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. 2022 Oct 18;13:6167. doi: 10.1038/s41467-022-33287-9

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© The Author(s) 2022

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a A schematic illustration of an integration gene circuit and BxB1 recombinase-mediated, site-specific integration in an engineered, adherent CHO cell line with a single landing pad (sLP). Positive integration control circuits had a central TU with an EF1α or CMV promoter driving mKate expression and two flanking dummy TUs with no gene expression in the same architecture. b The mKate signal intensities of the chromosomally integrated crisprTF promoter circuits in sLP-CHO cells relative to the integrated EF1α control circuit (one-way ANOVA with multiple comparisons corrected by Dunnett test; CMV, gRNA4 2×, 4×, and 6× BS without SI, and gRNA10 2×, 4×, and 16× BS without SI vs. EF1α: p < 0.0001, p = 0.0031, p = 0.0111, p = 0.0414, p = 0.0066, p = 0.0191, and p = 0.0006, respectively; gRNA10 8× and 16× BS with SI vs. EF1α: p < 0.0001 and p < 0.0001, respectively). c A schematic illustration of an integration circuit with the addition of a 3′ flanking selection marker (blasticidin) into dCas9-VPR, linked by a 2A self-cleavage peptide, to increase stability of target gene expression in long-term culture. A number of integration circuits from the gRNA4 and gRNA10 series were selected for the modification. d The mKate signal intensities of the chromosomally integrated crisprTF promoter circuits after dual selection with puromycin and blasticidin (one-way ANOVA with multiple comparisons corrected by Dunnett test; CMV and gRNA10 16× BS without SI vs. EF1α: p < 0.0001 and p = 0.0020, respectively; gRNA10 8× and 16× BS with SI vs. EF1α: p = 0.0446 and p < 0.0001, respectively). e The mKate expression levels with two of the strongest circuits from each gRNA series over the course of 4 weeks following dual selection. All four circuits examined displayed no noteworthy change in mKate signals at 2 or 4 weeks (all p > 0.05), similar to the EF1α control (two-way ANOVA with multiple comparisons corrected by Dunnett test; CMV: 0 vs. 2 weeks: p < 0.0001, 0 vs. 4 weeks: p < 0.0001). All data represent the mean ± SD (n = 3) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Source data are provided as Source Data files.