In vitro and in vivo nucleotide exchange directed by chimeric RNA/DNA oligonucleotides in Saccharomyces cerevisae

Targeted gene repair directed by chimeric RNA/DNA oligonucleotides has proven successful in eukaryotic cells including animal and plant models, In many cases, however, there has been a disparity in the levels of gene correction or frequency, While the delivery of these chimera into the nucleus and the long-term stability or purity of these molecules may contribute to this variability, understanding the molecular regulation of conversion is the key to improving or stabilizing frequency. To this end, we have identified genes that control targeted repair, using the genetically tractable organism, Saccharomyces cerevisae and a bank of yeast mutants. Results from experiments in cell-free extracts focused our attention on RAD52, RAD1 and RAD59 as central regulatory factors, RAD1 and RAD59 appear to be required for high levels of conversion whereas RAD52 appears to act, surprisingly, in a suppressive fashion. Results from the in vitro experiments were translated into targeting experiments in vivo. Here, mutations in a fusion construct, containing a marker gene, were converted to wild type, evidenced by the expression of green fluorescence in converted cells. Because the repaired fusion gene contains a corrected neomycin sequence, cells were subsequently placed under G418 selection and conversion confirmed at the genetic level, Taken together, these results establish, for the first time, genes that participate in the regulation of targeted gene repair and provide a novel system for evaluating true frequencies of correction, Importantly, this system enables visualization of corrected (green) and uncorrected (clear) cells enabling measurements of conversion in real time.