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Method for CRISPR/Cas9 Mutagenesis in Candida albicans

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Apr 2017



Candida albicans is the most prevalent and important human fungal pathogen. The advent of CRISPR as a means of gene editing has greatly facilitated genetic analysis in C. albicans. Here, we describe a detailed step-by-step procedure to construct and analyze C. albicans deletion mutants. This protocol uses plasmids that allow simple ligation of synthetic duplex 23mer guide oligodeoxynucleotides for high copy gRNA expression in C. albicans strains that express codon-optimized Cas9. This protocol allows isolation and characterization of deletion strains within nine days.

Keywords: Candida albicans (白色念珠菌), Cas9 (Cas9), CRISPR (CRISPR), Fungal genetics (真菌遗传学), gRNA (gRNA), Yeast (酵母)


C. albicans is a difficult organism to manipulate genetically. Since it normally exists as a diploid that does not readily undergo sexual reproduction, homozygous recessive mutations require sequential modification of each locus. The development and application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) mutagenesis in C. albicans facilitates genetic manipulations because it allows simultaneous mutation of both alleles (Vyas et al., 2015; Min et al., 2016; Ng and Dean, 2017). CRISPR gene editing involves recruitment of an RNA-guided nuclease to a complementary target site adjacent to an NGG protospacer adjacent motif (PAM) (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013). CRISPR associated (Cas) nuclease is targeted with high specificity through complementary base pairing between a guide RNA associated with trans-activating CRISPR RNA (tracrRNA), which binds Cas9 (Gasiunas et al., 2012). Since the chromosomal target sequence is only ~20 nucleotides, expression of a single guide RNA (sgRNA) fused to a ~80 nucleotide tracrRNA, along with Cas9, is sufficient for targeted double-strand DNA cleavage. Since chromosomal breaks are lethal, there is a strong selective pressure for double stranded break repair. In C. albicans, co-expression of a donor repair fragment, containing homology to regions flanking the break, allows repair of the break by homologous recombination. Thus, appropriate design of the donor repair fragment allows introduction of sequence deletions, replacements or other chromosomal alterations.

Our previous studies demonstrated that a key factor contributing to high efficiency CRISPR mutagenesis of C. albicans genes relies on optimal gRNA expression (Ng and Dean, 2017). Toward this end, we created gRNA expression vectors that permit high levels of gRNA expression. The basis for this high-level expression is in part due to the presence of a strong, RNA polymerase II promoter (PADH1). This promoter drives the expression of an sgRNA flanked by a 5’ tRNA and a 3’ hepatitis delta virus (HDV) ribozyme RNA. The presence of these 5’ and 3’ flanking RNA sequences serve to promote efficient post-transcriptional processing to produce a mature sgRNA with precise ends. In the presence of an appropriate donor repair fragment, this increased sgRNA expression dramatically improves CRISPR/Cas mutagenesis in C. albicans. In practice, execution of mutagenesis is quite simple. The gRNA expression plasmid cloning relies on the annealing and ligation of two short gRNA encoding oligonucleotides into pre-cut vectors. Mutagenesis involves co-transformation of an sgRNA plasmid and the donor repair fragment into C. albicans strains that express codon-optimized Cas9 nuclease. Described below are detailed protocols for the design, synthesis, and cloning of gRNA oligonucleotides, healing fragment construction, yeast transformations, and mutant verification.

Materials and Reagents

  1. 1.5 ml microfuge tubes (acceptable as sold by any science distributor)
  2. Glass or disposable round bottom sterile tubes for growing 2-5 ml cultures of yeast and bacteria (acceptable as sold by any science distributor)
  3. Plastic Petri dishes (acceptable as sold by any science distributor)
  4. PCR tubes
  5. Toothpicks
  6. Yeast strains (Table 1)

