In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides.

Abstract

Synthetic oligonucleotides have been used to direct base exchange and gene repair in a variety of organisms. Among the most promising vectors is chimeric oligonucleotide (CO), a double-stranded, RNA-DNA hybrid molecule folded into a double hairpin conformation: by using the cell's DNA repair machinery, the CO directs nucleotide exchange as episomal and chromosomal DNA. Systematic dissection of the CO revealed that the region of contiguous DNA bases was the active component in the repair process, especially when the single-stranded ends were protected against nuclease attack. Here, the utility of this vector is expanded into Saccharomyces cerevisiae. An episome containing a mutated fusion gene encoding hygromycin resistance and eGFP expression was used as the target for repair. Substitution, deletion and insertion mutations were corrected with different frequencies by the same modified single-stranded vector as judged by growth in the presence of hygromycin and eGFP expression. A substitution mutation was repaired the most efficiently followed by insertion and finally deletion mutants. A strand bias for gene repair was also observed; vectors designed to direct the repair of nucleotide on the non-transcribed (non-template) strand displayed a 5-10-fold higher level of activity. Expanding the length of the oligo-vector from 25 to 100 nucleotides increases targeting frequency up to a maximal level and then it decreases. These results, obtained in a genetically tractable organism, contribute to the elucidation of the mechanism of targeted gene repair.