Targeted assembly of ectopic kinetochores to induce chromosome‐specific segmental aneuploidies

Abstract

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. image In this study, individual human chromosomes were targeted for mitotic mis‐segregation to generate cells carrying chromosome‐specific segmental aneuploidies. This was achieved by targeting the kinetochore‐nucleating domain of CENP‐T to endogenous repeats using a nuclease‐dead Cas9. CENP‐T‐dCas9 can be specifically targeted to endogenous repeat sequences to form a functional ectopic kinetochore. The ectopic kinetochore is prone to form erroneous attachments with the mitotic spindle, resulting in spindle assembly checkpoint activation and metaphase delay, releasable by a short pulse of Mps1 inhibitor. Specific mis‐segregation of the target chromosome and subsequent segmental aneuploidies of chromosomes 1 and 9 were induced in human cells. Putative target repeat sequences and guide RNAs were identified for 18 of the human chromosomes.