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.
Bibliographical noteFunding Information:
The authors thank Kristi Pilat, Roli Hirata, and Raisa Stolitenko for technical assistance and Els Henckaerts for helpful discussions. This work was supported by grants from the US NIH to D.W.R. (HL53750, DK55759), D.S.K. (Minnesota Partnership for Biotechnology), and G.K. (NIH U01HL100395 and CIHR MOP126117). D.W.R. holds equity in Horizon Discovery and Universal Cells Inc. The other authors declare no competing financial interests.