Research on Techniques for Generating Cancer Mutations Using CRISPR-Cas9 Based Gene Editing Methods

Abstract

Cancer development is driven by mutations in key regulatory genes; thus, creating tumor models that reflect individual mutation profiles is essential. The aim of this study was to establish a foundation for developing tumor models that replicate genetic mutations observed in cholangiocarcinoma using CRISPR-Cas9-based gene-editing techniques. By employing various CRISPR-Cas9-based gene-editing methods, including homology-mediated end joining (HMEJ), prime editing-assisted insertion of nucleotide templates (PAINT), programmable addition via site-specific targeting elements (PASTE), and prime-assisted site-specific integration of genetic elements (PASSIGE), we compared the knock-in efficiency of reporter plasmids using a cell line engineered to express an mCherry fluorescence cassette. Among these methods, PASSIGE demonstrated the highest insertion efficiency (29%). Furthermore, GFP fluorescent protein reporters were successfully inserted into cholangiocarcinoma-related genes in HEK293T cells using the PASSIGE method, inducing knock- out of these genes and confirming the effectiveness of the technique. The PASSIGE method holds promise for developing personalized tumor models that reflect the unique genetic characteristics of individual cholangiocarcinoma patients. Additionally, it provides a valuable foundation for advancing research on cancer therapeutics and personalized treatment strategies.