PMC

Optimization of CRISPRi for genetic repression in C. albicans. (a) Promoter region of the ADE2 gene targeted with sgRNAs. Four sgRNAs were designed at four distinct loci upstream of the ADE2 start codon (−416, −129, −55, and −19 bp upstream). (b) Identifying a promoter region for CRISPRi targeting. C. albicans strains were generated, each of which contained a dCas9 plasmid and one of the four sgRNAs described for panel a. To determine the extent of ADE2 repression, growth was monitored by serial dilution spotting assays on SD media with or without supplemented adenine (Ade). Two strains (those with −129-bp and −55-bp sgRNAs) showed reduced growth on SD medium without adenine, suggesting that those strains successfully repressed ADE2.

Design and validation of a CRISPRi system for C. albicans. (a) dCas9 plasmid engineered for CRISPRi repression. This dCas9-based plasmid represents an all-in-one system for CRISPRi repression in C. albicans. All components have been codon optimized for C. albicans, and two nuclease mutations (D10A and N863A) have been introduced into Cas9 to render it nuclease-dead (dCas9). NEUT5L homology is present for integration into the C. albicans genome upon plasmid linearization with PacI. The two SapI cloning sites allow simple sgRNA N20 cloning to generate unique sgRNAs. (b) dCas9 is deficient with respect to its nuclease function. Side-by-side comparisons of C. albicans transformation plates were performed using a Cas9 and dCas9 plasmid with sgRNAs targeting the ADE2 ORF for Cas9-mediated DSB. The two strains were cotransformed with a repair donor DNA template harboring a frameshift mutation to generate a premature stop codon in the ADE2 gene, leading to loss of function, and a red phenotype. Absence of observed red colonies upon transformation of the dCas9 construct suggests that it was deficient with respect to its nuclease activity. CaCAS9, C. albicans Cas9. (c) The dCas9 plasmid integrated in the C. albicans genome does not affect growth. Growth curves were performed using a wild-type C. albicans strain and one with the dCas9 plasmid integrated in its genome at the NEUT5L locus. The dCas9-containing strain did not show a defect in growth compared to the wild-type (WT) strain.

CRISPRi repression with dCas9-repressor fusion constructs. (a) dCas9 fusion constructs for CRISPRi repression. The diagram depicts the three dCas9 constructs (dCas9, dCas9-Mig1, and dCas9-Mxi1) engineered for CRISPRi repression in C. albicans. sgRNAs 1, 2, and 4 are on the sense strand; sgRNA 3 is antisense. RNAP, RNA polymerase. (b) dCas9-Mxi1 enhances repression from the ADE2 locus. C. albicans strains were generated, with each containing a dCas9 plasmid (dCas9, dCas9-Mig1, or dCas9-Mxi1), each with the same sgRNA targeting ADE2. To determine the extent of ADE2 repression, growth was monitored by serial dilution spotting assays on SD media with or without supplemented adenine. While dCas9 and dCas9-Mig1 showed reduced growth on SD without adenine medium, dCas9-Mxi1 showed further growth reduction, suggesting that this construct most effectively repressed ADE2 expression. (c) Growth curves confirming dCas9-based repression from the ADE2 locus. Wild-type, dCas9, and dCas9-Mxi1 C. albicans strains were grown in liquid SD media with or without supplemented adenine, and growth kinetics were monitored over ∼18 h. Both CRISPRi strains showed reduced growth in the absence of adenine, and the dCas9-Mxi1 strains showed further growth reduction. (d) qRT-PCR confirmed the reduced ADE2 transcript levels. To validate the transcriptional repression via CRISPRi, qRT-PCR was performed on wild-type, dCas9, and dCas-Mxi1 strains. ADE2 transcripts were monitored and normalized to an ACT1 transcript as a housekeeping gene. Data were plotted as fold repression of ADE2 relative to the wild-type control strain. Error bars depict standard errors of the means (SEM).

CRISPRi-based repression of the essential gene HSP90 in C. albicans. (a) Reduced levels of HSP90 rendered C. albicans more sensitive to azoles in MIC assays. MIC assays were performed with a gradient of an azole drug, namely, fluconazole (from 40 µg/ml to 0 µg/ml) or miconazole (from 3 µg/ml to 0 µg/ml), in 2-fold serial dilutions. Growth of all strains was monitored across the drug gradients and in a no-drug control (ND). The strains tested for antifungal susceptibility are indicated as follows: WT (SC5314), WT (with dCas9-Mxi1) (strain containing only dCas9-Mxi1 with nontargeting sgRNA integrated at the NEUT5L locus [also known as fRS187]), and HSP90 CRISPRi 1 (fRS221) and HSP90 CRISPRi 2 (fRS222) (two independently generated HSP90 CRISPRi dCas9-Mxi1 strains, each with a unique sgRNA targeting HSP90 for repression). Growth was normalized relative to the no-drug control, and data were quantitatively visualized using TreeView3. (b) Reduced levels of HSP90 render C. albicans more sensitive to fluconazole in disk diffusion assays. Disk diffusion assays were performed using a 25-µg-fluconazole disk on Casitone agar plates. Growth of wild-type (WT) strains (including SC5314 WT and a WT strain containing only dCas9-Mxi1 with nontargeting sgRNA at the NEUT5L locus) and of two independent HSP90 CRISPRi dCas9-Mxi1 strains (each with a unique sgRNA targeting HSP90 for repression) was observed on these plates after 24 h and 48 h (48-h results are depicted here). Quantification of the zone of inhibition (measured using the diskImageR program []) is depicted in the graph. (c) Growth curves confirming sensitivity of HSP90 depletion strains. The dCas9-Mxi1 wild-type strain and both HSP90 CRISPRi strains of C. albicans were grown in liquid YPD media with no drug or with 2.5 µg/ml fluconazole, and growth kinetics were monitored over ∼25 h. Both CRISPRi strains showed reduced growth in the presence of fluconazole.