The study investigates the on- and off-target effects of paired CRISPR-Cas9 nickases in primary human cells, specifically keratinocytes derived from patients with epidermolysis bullosa (EB). The researchers developed a novel CAST-seq pipeline, dual CAST (D-CAST), to analyze chromosomal aberrations at three different loci: COL7A1, COL17A1, and LAMA3. Key findings include: 1. **Off-Target Effects**: Paired nickase editing significantly reduced chromosomal translocations compared to Cas9 nuclease editing, which were previously observed at off-target sites. 2. **On-Target Effects**: Despite the reduced off-target effects, paired nickase editing still induced substantial on-target chromosomal aberrations, such as large deletions and inversions, at the target sites. 3. **Safety Profile**: The double-nickase approach combined high on-target efficiency with an improved safety profile, as it did not trigger chromosomal translocations. 4. **CAST-Seq Analysis**: D-CAST was highly sensitive and specific in detecting chromosomal translocations and on-target aberrations, providing a comprehensive genome-wide analysis of CRISPR-Cas9 double-nickase-based editing. The study highlights the potential of double-nickase strategies in genome editing, offering a safer alternative to CRISPR-Cas9 by reducing off-target effects while maintaining high on-target efficiency.The study investigates the on- and off-target effects of paired CRISPR-Cas9 nickases in primary human cells, specifically keratinocytes derived from patients with epidermolysis bullosa (EB). The researchers developed a novel CAST-seq pipeline, dual CAST (D-CAST), to analyze chromosomal aberrations at three different loci: COL7A1, COL17A1, and LAMA3. Key findings include: 1. **Off-Target Effects**: Paired nickase editing significantly reduced chromosomal translocations compared to Cas9 nuclease editing, which were previously observed at off-target sites. 2. **On-Target Effects**: Despite the reduced off-target effects, paired nickase editing still induced substantial on-target chromosomal aberrations, such as large deletions and inversions, at the target sites. 3. **Safety Profile**: The double-nickase approach combined high on-target efficiency with an improved safety profile, as it did not trigger chromosomal translocations. 4. **CAST-Seq Analysis**: D-CAST was highly sensitive and specific in detecting chromosomal translocations and on-target aberrations, providing a comprehensive genome-wide analysis of CRISPR-Cas9 double-nickase-based editing. The study highlights the potential of double-nickase strategies in genome editing, offering a safer alternative to CRISPR-Cas9 by reducing off-target effects while maintaining high on-target efficiency.