Abstract

For decades, Baker’s yeast (Saccharomyces cerevisiae) has served as a tremendous model for biomedical research. Genome-scale engineering in yeast is feasible primarily due to prodigious homology-directed DNA repair, a plethora of genetic tools/selection markers, and simple conversion between haploid and diploid forms. However, with the emergence of yeast as a model eukaryote for systems and synthetic biology research, there is a need for highly efficient and scalable genome engineering strategies. Previously, using CRISPR-Cas9, our laboratory developed a method for one-step, marker-free editing of the yeast genome using Homology Directed Repair (HDR). In the first part of my work, I created CRISPR-Cas9 toolkits targeting several yeast loci and showed specific, efficient and targeted Double-strand Breaks (DSBs) followed by HDR. Next, by combining CRISPR-Cas9, DNA repair via HDR, and yeast mating and sporulation, I demonstrate a highly efficient gene drive (referred to as Cas9-induced Gene Drive or CGD) to perform precise, scar-free, and selection-less conversion of native yeast loci to heterologous engineered loci respectively. To test the efficiency of the gene drive, I convert the functional copy of the Ade2 gene to Ade2 null locus using a heterozygous diploid strain (Ade2 / Δade2::KanMX). First, I show the conversion of the Ade2 to Δade2::KanMX locus is near 100% efficient since the DSB-resistant KanMX copy of the homologous chromosome serves as a highly effective repair template for HDR. Next, I demonstrate the conversion of two or more heterozygous human-yeast loci to become homozygous for human genes at a comparable rate (i.e., ~100%). To show the feasibility and scalability of this method for assembling multi-gene biosynthetic pathways or complexes, I am testing the engineering of the entire carotenoid pathway and the orthologous human proteasome alpha core genes in yeast. Thus, CGD lays the foundation for large-scale combinatorial engineering of the entire heterologous biological processes in budding yeast.