Engineered S. cerevisiae Strain Generation

Standard molecular biology protocols were used to engineer all S. cerevisiae strains (Supplementary Table 1) used in this study⁵⁸ . The overall methodology for construction of the triple gene drive strain utilized both standard HR-based chromosomal integrations (sans any DSB) and Cas9-based editing (Supplementary Fig. 1). Briefly, DNA constructs were first assembled onto CEN-based plasmids (typically pRS315) using in vivo assembly in yeast^{59 (link)}. If necessary, point mutations were introduced using PCR mutagenesis^{60 (link)}. Next, the engineered cassette was amplified with a high-fidelity polymerase (KOD Hot Start, EMD Millipore), transformed into yeast using a modified lithium acetate method^{61 (link)}, and integrated at the desired genomic locus. PCR was used to diagnose proper chromosomal position for each integration event followed by DNA sequencing. The DNA maps for manipulated yeast loci are included in Supplementary Fig. 2. DNA plasmids used in this study can be found in Supplementary Table 2. Expression cassettes for sgRNA were based on a previous study^{62 (link)}, purchased as synthetic genes (Genscript), and sub-cloned to high-copy plasmids using unique flanking restriction sites. All vectors were confirmed by Sanger sequencing.