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. 2021 May 10;11:9865. doi: 10.1038/s41598-021-89312-2

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Genome editing at the PPP2R2B locus in human iPSCs. (A) A schematic diagram (not drawn to scale) showing PPP2R2B exon 7, donor construct, PiggyBac (PB) selection cassette, and primer locations. HA: homologous arms; Puro: puromycin N-acetyltransferase gene; DTK: truncated thymidine kinase gene. EGFP/Puro/TDK was driven by the CMV early enhancer/chicken β actin (CAG) promoter. TTAA site was used for PB cassette insertion and removal. (B) Timeline of genome editing, including positive/negative selection, screening, excision, and rescreening. (C-N) GFP expression during the process of genome editing. GFP signal from the unincorporated donor is stable for up to five days post electroporation. GFP expression level was similar at 48 h post electroporation across control, SN, and DN conditions (C–F). EGFP declines rapidly in the control and was only marginally detected by day 5 (G–J’). Percentage of GFP positive cells in F and J was quantified by flow cytometry. N = 3 different wells of cells per condition. (F) One-way ANOVA F(2,6) = 1210; p < 0.001; Tukey’s multiple comparison test; ***p < 0.001. (J) One-way ANOVA F(2,6) = 19,852; p < 0.001; Tukey’s multiple comparison test; ***p < 0.001. (K–N)The number of GFP positive and puromycin resistant clones was substantially greater in cells following DN than following SN, or control treatment. Colony counting was performed at 2X magnification. N = 3 different fields per condition. Mean ± SEM. One-way ANOVA F(2,6) = 253.1; p < 0.001; Tukey’s multiple comparison test. *p < 0.05; ***p < 0.001; ***p < 0.001.