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. 2024 Jul 26;14:17233. doi: 10.1038/s41598-024-68107-1

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© The Author(s) 2024

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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The Cas9 hybrids do not edit DNA in human cells. (A) Schematic representation of the GFP gene construction used for the editing assay using Cas9 nucleases. Each position targeted by the sgRNA used are shown with different triangles. This representation is not to scale. CMV-TetON is a doxycycline-inducible promoter. (B) Timeline of experiment used for the GFP assay. (C) Percentage of GFP negative cells for the 4 targets used at day 6 (green), 8 (orange) and 10 (yellow) for Sp, SaSp Merry and SaSp Gollum. (D) T7 endo I assay results for targets 1 and 3. The white arrows indicate the cleavage products indicative of efficient editing. Uncropped gels are found in Supplementary Fig. 7. (E) Percentage of GFP positive cells for each condition. Dashed lines represent the gate containing the brightest 1% of the cells transfected with Cas9 only.