CRISPR-Cas off-target DNA site (OTS) (OTS)

Mismatched or non-canonical PAM double-stranded DNA off-target sites are genomic sequences unintentionally recognized and modified by CRISPR-Cas systems or other programmable nucleases (Fu et al., 2013, Nature Biotechnology). These sites typically possess high sequence similarity to the intended target site but contain one or more base pair mismatches or utilize a non-canonical protospacer adjacent motif (PAM) that the nuclease can still recognize (Zhang et al., 2015, Science). While the primary goal of gene editing is to modify a specific on-target locus, the inherent flexibility of nuclease binding can lead to these off-target effects, posing significant risks to genomic integrity (Tsai et al., 2015, Nature Biotechnology). In a therapeutic context, such as the use of Exagamglogene autotemcel for sickle cell disease, identifying and minimizing these sites is crucial for safety (FDA, 2023, Cellular, Tissue, and Gene Therapies Advisory Committee Briefing Document). Unintended cleavage at these locations can result in permanent mutations, chromosomal translocations, or the activation of oncogenes, potentially leading to cellular transformation (Kosicki et al., 2018, Nature Biotechnology). These sites are characterized by the recruitment of DNA repair machinery, such as non-homologous end joining (NHEJ), which can introduce stochastic insertions or deletions (Hsu et al., 2013, Nature Biotechnology). Advanced sequencing methods like GUIDE-seq and CIRCLE-seq are employed to map these sites across the genome to ensure the specificity of therapeutic interventions (Tsai et al., 2017, Nature Methods). Consequently, these sites are not therapeutic targets themselves but are critical safety liabilities that must be rigorously characterized during the development of gene-editing drugs.

Other names

Off-target genomic sitesNon-specific cleavage sitesMismatched protospacersNon-canonical PAM sitesCRISPR off-targets

Unintended nuclease-mediated DNA cleavage or modification at genomic locations with high sequence homology to the guide RNA (Fu et al., 2013, Nature Biotechnology).