Comprehensive off-target assessment strategies for CRISPR-based genome editing therapeutics: integrating biochemical, bioinformatic, and unbiased approaches​

Comprehensive assessment of gene editing specificity is key for developing genetic medicines. This presentation shows that a multi-layered nomination strategy improves detection of off-target sites across multiple editing modalities (nuclease, adenosine, and cytosine base editors) compared with any single method. Off-target profiles vary significantly by cell type, editing modality, and editor engineering, requiring evaluation in therapeutically relevant models using both male and female primary cells. This multi-assay approach is aligned with current regulatory guidance for the comprehensive assessment of genome editing safety. Our approach merges orthogonal nomination methods with deep sequencing validation and clinical variant analysis to systematically assess off-target editing risks for CRISPR therapeutics.