Recombinase-Mediated Cassette Exchange (RMCE) for Gene-Stacking in Saccharomyces cerevisiae

Recombinase-mediated cassette exchange (RMCE) is a genomic engineering tool that allows for precise integration of a gene-of-interest (GOI) and excision of extraneous DNA using site-specific recombinases. Working with Saccharomyces cerevisiae, multiple GOIs are incorporated at a targeted locus (TAG site) through multiple rounds of RMCE with different recombinases. First, TAG lines are developed for use with Bxb1, PhiC31, CinH and ParA recombinases by genomic integration of recombinase recognition sequences (RRS; attP and res sites) bordering a selection gene. All recombinases are functional in S. cerevisiae with Bxb1 and PhiC31 promoting integration, and CinH and ParA promoting excision. After initial tests of recombinase function, Bxb1, PhiC31 and CinH were selected for use in a gene-stacking tool. To achieve gene-stacking, an EXCH vector is introduced carrying the first GOI, bordered by Bxb1 attB and CinH res for RMCE at the TAG site, and an additional PhiC31 attB site for a subsequent integration event. The next GOI is introduced with a second EXCH vector carrying the GOI, bordered by PhiC31 attP and CinH res for RMCE between complementary RRS. This EXCH vector also carries a Bxb1 attP site for the next integration event. Gene-stacking continues by switching between EXCH vectors that use alternate integration recombinases (Bxb1 or PhiC31), paired with CinH for excision. Currently, this technology is being used to incorporate multiple GOIs into S. cerevisiae for a trait enhancement study, with future applications to crop systems in development.