A motor-based approach to induce chromosome-specific mis-segregations in human cells

Abstract

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.

KaryoCreate: a new CRISPR-based technology to generate chromosome-specific aneuploidy by targeting human centromeres

“…Most of the methods to obtain chromosome losses involve the use of catalytically active Cas9 to eliminate chromosomes or parts of chromosomes, introducing DNA double strand breaks and potentially instigating undesired genomic instability (see for example (Rayner et al, 2019)). Of particular interest are two methods that were recently described to induce chromosome-specific gains and, mostly, losses (Tovini et al, 2022; Truong et al, 2022). Both methods target non-centromeric repeats and have been so far successful for two chromosomes: chromosome 1, using a sub-telomeric repeat, and chromosome 9 using a peri-centromeric repeat.…”

“…Of particular interest are two methods that were recently described to induce chromosome-specific gains and, mostly, losses (Tovini et al, 2022;Truong et al, 2022).…”

“…Notably, compared to existing technologies Karyocreate has a high capability of inducing specific aneuploidies. In the recent study by (Truong et al, 2022) the authors applied their method to chromosome 9 and showed the induction of arm-level losses in about ~16% of cells for 9q and~3% for 9p, and arm-level gains of~3% for 9q and <1% for 9p; it was unclear whether any whole chromosome loss or gain was induced, likely due to the formation of dicentric chromosomes. Although it is very to hard to compare methods especially due to the use of different cell lines, the efficiency of induction of chromosome gains and losses for KaryoCreate was on average 9% for arm-level gains, 13% for arm-level losses, 8% for whole chromosome-chromosome gains and 11% for whole-chromosome losses.…”

“…Other groups have used CRISPR/Cas9 to cut in non-coding regions to generate whole chromosome losses, but this method has only been successful with the acrocentric chromosomes Y, 14, and 21 (Rayner et al, 2019; Zuo et al, 2017). Finally, additional methods were recently described that use non-centromeric repeats to induce specific losses and, to a lower extent, gains of only chromosomes 1 and 9, with losses occurring mostly at the arm-level (Tovini et al, 2022; Truong et al, 2022).…”

“…Other groups have used CRISPR/Cas9 to cut in non-coding regions to generate whole chromosome losses, but this method has only been successful with the acrocentric chromosomes Y, 14, and 21 (Rayner et al, 2019;Zuo et al, 2017). Finally, additional methods were recently described that use non-centromeric repeats to induce specific losses and, to a lower extent, gains of only chromosomes 1 and 9, with losses occurring mostly at the arm-level (Tovini et al, 2022;Truong et al, 2022).Human centromeres are composed of highly repetitive AT-rich repeats, also known as αsatellite DNA. Centromeric sequences are hierarchically organized in megabase-long arrays (HOR) of alphoid DNA that directly binds to CENPA, a histone H3 variant, critical to kinetochore position and assembly (Barra and Fachinetti, 2018;Hayden, 2012;Schueler and Sullivan, 2006;Whinn et al, 2019).…”