Plasmids for CaCas9 Duet and Solo systems are listed in the Supplementary Materials. The CaCas9 DNA was synthesized by BioBasic, with codons optimized for expression in both C. albicans and Saccharomyces cerevisiae. All key components were verified by sequencing and restriction analysis, and vector sequences will be provided upon request. Solo and/or Duet vectors (5 to 10 μg) were linearized by digesting with Kpn1 and Sac1 before transformation for efficient targeting to the ENO1 and/or the RP10 locus. Purified repair templates (3 μg) were transformed along with the guide expression plasmids for the Solo or Duet systems. Repair templates were generated with 60-bp oligonucleotide primers containing 20-bp overlap at their 3′ ends centered on the desired mutation point. Primers were extended by thermocycling with ExTaq. Most guides were either immediately adjacent to or within 15 bp of the desired mutagenesis point. Phosphorylated and annealed guide sequence–containing primers were ligated into CIP (calf intestinal phosphatase)–treated BsmBI-digested parent vectors as depicted in Fig. 1C. Correct clones were identified by sequencing.