Off-target genomic DNA sites with partial guide complementarity and compatible PAM (Off-target sites)

Off-target genomic DNA sites are sequences within the genome that exhibit partial complementarity to a synthetic guide RNA (gRNA) used in programmable nuclease systems like CRISPR-Cas9 (Fu et al., 2013, Nature Biotechnology). These sites are characterized by the presence of a compatible Protospacer Adjacent Motif (PAM), which allows the Cas protein to bind and potentially induce double-strand breaks (DSBs) despite mismatches between the gRNA and the DNA sequence (Zhang et al., 2015, Molecular Therapy - Nucleic Acids). While the primary goal of gene editing is to modify a specific on-target site, the recognition of these off-target sites can lead to unintended mutations, including insertions, deletions, or chromosomal translocations (Tsai et al., 2015, Nature Biotechnology). In a clinical context, such as the use of exagamglogene autotemcel (Casgevy), these sites represent a significant safety concern due to the risk of genotoxicity or the activation of oncogenes (FDA, 2023, Summary Basis for Regulatory Action). Consequently, identifying and minimizing activity at these sites is a critical component of therapeutic development, utilizing both computational prediction and empirical genome-wide assays like GUIDE-seq or DISCOVER-seq to ensure genomic integrity (Wienert et al., 2019, Science).

Other names

CRISPR off-targetsNon-specific genomic cleavage sitesOff-target effectsOTEsMismatched protospacersUnintended genomic modifications

Unintended binding and enzymatic cleavage of DNA by a programmable nuclease (e.g., Cas9) at sequences with partial complementarity to the guide RNA and a compatible PAM sequence, leading to double-strand breaks and subsequent error-prone repair.