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CRISPR IPS

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https://www.readbyqxmd.com/read/28107504/sox10-nano-lantern-reporter-human-ips-cells-a-versatile-tool-for-neural-crest-research
#1
Tomoko Horikiri, Hiromi Ohi, Mitsuaki Shibata, Makoto Ikeya, Morio Ueno, Chie Sotozono, Shigeru Kinoshita, Takahiko Sato
The neural crest is a source to produce multipotent neural crest stem cells that have a potential to differentiate into diverse cell types. The transcription factor SOX10 is expressed through early neural crest progenitors and stem cells in vertebrates. Here we report the generation of SOX10-Nano-lantern (NL) reporter human induced pluripotent stem cells (hiPS) by using CRISPR/Cas9 systems, that are beneficial to investigate the generation and maintenance of neural crest progenitor cells. SOX10-NL positive cells are produced transiently from hiPS cells by treatment with TGFβ inhibitor SB431542 and GSK3 inhibitor CHIR99021...
2017: PloS One
https://www.readbyqxmd.com/read/28102837/precise-and-efficient-scarless-genome-editing-in-stem-cells-using-correct
#2
Dylan Kwart, Dominik Paquet, Shaun Teo, Marc Tessier-Lavigne
CRISPR/Cas9 is a promising tool for genome-editing DNA in cells with single-base-pair precision, which allows novel in vitro models of human disease to be generated-e.g., in pluripotent stem cells. However, the accuracy of intended sequence changes can be severely diminished by CRISPR/Cas9's propensity to re-edit previously modified loci, causing unwanted mutations (indels) alongside intended changes. Here we describe a genome-editing framework termed consecutive re-guide or re-Cas steps to erase CRISPR/Cas-blocked targets (CORRECT), which, by exploiting the use of highly efficacious CRISPR/Cas-blocking mutations in two rounds of genome editing, enables accurate, efficient and scarless introduction of specific base changes-for example, in human induced pluripotent (iPS) stem cells...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28090699/modelling-irf8-deficient-human-hematopoiesis-and-dendritic-cell-development-with-engineered-ips-cells
#3
Stephanie Sontag, Malrun Förster, Jie Qin, Paul Wanek, Saskia Mitzka, Herdit M Schüler, Steffen Koschmieder, Stefan Rose-John, Kristin Seré, Martin Zenke
Human induced pluripotent stem (iPS) cells can differentiate into cells of all three germ layers, including hematopoietic stem cells and their progeny. Interferon regulatory factor 8 (IRF8) is a transcription factor, which acts in hematopoiesis as lineage determining factor for myeloid cells, including dendritic cells (DC). Autosomal recessive or dominant IRF8 mutations occurring in patients cause severe monocytic and DC immunodeficiency. To study IRF8 in human hematopoiesis we generated human IRF8-/- iPS cells and IRF8-/- embryonic stem (ES) cells using RNA guided CRISPR/Cas9n genome editing...
January 16, 2017: Stem Cells
https://www.readbyqxmd.com/read/27825983/accelerating-glioblastoma-drug-discovery-convergence-of-patient-derived-models-genome-editing-and-phenotypic-screening
#4
Eoghan O'Duibhir, Neil Carragher, Steven M Pollard
Patients diagnosed with glioblastoma (GBM) continue to face a bleak prognosis. It is critical that new effective therapeutic strategies are developed. GBM stem cells have molecular hallmarks of neural stem and progenitor cells and it is possible to propagate both non-transformed normal neural stem cells and GBM stem cells, in defined, feeder-free, adherent culture. These primary stem cell lines provide an experimental model that is ideally suited to cell-based drug discovery or genetic screens in order to identify tumour-specific vulnerabilities...
