CRISPR-Mediated Genetic Manipulation of Candida albicans

C. albicans strain SC5314 was used for all experiments unless otherwise noted. The fluconazole-resistant C. albicans strain Can90 was provided by the Massachusetts General Hospital. Yeast strains were grown in YPD medium supplemented with 0.27 mM uridine and selected using Nat at a concentration of 200 μg/ml. Transformations were performed using the lithium acetate method (27 (link)). Flipout of Nat^R gene from Cas9-expressing Duet vector pV1025 was done by induction of flippase by growth in Difco yeast carbon base with bovine serum albumin, and screening for isolates that had lost the Nat^R gene. Filamentation experiments were performed with yeast grown overnight in liquid YPD, washed twice in RPMI 1640 medium (cat. #22400-105, Life Technologies) supplemented with 10% FBS, and incubated in RPMI + 10% FBS for the indicated time at a starting optical density (OD) of 0.1. Growth curves were performed in a clear-bottom 96-well plate and incubated with shaking at 30°C in a Tecan Saphire² plate reader, reading OD at 600 nm every 5 min for the indicated time. YPD-grown overnight yeast cultures were used to inoculate these wells to an initial OD of 0.05. CRISPR-mutagenized loci were verified by sequence analysis of PCR products amplified from the target locus and by restriction digest where applicable.