Abstract Chromosome gains and losses are found in nearly all human cancers. However, the functional importance of these massive gene dosage imbalances remains poorly understood. Notably, large chromosomal segments are technically challenging to manipulate, which has limited our ability to precisely model and interrogate the consequences of specific aneuploidies. The relationship between aneuploidy and cancer development is of particular importance for individuals with Trisomy 21/Down syndrome, as children with this condition exhibit a 150-fold increased risk of developing acute myeloid leukemia (AML). In the parent grant for this application (1R01CA276666), we described a novel CRISPR-mediated chromosome engineering methodology that allows us to eliminate specific aneuploid chromosomes from human cancer cells. We proposed to apply this approach to interrogate the consequences of chromosome 1q aneuploidies in a genetically-controlled setting. In this supplemental application, we plan to leverage these same chromosome engineering techniques to study gains of chromosome 21 in leukemia. In particular, we will eliminate single copies of chromosome 21 from human AML cell lines that are aneuploid for this chromosome, thereby creating genetically-matched cells that are either disomic or trisomic for chromosome 21. We will then examine how loss of the chromosome 21 trisomy impacts various cancer-related phenotypes, including gene expression, cell fitness, and drug sensitivity. Additionally, we will over-express individual chromosome 21 genes in our engineered chromosome 21-loss cell lines, which will allow us to identify specific genes encoded on this chromosome that impact specific leukemic phenotypes. These assays will address the NCI’s INCLUDE program goal of uncovering genetic factors associated with trisomy 21 that lead to the distinct biological and clinical behavior of Down syndrome-associated leukemias.