliang Wang, Peng Xu, Yunwei Zhang, Ping Hu, Xiao-Dong Zhang, Run-Lei Du, Hui Wang
Hepatic ischemia/reperfusion injury (IRI) is a common cause of morbidity and mortality in liver transplantation settings and involves severe cell death and inflammatory responses. MicroRNA-191 has recently been reported to be abnormally expressed in hepatocellular carcinoma and other liver diseases in the regulation of important cellular processes. However, little is known about its function and molecular mechanism in IRI. Here, we demonstrate that miR-191 is significantly upregulated in a cultured cell line during hypoxia/reperfusion (H/R) and in liver tissue during IRI in mice...
May 16, 2018: Cell Death and Differentiation
https://www.readbyqxmd.com/read/29769349/hiv-1-activation-of-innate-immunity-depends-strongly-on-the-intracellular-level-of-trex1-and-sensing-of-incomplete-reverse-transcription-products
#7
Swati Kumar, James H Morrison, David Dingli, Eric Poeschla
TREX1 has been reported to degrade cytosolic immune-stimulatory DNA, including viral DNA generated during HIV-1 infection, but the dynamic range of its capacity to suppress innate immune stimulation is unknown and its full role in the viral life cycle remains unclear. A main purpose of our study was to determine how the intracellular level of TREX1 affects HIV-1 activation and avoidance of innate immunity. Using stable over-expression and CRISPR-mediated gene disruption, we engineered a range of TREX1 levels in human THP-1 monocytes...
May 16, 2018: Journal of Virology
https://www.readbyqxmd.com/read/29769309/etoposide-induced-protein-2-4-functions-as-a-regulator-of-the-calcium-atpase-and-protects-pancreatic-%C3%AE-cell-survival
#8
Lin Yuan, Huiyu Wang, Qi Liu, Zhe Wang, Mingshu Zhang, Yan Zhao, Kuo Liang, Liangyi Chen, Tao Xu, Pingyong Xu
Calcium homeostasis is essential for maintaining the viability and function of pancreatic β cells and plays a key role in preventing the development of diabetes. Decreased levels of ATPase sarco-plasmic/endoplasmic reticulum Ca2+ -transporting 2 (ATP2a2), the main calcium pump in β cells, are often found in individuals with diabetes and in diabetic animal models. However, the regulators of ATP2a2 and the molecular mechanisms responsible for controlling ATP2a2 activity remain unclear. Etoposide-induced protein 2...
May 16, 2018: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29766028/a-non-integrating-lentiviral-approach-overcomes-cas9-induced-immune-rejection-to-establish-an-immunocompetent-metastatic-renal-cancer-model
#9
Junhui Hu, Shiruyeh Schokrpur, Maani Archang, Kip Hermann, Allison C Sharrow, Prateek Khanna, Jesse Novak, Sabina Signoretti, Rupal S Bhatt, Beatrice S Knudsen, Hua Xu, Lily Wu
The CRISPR-based technology has revolutionized genome editing in recent years. This technique allows for gene knockout and evaluation of function in cell lines in a manner that is far easier and more accessible than anything previously available. Unfortunately, the ability to extend these studies to in vivo syngeneic murine cell line implantation is limited by an immune response against cells transduced to stably express Cas9. In this study, we demonstrate that a non-integrating lentiviral vector approach can overcome this immune rejection and allow for the growth of transduced cells in an immunocompetent host...
June 15, 2018: Molecular Therapy. Methods & Clinical Development
https://www.readbyqxmd.com/read/29764210/curative-ex-vivo-hepatocyte-directed-gene-editing-in-a-mouse-model-of-hereditary-tyrosinemia-type-1
#10
Caitlin VanLith, Rebekah Guthman, Clara T Nicolas, Kari Allen, Zeji Du, Dong Jin Joo, Scott L Nyberg, Joseph B Lillegard, Raymond Daniel Hickey
Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder caused by deficiency of fumarylacetoacetate hydrolase (FAH). It has been previously shown that ex vivo hepatocyte-directed gene therapy using an integrating lentiviral vector to replace the defective Fah gene can cure liver disease in small and large animal models of HT1. In this study, we hypothesized that ex vivo hepatocyte-directed gene editing using CRISPR-Cas9 could be used to correct a mouse model of HT1, in which a single point mutation results in loss of FAH function...
