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

Abstract

SummaryTargeted 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.

Optimising gene repair strategies in cell culture

“…Therefore to optimise gene correction, we created a reporter system that allows us to measure gene repair directly in living cells. Gene repair reporters have proved effective [11][12][13][15][16][17][18][19][20][21][22] and we hope that this system will allow application of this technology to living cells (see also Liu et al 14 ). A single base substitution was introduced into the EGFP gene (from Clontech's pHygEGFP vector (Clontech, Palo Alto, CA, USA)) to create a premature stop codon (GFP W399X) which inactivates the green fluorescence of EGFP.…”

Delivery and mechanistic considerations for the production of knock-in mice by single-stranded oligonucleotide gene targeting

“…Oligonucleotides were single-stranded 53-mers with the last four bases on each end protected by phosphorothioate linkages (Figure 1). The length and ends were based on the optimization in Igoucheva et al 4 Figure 1) similar to the approach of Rice et al 30 Plasmids were linearized at the MfeI site, purified by phenol chloroform extraction, and electroporated into AB2.2 ESC cells at 30 mg per 8 million cells. The electroporated cells were plated in dishes for selection.…”

Multiple roles for MSH2 in the repair of a deletion mutation directed by modified single-stranded oligonucleotides

“…The repair phase itself may consist of two separate events: the first, the correction of the targeted strand is followed by a second reaction in which the newly modified strand serves as a template to alter the non-targeted strand. Mechanistic studies of TNE have revealed that a number of repair proteins play a role in the correction process by acting through various pathways that include mismatch repair (MMR), and nucleotide excision repair (NER) (ColeStrauss et al, 1999;Dekker et al, 2003;Rice et al, 2001). …”