Off-target genomic DNA loci with partial complementarity to the MACF1-directed sgRNA (MACF1 CRISPR off-targets)

Off-target genomic DNA loci with partial complementarity to the MACF1-directed sgRNA represent unintended sites in the genome where CRISPR-Cas9 complexes may bind and induce double-strand breaks. These sites possess sequence similarity to the primary target sequence within the Microtubule-actin cross-linking factor 1 (MACF1) gene, leading to potential 'off-target' effects during gene editing procedures (Fu et al., 2013). While MACF1 itself is a target for research in bone health and cancer due to its role in cytoskeletal dynamics, these off-target loci are considered significant safety risks rather than therapeutic targets (Yin et al., 2019). The interaction occurs when the sgRNA-Cas9 complex tolerates mismatches between the guide RNA and the genomic DNA, resulting in permanent mutations such as insertions or deletions (indels) at these locations (Tsai et al., 2015). Such unintended modifications can lead to genomic instability, the disruption of essential genes, or the activation of oncogenes, posing a major hurdle for the clinical translation of MACF1-targeted therapies. Monitoring these loci using high-throughput sequencing methods like GUIDE-seq or CIRCLE-seq is critical for assessing the specificity and safety of CRISPR-based interventions (Tsai et al., 2015). These loci are not intentionally targeted but are a byproduct of the inherent limitations in the specificity of RNA-guided nuclease systems. Understanding the landscape of these off-target sites is essential for designing more precise sgRNAs with reduced risk of genotoxicity.

Other names

CRISPR off-target sitesNon-specific genomic cleavage sitesMACF1 sgRNA off-targetsUnintended MACF1-related DNA cleavage sites

Unintended binding and cleavage of DNA by the Cas9 nuclease guided by an sgRNA with sequence homology to non-target genomic regions (Fu et al., 2013).