Off-target genomic DNA loci with partial sgRNA complementarity (CRISPR off-targets)

Off-target genomic DNA loci with partial sgRNA complementarity represent unintended sites within the genome where CRISPR-Cas nucleases bind and induce double-strand breaks (Fu et al., 2013). This phenomenon occurs because the Cas enzyme, guided by a single-guide RNA (sgRNA), can tolerate a limited number of base-pair mismatches, particularly outside the 'seed' region adjacent to the Protospacer Adjacent Motif (PAM) (Zhang et al., 2015). While CRISPR-based therapeutics like Exagamglogene autotemcel are designed for high specificity, these off-target events can lead to permanent genetic alterations, including insertions, deletions, or complex chromosomal rearrangements (Tsai et al., 2015). Such unintended modifications pose significant safety risks, including the potential for oncogene activation or the disruption of tumor suppressor genes, which could lead to malignant transformation (Kosicki et al., 2018). Consequently, identifying and minimizing off-target activity through advanced sequencing techniques like GUIDE-seq or CIRCLE-seq is a fundamental requirement for the clinical development and regulatory approval of gene-editing drugs (Wienert et al., 2019).

Other names

CRISPR off-target sitesNon-specific cleavage sitesUnintended genomic modificationssgRNA off-targetsOff-target effects

Unintended binding and cleavage of DNA by CRISPR-Cas complexes at sites with partial sequence homology to the sgRNA, followed by error-prone DNA repair mechanisms such as non-homologous end joining (NHEJ) (Fu et al., 2013).