Khalil, Yara2025-08-132025-08-132025-08-13http://hdl.handle.net/10393/50760https://doi.org/10.20381/ruor-31315Saccharomyces cerevisiae's well-characterized genome and efficient homologous recombination capabilities make it a powerful model organism for genetic engineering, enabling the development of precise genome modification techniques. However, simultaneous multi-locus gene integration (multiplexing) remains challenging, often requiring extensive and tedious in vitro cloning. This thesis hypothesizes that a novel yeast integrating plasmid, envisioned as an EasyClone Expansion Pack, can be designed to enable two-step multiplexing leveraging homologous recombination and bypassing in vitro cloning. To accomplish this, we developed a clone-less in vivo DNA assembly method, demonstrating a 20-fold increase in transformation efficiency with CRISPR-Cas9-mediated DNA breaks. A cost-effective multiplexing workflow was established using 200 bp chromosomal homology regions and 30 bp overhangs on transgenes. The foundational design of the expansion pack plasmid, including one-step linearizability and unique homology regions, was validated. This research offers a cost-effective and streamlined approach for strain engineering for use in diverse bioproduction and biomanufacturing applications.enGenetic EngineeringCRISPR-Cas9YeastThesis