Engineering and characterisation of AAVS1 FRT cell lines for inducible and dose-dependent gene expression

Investigating biological function with high reproducibility requires the generation of stable cell lines that offer precise and inducible transgene expression. However, traditional methods often suffer from the limitations of random genomic integration, including unpredictable expression levels, leaky basal activity, and disruption of endogenous loci. To address these challenges, we engineered a versatile RPE1 cell line by integrating a TetOn3G-FRT cassette into the AAVS1 safe-harbour locus using CRISPR/Cas9(D10A) nickase. This platform utilises the Flp-FRT recombination system to facilitate rapid, site-specific "plug-and-play" insertion of transgenes, combined with the Tet-On 3G system for tight, dose-dependent expression control. The resulting RPE1 AAVS1 FRT8 line enables highly consistent transgene induction across independent clones, behaving as a monoclonal population. We highlight the importance of rigorous cell line characterisation through a multi-omics approach. Whole genome sequencing identified precise heterozygous integration at the target locus and de novo genomic duplications on other chromosomes. By integrating WGS with high-resolution proteomics and cell cycle analysis, we confirmed that these alterations did not significantly perturb cellular homeostasis or phenotypic fitness. This work establishes a robust and reliable platform for the efficient generation of inducible AAVS1 FRT stable cell lines, facilitating reproducible research in modern molecular biology.