Off-target genomic loci (Perforin 1 guide RNA)

Off-target genomic loci represent unintended DNA sequences where a CRISPR-Cas9 complex may bind and induce double-strand breaks (Fu et al., 2013, Nature Biotechnology). In the context of Perforin 1 (PRF1) gene editing, these sites are characterized by partial sequence complementarity to the PRF1-specific guide RNA (gRNA) and the presence of a 5'-NGG-3' Protospacer Adjacent Motif (PAM) (Mojica et al., 2009, Microbiology). While the intended target is the PRF1 gene, which encodes a pore-forming protein essential for the cytotoxic activity of T cells and Natural Killer cells (UniProt KB: P14222), Cas9 can tolerate several mismatches between the gRNA and the genomic DNA. This lack of absolute specificity can lead to permanent mutations at non-target sites, potentially causing genomic instability or the disruption of tumor suppressor genes (National Institutes of Health, NIH). Identifying these loci through methods like GUIDE-seq or CIRCLE-seq is a fundamental requirement for the clinical development of gene therapies targeting PRF1-related disorders, such as Familial Hemophagocytic Lymphohistiocytosis (Tsai et al., 2015, Nature Biotechnology).

Other names

CRISPR off-target sitesUnintended genomic cleavage sitesNon-specific PRF1 gRNA binding sitesPRF1 gRNA off-targets

Unintended DNA double-strand breaks followed by error-prone repair (NHEJ) or homology-directed repair (HDR) at sites with partial gRNA complementarity and a compatible PAM.