Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY, USA.
Department of Experimental Oncology, IEO European Institute of Oncology IRCCS, Milan, Italy.
Cell. 2023 Apr 27;186(9):1985-2001.e19. doi: 10.1016/j.cell.2023.03.029. Epub 2023 Apr 18.
Aneuploidy, the presence of chromosome gains or losses, is a hallmark of cancer. Here, we describe KaryoCreate (karyotype CRISPR-engineered aneuploidy technology), a system that enables the generation of chromosome-specific aneuploidies by co-expression of an sgRNA targeting chromosome-specific CENPA-binding ɑ-satellite repeats together with dCas9 fused to mutant KNL1. We design unique and highly specific sgRNAs for 19 of the 24 chromosomes. Expression of these constructs leads to missegregation and induction of gains or losses of the targeted chromosome in cellular progeny, with an average efficiency of 8% for gains and 12% for losses (up to 20%) validated across 10 chromosomes. Using KaryoCreate in colon epithelial cells, we show that chromosome 18q loss, frequent in gastrointestinal cancers, promotes resistance to TGF-β, likely due to synergistic hemizygous deletion of multiple genes. Altogether, we describe an innovative technology to create and study chromosome missegregation and aneuploidy in the context of cancer and beyond.
非整倍体，即染色体的获得或丢失，是癌症的一个标志。在这里，我们描述了 KaryoCreate（染色体 CRISPR 工程非整倍体技术），这是一种通过共表达靶向染色体特异性着丝粒结合ɑ-卫星重复序列的 sgRNA 与融合到突变 KNL1 的 dCas9 的方法来产生染色体特异性非整倍体的系统。我们为 24 条染色体中的 19 条设计了独特且高度特异性的 sgRNA。这些构建体的表达导致靶向染色体的错误分离，并在细胞后代中诱导获得或丢失，在 10 条染色体中验证的获得效率平均为 8%，丢失效率为 12%（高达 20%）。在结肠上皮细胞中使用 KaryoCreate，我们表明胃肠道癌症中常见的 18q 染色体丢失促进了对 TGF-β 的耐药性，这可能是由于多个基因的协同半合子缺失所致。总之，我们描述了一种创新的技术，用于在癌症及其他情况下创建和研究染色体的错误分离和非整倍体。
