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

Eva Lentsch,Christian Pilarsky,Susanne Pfeffer,Robert Grützmann,Lifei Li,Arif B Ekici,Leila Taher

doi:10.3390/ijms20225706

Abstract

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.

Highlights

Pancreatic cancer (PaCa) is the fourth leading cause of all cancer death in the United States of America and Germany
Expression of KrasG12D and Total Ras after clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-Mediated Knock-Out in PaCa Cell Lines To verify the role of mutant Kras in pancreatic cancer cell lines, two human (Panc-1 and SUIT-2)
TB32047 RNA Sequencing Results Since the edited TB32047 cells demonstrated some changes in the expression levels of key signal transduction proteins, we further investigated the differential gene expression of the CRISPR/Cas9 knock-out in this model system

Summary

Introduction

Pancreatic cancer (PaCa) is the fourth leading cause of all cancer death in the United States of America and Germany. Chronic pancreatitis, heavy alcohol consumption or genetic predispositions, such as Lynch syndrome, are only a small variety of risk factors. Common symptoms, including weight loss, abdominal or back pain, mean that identification during the early stages can be difficult. With its disastrous five-year survival rate of about 8%, the chances of survival after diagnosis are remote. Besides chemotherapy or radiation therapy as therapeutic options to alleviate pain or extend survival, the only potentially curative prospect remains resection [1,2,3,4]. A better understanding of the functions of the tumor is fundamental to develop new treatment strategies and identify new therapeutic targets

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Conclusion