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

Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type-specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis-segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease-dead Cas9 to link a microtubule minus-end-directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore-attached microtubules. This tug-of-war resulted in chromosome-specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single-cell whole-genome sequencing, we established that kinesin-induced targeted mis-segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin-based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue-specific aneuploidy patterns evolve.</p

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

Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type-specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis-segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease-dead Cas9 to link a microtubule minus-end-directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore-attached microtubules. This tug-of-war resulted in chromosome-specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single-cell whole-genome sequencing, we established that kinesin-induced targeted mis-segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin-based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue-specific aneuploidy patterns evolve

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

Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type-specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis-segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease-dead Cas9 to link a microtubule minus-end-directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore-attached microtubules. This tug-of-war resulted in chromosome-specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single-cell whole-genome sequencing, we established that kinesin-induced targeted mis-segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin-based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue-specific aneuploidy patterns evolve

Reconstructing single-cell karyotype alterations in colorectal cancer identifies punctuated and gradual diversification patterns

Central to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq—a protocol that integrates live-cell imaging of tumor organoid outgrowth and whole-genome sequencing of each imaged cell to reconstruct evolving tumor cell karyotypes across consecutive cell generations. Using patient-derived colorectal cancer organoids and fresh tumor biopsies, we demonstrate that karyotype alterations of varying complexity are prevalent and can arise within a few cell generations. Sub-chromosomal acentric fragments were prone to replication and collective missegregation across consecutive cell divisions. In contrast, gross genome-wide karyotype alterations were generated in a single erroneous cell division, providing support that aneuploid tumor genomes can evolve via punctuated evolution. Mapping the temporal dynamics and patterns of karyotype diversification in cancer enables reconstructions of evolutionary paths to malignant fitness

Nuclear chromosome locations dictate segregation error frequencies

Chromosome segregation errors during cell divisions generate aneuploidies and micronuclei, which can undergo extensive chromosomal rearrangements such as chromothripsis [1, 2, 3, 4, 5]. Selective pressures then shape distinct aneuploidy and rearrangement patterns—for example, in cancer [6, 7] —but it is unknown whether initial biases in segregation errors and micronucleation exist for particular chromosomes. Using single-cell DNA sequencing [8] after an error-prone mitosis in untransformed, diploid cell lines and organoids, we show that chromosomes have different segregation error frequencies that result in non-random aneuploidy landscapes. Isolation and sequencing of single micronuclei from these cells showed that mis-segregating chromosomes frequently also preferentially become entrapped in micronuclei. A similar bias was found in naturally occurring micronuclei of two cancer cell lines. We find that segregation error frequencies of individual chromosomes correlate with their location in the interphase nucleus, and show that this is highest for peripheral chromosomes behind spindle poles. Randomization of chromosome positions, Cas9-mediated live tracking and forced repositioning of individual chromosomes showed that a greater distance from the nuclear centre directly increases the propensity to mis-segregate. Accordingly, chromothripsis in cancer genomes [9] and aneuploidies in early development [10] occur more frequently for larger chromosomes, which are preferentially located near the nuclear periphery. Our findings reveal a direct link between nuclear chromosome positions, segregation error frequencies and micronucleus content, with implications for our understanding of tumour genome evolution and the origins of specific aneuploidies during development. 1. van Jaarsveld, R. H. &amp; Kops, G. J. P. L. Difference makers: chromosomal instability versus aneuploidy in cancer. Trends Cancer 2, 561–571 (2016). 2. Compton, D. A. Mechanisms of aneuploidy. Curr. Opin. Cell Biol. 23, 109–113 (2011). 3. Zhang, C. Z. et al. Chromothripsis from DNA damage in micronuclei. Nature 522, 179–184 (2015). 4. Ly, P. et al. Chromosome segregation errors generate a diverse spectrum of simple and complex genomic rearrangements. Nat. Genet. 51, 705–715 (2019). 5. Shoshani, O. et al. Chromothripsis drives the evolution of gene amplification in cancer. Nature 591, 137–141 (2021). 6. Davoli, T. et al. Cumulative haploinsufficiency and triplosensitivity drive aneuploidy patterns and shape the cancer genome. Cell 155, 948–962 (2013). 7. Knouse, K. A., Davoli, T., Elledge, S. J. &amp; Amon, A. Aneuploidy in cancer: seq-ing answers to old questions. Annu. Rev. Cancer Biol. 1, 335–354 (2017). 8. Bolhaqueiro, A. C. F. et al. Ongoing chromosomal instability and karyotype evolution in human colorectal cancer organoids. Nat. Genet. 51, 824–834 (2019). 9. Cortés-Ciriano, I. et al. Comprehensive analysis of chromothripsis in 2, 658 human cancers using whole-genome sequencing. Nat. Genet. 52, 331–341 (2020). 10. McCoy, R. C. et al. Evidence of selection against complex mitotic-origin aneuploidy during preimplantation development. PLoS Genet. 348, 235–238 (2015)

Human chromosome‐specific aneuploidy is influenced by DNA ‐dependent centromeric features

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Reconstructing single-cell karyotype alterations in colorectal cancer identifies punctuated and gradual diversification patterns

Central to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq—a protocol that integrates live-cell imaging of tumor organoid outgrowth and whole-genome sequencing of each imaged cell to reconstruct evolving tumor cell karyotypes across consecutive cell generations. Using patient-derived colorectal cancer organoids and fresh tumor biopsies, we demonstrate that karyotype alterations of varying complexity are prevalent and can arise within a few cell generations. Sub-chromosomal acentric fragments were prone to replication and collective missegregation across consecutive cell divisions. In contrast, gross genome-wide karyotype alterations were generated in a single erroneous cell division, providing support that aneuploid tumor genomes can evolve via punctuated evolution. Mapping the temporal dynamics and patterns of karyotype diversification in cancer enables reconstructions of evolutionary paths to malignant fitness