Falgowski K, Falgowski C, York-Vickers C, Kmiec EB
Nucleic Acids Res. 2011 Jun;39(11):4783-94
PMID: 21343181
Abstract
Gene editing directed by modified single-stranded DNA oligonucleotides has been used to alter a single base pair in a variety of biological systems. It is likely that gene editing is facilitated by the direct incorporation of the oligonucleotides via replication and/or by direct conversion, most likely through the DNA mismatch repair pathway. The phenomenon of strand bias, however, as well as its importance to the gene editing reaction itself, has yet to be elucidated in terms of mechanism. We have taken a reductionist approach by using a genetic readout in Eschericha coli and a plasmid-based selectable system to evaluate the influence of strand bias on the mechanism of gene editing. We show that oligonucleotides (ODNs) designed to anneal to the lagging strand generate 100-fold greater ‘editing’ efficiency than ‘those that anneal to’ the leading strand. The majority of editing events (∼70%) occur by the incorporation of the ODN during replication within the lagging strand. Conversely, ODNs that anneal to the leading strand generate fewer editing events although this event may follow either the incorporation or direct conversion pathway. In general, the influence of DNA replication is independent of which ODN is used suggesting that the importance of strand bias is a reflection of the underlying mechanism used to carry out gene editing.