Silent IL2RG Gene Editing in Human Pluripotent Stem Cells

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Differentiation
  • Cell Survival / genetics
  • Collagen Type I / genetics
  • Collagen Type I, alpha 1 Chain
  • Epigenesis, Genetic
  • Gene Editing*
  • Gene Knockout Techniques
  • Gene Silencing*
  • Gene Targeting
  • Genetic Loci
  • Humans
  • Interleukin Receptor Common gamma Subunit / genetics*
  • Killer Cells, Natural / cytology
  • Pluripotent Stem Cells / cytology
  • Pluripotent Stem Cells / metabolism*
  • Promoter Regions, Genetic
  • T-Lymphocyte Subsets / cytology
  • Transgenes
  • X-Linked Combined Immunodeficiency Diseases / genetics

Substances

  • Collagen Type I
  • Collagen Type I, alpha 1 Chain
  • IL2RG protein, human
  • Interleukin Receptor Common gamma Subunit