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.

Various cancer types exhibit highly characteristic and recurrent aneuploidy patterns. The origin of these cancer type-specific karyotypes, and the extent to which they contribute to cancer progression, remains to be elucidated, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to mis-segregate specific chromosomes at will in different human cell types. We employed Tet repressor (TetR) or nuclease dead Cas9 (dCas9) to link a plant-derived microtubule minus-end-directed kinesin (Physcomitrella patens Kinesin14VIb) 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 often 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. Using chromosome-specific FISH and single-cell whole genome sequencing, we established that motor-induced mis-segregations result in specific arm-level, and to a lesser extent, whole chromosome aneuploidies, after a single cell division. Our kinesin-based strategy to manipulate individual mitotic chromosomes opens up the possibility to investigate the immediate cellular responses to specific (arm level) aneuploidies in different cell types; an important step towards understanding how recurrent aneuploidy patterns arise in different cancer types.