Abstract LB443: Unlocking cell cycle dynamics: Gene regulation of CRISPR-Cas9 and siRNA phenotypes using cell painting

Abstract In cancer research, perturbing gene function is crucial for understanding disease mechanisms and developing treatments. Techniques such as RNA interference and CRISPR-knockout, -interference, or -activation are commonly used. In this recent study we compared the phenotypic effects of cellular morphology by knocking out or knocking down key cell cycle regulators, Aurora kinase B (AURKB), Geminin (GMNN), and Polo-like kinase 1 (PLK1), which are known key critical proteins involved in cell cycle regulation, and their dysfunction linked to cancer progression. In this in vitro osteosarcoma cell line (U 2-OS) model, we inhibited these proteins using CRISPR-Cas9 for gene knockout or siRNA for mRNA knockdown in a comparative study not previous reported. Employing Cell Painting methodology (6 fluorescent probes, high content screening (HCS) confocal imaging, and multivariant analysis) we imaged and profiled the morphological phenotypes following gene perturbations. Principal component analysis (PCA) analysis of hundreds of HCS image phenotypic feature measurements revealed significant morphological changes and reduced cell numbers from AURKB, GMNN, and PLK1 inhibition in U 2-OS cells caused significant phenotypic changes and reduced cell numbers suggesting alteration of cell cycle progression. Interestingly, in the GMNN inhibition, the siRNA-treated cells showed a higher percentage of large nuclei compared to small nuclei from CRISPR-treated cells. These findings suggest siRNA-mediated knockdown has a faster inhibitory effect on GMNN than CRISPR-treated conditions. This study demonstrates the complementary nature of CRISPR-Cas9 and siRNA gene modulation and underscores the importance of orthogonal validation for robust genetic studies with key cell cycle regulators. Citation Format: Alexander Schreiner, Angelika Foitzik, Josien Levenga, Karin Boettcher, Barbara Sonnenberg, Žaklina Strezoska, Joe Trask. Unlocking cell cycle dynamics: Gene regulation of CRISPR-Cas9 and siRNA phenotypes using cell painting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB443.