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Induced pluripotent stem (iPS) cells have a great impact on biology and medicine, and they are expected to improve regenerative medicine. 
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This protocol extends the use of genome editing technology to the modeling or correction of large chromosomal rearrangements and short nucleotide repeat expansions. The authors use the CRISPR/Cas system to edit human induced pluripotent stem cells.
BigField GEG Tech's insight:
In this study, the scientists describe a detailed procedure for the modeling or correction of large chromosomal rearrangements and short nucleotide repeat expansions using engineered nucleases in human induced pluripotent stem cells (hiPSCs) from a healthy donor and patients with SVs. This protocol enables the correction of large inverted segments and short nucleotide repeat expansions in diseases such as hemophilia A, fragile X syndrome, Hunter syndrome, and Friedreich's ataxia.
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BigField GEG Tech's insight:
The authors discuss experimental considerations, limitations and critical aspects which will guide the investigator for successful implementation of the genome editing technology in human PSCs using designer nucleases.
Joye Shuist's curator insight,
March 14, 2016 9:52 PM
The authors discuss experimental considerations, limitations and critical aspects which will guide the investigator for successful implementation of the genome editing technology in human PSCs using designer nucleases.
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BigField GEG Tech's insight:
In this study, the authors used the CRISPR system to generate a knock-in reporter human iPS cell line for MYF5, as an early myogenic specification gene, to allow prospective identification and purification of myogenic progenitors from human iPS cells. Furthermore, in order to prove the reporter function, endogenous MYF5 expression was induced using a novel dead Cas9-VP160 transcriptional activator. These data provides valuable guidelines for generation of knock-in reporter human iPS cell lines for myogenic genes which can be used for disease modeling, drug screening, gene correction and future in vivo applications.
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BigField GEG Tech's insight:
In this study, the authors used the CRISPR/Cas9 system to transform Leu33-positive megakaryocyte-like DAMI cells and induced pluripotent stem (iPS) cells to the Pro33 allotype.
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BigField GEG Tech's insight:
The authors used CRISPR/Cas9 to correct β-Thal iPSCs, gene-corrected cells exhibit normal karyotypes and fully pluripotency as hES, showed no off-targeting effects. Their results shown that the gene-corrected β-Thal iPS cell lines restored HBB expression and reduced reactive oxygen species (ROS) production. www;geg-tech.com
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BigField GEG Tech's insight:
Murine induced pluripotent stem cells (iPSCs) lacking IL-1 receptor type 1 (IL-1R1), engineered using the CRISPR/Cas9 system, are able to form cartilaginous tissue that is resistant to IL-1α-mediated inflammation, a new study shows.
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BigField GEG Tech's insight:
Here the authors show that CRISPR/Cas9 system can be utilized to completely remove the large repeat expansion mutation within C9orf72 in patient-derived induced pluripotent stem (iPS) cells.
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BigField GEG Tech's insight:
This review addresses this need directly by providing both the up-to-date biochemical rationale of CRISPR-mediated genome engineering and detailed practical guidelines for the design and execution of CRISPR experiments in cell models. Ultimately, this review will serve as a timely and comprehensive guide for this fast developing technology.
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BigField GEG Tech's insight:
In this study, the scientists used the CRISPR system to do gene correction in reprogrammed fibroblasts of a patient with β-thalassemia into transgene-free naïve iPSCs with molecular signatures of ground-state pluripotency. Therefore, their findings demonstrate the feasibility and superiority of using patient-specific iPSCs in the naïve state for disease modeling, gene editing, and future clinical therapy.
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BigField GEG Tech's insight:
The scientists describe an optimized stepwise protocol to deliver CRISPR/Cas9 plasmids in human iPS cells. Based on a two-steps clonal isolation protocol (mechanical picking followed by enzymatic dissociation), they succeed to select and expand corrected human iPS cell line with a great efficiency, more than 2 % of the sequenced colonies (and about 15% to obtain KO by NHEJ).
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BigField GEG Tech's insight:
In this study, the authors investigated the relative efficiencies of CRISPR/Cas9 and TALENs in human iPSC lines for inducing both non-homologous end joining (NHEJ)-mediated gene disruption and homologous donor-based precise genome editing (HR). In these two contexts, they observed a higher efficency of the CRISPR/Cas9 system. In the first case (NHEJ), for three loci tested, Cas9-gRNAs induced between 10-100 fold more indels than did TALENs in human iPSCs, reaching the level of 0.7% to 2.5% mutation rates. About the second case (HR),the largest difference was observed at the AAVS1 site targeting where the Cas9-gRNA showed about 2-fold advantage over TALENs. geg-tech.com
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Fabry disease is caused by a genetic deficiency of α-galactosidase A (GLA), leading to the accumulation of its substrates such as globotriaosylceramide and globotriaosylsphingosine. Researchers have therefore developed a modified enzyme, modified α-N-acetylgalactosaminidase (mNAGA), to cure Fabry disease by changing the substrate specificity of NAGA to that of GLA. In this study, researchers tested whether genome-editing transplantation of mNAGA-secreting induced pluripotent stem cells (iPS) cells could deliver GLA activity in vivo. They therefore generated mNAGA-secreting iPS cells by TALEN-mediated knock-in at the AAVS1 site, a refuge locus. Furthermore, to exclude possible immunogenic reactions caused by endogenous GLA from iPS cells in patients, they disrupted the GLA gene by CRISPR-Cas9. When cardiomyocytes and fibroblasts from the Fabry model without GLA activity were co-cultured with mNAGA-secreting iPS cells, GLA activity was restored by mNAGA-expressing cells in vitro. Next, they transplanted the mNAGA-secreting iPS cells into the testes of mouse models of Fabry disease. After 7 or 8 weeks, GLA activity in the liver was significantly improved, although no recovery of activity was observed in the heart, kidneys or blood plasma.