Genome-wide off-target DNA sites (Off-target sites)

Genome-wide off-target DNA sites are unintended genomic locations where programmable nucleases or DNA-binding molecules interact with the genome due to sequence similarity with the intended target. These interactions are a primary concern in the development of gene-editing therapies, such as CRISPR-Cas9, as they can lead to permanent, deleterious mutations in non-target genes (Nature, 2013, doi:10.1038/nbt.2623). The biological consequence of activity at these sites includes double-strand breaks, which the cell may repair via error-prone pathways, potentially resulting in chromosomal translocations or the disruption of tumor suppressor genes (PubMed, 2019, PMID: 31142833). Consequently, identifying and quantifying off-target activity is essential for ensuring the safety and precision of genomic medicines (NIH, 2021). Various high-throughput sequencing methods, such as GUIDE-seq and CIRCLE-seq, have been developed to map these sites across the entire genome to mitigate risks during drug development (Nature Methods, 2015, doi:10.1038/nmeth.3284).

Other names

Off-target effectsNon-specific DNA binding sitesUnintended genomic modificationsCRISPR off-targetsOff-target cleavage sites

Unintended binding and enzymatic modification (cleavage, deamination, or integration) of DNA sequences that share partial homology with the intended target sequence, often mediated by guide RNA mismatch tolerance.