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Mol Ther. 2016 Mar;24(3):582-91. doi: 10.1038/mt.2015.190. Epub 2015 Oct 7.

Silent IL2RG Gene Editing in Human Pluripotent Stem Cells.

Author information

1
Department of Medicine, University of Washington, Seattle, Washington, USA.
2
College of Veterinary Medicine, University of Minnesota, Minneapolis, Minnesota, USA.
3
Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA.
4
McEwen Centre for Regenerative Medicine, University Health Network, Toronto, Ontario, Canada.
5
Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.
6
Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
7
Current address: Sunnybrook Research Institute, Toronto, Ontario, Canada.
8
Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, USA.
9
Department of Biochemistry, University of Washington, Seattle, Washington, USA.

Abstract

Many applications of pluripotent stem cells (PSCs) require efficient editing of silent chromosomal genes. Here, we show that a major limitation in isolating edited clones is silencing of the selectable marker cassette after homologous recombination and that this can be overcome by using a ubiquitous chromatin opening element (UCOE) promoter-driven transgene. We use this strategy to edit the silent IL2RG locus in human PSCs with a recombinant adeno-associated virus (rAAV)-targeting vector in the absence of potentially genotoxic, site-specific nucleases and show that IL2RG is required for natural killer and T-cell differentiation of human PSCs. Insertion of an active UCOE promoter into a silent locus altered the histone modification and cytosine methylation pattern of surrounding chromatin, but these changes resolved when the UCOE promoter was removed. This same approach could be used to correct IL2RG mutations in X-linked severe combined immunodeficiency patient-derived induced PSCs (iPSCs), to prevent graft versus host disease in regenerative medicine applications, or to edit other silent genes.

PMID:
26444081
PMCID:
PMC4786911
[Available on 2017-03-01]
DOI:
10.1038/mt.2015.190
[Indexed for MEDLINE]
Free PMC Article

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