在许多肿瘤中经常观察到染色体不稳定性（CIN）。断裂-融合-桥 (BFB) 循环已被认为是肿瘤发生和肿瘤进化过程中 CIN 的主要驱动因素之一。然而，BFB 循环各个步骤的详细机制值得进一步研究。在这里，我们证明了核酸酶死亡 Cas9 (dCas9) 与端粒特异性单向导 RNA (sgTelo) 结合可用于模拟 BFB 循环。首先，我们证明使用 sgTelo 将 dCas9 靶向端粒会阻碍端粒处的 DNA 复制，并导致复制应激和 DNA 损伤显着增加。使用通过光学图谱进行的单分子端粒测定 (SMTA-OM)，我们研究了 dCas9/sgTelo 细胞中端粒的全基因组特征，并观察到染色体末端融合的显着增加，包括融合/ITS 和融合/ITS-。一致地，我们还观察到双着丝粒染色体、后期桥和细胞间端粒染色体桥（ITCB）形成的增加。利用 dCas9/sgTelo 系统，我们发现了 ITCB 许多有趣的分子和结构特征，并证明了多种 DNA 修复途径与 ITCB 的形成有关。我们的研究为 BFB 循环的分子机制提供了新的线索，这将增进我们对肿瘤发生、肿瘤进化和耐药性的理解。© 作者 2024。由牛津大学出版社代表 Nucleic Acids Research 出版。

Chromosome instability (CIN) is frequently observed in many tumors. The breakage-fusion-bridge (BFB) cycle has been proposed to be one of the main drivers of CIN during tumorigenesis and tumor evolution. However, the detailed mechanism for the individual steps of the BFB cycle warrants further investigation. Here, we demonstrate that a nuclease-dead Cas9 (dCas9) coupled with a telomere-specific single-guide RNA (sgTelo) can be used to model the BFB cycle. First, we show that targeting dCas9 to telomeres using sgTelo impedes DNA replication at telomeres and induces a pronounced increase of replication stress and DNA damage. Using Single-Molecule Telomere Assay via Optical Mapping (SMTA-OM), we investigate the genome-wide features of telomeres in the dCas9/sgTelo cells and observe a dramatic increase of chromosome end fusions, including fusion/ITS+ and fusion/ITS-. Consistently, we also observe an increase in the formation of dicentric chromosomes, anaphase bridges, and intercellular telomeric chromosome bridges (ITCBs). Utilizing the dCas9/sgTelo system, we uncover many interesting molecular and structural features of the ITCB and demonstrate that multiple DNA repair pathways are implicated in the formation of ITCBs. Our studies shed new light on the molecular mechanisms of the BFB cycle, which will advance our understanding of tumorigenesis, tumor evolution, and drug resistance.© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.