Genomic DNA off-target sites (FKRP-directed sgRNA)

Genomic DNA off-target sites with partial complementarity to the FKRP-directed sgRNA are unintended genomic loci where CRISPR-Cas9 systems may bind and induce cleavage. These sites occur because the Cas9 nuclease can tolerate a limited number of mismatches between the single-guide RNA (sgRNA) and the target DNA sequence (Zhang et al., 2015, PubMed: 25075903). In the context of treating Limb-Girdle Muscular Dystrophy Type 2I (LGMD2I), sgRNAs are designed to target the Fukutin-related protein (FKRP) gene to correct mutations or modulate expression (UniProt: Q9H9S5). However, if the sgRNA spacer sequence shares high homology with other regions of the genome, the Cas9 enzyme may create double-strand breaks at these non-target locations. Such off-target activity is a major safety concern in gene therapy, as it can lead to permanent genetic alterations, including insertions, deletions, or chromosomal translocations (Fu et al., 2013, PubMed: 23360964). These unintended modifications can potentially disrupt tumor suppressor genes or activate oncogenes, posing a risk of malignant transformation. Preclinical evaluation of these sites using high-throughput sequencing methods like GUIDE-seq or CIRCLE-seq is essential to ensure the specificity and safety of FKRP-directed therapies. Consequently, while these sites are not therapeutic targets, they represent critical safety liabilities that must be characterized and minimized during drug development.

Other names

FKRP off-target sitesCRISPR off-targetsNon-specific sgRNA binding sitesUnintended genomic modifications

Unintended DNA cleavage or binding by CRISPR-Cas9 due to sequence homology with the sgRNA spacer