More recently, ssODNs have been used in place of targeting plasmids for short modifications within a defined locus without cloning32. To achieve high HDR efficiencies, ssODNs contain flanking sequences of at least 40 bp on each side that are homologous to the target region, and they can be oriented in either the sense or antisense direction relative to the target locus. It is worth noting that targeting efficiencies can vary widely depending on cell type, target locus, type of repair donor and location of modification relative to the DSB site. As a rule of thumb, single-base correction rates drop approximately fourfold at 100 bp away from the DSB site, and beyond 200 bp away drug selection markers may be required58.
