Genomic DNA at unintended off-target Cas9 recognition sites (Cas9 off-target sites)

Genomic DNA at unintended off-target Cas9 recognition sites refers to specific sequences within the genome that are inadvertently targeted and cleaved by the CRISPR-Cas9 system (Zhang et al., 2015). This phenomenon occurs because the Cas9 nuclease, directed by a single-guide RNA (sgRNA), can recognize and bind to DNA sequences that possess several mismatches relative to the intended target sequence (Fu et al., 2013). Such off-target activity results in double-strand breaks (DSBs) at non-target loci, which are typically repaired by the cell's error-prone non-homologous end joining (NHEJ) pathway, leading to permanent mutations (Tsai et al., 2015). In a clinical context, these unintended modifications pose a significant safety risk, as they may disrupt functional genes or regulatory elements, potentially leading to oncogenic transformation or other deleterious cellular phenotypes (FDA, 2023). Minimizing off-target effects is a primary focus of CRISPR therapeutic design, involving the use of high-fidelity Cas variants and sophisticated bioinformatic and experimental validation tools like GUIDE-seq.

Other names

CRISPR off-targetsNon-specific Cas9 cleavage sitesUnintended genomic modificationsOff-target DNA sitesCas9 off-target activity

Unintended Cas9-mediated double-strand breaks (DSBs) at genomic loci with sequence homology to the guide RNA, followed by error-prone DNA repair.