S1 Fig. CRISPR/Cas9-mediated gene inactivation of GDH2 and construction of a GDH2-GFP reporter strain.

(A) A purified KpnI/SacI fragment from pFS108, harboring GDH2-specific sgRNA, and PCR generated repair template (RT) were introduced into wildtype strain SC5314 by electroporation. Nou^R transformants were pre-screened in YNB+Arg medium containing the pH indicator bromocresol purple; the initial pH was 4.0. Three Nou^R colonies were picked for further analysis. Clones #1 and #2 grew poorly and were unable to alkalinize the media; clone #3 grew and alkalinized the media. (B) Genomic DNA, isolated from the three clones, was used as template for PCR amplification of the targeted GDH2 locus; ddH₂O was used as negative control. Restriction of the amplified ≈900 bp fragment by XhoI is diagnostic for successful mutagenesis (primers p5/p6; S2 Table). Strains, clone #1 (CFG277) and clone #2 (CFG278), carry inactivated gdh2-/- alleles. (C) GDH2 is not essential but required for robust growth on glutamate or proline as sole nitrogen source. Five microliters of serially diluted wildtype (SC5314), gdh2-/- NAT^R (CFG277), gdh2-/- NAT^S (CFG279), and control (CFG182) cells were spotted on yeast peptone (YP), synthetic glutamate (SE) and synthetic proline (SP) media containing either 2% glucose (YPD, SED, SPD) or 1% glycerol (YPG, SEG, SPG) as carbon source. The plates were incubated for 48 h at 30 °C and photographed. (D) Fresh colonies of SC5314 (PLC005; WT), CFG279 (gdh2-/-), CASJ041 (cph1-/- efg1-/-) and CFG352 (cph1-/- efg1-/- gdh2-/-) were individually resuspended in YNB+CAA medium and incubated for 24 h at 37 °C. (E) The insertion of GFP in strain CFG273 (GDH2-GFP) was verified by PCR, the expected 1695 bp fragment was amplified using primers (p24/p25; S2 Table); strain CAI4 served as untagged control (middle left panel). CFG273 was transformed with the CRISPR/Cas9 cassette to inactivate GDH2. Putative gdh2-/- clones were identified as described and verified by PCR-RD (p13/p6; S2 Table) resulting in strain CFG400. Strains CFG273 (GDH2/GDH2-GFP) and CFG400 (gdh2/gdh2-GFP) were grown at a starting OD₆₀₀ ≈ 2 in SE medium with 0.2% glucose and 1 mM proline (SED 0.2%+Pro) for 2 h. In contrast to CFG273, strain CFG400 failed to express Gdh2-GFP as assessed by immunoblot (middle right panel) and microscopy (lower panels; Scale bar = 5 μm), demonstrating the specificity of CRISPR/Cas9. https://doi.org/10.1371/journal.ppat.1009877.s001.

https://doi.org/10.1371/journal.ppat.1010008.s001

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