Engineering Chromosome Bridges Through CRISPR/Cas9 to Decipher the Impact of Intercentromeric Distance on Resolution...

Resolution of chromosome bridges during mitosis is a critical yet incompletely understood process with implications for genomic stability and cancer development. In this study, we investigated the impact of the bridging chromatin length on the timing and mechanism of chromosome bridge resolution. Using CRISPR/Cas9 technology, we engineered chromosome bridges with precisely defined intercentromeric distances in human RPE-1 cells. Our study revealed a decline in the frequency of chromosome bridges as cells progressed from early anaphase to late telophase, indicating resolution during mitosis. Moreover, the longer the bridging chromatin length, the higher the frequency of chromosome bridges observed at the mitotic exit, demonstrating that the size of the bridge influences its resolution during mitosis. Additionally, the separation between the bridge kinetochores needed for bridge breakage was strongly dependent on the megabase length of the bridging chromatin, with longer chromosome bridges requiring greater separation for their resolution. Given that chromosome bridge resolution occurs in a concerted manner with spindle elongation and is influenced by the length of the bridging chromatin, we posit that the traction forces generated by microtubules attaching to dicentric chromosomes play a significant role in resolving chromosome bridges during mitosis. Our study underscores the intricate interplay between chromosome bridge geometry and mechanical forces in mitotic chromosome bridge resolution. Our model offers a valuable framework for future investigations into the molecular mechanisms underlying chromosome bridge resolution, with potential implications for cancer biology and genomic stability maintenance.