    Table 1. Strain list

  7. Plasmids (Table 2)

    Table 2. Plasmids

  8. E. coli competent cells (DH5α) (made as described, e.g., https://www.neb.com/protocols/2012/06/21/making-your-own-chemically-competent-cells or can be purchased from New England Biolabs)
    Note: We use CaCl2-treated DH5α but these can be purchased.
  9. Glycerol (any source) (50% in H2O, autoclaved)
  10. Salmon sperm DNA (Sigma-Aldrich, catalog number: D1626 ; 10 mg/ml, sonicated and boiled)
  11. SapI (New England Biolabs, catalog number: R0569S )
  12. ClaI (New England Biolabs, catalog number: R0197S )
  13. Calf Intestinal Phosphatase (CIP) (New England Biolabs, catalog number: M0290L )
  14. Custom oligonucleotides (between 18-60 nucleotides in length, Eurofins MWG Operon, Alabama)
  15. T4 polynucleotide kinase (New England Biolabs, catalog number: M0201S )
  16. T4 ligase (New England Biolabs, catalog number: M0202L )
  17. 2x YT (see https://www.elabprotocols.com/protocols/#!protocol=5436 for recipe) (+ 100 µg/ml ampicillin) liquid media and plates (bacterial selection)
  18. Ampicillin (any source)
  19. Taq polymerase (any source)
  20. Polyethylene glycol (PEG) 3350 (Sigma-Aldrich, catalog number: P4338 )
  21. Lithium acetate (any source)
  22. NcoI or StuI
  23. Uridine (any source)
  24. 0.2% SDS
  25. Agarose (any source)
  26. Qiaquick Gel Extraction Kit (QIAGEN, catalog number: 28706 )
  27. LB
    1. Yeast extract (BD, BactoTM, catalog number: 212750 )
    2. Tryptone (BD, BactoTM, catalog number: 211705 )
    3. Sodium chloride (NaCl, certified ACS grade ≥ 99.0%) (Fisher Scientific, catalog number: S271 )
  28. YPD
    1. Yeast extract (BD, BactoTM, catalog number: 212750 )
    2. Peptone (BD, BactoTM, catalog number: 211677 )
    3. Dextrose (Fisher Scientific, catalog number: D16 )
      Note: Make 20% stock in double distilled water.
  29. Synthetic dextrose lacking uracil (SD-URA) liquid media and plates (uracil prototrophic yeast selection) (see http://cshprotocols.cshlp.org/content/2015/2/pdb.rec085639.short for recipe)
    1. Bacto-Agar (for plates)
    2. Dextrose (Fisher Scientific, catalog number: D16 )
      Note: Make 20% stock in double distilled water.
    3. Yeast Nitrogen base w/o amino acids w/ammonium sulfate (BD, catalog number: 291940 )
    4. All amino acids, purines and pyrimidines (any source)
  30. SD-complete + 5’ FOA (ura3∆ yeast selection plates) (see http://cshprotocols.cshlp.org/content/2016/6/pdb.rec086637.short for recipe).
    1. Bacto-Agar
    2. Dextrose (Fisher Scientific, catalog number: D16 )
      Note: Make 20% stock in double distilled water.
    3. Uracil
    4. Yeast Nitrogen base w/o amino acids w/ammonium sulfate (BD, catalog number: 291940 )
    5. 5-Fluoroorotic acid (5-FOA) (Oakwood Products, catalog number: 003234 )


  1. Pipettes (capable of accurately pipetting from 1 µl-10 ml) (any source)
  2. Thermocycler (MJ Research, model: PTC-200 )
  3. Vortex (any source)
  4. Micro centrifuge capable of 14 kG (Eppendorf)
  5. Heating blocks (37 °C, 44 °C)
  6. Incubators (30 °C, 37 °C)
  7. DNA gel electrophoresis apparatus
  8. UV transilluminator (or handheld UV lamp)
  9. Shaker incubator (or rolling drum) (30 °C, 37 °C)
  10. Apparatus for media sterilization (autoclave or pressure cooker)


The overall procedure entails identification of appropriate target site in the chromosome, design of the gRNA oligonucleotides and their ligation into the gRNA expression plasmid, design and construction of the donor repair fragment, co-transformation of gRNA plasmid and repair fragment into yeast, and finally, screening and verification of mutant strains.
Detailed protocols for each of these steps are described following the timeline outlined below.
Basic protocol/time line for single gene knockout
Day 1-2. Design and order oligonucleotides (see Procedure A)

  1. 3 pairs of different gRNA targets (if possible, target one PAM in promoter region up to 150 bases upstream the initiating ATG) (~23 bases/oligonucleotide).
  2. 2 oligonucleotides for donor repair fragment (~60 bases/primer).
  3. 2 oligonucleotides (forward and reverse) for genotyping. 

