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

??Aneuploidy, the presence of chromosome gains or losses, is a hallmark of cancer and congenital syndromes. Here, we describe KaryoCreate (Karyotype CRISPR Engineered Aneuploidy Technology), a system that enables generation of chromosome-specific aneuploidies by co-expression of a sgRNA targeting chromosome-specific CENPA-binding ?-satellite repeats together with dCas9 fused to a mutant form of KNL1. We designed unique and highly specific sgRNAs for 19 out of 24 chromosomes. Expression of these sgRNAs with KNL1Mut-dCas9 leads to missegregation and induction of gains or losses of the targeted chromosome in cellular progeny with an average efficiency of 8% and 12% for gains and losses, respectively (up to 20%), tested and validated across 9 chromosomes. Using KaryoCreate in colon epithelial cells, we show that chromosome 18q loss, a frequent occurrence in gastrointestinal cancers, promotes resistance to TGFß, likely due to synergistic hemizygous deletion of multiple genes. Altogether, we describe a novel technology to create and study chromosome missegregation and aneuploidy in the context of cancer and beyond. Overall design: KaryoCreate was used to generate an hCEC clone with (clone 14) and without (clone 13) chromosome 18q loss. Bulk RNA- sequencing was performed on these two clones (2 biological replciates each). Differential expression analysis using DESeq2 and gene set enrichment analysis (GSEA) were then performed.