Off-target genomic loci (R553X sgRNA) (None)

Off-target genomic loci with partial complementarity to the R553X sgRNA represent unintended DNA sequences that may be recognized and cleaved by CRISPR-Cas9 systems designed to target the R553X mutation in the CFTR gene (Cystic Fibrosis Foundation, 2023). The R553X mutation (Arg553Ter) is a common nonsense mutation responsible for Cystic Fibrosis, and gene-editing therapies aim to correct this defect using specific single guide RNAs (sgRNAs) to direct Cas9 to the site. Because Cas9 can tolerate a limited number of mismatches between the sgRNA and the genomic DNA, sites with high sequence similarity and a compatible Protospacer Adjacent Motif (PAM) are at risk of being modified (Fu et al., Nature Biotechnology, 2013). These off-target effects are a primary safety concern in the development of genetic medicines, as they can lead to permanent mutations in unrelated genes or regulatory regions (Tsai et al., Nature Biotechnology, 2015). Potential consequences include the activation of oncogenes, the inactivation of tumor suppressor genes, or complex chromosomal translocations (Kosicki et al., Nature Biotechnology, 2018). Rigorous identification and monitoring of these loci using sensitive assays like GUIDE-seq or CIRCLE-seq are essential for ensuring the safety and precision of R553X-targeted therapies. Consequently, these loci are not therapeutic targets themselves but are critical parameters in the risk-benefit analysis of gene-editing drugs.

Other names

CRISPR off-target sitesUnintended genomic modificationssgRNA R553X off-targetsNon-specific Cas9 cleavage sites

Unintended DNA cleavage and subsequent repair (via Non-Homologous End Joining or Homology-Directed Repair) at non-target genomic locations due to sequence homology with the R553X sgRNA and the presence of a compatible Protospacer Adjacent Motif (PAM).