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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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Bing Song, Yong Fan, Wenyin He, Detu Zhu, Xiaohua Niu, Ding Wang, Zhanhui Ou, Min Luo, Xiaofang Sun
The generation of beta-thalassemia (β-Thal) patient-specific induced pluripotent stem cells (iPSCs), subsequent homologous recombination-based gene correction of disease-causing mutations/deletions in the β-globin gene (HBB), and their derived hematopoietic stem cell (HSC) transplantation offers an ideal therapeutic solution for treating this disease. However, the hematopoietic differentiation efficiency of gene-corrected β-Thal iPSCs has not been well evaluated in the previous studies. In this study, we used the latest gene-editing tool, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), to correct β-Thal iPSCs; gene-corrected cells exhibit normal karyotypes and full pluripotency as human embryonic stem cells (hESCs) showed no off-targeting effects...
May 1, 2015: Stem Cells and Development
E-Pien Tan, Yilong Li, Martin Del Castillo Velasco-Herrera, Kosuke Yusa, Allan Bradley
The CRISPR-Cas9 system consists of a site-specific, targetable DNA nuclease that holds great potential in gene editing and genome-wide screening applications. To apply the CRISPR-Cas9 system to these assays successfully, the rate at which Cas9 induces DNA breaks at undesired loci must be understood. We characterized the rate of Cas9 off-target activity in typical Cas9 experiments in two human and one mouse cell lines. We analyzed the Cas9 cutting activity of 12 gRNAs in both their targeted sites and ∼90 predicted off-target sites per gRNA...
February 2015: Genesis: the Journal of Genetics and Development
Xuefei Gao, Jason C H Tsang, Fortis Gaba, Donghai Wu, Liming Lu, Pentao Liu
The transcription activator-like effectors (TALEs) and the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR) associated protein (Cas9) utlilize distinct molecular mechanisms in targeting site recognition. The two proteins can be modified to carry additional functional domains to regulate expression of genomic loci in mammalian cells. In this study, we have compared the two systems in activation and suppression of the Oct4 and Nanog loci by targeting their enhancers. Although both are able to efficiently activate the luciferase reporters, the CRISPR/dCas9 system is much less potent in activating the endogenous loci and in the application of reprogramming somatic cells to iPS cells...
November 10, 2014: Nucleic Acids Research
Young Tae Heo, Xiaoyuan Quan, Yong Nan Xu, Soonbong Baek, Hwan Choi, Nam-Hyung Kim, Jongpil Kim
Efficient and precise genetic engineering in livestock such as cattle holds great promise in agriculture and biomedicine. However, techniques that generate pluripotent stem cells, as well as reliable tools for gene targeting in livestock, are still inefficient, and thus not routinely used. Here, we report highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease. First, we generate induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by the ectopic expression of yamanaka factors and GSK3β and MEK inhibitor (2i) treatment...
February 1, 2015: Stem Cells and Development
Takuro Horii, Daiki Tamura, Sumiyo Morita, Mika Kimura, Izuho Hatada
Genome manipulation of human induced pluripotent stem (iPS) cells is essential to achieve their full potential as tools for regenerative medicine. To date, however, gene targeting in human pluripotent stem cells (hPSCs) has proven to be extremely difficult. Recently, an efficient genome manipulation technology using the RNA-guided DNase Cas9, the clustered regularly interspaced short palindromic repeats (CRISPR) system, has been developed. Here we report the efficient generation of an iPS cell model for immunodeficiency, centromeric region instability, facial anomalies syndrome (ICF) syndrome using the CRISPR system...
2013: International Journal of Molecular Sciences
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