Research ArticleMOLECULAR GENETICS

A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families

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Science Advances  03 Apr 2015:
Vol. 1, no. 3, e1500248
DOI: 10.1126/sciadv.1500248
  • Fig. 1 Candida CRISPR expression constructs.

    (A) Duet system consists of two plasmids, pV1025 (top before and after flipout), which targets ENO1, and pV1090, which targets RP10. (B) Solo system consists of one plasmid, pV1093, which targets ENO1. Figures are not drawn to scale. (C) Both Solo and Duet guide expression systems permit rapid cloning by digestion with BsmBI followed by ligation of annealed oligos (shaded sequences) with desired guide sequence (ADE2 guide sequence in red box). (D) Schematic of Cas9 mutagenesis method. The system can create homozygous mutations in the gene (*) and simultaneously mutate sequences (for example, the PAM) to prevent repeated cleavage subsequent to integration.

  • Fig. 2 C. albicans CRISPR is an efficient mutagenesis system.

    (A) Candida CRISPR efficiently mutagenizes both ADE2 loci in SC5314. The transformation mix contained pV1081 and a mutagenic repair template (see Materials and Methods and the Supplementary Materials). Omission of Cas9, sgRNA, or a repair template with homology to the guide resulted in failure to obtain ade2 mutants. (B) Sequence of the ADE2 locus in wild-type (WT) and mutant isolates. (C) Assay for ura3/ura3 transformant on 5-fluoroorotic acid (FOA) plates. FOA permits growth of ura3/ura3 but not URA3+ strains. (D) Wrinkled colony morphology of RAS1V13 on transformation plates (top) and glycogen accumulation defect/wrinkled colony morphology of RAS1V13 (bottom). Glycogen accumulation is visualized by exposing yeast to iodine vapors, which stains glycogen red. WT (left) has a smooth morphology and stains red due to accumulated glycogen (left), whereas RAS1V13 (right) has a wrinkled morphology and fails to stain. (E) Truncation of RAS1 at position 13 [ras1(TAA)13] reduces growth rate.

  • Fig. 3 CRISPR permits simultaneous targeting of CDR1 and CDR2, which mediate resistance to fluconazole and cycloheximide.

    (A) Sequence of CDR1 and CDR2 loci and verification by digestion. (B) Mutation of CDR1 and CDR2 sensitizes SC5314 (left) and fluconazole-resistant clinical isolate Can90 (right) to fluconazole (0.41 μg/ml for SC5314, 200 μg/ml for Can90). Different fluconazole concentrations were used for each strain background, because the Can90 isolate had much greater resistance. Solid lines indicate medium without fluconazole, and dotted lines indicate medium with fluconazole. (C) Simultaneous mutation of three genes (six sites) in a single transformation, and the resulting phenotypes. Left panel is YPD, and right panel is YPD plus cycloheximide at 400 μg/ml. The poorer growth on petri plates of the ade2 cdr1 cdr2 triple mutants is reflected in liquid growth on fluconazole. The ade2 CDR1 CDR2 has a doubling time of 6 hours, whereas the ade2 cdr1 cdr2 mutant has a doubling time of 12 hours when grown in fluconazole (1.2 μg/ml).

  • Fig. 4 The Candida CRISPR system allows efficient isolation of mutations in essential functions.

    (A) SC5314 of the indicated genotype was grown at 37° or 16°C. (B) Strains were grown on YPD with the indicated carbon source at 37°C for 3 days. (C) Strains were grown in YPD at the indicated temperatures. (D) Overnight YPD cultures were diluted into RPMI + 10% fetal bovine serum (FBS) and grown for 2 hours at 37°C. Scale bars, 5 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/3/e1500248/DC1

    CaCas9 protein sequence

    Fig. S1. Recyclable Solo system vector pV1200 permits serial mutagenesis.

    Fig. S2. Candida CRISPR-Duet system requires Cas9, sgRNA expression, and a mutagenic repair template.

    Fig. S3. Candida CRISPR-Solo system requires a mutagenic repair template, but does not require selection for system components.

    Fig. S4. Candida CRISPR permits the isolation of homozygous mutants at multiple loci, including MtlA1, Mtlα2, TPK2, and DCR1.

    Fig. S5. Mutation of CDR1 and CDR2 creates pleiotropic drug sensitivity.

    Fig. S6. Mutation of SNF1 in Candida.

    Table S1. Plasmids used in this study.

    Supplementary Data Files (separate zip file)

  • Supplementary Materials

    This PDF file includes:

    • CaCas9 protein sequence
    • Fig. S1. Recyclable Solo system vector pV1200 permits serial mutagenesis.
    • Fig. S2. Candida CRISPR-Duet system requires Cas9, sgRNA expression, and a mutagenic repair template.
    • Fig. S3. Candida CRISPR-Solo system requires a mutagenic repair template, but does not require selection for system components.
    • Fig. S4. Candida CRISPR permits the isolation of homozygous mutants at multiple loci, including MtlA1, Mtla2, TPK2, and DCR1.
    • Fig. S5. Mutation of CDR1 and CDR2 creates pleiotropic drug sensitivity.
    • Fig. S6. Mutation of SNF1 in Candida.
    • Table S1. Plasmids used in this study.

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