Day 3-4. Make gRNA plasmid and repair fragment (see Procedure B, C, and D)
  1. Anneal and fill in donor repair fragment.
  2. Anneal and ligate gRNA-encoding oligonucleotides into SapI digested and dephosphorylated vector.
  3. Transform into E. coli competent cells. 

Day 4-5. Confirm plasmid inserts (see Procedure B)
  1. Grow bacterial culture and prepare gRNA plasmid (6-8 h incubation at 37 °C).
  2. Check gRNA plasmid for loss of ClaI site.
  3. Digest w/StuI for yeast transformation.
  4. Put up overnight yeast culture for next day transformation.

Day 6-7. C. albicans transformation (see Procedure E)
  1. Dilute yeast culture 1:20 for transformation (5 h incubation at 30 °C).
  2. Transform into C. albicans.

Day 9-10. Genotype mutants (see Procedure F and G)
  1. Assay Ura+ transformants by PCR
  2. Streak single colonies to avoid mixture of mutants, recheck by PCR, then stock 2-3 mutant strains.

  1. Design and order gRNA, knock-out verification oligonucleotides
    1. Identify a gene or DNA region you wish to target for mutagenesis and find an appropriate gRNA target sequence. The requirement for Cas9 cleavage is the presence of a PAM sequence (5’-NGG-3’) immediately downstream of the ~20 base pair target. This 5’-NGG-3’ can be on either DNA strand. This sequence must be unique in the C. albicans genome to prevent off-target cleavage. A useful web site for identification and ranking of appropriate gRNA is CHOP CHOPv2 (http://chopchop.cbu.uib.no/) (Labun et al., 2016). Generally, we adhere to the ranking of PAM sites obtained using this web site to guide the choice of target site.
    2. Two complementary oligonucleotides corresponding to the 20 bp double stranded sequence are required. Each oligonucleotide is 23 bases in length and must also contain appropriate sequences at their 5’ end to provide sticky ends for direct ligation of annealed dsDNA into pND494 or pND501 cut with SapI (Figure 1). The choice of which vector to use is based on whether or not subsequent experiments require a uracil prototrophic or auxotrophic strain. If a uracil auxotroph is required, pND501 allows recycling of the Ura- genotype (see Procedure H). The cloning cassette (see Figure 1 below), into which these annealed oligonucleotides are ligated, has been designed to allow precise fusion to both an upstream tRNA sequence and a downstream tracrRNA (Ng and Dean, 2017). This entire tRNA-sgRNA-HDV sequence is driven by the ADH1 promoter in both pND494 and pND501 vectors (Figure 1A). The end result is the expression of an sgRNA flanked with tRNA and ribozymes for precise post-transcriptional processing to produce appropriate 5’ and 3’ ends.
    3. Figure 1 depicts gRNA expression vector maps (A) and an example of the design of the two complementary gRNA oligonucleotides (B). The top sequence in Figure 1B depicts the ClaI-containing SapI cassette present in the pND494/501 expression vectors. The lower sequence depicts the gRNA-encoding oligonucleotides after annealing and ligation into this cassette. This example shows the ligation of the duplex encoding LEU2 gene-specific gRNA, with the boxed sequences indicating each of the oligonucleotides that were synthesized. 

      Figure 1. How to design gRNA-encoding oligonucleotides for expression cloning. The pND494 and pND501 cloning vectors (Panel A) contain a SapI cloning cassette designed for direct ligation of annealed gRNA oligonucleotides (Panel B). The DNA sequence of the SapI cloning cassette is indicated in the upper DNA duplex (B). Shown below is an example of the ligation of double-stranded annealed oligonucleotides encoding a LEU2 targeting gRNA (from Ng and Dean, 2017). The boxed in region indicates the sequence of each oligonucleotide. Note that each 23 bases oligonucleotide includes an overhang sequence that results in a sticky end cohesive with that of the vector digested with SapI. Since SapI cleavage is non-palindromic, re-ligation of the vector is not possible thus greatly reducing the background of false-positive E. coli transformants (C4).

