The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast

Abstract

In vivo genome manipulation through site-directed mutagenesis and chromosome rearrangements has been hindered by the difficulty in achieving high frequencies of targeting and the intensive labor required to create altered genomes that do not contain any heterologous sequence. Here we describe our approach, referred to as delitto perfetto, that combines the versatility of synthetic oligonucleotides for targeting with the practicality of a general selection system. It provides for an enormously wide variety of genome modifications via homologous recombination. Exceptional high frequencies of mutations are reached when a site-specific double-strand break (DSB) is induced within the locus targeted by the synthetic oligonucleotides. Presented in this chapter is an in-depth description of a series of applications of the delitto perfetto strategy for mutagenesis and chromosome modification both with and without the induction of a DSB, along with the procedures and materials.