Off-target genomic DNA sequences (Off-target sites)

Off-target genomic DNA sequences are unintended locations within the genome where gene-editing tools, such as CRISPR-Cas9, bind and induce modifications due to sequence similarity with the intended target. This phenomenon occurs when a non-target site possesses a compatible protospacer adjacent motif (PAM) and sufficient complementarity to the guide RNA (gRNA) (Fu et al., 2013, Nature Biotechnology). These unintended edits are a major safety concern in the development of genetic medicines, as they can lead to permanent mutations, chromosomal translocations, or the disruption of tumor suppressor genes (Zhang et al., 2015, Science). While these sequences are not therapeutic targets, their identification and characterization are mandatory for the regulatory approval of CRISPR-based therapies like exagamglogene autotemcel (Casgevy). Various high-throughput sequencing methods, such as GUIDE-seq and CIRCLE-seq, have been developed to detect these off-target events with high sensitivity (Tsai et al., 2015, Nature Biotechnology). Minimizing off-target activity through gRNA optimization and the use of high-fidelity Cas variants is a primary focus of current biotechnological research. Ultimately, the management of off-target risks is essential to ensure the long-term safety and clinical viability of genome-editing platforms.

Other names

Off-target effectsNon-specific genomic sitesCRISPR off-targetsUnintended cleavage sites

Unintended DNA cleavage and mutagenesis at non-target loci mediated by programmable nucleases followed by error-prone DNA repair.