Modeling specific aneuploidies: from karyotype manipulations to biological insights

Abstract

An abnormal chromosome number, or aneuploidy, underlies developmental disorders and is a common feature of cancer, with different cancer types exhibiting distinct patterns of chromosomal gains and losses. To understand how specific aneuploidies emerge in certain tissues and how they contribute to disease development, various methods have been developed to alter the karyotype of mammalian cells and mice. In this review, we provide an overview of both classic and novel strategies for inducing or selecting specific chromosomal gains and losses in human and murine cell systems. We highlight how these customized aneuploidy models helped expanding our knowledge of the consequences of specific aneuploidies to (cancer) cell physiology.

Keywords: Aneuploidy; CIN; CRISPR/Cas9; cancer; chromosome.

Fig. 1

Strategies to introduce an additional chromosome into mammalian cells and mice. a Microcell-mediated chromosome transfer consists of 2 steps: (1) micronuclei formation in donor cells by colcemid treatment or irradiation; and (2) fusion of micronuclei to recipient cells of choice. Hybrid Chinese hamster ovary (CHO) or mouse A9 cells containing a single human chromosome are mostly used as donor cells (Tanabe et al. 2000). A positive selection marker, (often puromycin or neomycin resistance genes), is usually integrated into the chromosome of interest to facilitate the recovery of recipient cells with specific chromosomal gains. b Crossings of mice carrying Robertsonian chromosomes can generate mice and mouse ESCs with specific trisomies (Williams et al. 2008). As starting point, two mouse strains are crossed each carrying a different Robertsonian translocation involving the chromosome of interest (e.g., Rob (13;16) and Rob (11;13)). c CRISPR/Cas9-induced targeted chromosome fusions can generate either Robertsonian-like metacentric chromosomes (Zhang et al. 2022) or a large telocentric fused chromosome (Wang et al. 2022) (Supplemental Table 2). This could be applied to create parental mouse strains carrying specific (viable) chromosome fusions for crossings that eventually generate specific trisomies

Fig. 2

Methods to eliminate specific chromosomes. a Two inverted LoxP sites are integrated into a chromosome arm of interest. Upon expression of Cre recombinase after S phase, the two sister chromatids can recombine, generating dicentric and acentric chromosomes that are eventually lost after one or multiple mitoses. An antibiotic resistance gene such as pac (puromycin resistance) or neo (neomycin resistance) is usually inserted between the two loxP sites to facilitate selection of cells harboring loxP integration. To efficiently recover cells with the targeted chromosomal loss, a number of transgenes can be inserted, including ones encoding for fluorescent proteins (FP), cell-surface proteins such as human (h)CD2, or a suicide gene such as herpes simplex virus thymidine kinase (HSV-tk), allowing for FACS sorting or Ganciclovir (GCV)-induced negative selection, respectively. b (i) With one or multiple chromosome-specific sgRNAs, one or multiple DNA double stranded breaks (DSBs) are induced either in the arm or in the (peri-)centromere of the targeted chromosome by CRISPR/Cas9, leading to either whole or partial loss of the targeted chromosome. Integration of a suicide gene (i.e., HSV-tk) in the arm of the targeted chromosome can facilitate selection of cells that have lost the targeted chromosome (arm). Alternatively, CRISPR/Cas9 or TALEN can be used to induce two DSBs flanking the chromosomal region to be deleted. This will lead to ligation of the endogenous telomere to the centromere-proximal break site, leading to specific segmental arm loss. (ii) Telomere-mediated chromosome truncation: CRISPR/Cas9 induces a single DSB near the centromere, and the break is repaired using a repair template containing a positive selection marker (pac or L-histidinol dihydrochloride, hisD), a human telomere sequence (telo), and frequently homology arms (HA) overlapping the break site. The incorporation of a synthetic S. cerevisiae cytosine deaminase-uracil phosphoribosyl transferase fusion gene (Fcy::Fur) outside the HA, can be used to eliminate cells with an off-target integration by 5-fluorocytosine

Fig. 3

Approaches to induce, detect, and isolate cells with specific aneuploidies. Non-transformed or transformed near-diploid cells with or without functional TP53 can acquire heterogeneous aneuploid karyotypes after transient or chronic induction of CIN by compounds that disrupt the chromosome segregation machinery. In addition, the mere knockout (KO) or knockdown (KD) of TP53 in hTERT-RPE1 cells or sequential mutation of APC, TP53, KRAS and SMAD4 (APKS) in colorectal organoids is sufficient to increase CIN. FACS-based single-cell sorting followed by WG DNA or RNA sequencing shortly after CIN induction permits assessment of karyotype heterogeneity in the initial aneuploidy landscape thereby revealing potential mis-segregation biases. At the same time, single cell culture after CIN induction can generate monoclonal lines harboring specific monosomies or trisomies. These can be subjected to bulk WG DNA or RNA sequencing to reveal their (altered) karyotypes. Finally, the initially heterogeneous and mosaic aneuploid population can be further cultured under standard conditions or under certain challenging conditions such as anti-cancer drugs. Aneuploidy patterns that eventually emerge can be detected by single-cell WG DNA or RNA sequencing. Of note, WG RNA sequencing, carried out either shortly after CIN induction or after prolonged culture, also allows for analyses of cellular responses to specific chromosomal gains and losses. i, inhibitor. This figure was partially created with Biorender

Fig. 4

dCas9-based methods for mis-segregating specific human chromosomes. a–c dCas9 fused to either P. patens Kinesin14VIb (Kin14VIb) (b), or human CENP-T^1-243 (c) has been used in combination with a sgRNA targeting chromosome-specific DNA repeats in chr1 and 9 for inducing chromosome-specific mis-segregation and aneuploidy. d A third strategy targets chromosome-specific higher order repeats (HOR) of centromeres with unique sgRNAs to recruit dCas9 fused to either KNL1^{1-86/S24A;S60}, KNL1^{1-86/RVSF/AAAA}, or NDC80^{1-207/6xS/T>A}. We propose that dCas9-KNL1^{-86/RVSF/AAAA} and dCas9-NDC80^1-207/6xST>A together with a centromere-specific sgRNA create an ectopic microtubule binding site on the centromere, which can lead to (pseudo-) merotelic attachment of the targeted chromosome during mitosis. Whole chromosome and chromosomal arm aneuploidies for chr6, 7, 8, 9, 12, 16, 18, and X could be generated using this approach. Number of predicted sgRNA binding sites (b.s.) based on the T2T genome assembly are indicated in a, d. AurB, Aurora B