Delitto perfetto

Delitto perfetto (Italian: [deˈlitto perˈfɛtto]) is a genetic technique for in vivo site-directed mutagenesis in yeast.

This name is the Italian term for "perfect murder", and it refers to the ability of the technique to create desired genetic changes without leaving any foreign DNA in the genome.

The primary advantage of this technique is its ability to eliminate any foreign DNA from the genome after the mutagenesis process.

This ensures there are no selectable markers or exogenous sequences used for targeting left in the genome that may cause unforeseen effects.

Other methods require multiple cloning steps and extensive DNA sequencing to confirm mutagenesis, which is often a complicated and inefficient process.

[1][2][3] There is great flexibility in this approach because after the CORE cassette is inserted (see Method Overview for details), multiple mutations in the gene of interest can be made easily and quickly.

This method can be applied to other organisms where homologous recombination is efficient, such as the moss Physcomitrella patens, DT40 chicken cells, or E. coli.

In Saccharomyces cerevisiae, the RAD52 gene is essential for homologous recombination, and thus is required for the delitto perfetto method.

The counterselectable marker allows for the selection of yeast cells that lose the CORE cassette by the integration of the mutated oligonucleotide during the second step of the process.

[4] CORE-containing yeast cells are transformed with oligonucleotides containing the desired mutation such that they lead to the loss of the CORE cassette during homologous recombination.

This feature allows for the expression of SceI, an endonuclease that recognizes a highly unique 18 nucleotide sequence unlikely to occur anywhere else in the S. cerevisiae genome.

In contrast, this number increases up to 100 transformants per 107 viable cells when a double stranded break is generated.