Chromosome-Specific Aneuploidy Engineering via dCas9-Induced Centromeric Chromatin Relaxation

Aneuploidy, characterised by an imbalanced chromosome copy number, is the cause of chromosomal disorders ¹ as well as a hallmark of cancer ² . Beyond pathological conditions, recent studies highlight the presence of lineage-specific recurrent aneuploidies in normal tissues ^3–5 . Despite its prevalence, the functional significance of aneuploidies in cellular dysfunction and diseases remains elusive, hindered by experimental challenges in engineering relevant aneuploidy models for mechanistic investigation. Here, we discover that the centromeric recruitment of catalytically dead Cas9 (dCas9) induces efficient chromosome-specific mis-segregation, applicable to all 24 human chromosomes. We show that the mis-segregation phenotype is driven by dCas9-induced kinetochore chromatin relaxation, resulting in whole chromosome aneuploidy or arm-level aneuploidy with centromeric breakage. In induced pluripotent stem cells, we demonstrate aneuploidy manipulation for chromosome 13, 21, X, and Y. In primary renal epithelial cells, we engineered chromosome 3(p) loss, the tumour-initiating event in clear cell renal cell carcinoma (ccRCC) ⁶ .. Overall, we describe a simple, efficient, and versatile approach for chromosome-specific aneuploidy generation that can facilitate preclinical aneuploid model development for functional interrogation of aneuploidy.