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Open AccessArticle

CRISPR/Cas9-Mediated Knock-Out of KrasG12D Mutated Pancreatic Cancer Cell Lines

1
Department of Surgery, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) and Universitätsklinikum Erlangen, 91054 Erlangen, Germany
2
Division of Bioinformatics, Department of Biology, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany
3
Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) and Universitätsklinikum Erlangen, 91054 Erlangen, Germany
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(22), 5706; https://doi.org/10.3390/ijms20225706
Received: 5 September 2019 / Revised: 31 October 2019 / Accepted: 9 November 2019 / Published: 14 November 2019
In 90% of pancreatic ductal adenocarcinoma cases, genetic alteration of the proto-oncogene Kras has occurred, leading to uncontrolled proliferation of cancerous cells. Targeting Kras has proven to be difficult and the battle against pancreatic cancer is ongoing. A promising approach to combat cancer was the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, which can be used to genetically modify cells. To assess the potential of a CRISPR/CRISPR-associated protein 9 (Cas9) method to eliminate Kras mutations in cells, we aimed to knock-out the c.35G>A (p.G12D) Kras mutation. Therefore, three cell lines with a heterozygous Kras mutation (the human cell lines SUIT-2 and Panc-1 and the cell line TB32047 from a KPC mouse model) were used. After transfection, puromycin selection and single-cell cloning, proteins from two negative controls and five to seven clones were isolated to verify the knock-out and to analyze changes in key signal transduction proteins. Western blots showed a specific knock-out in the KrasG12D protein, but wildtype Kras was expressed by all of the cells. Signal transduction analysis (for Erk, Akt, Stat3, AMPKα, and c-myc) revealed expression levels similar to the wildtype. The results described herein indicate that knocking-out the KrasG12D mutation by CRISPR/Cas9 is possible. Additionally, under regular growth conditions, the knock-out clones resembled wildtype cells. View Full-Text
Keywords: CRISPR/Cas9 system; G12D; Kras; Suit-2; Panc-1; TB32047 CRISPR/Cas9 system; G12D; Kras; Suit-2; Panc-1; TB32047
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Lentsch, E.; Li, L.; Pfeffer, S.; Ekici, A.B.; Taher, L.; Pilarsky, C.; Grützmann, R. CRISPR/Cas9-Mediated Knock-Out of KrasG12D Mutated Pancreatic Cancer Cell Lines. Int. J. Mol. Sci. 2019, 20, 5706.

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