Fig. 7

Strategies for targeted gene insertions. A The reporter strain carries the pyrG gene flanked by two I-SceI recognition sites (shaded boxes) and two halves of a reporter genes. Transient expression of the I-SceI leads to doubles strand breaks (DSB). An expression cassette can be integrated via homologous recombination (HR) using homologous flanking regions (3’ and 5’ FR). B The gene of interest (GOI) is inserted together with the pyr2 marker into the ade2 locus via HR, resulting in a gene deletion. In this system, the pyr2 gene is flanked by direct repeats that may be used for maker recycling. C For the insertion of a GOI into the locus of an auxotrophic selection marker (SM), first the SM gene needs to be deleted. Then, an integration cassette containing the GOI and the previously deleted SM, are integrated into genome of the auxotrophic strain, reestablishing prototrophy