Genome-wide Mapping of Off-Target Events in Single-Stranded Oligodeoxynucleotide-Mediated Gene Repair Experiments.

Short single-stranded oligodeoxynucleotides are versatile molecular tools used in different applications. They enable gene repair and genome editing, and they are central to the antisense technology. Because the usability of single-stranded oligodeoxynucleotides depends on their efficiencies, as well as their specificities, analyzing their genotoxic off-target activities is important. Thus, we have developed a protocol that follows the fate of a biotin-labeled single-stranded oligodeoxynucleotide in human cells based on its physical incorporation into the targeted genome. Affected chromosomal fragments are enriched and preferably sequenced by nanopore sequencing. This protocol was validated in gene repair experiments without intentionally inducing a DNA double-strand break. For a 21-nucleotide-long phosphorothioate-modified oligodeoxynucleotide, we compiled a broad array of error-free incorporations, point mutations, indels, and structural rearrangements from actively dividing HEK293-derived cells. Additionally, we demonstrated the usefulness of this approach for primary cells by treating human CD34