November 4, 2016: Molecular and Cellular Neurosciences
https://www.readbyqxmd.com/read/27792189/non-smc-element-2-nsmce2-of-the-smc5-6-complex-helps-to-resolve-topological-stress
#5
Dideke E Verver, Yi Zheng, Dave Speijer, Ron Hoebe, Henk L Dekker, Sjoerd Repping, Jan Stap, Geert Hamer
The structural maintenance of chromosomes (SMC) protein complexes shape and regulate the structure and dynamics of chromatin, thereby controlling many chromosome-based processes such as cell cycle progression, differentiation, gene transcription and DNA repair. The SMC5/6 complex is previously described to promote DNA double-strand breaks (DSBs) repair by sister chromatid recombination, and found to be essential for resolving recombination intermediates during meiotic recombination. Moreover, in budding yeast, SMC5/6 provides structural organization and topological stress relief during replication in mitotically dividing cells...
October 26, 2016: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/27768763/reactivation-of-fmr1-by-crispr-cas9-mediated-deletion-of-the-expanded-cgg-repeat-of-the-fragile-x-chromosome
#6
Nina Xie, He Gong, Joshua A Suhl, Pankaj Chopra, Tao Wang, Stephen T Warren
Fragile X syndrome (FXS) is a common cause of intellectual disability that is most often due to a CGG-repeat expansion mutation in the FMR1 gene that triggers epigenetic gene silencing. Epigenetic modifying drugs can only transiently and modestly induce FMR1 reactivation in the presence of the elongated CGG repeat. As a proof-of-principle, we excised the expanded CGG-repeat in both somatic cell hybrids containing the human fragile X chromosome and human FXS iPS cells using the CRISPR/Cas9 genome editing. We observed transcriptional reactivation in approximately 67% of the CRISPR cut hybrid colonies and in 20% of isolated human FXS iPSC colonies...
2016: PloS One
https://www.readbyqxmd.com/read/27143231/highly-efficient-baculovirus-mediated-multigene-delivery-in-primary-cells
#7
Maysam Mansouri, Itxaso Bellon-Echeverria, Aurélien Rizk, Zahra Ehsaei, Chiara Cianciolo Cosentino, Catarina S Silva, Ye Xie, Frederick M Boyce, M Wayne Davis, Stephan C F Neuhauss, Verdon Taylor, Kurt Ballmer-Hofer, Imre Berger, Philipp Berger
Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including, synthetic and structural biology, cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity, thus impeding unrestricted multigene expression. We developed MultiPrime, a modular, non-cytotoxic, non-integrating, baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters...
2016: Nature Communications
https://www.readbyqxmd.com/read/26826926/generation-and-validation-of-pax7-reporter-lines-from-human-ips-cells-using-crispr-cas9-technology
#8
Jianbo Wu, Samuel D Hunt, Haipeng Xue, Ying Liu, Radbod Darabi
Directed differentiation of iPS cells toward various tissue progenitors has been the focus of recent research. Therefore, generation of tissue-specific reporter iPS cell lines provides better understanding of developmental stages in iPS cells. This technical report describes an efficient strategy for generation and validation of knock-in reporter lines in human iPS cells using the Cas9-nickase system. Here, we have generated a knock-in human iPS cell line for the early myogenic lineage specification gene of PAX7...
March 2016: Stem Cell Research
https://www.readbyqxmd.com/read/26657769/oct4-activity-during-conversion-of-human-intermediately-reprogrammed-stem-cells-to-ipscs-through-mesenchymal-epithelial-transition
#9
Rika Teshigawara, Kunio Hirano, Shogo Nagata, Justin Ainscough, Takashi Tada
To facilitate understanding the mechanisms of somatic reprogramming to human induced pluripotent stem cells (iPSCs), we have established intermediately reprogrammed stem cells (iRSCs), human mesenchymal cells that express exogenous Oct4, Sox2, Klf4 and c-Myc (OSKM) and endogenous SOX2 and NANOG. iRSCs can be stably maintained at low density. At high density, however, they are induced to enter mesenchymal-epithelial transition (MET), resulting in reprogramming to an iPSC state. Morphological changes through MET correlate with silencing of exogenous OSKM, and upregulation of endogenous OCT4...
