Modeling early genomic events of pancreatic cancer using CRISPR/Cas9

Abstract

Background: Duct epithelial cells transform into pancreatic cancer based on specific genetic alterations. Regards the lack of untransformed duct epithelial cell lines, no conclusive in vitro carcinogenesis model exists. A sensitive immortalization strategy (hTERT) was used to establish a new duct epithelial cell line. Subsequently CRISPR/Cas9 genome editing was applied modeling an early genomic event of pancreatic cancer. Methods: Non-neoplastic pancreatic ducts were isolated and immortalized using a lentiviral transfer-vector containing the hTERT gene. IHC and FISH analyses assessed telomerase expression. Growth rate and cellular kinetics were monitored. Single guide(sg)RNA close to target region were sub-cloned into Cas9n plasmid constructs. These constructs were transfected together with KRASG12D homology donor. FACS sorted clones are screened using Sanger sequencing technique. Results: Lentiviral transduction resulted in hTERT expressing cells. After selection, cytokeratin expressing duct cells amplified in a cobblestone pattern with robust cellular kinetics calling for a new Primary Duct Cell Line (PDCL). IHC- and FISH-analysis indicated sustained telomerase activity. Cells were successfully transfected with sgRNA and homology donor, targeting KRASG12D. Following FACS sorting, monoclonal cells were screened for KRASG12D clones. Conclusion: We successfully introduced KRASG12D sgRNA constructs into a new PDCL. This might be the baseline for modeling tumorigenesis in vitro of pancreatic cancer.