Unrepaired double-strand breaks (DSBs) in chromosomal DNA can lead to mutagenesis, loss of genetic information, chromosomal aberrations, cell death and disease. The repair of chromosomal DSBs can be accomplished through homologous recombination in most organisms. Several approaches have been taken to address the consequences of a DSB and mechanisms of repair including the introduction of a break at a unique site placed into the genome of the yeast Saccharomyces cerevisiae and other organisms. The systems typically use the yeast mating-type switching enzyme HO endonuclease, or the mitochondrial I-SceI endonuclease that cuts at a unique 18-bp sequence, creating four-base 3? overhangs. DSB recombinational repair mechanisms have also been investigated by transfecting into cells linear or gapped plasmids where the ends, similarly to ends of a chromosomal DSB, are highly recombinagenic with homologous sequences, and can provide targeted integration and replacement of chromosomal DNAs. Recombination of gapped plasmids with long linear molecules or short oligonucleotide is very efficient and has been used for rapid plasmid modification in yeast and in vivo cloning of large chromosomal fragments. Oligonucleotide targeting to homologous chromosomal sequences has also been reported in yeast with systems that relied on a selectable phenotype of recombinants. Using a different procedure referred to as delitto perfetto we developed a system that provides direct selection for every recombinant. The cloning-free system, provided for rapid in vivo creation of products having only the desired mutation. We showed that single and multiple base substitutions, insertions and deletions of 1 to 16000 bp, could be accomplished with high efficiency and accuracy. The approach relies on the integration and successive precise excision (delitto perfetto) of a COunterselectable-REporter (CORE) cassette and exploits the proficient homologous recombination system of yeast. Transformation by oligonucleotides that have homology to small regions around the CORE provide for targeted replacement of the CORE cassette. By varying the sequence of the oligonucleotides, precise mutations including substitutions and small and large deletions can be accomplished. While the technique has many applications and the underlying mechanisms are interesting for studies of recombination and genome mutagenesis, there have been limitations. This led to the investigation of a DSB mediated process. We found that exogenous oligonucleotides can efficiently target for repair a single DSB induced in a chromosome of yeast. The efficiency of recombinational targeting leading to a desired DNA change can reach nearly 20% of all cells in the population during DNA transformation. The DSB was generated either by a regulatable I-SceI endonuclease just prior to transformation or the DSB appeared spontaneously at the site of a long inverted repeat composed of human Alu sequences. The break-mediated delitto perfetto system is a modification of the delitto perfetto method that provides a dramatic improvement with an over 1000-fold increase in efficiency, resulting in considerably greater versatility and high throughput generation of genetic alterations. Using this system we have addressed several issues relating to DSB recombinational repair, including the role of single-strand DNA, targeting at a naturally occurring DSB generated by a large inverted repeat, and the utility of oligonucleotides for rapidly creating a variety of genome modifications.
