Gene manipulation serves the purpose of providing a better understanding of the function of specific genes as well as for developing novel variants of the genes of interest. The generation of knockout genes, the alteration, depletion, or enhancement of a particular gene function through the generation of specific gene mutations, or the generation of random mutations in a gene are all essential processes for gene manipulation. The genome of the yeast Saccharomyces cerevisiae is relatively easy to modify, owing to its efficient homologous recombination (HR) system. Gene knockout can be a very simple, one-step approach to eliminate a gene by substituting its DNA sequence with that of a genetic marker. Differently, desired mutations can be introduced into a gene by replacing the sequence of the normal gene with that of the mutated gene. Recombinant DNA can be created in vitro and then introduced into cells, most often exploiting the endogenous recombination system of the cells. However, unless the desired mutation gives a particular phenotype, a bottleneck of 'recombineering' is the requirement of a selection system to identify the recombinant clones among those unmodified. Even in an organism like yeast where the level of HR is highly above the incidence of random integration, the frequency of homologous targeting is in the range of 10(-4)-10(-6) depending on the length of the homology used (Wach et al., 1994). Thus, a selection system is always required to identify the targeted clones. Counterselectable markers, such as URA3, LYS2, LYS5, MET15, and TRP1 (Bach and LaCroute, 1972; Chattoo et al., 1979; Singh and Sherman, 1974; Toyn et al., 2000), are widely utilized in yeast and can be recycled for additional usage in the same yeast strain. If the marker is not eliminated or it is popped out via site-specific recombination between direct repeats, such as in the Flp/FRT or Cre/Lox systems, a heterologous sequence is left as a scar at the site of the modified DNA (Storici et al., 1999; Sauer, 1987). The presence of such scars can threaten the genomic stability of the strain and/or limit the number of successive genetic manipulations for that strain. Here, we describe the delitto perfetto approach for in vivo mutagenesis that combines the practicality of a general selection system with the versatility of synthetic oligonucleotides for targeting (Storici et al., 2001). It provides for generation of gene knockouts and almost any sort of mutation and genome rearrangement via HR. The delitto perfetto in vivo mutagenesis technique is designed for efficient and precise manipulation of yeast strains in a two-step process spanning ~2 weeks. Here, we present the theory and procedures of the delitto perfetto technique.

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Stuckey S, Storici F

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