The CRISPR-Cas9 system is a powerful tool for studying gene functions and holds potential for disease treatment. However, precise genome editing requires thorough assessments to min-imize unintended on-and off-target effects. Here, we report an unexpected 283-kb deletion on Chromosome 10 (10q23.31) in chronic myelogenous leukemia-derived HAP1 cells, which are frequently used in CRISPR screens. The deleted region encodes regulatory genes, including PAPSS2, ATAD1, KLLN, and PTEN. We found that this deletion was not a direct consequence of CRISPR-Cas9 off-targeting but rather occurred frequently during the generation of CRISPR-Cas9–modified cells. The deletion was associated with global changes in histone acetylation and gene expression, affecting fundamental cellular processes such as cell cycle and DNA replication. We detected this deletion in cancer patient genomes. As in HAP1 cells, the deletion contributed to similar gene expression patterns among cancer patients despite interindividual differences. Our findings suggest that the unintended deletion of 10q23.31 can confound CRISPR-Cas9 studies and underscore the importance to assess unintended genomic changes in CRISPR-Cas9–modified cells, which could impact cancer research.