A study evaluates the on- and off-target effects of paired CRISPR-Cas9 nickases in primary human keratinocytes derived from patients with epidermolysis bullosa (EB). The research demonstrates that while paired-nickase strategies reduce off-target mutagenesis compared to traditional Cas9 nucleases, they still induce significant chromosomal aberrations at on-target sites. The study uses a novel CAST-seq pipeline, dual CAST, to analyze chromosomal rearrangements at three loci (COL7A1, COL17A1, and LAMA3). Targeting these loci with Cas9 nucleases resulted in previously unknown chromosomal rearrangements, while paired-nickase editing did not detect chromosomal translocations. However, paired-nickase editing led to a higher proportion of insertions and large deletions/inversions compared to nucleases. The study confirms that paired-nickase approaches combine efficient editing with reduced off-target effects but still cause substantial chromosomal aberrations at on-target sites. The findings highlight the importance of assessing both on- and off-target effects in genome editing to ensure safety and efficacy. The study also shows that the use of a repair template can influence the types and frequencies of chromosomal aberrations. Overall, the results suggest that while paired-nickase strategies are safer than traditional nucleases, they still pose risks of chromosomal damage that need to be carefully evaluated. The study provides a comprehensive analysis of chromosomal aberrations associated with CRISPR-Cas9 double-nickase-based editing in clinically relevant human cells.A study evaluates the on- and off-target effects of paired CRISPR-Cas9 nickases in primary human keratinocytes derived from patients with epidermolysis bullosa (EB). The research demonstrates that while paired-nickase strategies reduce off-target mutagenesis compared to traditional Cas9 nucleases, they still induce significant chromosomal aberrations at on-target sites. The study uses a novel CAST-seq pipeline, dual CAST, to analyze chromosomal rearrangements at three loci (COL7A1, COL17A1, and LAMA3). Targeting these loci with Cas9 nucleases resulted in previously unknown chromosomal rearrangements, while paired-nickase editing did not detect chromosomal translocations. However, paired-nickase editing led to a higher proportion of insertions and large deletions/inversions compared to nucleases. The study confirms that paired-nickase approaches combine efficient editing with reduced off-target effects but still cause substantial chromosomal aberrations at on-target sites. The findings highlight the importance of assessing both on- and off-target effects in genome editing to ensure safety and efficacy. The study also shows that the use of a repair template can influence the types and frequencies of chromosomal aberrations. Overall, the results suggest that while paired-nickase strategies are safer than traditional nucleases, they still pose risks of chromosomal damage that need to be carefully evaluated. The study provides a comprehensive analysis of chromosomal aberrations associated with CRISPR-Cas9 double-nickase-based editing in clinically relevant human cells.