title: Silent IL2RG Gene Editing in Human Pluripotent Stem Cells.
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.
authors.affiliation: Department of Medicine, University of Washington, Seattle, Washington, USA.
authors.forename: Li B
authors.initials: LB
authors.lastname: Li
authors.affiliation: College of Veterinary Medicine, University of Minnesota, Minneapolis, Minnesota, USA.|Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Chao
authors.initials: C
authors.lastname: Ma
authors.affiliation: McEwen Centre for Regenerative Medicine, University Health Network, Toronto, Ontario, Canada.|Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.|Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.|Current address: Sunnybrook Research Institute, Toronto, Ontario, Canada.
authors.forename: Geneve
authors.initials: G
authors.lastname: Awong
authors.affiliation: McEwen Centre for Regenerative Medicine, University Health Network, Toronto, Ontario, Canada.|Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.|Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Marion
authors.initials: M
authors.lastname: Kennedy
authors.affiliation: Department of Medicine, University of Washington, Seattle, Washington, USA.
authors.forename: German
authors.initials: G
authors.lastname: Gornalusse
authors.affiliation: McEwen Centre for Regenerative Medicine, University Health Network, Toronto, Ontario, Canada.|Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.|Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Gordon
authors.initials: G
authors.lastname: Keller
authors.affiliation: Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA.|Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Dan S
authors.initials: DS
authors.lastname: Kaufman
authors.affiliation: Department of Medicine, University of Washington, Seattle, Washington, USA.|Department of Biochemistry, University of Washington, Seattle, Washington, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Russell
mesh_terms: D002454:Cell Differentiation; D002470:Cell Survival / Q000235:genetics; D024042:Collagen Type I / Q000235:genetics; D000090602:Collagen Type I, alpha 1 Chain; D044127:Epigenesis, Genetic; D000072669:Gene Editing; D055786:Gene Knockout Techniques; D020868:Gene Silencing; D018390:Gene Targeting; D056426:Genetic Loci; D006801:Humans; D053631:Interleukin Receptor Common gamma Subunit / Q000235:genetics*; D007694:Killer Cells, Natural / Q000166:cytology; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D016176:T-Lymphocyte Subsets / Q000166:cytology; D019076:Transgenes; D053632:X-Linked Combined Immunodeficiency Diseases / Q000235:genetics
chemical_list: D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; C507941:IL2RG protein, human; D053631:Interleukin Receptor Common gamma Subunit