| Gene editing directed by modified single-stranded DNA oligonucleotides has been used to alter a single base pair in a variety of microbial and animal model systems. This technology could eventually be used as a gene repair treatment for inherited disorders, reversing deleterious single base mutations in affected genes. Gene repair reactions utilize short single-stranded pieces of DNA termed oligonucleotides (ODNs) to induce DNA repair events by the conversion of a single base pair. This reaction can be achieved by an ODN designed to be complementary to the target gene, except for a central mismatched base. Nucleotide conversion efficiencies can vary depending on the ODN length, modifications to prevent degradation, and the model system. The mechanism of ODN-mediated gene repair has yet to be fully understood; however, the two proposed mechanisms for resolving a gene repair event, which have gained the most support are the incorporation via replication and direct conversion pathways. In addition to ongoing research to understand the mechanisms involved in the ODN-mediated gene repair reaction, an interesting phenomenon of strand bias in which one strand of DNA (transcribed, T or non-transcribed, NT) is corrected at significantly higher correction frequencies than the other strand, has been a common observation in the field. Therefore, studies were conducted in an E. coli episomal system in an attempt to determine the underlying gene repair mechanisms involved to produce the strand bias effect. These studies have suggested that not only was strand bias a reflection of the underlying mechanism of repair, but a mechanism bias exists with respect to targeting the leading or lagging strand of DNA.;In addition to conducting mechanistic studies of ODN-mediated gene repair, this strategy was adapted to investigate gene repair capabilities of Caenorhabditis elegans. Current techniques to induce genetic alterations in C. elegans have been plagued by their inability to induce precise, directed alterations into the worm genome. ODN-mediated gene repair can be a technique adapted to direct specific single base modifications in C. elegans to generate novel strains in order to conduct extensive gene function studies. Several worm models and assay systems were designed to investigate gene repair capabilities in C. elegans. Unfortunately, no ODN-mediated gene repair events could be detected or confirmed in the developed systems. |
