Simplifying Multiplex Genome Engineering in Saccharomyces cerevisiae with Intron-mediated Random Assembly and INtegration (RAIN).

RAIN enables multiplex genome engineering in Saccharomyces cerevisiae with up to five integrations without cloning expression cassettes, streamlining pathway optimization.

• Why it matters: Current methods require extensive cloning of promoters and ORFs, which complicates and prolongs the engineering process. Simplifying this step can accelerate the development of engineered strains for various applications.
• What they did: The team developed RAIN, a system using intron-mediated homologous recombination to assemble and integrate promoter and ORF libraries directly in vivo, targeting over a hundred loci with consensus homology arms, and tested it through multiple integrations and strain modifications.
• The result: The approach successfully achieved markerless and antibiotic-selected integrations, generated methylotrophic strains capable of methanol growth, and laid the groundwork for more efficient, versatile genome engineering tools in yeast.