MATERIALS AND METHODSStrains and media. All C. parapsilosis, C. metapsilosis, and C. tropicalis strains used in this study (see Table S1 at https://doi.org/10.6084/m9.figshare.7776833) were grown in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) or on YPD plates (YPD plus 2% agar) at 30°C. Transformants were selected on YPD agar supplemented with 200 μg/ml nourseothricin (Werner Bioagents, Jena, Germany). Auxotrophies were confirmed by growing mutant strains on synthetic complete (SC) dropout media (0.19% yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 2% glucose, 0.075% amino acid dropout mix, 2% agar). All the plasmids used in this study (see Table S3 at https://doi.org/10.6084/m9.figshare.7776845) were propagated in Escherichia coli DH5α cells (NEB, United Kingdom) by growing cells in LB media without NaCl (Formedium) supplemented with 100 μg/ml ampicillin (Sigma).Construction of the pCP-tRNA series plasmids. The synthetic construct GAPDHp-tRNA-SapI-HDV (Eurofins MWG; see Fig. S2 at https://doi.org/10.6084/m9.figshare.7776818) was designed to include the C. parapsilosis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (GAPDH) (CPAR2_808670) promoter, a C. parapsilosis tRNAAla sequence, two tandem SapI/BspQI sites for the cloning of the guide, a hepatitis delta virus (HDV) ribozyme, and the GAPDH terminator. The cassette was cloned by Gibson assembly (primers are listed in Table S2 at https://doi.org/10.6084/m9.figshare.7776842) into NruI-digested pSAT3 plasmid, which differs from the published pSAT1 plasmid (29) only in that it does not contain any SapI/BspQI sites. The guide RNA was generated by annealing of two 23-bp oligonucleotides carrying appropriate overhanging ends and was cloned into the SapI-digested pCP-tRNA plasmid (see Table S2 at https://doi.org/10.6084/m9.figshare.7776842 and Table S3 at https://doi.org/10.6084/m9.figshare.7776845).Construction of pCT-tRNA plasmids. pAYCU268, from Defosse et al. (42), was used as a starting vector. The green fluorescent protein gene (GFP) was replaced with CAS9 from pV1326 (27) by Gibson assembly using primers jpGA_pAYCU268.fw and GA_pAYCU.CAS9.rv to amplify the backbone and primers jpGA_CAS9.1326.fw and pGA_Cas9.1326.rv to amplify CAS9, producing the plasmid GA_pAYCU.CAS9.1326. CaARS2 (43) was amplified using V3_GA.CaARS2.1326.fw and V3_GA.CaARS2.1326.rv and was introduced into the backbone amplified with the primers GA_pAYCUCAS9.1326.fw and GA_pAYCUCAS9.1326.rv by Gibson assembly. A SapI site in the plasmid backbone was removed in this step. A synthetic construct (Eurofins MWG; see Fig. S2 at https://doi.org/10.6084/m9.figshare.7776818) was designed as described for pCP-tRNA, except that expression of the sgRNA was driven from the A. gossypii TEF1 promoter and the CYC1 terminator from S. cerevisiae. The AgeI and SpeI restriction sites flanking the cassette were used for cloning into doubly digested GA_pAYCUCAS9.1326.ARS vector. C. tropicalis guide RNAs were designed using CHOPCHOP v2 (45, 46).Transformation of Candida strains. C. parapsilosis CLIB214 and C. metapsilosis SZMC8093 strains were transformed using the lithium acetate method as described in reference 29, with minor modifications. Each repair template was generated by primer extension of overlapping oligonucleotides (see Table S1 at https://doi.org/10.6084/m9.figshare.7776833 and Table S2 at https://doi.org/10.6084/m9.figshare.7776842), and 25 μl of unpurified product was used to transform yeast cells. Transformation of C. tropicalis strains was performed by using a modified electroporation protocol (12, 47, 48). C. tropicalis cells were grown to an A600 of 5 to 10 and then resuspended in 0.1 M lithium acetate–10 mM Tris-HCl (pH 8.0)–1 mM EDTA–10 mM dithiothreitol (DTT) and incubated at room temperature for 1 h. Cells were washed twice with ice-cold water and once in 1 M ice-cold sorbitol. The sorbitol wash was decanted, and cells were resuspended in the remaining liquid. Approximately 40 to 50 μl of cells was used per transformation, with 5 μg of plasmid together with 5 μg of purified repair template. Cells and DNA were electroporated at 1.8 kV by using a Bio-Rad Pulser XCell Electroporator and immediately resuspended in 1 ml of cold 1 M sorbitol. Cells were subsequently resuspended in 1 ml of YPD and allowed to recover for 4 h at 30°C before plating was performed on selective media (YPD plus 200 μg/ml nourseothricin). Nourseothricin-resistant transformants were patched onto YPD (and SC medium lacking adenine where indicated) and screened by colony PCR. The mutation efficiency was calculated as follows: (edited transformants on the plate) × 100/(total number of transformants on the plate). Representative mutants were sequenced by Sanger sequencing (Eurofins MWG). Loss of the plasmid was induced by patching transformants onto YPD agar without selection and repatching every 48 h until they no longer grew on YPD agar plates containing 200 μg/ml nourseothricin.
The pCP/CT-tRNA systems can be used for CRISPR-Cas9-mediated gene editing in all three members of theC. parapsilosissensu latocomplex, and inC. tropicalis. CRISPR editing can also be used to reconstitute wild-type alleles and to generate heterozygous mutations.