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. 2023 Sep 11;13:14988. doi: 10.1038/s41598-023-41367-z

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© The Author(s) 2023

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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Logic gate to identify only cancer cells of epithelial origin using halves of split Cas9 under a universal (pCMV) and a cell type-specific (phCEA) promoters. The expression of the green fluorescent protein reporter is observed only in cancer cells of epithelial origin. All cells were co-transfected with _phCEA-Cas4.5_N, pCMV-Cas4.5_C/gRNA, the EGxxFP reporter and pCMV-mCherry (transfection control) constructs. Co-expression of the split Cas9 halves resulted in holo-enzyme formation via inteins, followed by Cas9-induced homologous recombination of EGFP. Only cancer cells of epithelial origin expressed both halves since the expression of Cas4.5_N is restricted to these cells. Neither non-epithelial cell lines: human skin fibroblasts (HSF), murine myoblasts (C2C12), nor normal (non-cancerous) epithelial cells: CCD 841 CoTr and GC-2, showed any expression of the reporter (EGFP) (A), while all cancer cells with epithelial origin tested (H1299, SW480, A375, HeLa, and U-2 OS cells) expressed EGFP (B).