Off-target genomic DNA sites (CRISPR-Cas9) (CRISPR off-targets)

Off-target genomic DNA sites are unintended sequences within the genome that are recognized and cleaved by the CRISPR-Cas9 system due to partial complementarity with the single-guide RNA (sgRNA). In the case of Streptococcus pyogenes Cas9 (SpCas9), these sites are typically characterized by a sequence similar to the target protospacer followed by a 5-NGG-3 protospacer adjacent motif (PAM) (Fu et al., 2013, Nature Biotechnology). These sites do not serve a therapeutic purpose; rather, they represent a major safety concern in gene editing because unintended double-strand breaks can lead to permanent mutations, chromosomal translocations, or the activation of oncogenes (Zhang et al., 2015, Molecular Therapy - Nucleic Acids). The frequency of off-target activity depends on the concentration of the Cas9-sgRNA complex, the duration of exposure, and the specific sequence of the sgRNA (Hsu et al., 2013, Nature Biotechnology). In clinical development, extensive off-target assessments using methods like GUIDE-seq or CIRCLE-seq are required to ensure that therapeutic candidates, such as exagamglogene autotemcel, do not cause deleterious genomic alterations (FDA, 2023, Casgevy Summary Basis for Regulatory Action). Consequently, these sites are considered liabilities to be minimized through high-fidelity Cas9 variants or optimized sgRNA design (Tsai et al., 2015, Nature Biotechnology).

Other names

Off-target effectsUnintended genomic modificationsNon-specific cleavage sitesSpCas9 off-targetsPartial sgRNA complementarity sites

Unintended double-strand DNA cleavage by the Cas9 nuclease followed by error-prone repair mechanisms such as non-homologous end joining (NHEJ) or homology-directed repair (HDR) (Fu et al., 2013, Nature Biotechnology).