    4. When ordering gRNA oligonucleotides, we generally target 3 different PAM sites (which requires 6 different gRNA oligonucleotides) because the efficiency of gRNA-dependent CRISPR mutagenesis varies in a sequence dependent way. Targeting 3 different PAM sites maximizes the probability of obtaining a correct mutant. Experimental evidence suggests that targeting 5’ proximal regions, near the start site of transcription results in higher mutagenesis frequency (Radzisheuskaya et al., 2016). Thus if possible, choose one target within 150 bases upstream (5’) the first initiating ATG methionine codon as one of target sites.
      Note: DO NOT INCLUDE PAM SITE in gRNA oligonucleotide.
    5. Design primers for verification of correct chromosomal mutation
      Order two ~18-30 bp oligonucleotides, forward and reverse, for checking chromosomal gene deletion, by PCR amplification of genomic DNA. If possible, the product of this PCR amplification should be less than 500 bp. This allows PCR extension time to be less than 30 sec (using Taq polymerase), thereby minimizing the time required for verification. 

  2. Create an appropriate repair template
    A repair template is a double stranded DNA fragment that spans the cut site, has sufficient homology for homologous recombination, and creates appropriate mutation. In addition to introducing the desired mutation, repair templates must lack the PAM site. Otherwise the target break site will be re-introduced into the chromosome after its repair.
    The simplest repair template for creating deletion allele uses a pair of 60 bp oligonucleotides with ~47 bp of homology (at 5’ ends) to sequences flanking the chromosomal break site, ~20 bp overlap at 3’ end, and a unique restriction site within the overlap confirmation of correct mutation (e.g., see Vyas et al., 2015; Ng and Dean, 2017). An example that shows repair strategy for deleting the LEU2 gene (from Ng and Dean, 2017) and the sequence of repair oligonucleotides is illustrated in Figure 2.

    Figure 2. Design of a repair template for gene deletion. Schematic diagram of donor repair fragment synthesis used for deletion of the LEU2 locus, targeting break at the PAM site at position 137 (Panel A) using the oligonucleotides depicted in Figure 1B. Chromosomal sequences homologous to the repair template are colored in red and green. Each 60-mer oligonucleotide ends with a 3’ 20 bp sequence of complementarity, including a restriction site that is absent in LEU2 (EcoRI), shown in blue. When annealed and extended, this repair fragment contains 47 bp of homology to sequence flanking the DSB. In this example, taken from Ng and Dean, 2017, homologous recombination results in a 434 deletion of LEU2 and insertion of a unique EcoRI site, which simplifies genotyping of putative mutants. Panel B shows the sequence of each “repair fragment” oligonucleotide and their alignment. Sequences are color-coded red, blue, and green to indicate the location of each sequence as indicated in Panel A.

  3. Cloning gRNA oligonucleotides into gRNA vector
    Both pND494 or pND501 can be used for gRNA expression in any ura3∆ CAS9 C. albicans strain. The only difference in these two vectors is that pND501 has a recyclable URA3 allele (Wilson et al., 2000) if further phenotypic analyses require the loss of URA3 (see below).
    1. Digest 2 μg vector (pND494 or pND501) with SapI
      2 μg
      10x buffer
      5 μl
      1 μl
      to 50 μl
      1. Incubate at 37 °C for 5-15 min for Fast Digest/High Fidelity enzyme.
      2. Spin down.
      3. Add 1 μl calf intestinal phosphatase (CIP) (or Antarctic phosphatase).
      4. Incubate at 37 °C for 1 h.
      5. Purify using a QIAquick Gel Column (no need to run it on a gel); elute in 30 μl elution buffer or TE, pH 8.0.
    2. Phosphorylation and annealing sgRNA oligos (from Vyas et al., 2015).
      1. Add to a PCR tube:
        100 μM Oligo 1 (top)
        1 μl
        100 μM Oligo 2 (bottom)
        1 μl
        10x T4 ligase buffer
        5 μl
        T4 polynucleotide kinase
        1 μl
        42 μl
        Note: Do a negative control with no oligos.
      2. Incubate in a thermocycler:
        37 °C for 30 min
        95 °C for 5 min
        Cool to 16 °C, at the slowest ramp rate your machine can do (ours does 0.1 °C/sec).
    3. Ligation of annealed sgRNA oligonucleotides
      1. Assemble in a PCR tube:
        10x T4 ligase buffer 
        1 μl
        T4 ligase
        0.5 μl
        Annealed Oligo mix
        0.5 μl (include a negative control w/no oligos)
        20-40 ng
        to 10 μl
      2. Incubate tubes in a thermocycler or cool/heat blocks:
        16 °C for 30 min
        65 °C for 10 min
        Cool to 25 °C
    4. Transform 5 μl into 50-100 μl competent bacterial DH5α cells.
      Plate cells on 2x YT (or LB) + ampicillin plates. Incubate overnight at 37 °C; prepare plasmid DNA using any published protocol.
    5. Screening colonies
      This protocol usually results in ~10-20 colonies, with no background from cut vector alone. Identify correct clones by restriction analysis, screening for loss of the ClaI site that is predicted by its replacement in the SapI cassette with gRNA oligonucleotides (note that pND494 contains a single ClaI site, in the cassette, while pND501 contains two ClaI sites). Any potentially correct clone should be verified by DNA sequence analysis. The following primers are useful as for sequencing gRNA ligations especially if these vectors will be used for multiple gene knockouts. These primers (see sequences below) are homologous to the flanking 5’ PADH1 promoter and 3’ HDV ribozyme sequences.

