Constructing humanized yeast strains with delitto perfetto system. A. Replacing yeast MMR (yMMR) genes (i.e. yMLH1 and yPMS1) with their human orthologs (hMMR). First, the CORE cassette was introduced into yeast chromosome (line) after the stop codon of a yeast MMR gene, enabling the yeast gene to be still active during the second step of the process. Next, together with CORE cassette, the yeast gene was replaced with its human ortholog. B. Introducing missense alterations into hMLH1 gene in strain co-expressing hMLH1 and hPMS2 genes. First, the CORE cassette was introduced into hMLH1, replacing nucleotide of interest (black vertical line). Next, the CORE cassette was replaced by DNA fragment harbouring a single-nucleotide alteration (yellow vertical line). Genetic technique, delitto perfetto, is based on recombination event (grey area) between two identical strand of DNA.