Off-target genomic DNA sites (Staphylococcus aureus Cas9) (SaCas9 off-targets)

Off-target genomic DNA sites are unintended loci within the genome where the CRISPR-Cas9 system, specifically the Staphylococcus aureus Cas9 (SaCas9) ortholog, binds and induces DNA modifications. These sites are characterized by the presence of a Protospacer Adjacent Motif (PAM)—canonically NNGRRT for SaCas9—and a DNA sequence that shares partial complementarity with the guide RNA (gRNA) (Ran et al., 2015). While SaCas9 is widely utilized in gene therapy due to its small size, which allows for efficient packaging into adeno-associated virus (AAV) vectors, its potential for off-target activity remains a significant safety concern (Kleinstiver et al., 2015). Unintended cleavage at these sites can lead to permanent genomic alterations, such as insertions, deletions, or chromosomal translocations, which may result in the disruption of tumor suppressor genes or the activation of oncogenes (Tsai et al., 2015). Consequently, these sites are not therapeutic targets but are critical safety liabilities that must be identified and minimized during the development of CRISPR-based medicines. Preclinical assessment of these sites typically involves high-throughput sequencing methods like GUIDE-seq or CIRCLE-seq to ensure the specificity of the therapeutic intervention (Tsai et al., 2015).

Other names

CRISPR off-target sitesNon-specific cleavage sitesSaCas9 off-target lociUnintended genomic modifications

Unintended double-strand DNA breaks and subsequent non-homologous end joining (NHEJ) or homology-directed repair (HDR) at loci with partial gRNA complementarity and a valid PAM.