May 15, 2018: Human Gene Therapy
https://www.readbyqxmd.com/read/29761864/high-throughput-detection-and-screening-of-plantsmodified-by-geneediting-using-quantitative-real-time-pcr
#11
Cheng Peng, Hua Wang, Xiaoli Xu, Xiaofu Wang, Xiaoyun Chen, Wei Wei, Yongmin Lai, Guoquan Liu, Godwin Ian, Jieqin Li, Ling Zhang, Junfeng Xu
Gene editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been developed to detect gene-edited organisms, these techniques are time- and labour-intensive. Meanwhile, few studies have investigated high-throughput detection and screening strategies for plants modified by gene editing. In this study, we developed a simple, sensitive and high-throughput quantitative real-time(qPCR)-based method. The qPCR-based methodexploits two differently labelled probes that are placed within one amplicon at the gene editing target site to simultaneously detect the wild type and a gene-edited mutant...
May 15, 2018: Plant Journal: for Cell and Molecular Biology
https://www.readbyqxmd.com/read/29760402/roles-of-enhancer-rnas-in-rankl-induced-osteoclast-differentiation-identified-by-genome-wide-cap-analysis-of-gene-expression-using-crispr-cas9
#12
Yukako Sakaguchi, Keizo Nishikawa, Shigeto Seno, Hideo Matsuda, Hiroshi Takayanagi, Masaru Ishii
Bidirectional transcription has been proposed to play a role associated with enhancer activity. Transcripts called enhancer RNAs (eRNAs) play important roles in gene regulation; however, their roles in osteoclasts are unknown. To analyse eRNAs in osteoclasts comprehensively, we used cap-analysis of gene expression (CAGE) to detect adjacent transcription start sites (TSSs) that were distant from promoters for protein-coding gene expression. When comparing bidirectional TSSs between osteoclast precursors and osteoclasts, we found that bidirectional TSSs were located in the 5'-flanking regions of the Nrp2 and Dcstamp genes...
May 14, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29755980/importance-of-validating-antibodies-and-small-compound-inhibitors-using-genetic-knockout-studies-t-cell-receptor-induced-cyld-phosphorylation-by-ikk%C3%AE%C2%B5-tbk1-as-a-case-study
#13
Marie Lork, Marja Kreike, Jens Staal, Rudi Beyaert
CYLD is a deubiquitinating enzyme that plays a crucial role in immunity and inflammation as a negative regulator of NF-κB transcription factor and JNK kinase signaling. Defects in either of these pathways contribute to the progression of numerous inflammatory and autoimmune disorders. Therefore, we set out to unravel molecular mechanisms that control CYLD activity in the context of T cell receptor (TCR) signaling. More specifically, we focused on CYLD phosphorylation at Ser418, which can be detected upon immunoblotting of cell extracts with phospho(Ser418)-CYLD specific antibodies...
2018: Frontiers in Cell and Developmental Biology
https://www.readbyqxmd.com/read/29755367/the-transcription-factor-hif-1-enhances-the-radio-resistance-of-mouse-mscs
#14
Irene Calvo-Asensio, Eugène T Dillon, Noel F Lowndes, Rhodri Ceredig
Mesenchymal stromal cells (MSCs) are multipotent progenitors supporting bone marrow hematopoiesis. MSCs have an efficient DNA damage response (DDR) and are consequently relatively radio-resistant cells. Therefore, MSCs are key to hematopoietic reconstitution following total body irradiation (TBI) and bone marrow transplantation (BMT). The bone marrow niche is hypoxic and via the heterodimeric transcription factor Hypoxia-inducible factor-1 (Hif-1), hypoxia enhances the DDR. Using gene knock-down, we have previously shown that the Hif-1α subunit of Hif-1 is involved in mouse MSC radio-resistance, however its exact mechanism of action remains unknown...
2018: Frontiers in Physiology
https://www.readbyqxmd.com/read/29754960/high-level-precise-knockin-of-ipscs-by-simultaneous-reprogramming-and-genome-editing-of-human-peripheral-blood-mononuclear-cells
#15
Wei Wen, Xinxin Cheng, Yawen Fu, Feiying Meng, Jian-Ping Zhang, Lu Zhang, Xiao-Lan Li, Zhixue Yang, Jing Xu, Feng Zhang, Gary D Botimer, Weiping Yuan, Changkai Sun, Tao Cheng, Xiao-Bing Zhang
We have developed an improved episomal vector system for efficient generation of integration-free induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells. More recently, we reported that the use of an optimized CRISPR-Cas9 system together with a double-cut donor increases homology-directed repair-mediated precise gene knockin efficiency by 5- to 10-fold. Here, we report the integration of blood cell reprogramming and genome editing in a single step. We found that expression of Cas9 and KLF4 using a single vector significantly increases genome editing efficiency, and addition of SV40LT further enhances knockin efficiency...
