TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs

Suzuki, Shingo; Sargent, R. Geoffrey; Illek, Beate; Fischer, Horst; Esmaeili-Shandiz, Alaleh; Yezzi, Michael J.; Lee, Albert; Yang, Yanu; Kim, Soya; Renz, Peter F.; Qi, Zhongxia; Yu, Jingwei; Muench, Marcus O.; Beyer, Ashley I.; Guimaraes, Alessander O.; Ye, Lin; Chang, Judy C.; Fine, Eli J.; Cradick, Thomas J.; Bao, Gang; Rahdar, Meghdad; Porteus, Matthew H.; Shuto, Tsuyoshi; Kai, Hirofumi; Kan, Yuet Wai; Gruenert, Dieter C.

doi:10.1038/mtna.2015.43

Cited by 40 publications

(50 citation statements)

References 52 publications

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“…A major advantage of locus-specific gene correction over conventional gene therapy is that physiological regulation of gene expression by the endogenous promoter is retained [ 38 , 39 ]. Although it is not yet clear what the ultimate cellular target will be, nuclease-mediated gene editing at the CFTR locus has seen a surge of interest by the research community [ 18 , 40 , 41 ].…”

Section: Discussionmentioning

confidence: 99%

Targeted Integration of a Super-Exon into the CFTR Locus Leads to Functional Correction of a Cystic Fibrosis Cell Line Model

Bednarski

Tomczak²,

Hövel

et al. 2016

PLoS ONE

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In vitro disease models have enabled insights into the pathophysiology of human disease as well as the functional evaluation of new therapies, such as novel genome engineering strategies. In the context of cystic fibrosis (CF), various cellular disease models have been established in recent years, including organoids based on induced pluripotent stem cell technology that allowed for functional readouts of CFTR activity. Yet, many of these in vitro CF models require complex and expensive culturing protocols that are difficult to implement and may not be amenable for high throughput screens. Here, we show that a simple cellular CF disease model based on the bronchial epithelial ΔF508 cell line CFBE41o- can be used to validate functional CFTR correction. We used an engineered nuclease to target the integration of a super-exon, encompassing the sequences of CFTR exons 11 to 27, into exon 11 and re-activated endogenous CFTR expression by treating CFBE41o- cells with a demethylating agent. We demonstrate that the integration of this super-exon resulted in expression of a corrected mRNA from the endogenous CFTR promoter and used short-circuit current measurements in Ussing chambers to corroborate restored ion transport of the repaired CFTR channels. In conclusion, this study proves that the targeted integration of a large super-exon in CFTR exon 11 leads to functional correction of CFTR, suggesting that this strategy can be used to functionally correct all CFTR mutations located downstream of the 5’ end of exon 11.

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Section: Discussionmentioning

confidence: 99%

Targeted Integration of a Super-Exon into the CFTR Locus Leads to Functional Correction of a Cystic Fibrosis Cell Line Model

Bednarski

Tomczak²,

Hövel

et al. 2016

PLoS ONE

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“…For example, SSN-mediated correction of disease-causing mutations in LRKK2 that are associated in Parkinson’s disease revealed the transcriptional changes caused by disease-associated alleles in patient cells (Reinhardt et al, 2013). Likewise, genome editing of patient-specific iPSCs followed by in vitro differentiation was also used to generate an isogenic disease model for cystic fibrosis by correcting disease-relevant mutations in CFTR followed by differentiation into airway epithelium (Crane et al, 2015; Firth et al, 2015; Suzuki et al, 2016). …”

Section: Crispr/cas9: Everyone Can Edit Anythingmentioning

confidence: 99%

Induced Pluripotent Stem Cells Meet Genome Editing

2016

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It is extremely rare for a single experiment to be so impactful and timely that it shapes and forecasts the experiments of the next decade. Here, we review how two such experiments --the generation of human induced pluripotent stem cells (iPSCs) and the development of CRISPR/Cas9 technology-- have fundamentally reshaped our approach to biomedical research, stem cell biology and human genetics. We will also highlight the previous knowledge that iPSC and CRISPR/Cas9 technologies were built on as this groundwork demonstrated the need for solutions and the benefits that these technologies provided, and have set the stage for their success.

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“…Making precise alterations in the genome of cells is now relatively easy, but the isolation and selection of those cells is not necessarily as simple. Numerous protocols have been devised using selection markers that can be subsequently removed (Wang et al., ) or PCR‐based enrichment strategies to avoid the need for selectable markers (Suzuki et al., ).…”

Section: Design Cloning Testing Selection and Off‐targetmentioning

confidence: 99%

A beginner's guide to gene editing

Harrison

Hart

2018

Experimental Physiology

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Genome editing enables precise changes to be made in the genome of living cells. The technique was originally developed in the 1980s but largely limited to use in mice. The discovery that a targeted double-stranded break at a unique site in the genome, close to the site to be changed, could substantially increase the efficiency of editing raised the possibility of using the technique in a broader range of animal models and, potentially, human cells. But the challenge was to identify reagents that could create targeted breaks at a unique genomic location with minimal off-target effects. In 2005, the demonstration that programmable zinc finger nucleases (ZFNs) could perform this task led to a number of proof-of-concept studies, but a limitation was the ease with which effective ZFNs could be produced. In 2009, the development of TAL effector nucleases (TALENs) increased the specificity of gene editing and the ease of design and production. However, it was not until 2013 and the development of the clustered regularly interspaced short palindromic repeat (CRISPR) Cas9/guide RNA that gene editing became a research tool that any laboratory could use.

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TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs

Cited by 40 publications

References 52 publications

Targeted Integration of a Super-Exon into the CFTR Locus Leads to Functional Correction of a Cystic Fibrosis Cell Line Model

Targeted Integration of a Super-Exon into the CFTR Locus Leads to Functional Correction of a Cystic Fibrosis Cell Line Model

Induced Pluripotent Stem Cells Meet Genome Editing

A beginner's guide to gene editing

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