January 1, 2016: Development
https://www.readbyqxmd.com/read/26657644/modeling-alzheimer-s-disease-with-human-induced-pluripotent-stem-ips-cells
#10
REVIEW
Alison E Mungenast, Sandra Siegert, Li-Huei Tsai
In the last decade, induced pluripotent stem (iPS) cells have revolutionized the utility of human in vitro models of neurological disease. The iPS-derived and differentiated cells allow researchers to study the impact of a distinct cell type in health and disease as well as performing therapeutic drug screens on a human genetic background. In particular, clinical trials for Alzheimer's disease (AD) have been failing. Two of the potential reasons are first, the species gap involved in proceeding from initial discoveries in rodent models to human studies, and second, an unsatisfying patient stratification, meaning subgrouping patients based on the disease severity due to the lack of phenotypic and genetic markers...
June 2016: Molecular and Cellular Neurosciences
https://www.readbyqxmd.com/read/26634302/crispr-cas9-mediated-conversion-of-human-platelet-alloantigen-allotypes
#11
Nanyan Zhang, Huiying Zhi, Brian R Curtis, Sridhar Rao, Chintan Jobaliya, Mortimer Poncz, Deborah L French, Peter J Newman
Human platelet alloantigens (HPAs) reside on functionally important platelet membrane glycoproteins and are caused by single nucleotide polymorphisms in the genes that encode them. Antibodies that form against HPAs are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia and posttransfusion purpura. The HPA-1a/HPA-1b alloantigen system, also known as the Pl(A1)/Pl(A2) polymorphism, is the most frequently implicated HPA among whites, and a single Leu33Pro amino acid polymorphism within the integrin β3 subunit is responsible for generating the HPA-1a/HPA-1b alloantigenic epitopes...
February 11, 2016: Blood
https://www.readbyqxmd.com/read/26584543/simultaneous-reprogramming-and-gene-correction-of-patient-fibroblasts
#12
Sara E Howden, John P Maufort, Bret M Duffin, Andrew G Elefanty, Edouard G Stanley, James A Thomson
The derivation of genetically modified induced pluripotent stem (iPS) cells typically involves multiple steps, requiring lengthy cell culture periods, drug selection, and several clonal events. We report the generation of gene-targeted iPS cell lines following a single electroporation of patient-specific fibroblasts using episomal-based reprogramming vectors and the Cas9/CRISPR system. Simultaneous reprogramming and gene targeting was tested and achieved in two independent fibroblast lines with targeting efficiencies of up to 8% of the total iPS cell population...
December 8, 2015: Stem Cell Reports
https://www.readbyqxmd.com/read/26525194/efficient-genomic-correction-methods-in-human-ips-cells-using-crispr-cas9-system
#13
REVIEW
Hongmei Lisa Li, Peter Gee, Kentaro Ishida, Akitsu Hotta
Precise gene correction using the CRISPR-Cas9 system in human iPS cells holds great promise for various applications, such as the study of gene functions, disease modeling, and gene therapy. In this review article, we summarize methods for effective editing of genomic sequences of iPS cells based on our experiences correcting dystrophin gene mutations with the CRISPR-Cas9 system. Designing specific sgRNAs as well as having efficient transfection methods and proper detection assays to assess genomic cleavage activities are critical for successful genome editing in iPS cells...
May 15, 2016: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/26427458/personalized-medicine-cell-and-gene-therapy-based-on-patient-specific-ipsc-derived-retinal-pigment-epithelium-cells
#14
REVIEW
Yao Li, Lawrence Chan, Huy V Nguyen, Stephen H Tsang
Interest in generating human induced pluripotent stem (iPS) cells for stem cell modeling of diseases has overtaken that of patient-specific human embryonic stem cells due to the ethical, technical, and political concerns associated with the latter. In ophthalmology, researchers are currently using iPS cells to explore various applications, including: (1) modeling of retinal diseases using patient-specific iPS cells; (2) autologous transplantation of differentiated retinal cells that undergo gene correction at the iPS cell stage via gene editing tools (e...
2016: Advances in Experimental Medicine and Biology
https://www.readbyqxmd.com/read/26382709/mutations-in-the-noncoding-genome
#15
REVIEW
Cheryl A Scacheri, Peter C Scacheri
PURPOSE OF REVIEW: Clinical diagnostic sequencing currently focuses on identifying causal mutations in the exome, wherein most disease-causing mutations are known to occur. The rest of the genome is mostly comprised of regulatory elements that control gene expression, but these have remained largely unexplored in clinical diagnostics due to the high cost of whole genome sequencing and interpretive challenges. The purpose of this review is to illustrate examples of diseases caused by mutations in regulatory elements and introduce the diagnostic potential for whole genome sequencing...
