Geneticin g418

20 μL of prepared cells, 1–3 μg of drug resistant cassette DNA, CRISPR mix, and RNAse free water to a final volume of 45 μL was mixed and transferred to a BioRad Gene Pulser cuvette (0.2 cm gap). Cells were pulsed using an Eppendorf Eporator at 1500 V and immediately resuspended in 1 mL of ice-cold Sorbitol. Cells were then collected by centrifugation, resuspended in 1 mL of YPD media, and allowed to recover by incubation at 30°C at 250 rpm for 3–24 hours. Cells were then collected, resuspended in 100 μL of YPD, and plated onto drug selective media at the desired concentration. Nourseothricin (GoldBio) was used at a final concentration of 300 μg/mL for antibiotic selection of the NatMX cassette. Hygromycin B (Cayman) was used at a final concentration of 500 μg/mL antibiotic selection of the HphMX cassette. Geneticin (G418, GoldBio) was used at a final concentration of 800 μg/mL for antibiotic selection of the KanMX cassette. Zeocin (Cayman) was used at a final concentration of 600 μg/mL for antibiotic selection of the BleMX cassette in C. glabrata and 800 μg/mL in C. auris. Colonies were streaked onto fresh plates with the desired drug, and single colonies were selected and restreaked onto fresh YPD plates. Colonies were screened via colony PCR using primers indicated in Table S2. Three independent clones were verified by PCR and analyzed for phenotypic characterizations.