CRISPR off-target site (OTS) (OTS)

Off-target genomic DNA sites refer to locations in the genome where a CRISPR-Cas9 system, such as Staphylococcus aureus Cas9 (SaCas9), may bind and induce double-strand breaks despite having only partial complementarity to the guide RNA (gRNA) (Fu et al., 2013). In the context of SNCA-directed therapies for Parkinson's disease, these sites represent a significant safety concern as unintended modifications could lead to the disruption of essential genes or the activation of oncogenes (Polymeropoulos et al., 1997). The specificity of SaCas9 is determined by the 20-24 nucleotide gRNA sequence and the presence of a compatible Protospacer Adjacent Motif (PAM), which for SaCas9 is typically 5'-NNGRRT-3' (Ran et al., 2015). Identifying and minimizing these off-target effects is a critical step in the development of gene-editing therapeutics to ensure genomic integrity (Tsai et al., 2015). Techniques such as GUIDE-seq or in silico prediction are used to monitor these sites to ensure that the therapeutic intervention does not cause collateral damage to the host genome (Pickar-Oliver & Gersbach, 2019). These sites are essentially "anti-targets" that must be rigorously screened during the preclinical development of any CRISPR-based drug.

Other names

Off-target genomic DNA sites with partial complementarity to the SNCA-directed gRNA and compatible SaCas9 PAMNon-specific cleavage siteUnintended genomic modificationSaCas9 off-targetCRISPR off-target site

Unintended DNA cleavage mediated by SaCas9 when guided by a gRNA with partial sequence complementarity, followed by cellular DNA repair mechanisms (NHEJ/HDR) that introduce mutations (Fu et al., 2013).