The CRISPR/Cas9 system enables precise and efficient modification of eukaryotic genomes. Among its various applications, homology-directed repair (HDR) mediated knock-in (KI) is crucial for creating human disease models, gene therapy, and agricultural genetic enhancements. Despite its potential, HDR-mediated knock-in efficiency remains relatively low. This study investigated the impact of 5' end PEG10 modification on site-specific integration of the target gene. The HEK293 cell line is considered a highly attractive expression system for the production of recombinant proteins, with the construction of site-specific integration cell lines at the AAVS1 locus enabling stable protein expression. This study investigated the impact of the 5' end PEG10 modification on the site-specific integration of the target gene at the AAVS1 locus in the 293T cell line. Utilizing this 5' end PEG10 modification resulted in a 1.9-fold increase in knock-in efficiency for a 1.8 kb target fragment, improving efficiency from 26% to 49%. An optimized system was utilized to successfully establish a high-expression, site-specific integration 293T cell line for TAT-Cas9-EGFP, providing a reliable resource of seed cells for subsequent protein production.