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.

A kinesin‐based approach for inducing chromosome‐specific mis‐segregation in human cells

“…Therefore, we next studied the consequences of Kin14VIb binding to a chromosomal region more proximal to the kinetochore, by transducing a sgRNA specific to a pericentromeric DNA repeat of Chr9q12 (Chr9‐cen, Fig 6A ; Ma et al , 2015 ; Ma et al , 2018 ). Since this repeat is predicted to harbor ∼ 550,000 Chr9‐cen sgRNA binding sites, while the subtelomeric repeat in Chr1p is predicted to harbor ∼ 1,400 Chr1‐telo sgRNA binding sites (Tovini et al , 2023 , accompanying article), Chr9‐cen GFP foci appear larger than Chr1‐telo GFP foci (Figs 5B and 6A and Appendix Fig S4 G). Moreover, after rapalog addition, Chr9‐cen GFP foci localized near the centrosomes of monopolar spindles more often than Chr1‐telo GFP foci (Appendix Fig S4 H).…”

“…We developed an approach to enable both the visualization and manipulation of specific chromosomes during mitosis and that induced chromosome‐specific segmental aneuploidies in daughter cells after one round of cell division. In an accompanying article, Tovini et al employed dCas9‐CENP‐T fusion proteins to build an ectopic kinetochore on targeted chromosomes, a strategy that also increased the frequency of chromosome‐specific CIN and aneuploidies (Tovini et al , 2023 ). In addition, Bosco et al developed an elaborate computational pipeline to analyze the T2T human genome assembly for sgRNAs binding to chromosome‐specific centromere‐repeats (Bosco et al , 2023 ; Nurk et al , 2023 ).…”

“…These sgRNAs were used to tether a dCas9‐GFP‐Knl1 1‐86/RVSFmut fusion protein onto centromeres, a strategy that resulted in both segmental and whole‐chromosome‐specific aneuploidies of various chromosomes (Bosco et al , 2023 ). These three novel approaches rely on nuclease‐dead Cas9 and one unique sgRNA to tether a protein‐of‐interest to a chromosome‐specific highly repetitive sequence; sequences that are predominantly found in subtelomeric or (peri‐)centromeric chromosomal regions (Bosco et al , 2023 ; Tovini et al , 2023 ). Earlier methods employed nuclease‐active Cas9 to delete an entire mouse or human chromosome by inducing DNA double‐strand breaks in centromeres or along a chromosomal arm using multiple unique sgRNAs (Adikusuma et al , 2017 ; Zuo et al , 2017 ).…”

KaryoTap Enables Aneuploidy Detection in Thousands of Single Human Cells

“…These data can be used to reassemble the evolution of the tumor's cells, which can provide insights into how aneuploidy and chromosomal instability may drive tumorigenesis or inform treatments for therapeutic resistance 6,9 . Methods for modeling aneuploidies of specific chromosomes in cell culture have also emerged as powerful tools for studying the effects of aneuploidy in cancer [12][13][14] . These methods have benefitted from scDNA-seq, as sequencing individual cells enables the evaluation of the specificity and accuracy of the engineered karyotypes 13,14 .…”

“…Methods for modeling aneuploidies of specific chromosomes in cell culture have also emerged as powerful tools for studying the effects of aneuploidy in cancer [12][13][14] . These methods have benefitted from scDNA-seq, as sequencing individual cells enables the evaluation of the specificity and accuracy of the engineered karyotypes 13,14 .…”