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We present work on a nascent method for multiplex genome engineering in\u00a0Saccharomyces cerevisiae that negates the requirement for cloning of expression cassettes. Our system, Random Assembly and INtegration (RAIN), uses intron-mediated homologous recombination (HR) for random\u00a0in vivo assembly of exogenous promoter and ORF libraries, which are combined and cotransformed in a one-pot method. The libraries include consensus homology arms which target long terminal repeat regions of the Ty1 retrotransposon, providing over a hundred possible integration loci. In this way, our developmental system aims to negate the need for\u00a0in vitro combinatorial cloning of promoters and ORFs to generate expression cassettes, simplifying in vitro DNA preparation before multiplex genome engineering. This paper presents findings from a series of experiments to demonstrate a proof of concept for the RAIN system. These include: the first reported use of intron-mediated assembly of promoters and ORFs for expression of a functional gene product; up to three markerless genomic integrations; and up to five integrations with antibiotic selection. We also present a number of innovations to improve integration efficiency during multiplex engineering in S. cerevisiae including: SGS1 gene knockout; disruption of heteroduplex rejection; modified Cas9 expression architecture; and overexpression of HR genes RAD52, MRE11, and RAD59. To demonstrate how our system can be used for single transformation phenotype engineering of multiple strains, we also transformed a library of methylotrophy associated genes to generate four new strains that were able to grow on a solid minimal medium with methanol as the sole additional carbon source. Our findings contribute to the ongoing efforts to improve multiplex genome engineering tools in S. cerevisiae, and provide the foundations for further development of a novel toolbox for generating useful genetic diversity for metabolic pathway engineering.<\/jats:p>","DOI":"10.1093\/femsyr\/foag040","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T11:45:44Z","timestamp":1787226344000},"source":"Crossref","is-referenced-by-count":0,"title":["Simplifying multiplex genome engineering in\n                    <i>Saccharomyces cerevisiae<\/i>\n                    with intron-mediated Random Assembly and INtegration (RAIN)"],"prefix":"10.1093","volume":"26","author":[{"given":"Fergus S","family":"Harrison","sequence":"first","affiliation":[{"name":"Australian Research Council Centre of Excellence in Synthetic Biology, School of Natural Sciences, Macquarie University, Sydney , NSW 2109 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