Mismatched off-target double-stranded DNA sites (CRISPR off-targets)

Mismatched off-target double-stranded DNA sites refer to genomic sequences that are unintentionally recognized and cleaved by CRISPR-Cas9 systems (Hsu et al., 2013, Nature Biotechnology). These sites typically possess a Protospacer Adjacent Motif (PAM), such as the NGG sequence required by Streptococcus pyogenes Cas9 (SpCas9), and exhibit partial sequence complementarity to the guide RNA (gRNA) spacer (Fu et al., 2013, Nature Biotechnology). While CRISPR systems are designed to target specific sequences, the Cas9 enzyme can tolerate several mismatches, particularly in the distal region of the spacer, leading to unintended double-strand breaks at these off-target locations (Tsai et al., 2015, Nature Biotechnology). The occurrence of such off-target effects is a primary safety concern in gene therapy, as it can lead to permanent genomic alterations, including translocations, deletions, or the disruption of essential genes (Kosicki et al., 2018, Nature Biotechnology). These unintended modifications can potentially activate oncogenes or inactivate tumor suppressor genes, posing a risk of malignancy. Consequently, extensive bioinformatic prediction and experimental validation are required to minimize these risks during the development of CRISPR-based therapeutics like Exagamglogene autotemcel (Casgevy).

Other names

Off-target genomic sitesCRISPR-Cas9 off-targetsMismatched protospacersNon-specific DNA cleavage sites

Unintended double-strand break induction via Cas9 nuclease activity at non-target genomic loci.