CRISPR-Cas9 off-target genomic site (OTS)

Off-target genomic sites are DNA sequences that unintentionally interact with CRISPR-Cas systems due to partial sequence homology with the single-guide RNA (sgRNA) spacer (Fu et al., 2013, Nature Biotechnology). While the CRISPR-Cas9 system is designed to target a specific 20-nucleotide sequence, the Cas9 enzyme can tolerate several mismatches, particularly at the distal end of the protospacer, leading to unintended double-strand breaks (DSBs) at these off-target locations (Zhang et al., 2015, Molecular Therapy - Nucleic Acids). These sites must also possess a compatible Protospacer Adjacent Motif (PAM) to facilitate Cas9 binding and cleavage. The occurrence of off-target effects poses a significant safety risk in clinical gene editing, as unintended mutations can lead to the activation of oncogenes, inactivation of tumor suppressor genes, or large-scale chromosomal translocations (Tsai et al., 2015, Nature Biotechnology). Consequently, rigorous off-target assessment using both computational prediction and empirical genome-wide assays is a critical component of the regulatory approval process for CRISPR-based therapeutics like Exagamglogene autotemcel (FDA, 2023). Strategies to minimize these effects include the use of high-fidelity Cas variants, optimized sgRNA design, and transient delivery methods.

Other names

CRISPR off-targetNon-specific cleavage siteUnintended genomic modificationsgRNA-mismatched siteRNA-guided endonuclease off-target

RNA-guided endonuclease-mediated double-strand DNA cleavage