Genomic off-target DNA site (OTS)

Genomic off-target DNA sites are sequences within the genome that exhibit partial sequence complementarity to the guide RNA (gRNA) or DNA-binding components of programmable nuclease systems, such as CRISPR-Cas9, TALENs, or ZFNs (Zhang et al., 2015). Although these systems are engineered for site-specific modification, the nuclease complex can tolerate a limited number of mismatches, leading to unintended double-strand breaks (DSBs) at these off-target loci (Fu et al., 2013). The repair of these unintended breaks via non-homologous end joining (NHEJ) or homology-directed repair (HDR) can introduce permanent mutations, including deletions, insertions, or complex chromosomal rearrangements (Tsai et al., 2015). In a clinical context, off-target activity poses a substantial risk of genotoxicity, potentially resulting in the activation of proto-oncogenes or the disruption of tumor suppressor genes, which could lead to cellular transformation (FDA, 2023). Consequently, rigorous identification and quantification of off-target effects using methods like GUIDE-seq or CIRCLE-seq are mandatory for the safety assessment of gene-editing therapeutics (Casgevy FDA Briefing, 2023). Minimizing these risks involves optimizing gRNA specificity and utilizing high-fidelity enzyme variants to ensure the precision of the therapeutic intervention.

Other names

Off-target effectCRISPR off-targetNon-specific DNA cleavage siteOff-target locusUnintended genomic modification site

Unintended DNA cleavage or modification by programmable nucleases at loci with sequence homology to the guide RNA or DNA-binding domain (Zhang et al., 2015).