Abstract

Introduction: The concomitant presence of EGFR and KRAS mutations in non-small cell lung cancer (NSCLC) patients is relatively rare and their occurance is believed to be mutually exclusive. However, with advancing detection technologies such as liquid biopsy and next-generation sequencing, approximately 10% of patients with dual concomitant mutations are detected, and this can affect the administration of either EGFR or KRAS tyrosine kinase inhibitors. In order to address this issue, we aimed at generating an isogenic cell line with a dual mutation of EGFR and KRAS. The bacterial type II clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) protein systems were being used to introduce a KRAS G12C mutation into a KRAS wildtype, EGFR-mutant T790M lung cancer cell line and it started with cloning the KRAS-specific single-guide RNA (sgRNA) into an expression plasmid, PX330. Methods: Briefly, to clone the KRAS-targeting sgRNA into PX330, the plasmid was digested with BbsI restriction enzyme. The top and bottom sgRNA sequences were annealed and phosphorylated with antarctic phosphatase enzyme. They were ligated with the open-end PX330 with T4 PNK enzyme and T4 Ligase to create a complete closed plasmid, followed by transformation into the chemically competent DH5a Escherichia. coli (E.coli) strain. Following picking of the E. coli colonies, the plasmid was extracted and sent for Sanger sequencing to check for the presence of ligated sgRNA. Results: The KRAS-targeting sgRNA was successfully phosphorylated and ligated into an expression plasmid. In addition, it was successfully transfomed into a bacterial host for cloning. The sequencing results showed that the top and bottom sequences of KRAS-targeting sgRNA were successfully cloned into PX330 expression plasmid. Conclusion: The present study showed that the molecular cloning of KRAS-targeting sgRNA was a success and completed the first step in generating a dual mutation isogenic cell line.

Keywords: Bacterial transformation, CRISPR-Cas9, KRAS G12C