Cluster deletion and replacement of an essential gene using TREC-IN. (A) Schematic of replacement of the intervening ssrA essential gene (0158) upon cluster deletion in Mmc Syn1. In step 1, the target region (orange arrows) containing the ssrA gene and two adjacent non-essential clusters (0152-0157 and -159-0162) were replaced with the CORE6 cassette. In step 2, a knock-in module was integrated into the target site by co-transforming two PCR products. One of the amplicons contained the ssrA gene, 50 bp RS, and 50 bp DHR. The other amplicon included the 3’ region of the kanMX split marker gene component, 50 bp RS, and 50 bp homology region to the 5’ kanMX gene component in the CORE6 cassette. Homologous recombination resulted in full complementation of the kanMX gene component. Yeast colonies were selected for geneticin resistance and then grown on galactose. In step 3, galactose induces I-SceI expression, which produces double-strand break at the I-SceI site and enhances intra-molecular homologous recombination (dashed double-headed blue arrow) between the two RS, resulting in excision of the CORE6 cassette. Colonies were grown on 5-FOA for Uracil counter selection. (B) PCR screening to confirm cluster deletion and replacement of the ssrA essential gene. (a) DNA from four yeast colonies after selection on 5-FOA were amplified using diagnostic primers (dashed arrows) D0152/162-DGF and 0158-DGR (left junction; expected size, 239bp), and (b) D0152/162-DGF and D0152/162-DGR (expected size of ssrA replacement, 1.0kb).