Construction of C. albicans Knockout Strains

All of the C. albicans strains used in this study were derived from strain SC5314, and a complete list of the strains is provided in Table S2. nourseothricin-sensitive C. albicans strains were cultured in yeast extract-peptone-dextrose (YPD) liquid medium at 30°C and harvested at an optical density at 600 nm between 0.5 and 0.8 prior to transformation by a modified version of the standard lithium acetate protocol (10 ); see our detailed protocol in Text S1. After recovery in liquid YPD for 5 h, nourseothricin-resistant transformants were selected on YPD agar supplemented with 200 µg/ml nourseothricin (GoldBio). Subsequent removal of the CRISPR components was performed by single-colony isolation on synthetic defined (SD) agar medium minus leucine for the LEUpOUT method or by culturing overnight in YP-maltose liquid medium, followed by screening on YPD agar supplemented with 25 µg/ml nourseothricin for the FLP recombinase-mediated method (see Text S1 for details). The generation of homozygous URA3 deletion strains was confirmed by patching to SD minus uracil versus YPD plates; both medium types were supplemented with 200 µg/ml nourseothricin to maintain selection for strains that had integrated the CRISPR components. All E. coli strains were derived from DH5α and cultured at 37°C in LB medium supplemented with 100 µg/ml carbenicillin.