Site-directed mutagenesis systems in which specific DNA sequences are targeted for alteration in vitro have been instrumental in dissecting genetic pathways, gene regulation and in understanding structure-function relationships in proteins. Often there is a need for direct in vivo modification. However, the modification of genomic DNA within cells, such that no heterologous material is retained, is generally an elaborate and inefficient process, that includes various cloning steps for each mutant allele that is to be created. We developed an oligonucleotide targeting approach that eliminated many of the steps required to produce multiple changes in the genome. While developed in the yeast Saccharomyces cerevisiae where homologous recombination is highly efficient, the approach could be applied to other organisms. Briefly, the first step involves integration of a COunterselectable REporter (CORE) cassette that is targeted to a desired genomic locus. The gene modification step occurs by transformation with the appropriate transforming oligonucleotides such that the CORE cassette is lost and the appropriate change is generated. This versatile and accurate system for in vivo targeted mutagenesis has been referred to as delitto perfetto [Italian for perfect murder and idiomatic for prefect deletion] since there is complete elimination of the marker sequences that are used for selection. In this way, no heterologous sequence remains and multiple rounds of mutagenesis can be accomplished by what might be considered as a self-cloning process applicable to any target sequence. The system has been applied extensively in many studies in and beyond our laboratory to rapidly generate mutants of yeast genes or genes from other organisms cloned in yeast. A primary function of homologous recombination in the cells of all organisms is to repair double strand breaks (DSBs) that may occur during meiosis, programmed DNA rearrangements, faulty DNA metabolism and as a result of DNA damage. We proposed that an oligonucleotide might be capable of repairing a DSB and thereby provide a new tool to address DSB repair. A DSB-CORE cassette was created that contained a regulatable I-SceI endonuclease and an I-SceI double-strand cut site within the original CORE cassette described above. A site-specific DSB could, therefore, be generated by the I-SceI just prior to oligonucleotide transformation. We found that the DSB stimulated oligonucleotide targeting more than 1000-fold, with targeting efficiencies as high as 20% of all cells. This is over 2 orders of magnitude higher than any reported DNA integration frequency in yeast. We also found that a DSB can strongly stimulate recombination with single-strand DNA, without significant strand bias, suggesting new twists on present models of DSB repair. The extremely high transformation frequencies and versatility of the break-mediated delitto perfetto system, has resulted in new powerful tools for dissecting mechanisms of homologous recombination as well as rapid genome modification from point mutations to gross chromosome rearrangements such as chromosome circularization, chromosome fusion and reciprocal translocations. We demonstrated the mechanisms of DSB repair mediated by oligonucleotides by examining the effects of null mutations in all known DSB recombination/repair genes. We found that only the RAD52 gene, which is necessary in almost all homologous recombination events in yeast, is absolutely essential for the oligonucleotide targeting process. Rad52 can have two different roles in recombination: single-strand annealing (SSA) between complementary sequences in a Rad51 independent manner or facilitating Rad51-mediated strand invasion and recombination through recruitment of Rad51 protein. We demonstrated that oligonucleotide targeting to a DSB in haploid yeast is independent of Rad51. We also examined oligonucleotide targeting to a single DSB in diploid cells. Only 4% of repair events were due to recombination with the oligonucleotides, suggesting that most of the repair was via recombination with the unbroken homologous chromosome. Since DSB induced recombination between homologous chromosomes is prevented in a rad51 mutant, we examined oligonucleotide-targeted repair in rad51 homozygous cells. The efficiency of targeting increased nearly 14-fold and the vast majority of DSB repair events were due to oligonucleotide-mediated targeting. We propose that oligonucleotide targeting to a DSB occurs mainly via a Rad52-dependent SSA pathway. Rad51 constrains oligonucleotide targeting by directing the repair into recombination with a sister chromatid or a homologous chromosome and possibly by suppressing SSA. Based on these observations in yeast, we are currently pursuing the development oligonucleotide targeting in human cells.