May 4, 2018: Stem Cell Reports
https://www.readbyqxmd.com/read/29754237/crispr-cas9-mediated-genome-editing-and-transcriptional-control-in-yarrowia-lipolytica
#16
Cory Schwartz, Ian Wheeldon
The discovery and adaptation of RNA-guided nucleases has resulted in the rapid development of efficient, scalable, and easily accessible synthetic biology tools for targeted genome editing and transcriptional control. In these systems, for example CRISPR-Cas9 from Streptococcus pyogenes, a protein with nuclease activity is targeted to a specific nucleotide sequence by a short RNA molecule, whereupon binding it cleaves the targeted nucleotide strand. To extend this genome-editing ability to the industrially important oleaginous yeast Yarrowia lipolytica, we developed a set of easily usable and effective CRISPR-Cas9 episomal vectors...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29754234/rapid-control-of-genome-editing-in-human-cells-by-chemical-inducible-crispr-cas-systems
#17
Kaiwen Ivy Liu, Muhammad Nadzim Bin Ramli, Norfala-Aliah Binte Sutrisnoh, Meng How Tan
Genome editing using programmable DNA endonucleases enables the engineering of eukaryotic cells and living organisms with desirable properties or traits. Among the various molecular scissors that have been developed to date, the most versatile and easy-to-use family of nucleases derives from CRISPR-Cas, which exists naturally as an adaptive immune system in bacteria. Recent advances in the CRISPR-Cas technology have expanded our ability to manipulate complex genomes for myriad biomedical and biotechnological applications...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29753273/generation-of-an-induced-pluripotent-stem-cell-line-from-a-patient-with-non-syndromic-cln3-associated-retinal-degeneration-and-a-coisogenic-control-line
#18
Xiao Zhang, Dan Zhang, Shang-Chih Chen, Tina Lamey, Jennifer A Thompson, Terri McLaren, John N De Roach, Fred K Chen, Samuel McLenachan
We report the generation of the human iPSC line LEIi004-A from a patient with late-onset non-syndromic retinitis pigmentosa caused by compound heterozygous mutations in the CLN3 gene. Reprogramming of primary dermal fibroblasts was performed using episomal plasmids containing OCT4, SOX2, KLF4, L-MYC, LIN28, shRNA for p53 and mir302/367 microRNA. To create a coisogenic control line, one CLN3 variant was corrected in the patient-iPSC using CRISPR/Cas9 gene editing to generate the iPSC line LEIi004-A-1.
May 1, 2018: Stem Cell Research
https://www.readbyqxmd.com/read/29752948/functional-and-transcriptomic-insights-into-pathogenesis-of-r9c-phospholamban-mutation-using-human-induced-pluripotent-stem-cell-derived-cardiomyocytes
#19
Delaine K Ceholski, Irene C Turnbull, Chi-Wing Kong, Simon Koplev, Joshua Mayourian, Przemek A Gorski, Francesca Stillitano, Angelos A Skodras, Mathieu Nonnenmacher, Ninette Cohen, Johan L M Björkegren, Daniel R Stroik, Razvan L Cornea, David D Thomas, Ronald A Li, Kevin D Costa, Roger J Hajjar
Dilated cardiomyopathy (DCM) can be caused by mutations in the cardiac protein phospholamban (PLN). We used CRISPR/Cas9 to insert the R9C PLN mutation at its endogenous locus into a human induced pluripotent stem cell (hiPSC) line from an individual with no cardiovascular disease. R9C PLN hiPSC-CMs display a blunted β-agonist response and defective calcium handling. In 3D human engineered cardiac tissues (hECTs), a blunted lusitropic response to β-adrenergic stimulation was observed with R9C PLN. hiPSC-CMs harboring the R9C PLN mutation showed activation of a hypertrophic phenotype, as evidenced by expression of hypertrophic markers and increased cell size and capacitance of cardiomyocytes...
May 9, 2018: Journal of Molecular and Cellular Cardiology
https://www.readbyqxmd.com/read/29752286/genetic-and-cellular-sensitivity-of-caenorhabditis-elegans-to-the-chemotherapeutic-agent-cisplatin
#20
Francisco Javier García-Rodríguez, Carmen Martínez-Fernández, David Brena, Dmytro Kukhtar, Xènia Serrat, Ernest Nadal, Mike Boxem, Sebastian Honnen, Antonio Miranda-Vizuete, Alberto Villanueva, Julián Cerón
Cisplatin and derivatives are commonly used as chemotherapeutic agents. Although the cytotoxic action of cisplatin on cancer cells is very efficient, clinical oncologists need to deal with two major difficulties: (i) the onset of resistance to the drug, and (ii) the cytotoxic effect in patients. Here we use Caenorhabditis elegans to investigate factors influencing the response to cisplatin in multicellular organisms. In this hermaphroditic model organism, we observed that sperm failure is a major cause in cisplatin-induced infertility...
May 10, 2018: Disease Models & Mechanisms
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