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. 2021 Apr 8;29(5):505–518. doi: 10.1038/s41417-021-00326-4

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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 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. 1 — a Schema of cell selection. Gene-edited cells generated by transient transfection with gRNA, Cas9 complex, and ssODN, were sequentially selected by limiting dilution and ddPCR, yielding gene-edited monoclonal cells, blue and red wells, wild-type and KRAS mutant cells, respectively. b Determination of the levels of mutant KRAS (G12V) (genome-edited in cKRAS and HA-tagged mutant KRAS expression in vKRAS) and of related intracellular signaling molecules by Western blotting. The vKRAS construct has an HA-tag and is a slightly heavier molecule. Representative results from three independent experiments are shown. Band intensities of phosphorylated ERK (p-ERK) and phosphorylated AKT (p-AKT) are indicated below the images. c Determination of RAS activity by ELISA. Results are the average of three biological replicates; error bars represent the SD. *p < 0.05; **p < 0.01. d Cell growth curve. Cell numbers relative to those at day 0 are indicated. Results are the average of three biological replicates; error bars represent SD. **p < 0.01.