Targeted assembly of ectopic kinetochores to induce chromosome-specific segmental aneuploidies.
Targeted assembly of ectopic kinetochores to induce chromosome-specific segmental aneuploidies.
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DOI：
10.15252/embj.2022111587
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发表时间：
2023-05-15
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影响因子：
--
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癌细胞表现出持久的潜在染色体不稳定性，个别肿瘤类型有趣地表现出整体和亚染色体非整倍体的特征亚群。几乎不存在诱导特定非整倍体的方法，这阻碍了对反复发生的非整倍体的功能后果以及特定染色体错误分离的严重后果的研究。因此，我们研究了使用核酸酶死亡的CRISPR-Cas9(DCas9)作为特定基因组座位的运输载体来破坏特定染色体有丝分裂分离的可能性。我们招募着丝粒蛋白CENP-T的着丝粒核区在9号染色体着丝粒附近或1号染色体的端粒附近组装异位着丝粒。异位着丝粒组装导致目标染色体增加染色体不稳定性和部分非整倍体，为在一系列细胞类型中诱导特定的染色体错误分离事件提供了可能性。我们还对可能支持异位动粒形成的内源性重复序列进行了分析。总体而言，我们的发现为异位着丝粒生物学提供了新的见解，并代表着朝着研究特定的非整倍体和染色体错误分离事件在与非整倍体相关的疾病中的作用迈出了重要的一步。通过核酸酶死亡的CRISPR-Cas9-CENP-T结构破坏特定染色体的有丝分裂分离，应该有助于概括和研究特定疾病相关的非整倍体。
Cancer cells display persistent underlying chromosomal instability, with individual tumour types intriguingly exhibiting characteristic subsets of whole, and subchromosomal aneuploidies. Few methods to induce specific aneuploidies will exist, hampering investigation of functional consequences of recurrent aneuploidies, as well as the acute consequences of specific chromosome mis‐segregation. We therefore investigated the possibility of sabotaging the mitotic segregation of specific chromosomes using nuclease‐dead CRISPR‐Cas9 (dCas9) as a cargo carrier to specific genomic loci. We recruited the kinetochore‐nucleating domain of centromere protein CENP‐T to assemble ectopic kinetochores either near the centromere of chromosome 9, or the telomere of chromosome 1. Ectopic kinetochore assembly led to increased chromosome instability and partial aneuploidy of the target chromosomes, providing the potential to induce specific chromosome mis‐segregation events in a range of cell types. We also provide an analysis of putative endogenous repeats that could support ectopic kinetochore formation. Overall, our findings provide new insights into ectopic kinetochore biology and represent an important step towards investigating the role of specific aneuploidy and chromosome mis‐segregation events in diseases associated with aneuploidy. Sabotaging mitotic segregation of specific chromosomes via nuclease‐dead CRISPR‐Cas9‐CENP‐T constructs should facilitate recapitulating and studying specific disease‐associated aneuploidies.
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DOI：
10.1073/pnas.090013497
发表时间：
2000-05-09
影响因子：
11.1
作者：
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通讯作者：
Mandahl, N
影响因子：
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影响因子：
64.5
作者：
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DOI：
10.1083/jcb.200208092
发表时间：
2003-04-28
期刊：
The Journal of cell biology
影响因子：
--
作者：
Hauf S;Cole RW;LaTerra S;Zimmer C;Schnapp G;Walter R;Heckel A;van Meel J;Rieder CL;Peters JM
通讯作者：
Peters JM
影响因子：
45.3
作者：
Breems, Dimitri A.;Van Putten, Wim L. J.;Lowenberg, Bob
通讯作者：
Lowenberg, Bob