CRISPR-Cas9-mediated genome engineering exaggerates genomic deletion at 10q23.31 including the PTEN gene locus mimicking cancer profiles

The CRISPR-Cas9 system is a powerful tool for studying gene functions and has tremendous potential for disease treatment. However, precise genome editing requires thorough assessments to minimize unintended on- and off-target effects. Here, we report an unexpected deletion of a 287 kb region on Chromosome 10 (10q23.31) in chronic myelogenous leukemia 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 by the process of generating CRISPR-Cas9-modifed 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. Overall, our findings suggest that the unintended deletion of 10q23.31 can confound CRISPR-Cas9 studies, highlights the importance of assessing unintended genomic changes in CRISPR-Cas9-modified cells and may have clinical significance in cancer research. Highlights ### Competing Interest Statement The authors have declared no competing interest. * Δ : deletion 3D : three-dimension Chr : Chromosome ctrl : control clones BP : biological process Cas9 : CRISPR-associated protein 9 ChIP-seq : chromatin immunoprecipitation followed by sequencing CRISPR : clustered regularly interspaced short palindromic repeats DE : differentially expressed DAc : differentially acetylated DSB : double-strand break FC : fold-change FDR : false discovery rate gRNA : guide RNA H3K4me3 : histone 3 lysine 4 trimethylation H3K27ac : histone 3 lysine 27 acetylation iPSCs : induced pluripotent stem cells GO : gene ontology MF : molecular function PCA : Principal component analysis Pol II : polymerase II tRNA : transfer RNA TAD : topologically associating domain TSS : transcription start site