Genomic double-stranded DNA at partially complementary off-target protospacers with NGG PAMs (CRISPR off-target sites)

Genomic double-stranded DNA at partially complementary off-target protospacers represents the primary safety liability for CRISPR-Cas9-based gene editing technologies (Fu et al., 2013, Nature Biotechnology). These sites are genomic loci that contain a Protospacer Adjacent Motif (PAM)—specifically the NGG sequence for SpCas9—and exhibit high sequence similarity to the intended target sequence (Zhang et al., 2015, Molecular Therapy - Nucleic Acids). The Cas9-gRNA complex can hybridize to these sites despite the presence of several mismatches, leading to unintended binding and cleavage. When the Cas9 endonuclease induces double-strand breaks (DSBs) at these locations, the cell's repair machinery can introduce permanent mutations such as indels or large-scale chromosomal rearrangements (Kosicki et al., 2018, Nature Biotechnology). In a clinical context, off-target activity poses a significant risk of genotoxicity, which may lead to the activation of proto-oncogenes or the inactivation of tumor suppressor genes. Consequently, rigorous off-target analysis using both computational and experimental methods like GUIDE-seq is a critical component of the regulatory approval process for CRISPR-based therapeutics such as Exagamglogene autotemcel (Casgevy FDA Label, 2023).

Other names

Off-target genomic sitesCRISPR-Cas9 off-targetsNon-specific protospacersMismatched DNA targetsUnintended cleavage sites

Unintended RNA-guided endonuclease-mediated cleavage of DNA at sites with sequence homology to the guide RNA.