Frozen ez yeast transformation kit

Deletion mutants were generated in house using a CRISPR-Cas9-targeted integration replacing the desired open reading frame (ORF) by a nourseothricin (NAT) resistance cassette (Table S3). The deletion constructs were generated by PCR using ∼100-nucleotide-long primers designed to amplify NAT and also containing homology regions flanking the locus of interest. All primers used are listed in Table S4. Gene replacement by the deletion cassettes was performed using CRISPR as described previously (70 (link)). Briefly, cells were made competent for electroporation using the Frozen-EZ yeast transformation kit (Zymo Research) according to the manufacturer’s instructions and then electroporated with a Cas9-genomic RNA (gRNA) complex (Integrated DNA Technologies, Coralville, IA) and the DNA containing the deletion construct. Transformants were selected on NAT-containing plates and validated by PCR amplification and sequencing of the targeted locus using external primers (Table S4). At least two independent transformants were generated and analyzed for every deletion mutant. All primers were ordered from Integrated DNA Technologies, and all Sanger sequencing of the above-described constructs was done by Genewiz.

The following genes were subjected to deletion, catalase 1 (CAT1, CAGL0K10868g), glutathione oxidoreductase (GRX2, CAGL0K05813g), manganese superoxide dismutase (SOD2, CAGL0E04356g), and three transcription factors playing role in oxidative stress responses, namely SKN7 (CAGL0F09097g), MSN4 (CAGL0M13189g), and YAP1 (CAGL0H04631g), and ICL1 (CAGL0L09273g). Knock-out mutants were created in house using a previously described protocol^{65 (link)}, in which the open reading frame of the gene of interest was replaced by nourseothricin (NAT) resistance cassette. The knock-out construct was generated by using Ultramer primers (∼80–100 bps) containing homology regions with NAT and with regions flanking GOIs. Competent cells were created by log-phase grown C. glabrata cells using Frozen-EZ Yeast Transformation Kit (Zymo Research) and transformation followed an electroporation-based protocol described previously^{67 (link)}. The colonies growing on YPD plates containing NAT were subjected to PCR and sequencing using diagnostic primers listed in Supplementary Table 3 (the primers used in the current study were manufactured by Integrated DNA Technologies and Sanger Sequencing carried out by Genewiz). Two independent knock-out mutants were used for each experiment.