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

Abstract

ABSTRACTThe 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, includingPAPSS2, ATAD1, KLLN, andPTEN. 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.HighlightsCRISPR-Cas9-modified HAP1 cells carry an unexpected large genomic deletion at 10q23.31 encompassing four protein-coding genes frequently expressed across various cell types.The 10q23.31 deletion is accompanied by global changes in histone modification and transcriptomes.The generation of CRISPR-Cas9-modified cells rather than Cas9 activity increases the frequencies of the deletion at 10q23.31.The 10q23.31 deletion identified in HAP1 cells resembles a commonly occurring deletion pattern in cancer patients.