December 2015: Current Opinion in Pediatrics
https://www.readbyqxmd.com/read/26241222/the-egg-and-the-nucleus-a-battle-for-supremacy
#16
J B Gurdon
This brief introduction is followed by a published version of my Nobel Laureate lecture, re-published herein with the kind permission of the Nobel Foundation. Much has happened since my original research, for which that prize was awarded. Hence, I am pleased to offer a few thoughts about the future of my research and its possible impact on humankind.Although the original work on nuclear transfer and reprogramming was done over half a century ago, advances continue to be made. In particular the Takahashi and Yamanaka induced pluripotent stem cells (iPS) procedure has opened up the field of cell replacement to a great extent...
2015: Rambam Maimonides Medical Journal
https://www.readbyqxmd.com/read/26122099/pluripotent-stem-cells-for-gene-therapy-of-degenerative-muscle-diseases
#17
REVIEW
Mariana Loperfido, Heather B Steele-Stallard, Francesco Saverio Tedesco, Thierry VandenDriessche
Human pluripotent stem cells represent a unique source for cell-based therapies and regenerative medicine. The intrinsic features of these cells such as their easy accessibility and their capacity to be expanded indefinitely overcome some limitations of conventional adult stem cells. Furthermore, the possibility to derive patient-specific induced pluripotent stem (iPS) cells in combination with the current development of gene modification methods could be used for autologous cell therapies of some genetic diseases...
2015: Current Gene Therapy
https://www.readbyqxmd.com/read/26101162/crispr-mediated-genotypic-and-phenotypic-correction-of-a-chronic-granulomatous-disease-mutation-in-human-ips-cells
#18
Rowan Flynn, Alexander Grundmann, Peter Renz, Walther Hänseler, William S James, Sally A Cowley, Michael D Moore
Chronic granulomatous disease (CGD) is a rare genetic disease characterized by severe and persistent childhood infections. It is caused by the lack of an antipathogen oxidative burst, normally performed by phagocytic cells to contain and clear bacterial and fungal growth. Restoration of immune function can be achieved with heterologous bone marrow transplantation; however, autologous bone marrow transplantation would be a preferable option. Thus, a method is required to recapitulate the function of the diseased gene within the patient's own cells...
October 2015: Experimental Hematology
https://www.readbyqxmd.com/read/26071353/in%C3%A2-vitro-culture-and-in%C3%A2-vitro-fertilization-techniques-for-prairie-voles-microtus-ochrogaster
#19
Kengo Horie, Shizu Hidema, Takashi Hirayama, Katsuhiko Nishimori
Prairie vole (Microtus ochrogaster) is a highly social animal and is a commonly used animal model for neuropsychopharmacological and psychiatric studies. To date, only a few reports on the development of transgenic prairie voles which was primarily due to the suboptimal development of assisted reproductive technology (ART) in prairie voles. Limitations in ART further hinder the development of genetically modified prairie voles such as the application of conventional gene targeting technologies using embryonic stem (ES) or induced pluripotent stem (iPS) cells to generate chimeric prairie voles...
August 7, 2015: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/26059412/generation-of-isogenic-human-ips-cell-line-precisely-corrected-by-genome-editing-using-the-crispr-cas9-system
#20
Benjamin Grobarczyk, Bénédicte Franco, Kevin Hanon, Brigitte Malgrange
Genome engineering and human iPS cells are two powerful technologies, which can be combined to highlight phenotypic differences and identify pathological mechanisms of complex diseases by providing isogenic cellular material. However, very few data are available regarding precise gene correction in human iPS cells. Here, we describe an optimized stepwise protocol to deliver CRISPR/Cas9 plasmids in human iPS cells. We highlight technical issues especially those associated to human stem cell culture and to the correction of a point mutation to obtain isogenic iPS cell line, without inserting any resistance cassette...
October 2015: Stem Cell Reviews
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