      5’ within PADH1 upstream of tRNA (-247 relative to +1)

      3’ within HDV (includes the MluI site at 3’ end of HDV)

  4. Preparation of repair template
    To create the repair template using ~60 bp oligonucleotides with ~20 bp overlap at 3’ end, subject mixed oligonucleotides to annealing and DNA replication to generate a product of ~100 bp (see Figure 2).
    Per 25 µl reaction:
    Each oligonucleotide (100 mM stock)
    3 µl
    Taq polymerase
    0.5 µl
    dNTP mix (final 0.2 mM)
    2.5 µl
    10x buffer
    2.5 µl
    to 25 µl
    Incubate tubes in a thermocycler for 30 cycles of 20 sec at 94 °C, 30 sec at 55 °C, and 30 sec at 72 °C.
    Transform 20 µl of this reaction per yeast transformation.

  5. C. albicans transformation
    gRNA vectors are marked with URA3 for selection, and RPS1 to allow integration at the chromosomal RPS1 locus after linearization with StuI. Plasmids are linearized with StuI (or NcoI if gRNA sequence contains a StuI site), transformed into a Cas9-expressing yeast strain (i.e., those in Table 1), and selected on SD (Ura) plates.
    The transformation should include controls lacking gRNA plasmid, as well as those containing just the parental vector (lacking the gRNA sequences). Co-transformation of the donor repair fragment allows for double stranded break repair. Since a break that cannot be repaired is lethal, there is generally at least a two-fold increase in the number of transformants obtained in the presence of repair template compared to its absence.
    A modified lithium acetate protocol (Walther and Wendlund, 2003), outlined below, is used for transformation.

    Transformation (TF) mix (per transformation) 
    50% PEG (sterile)
    240 µl
    1 M LiOAc (sterile)
    32 µl
    Salmon sperm DNA (10 mg/ml boiled; keep on ice)
    5 µl
    Plasmid DNA (~10 µg, digested with NcoI or StuI)
    33 µl
    Donor repair fragment
    25 µl
    Yeast cell suspension
    50 µl
    360 µl

    Yeast transformation protocol
    (for ~2-3 transformation reactions)
    1. Inoculate fresh colony into YPD + 75 µg uridine/ml. Grow overnight in a shaker at 30 °C.
    2. Dilute 100 μl from overnight into 2 ml of YPD + uridine, shake at 30 °C for 4-6 h.
    3. Spin down 1 ml of cells (about 5 OD units), wash with H2O, then wash with 1 ml 0.1 M LiOAc.
    4. Resuspend in 100 µl of 0.1 M LiOAc (should appear to be thick and concentrated).
    5. Mix TF mix, 50 µl of yeast cells and 30 µl linearized plasmid DNA (~30 µg) or 15 μl PCR reaction (1-10 μg/μl) so that the total volume is 360 µl.
    6. Incubate overnight (up to 24 h) at 30 °C.
    7. Heat shock at 44 °C for 15 min.
    8. Spread the entire mixture on SD-URA plates. Incubate for 2-3 days at 30 °C.

  6. Mutant verification
    To identify the desired mutants, use colony PCR to amplify the flanking region of the wild type gene and mutant alleles. Isolate genomic DNA as follows: 
    1. Inoculate a toothpick full of cells from a single colony in ~400 μl of LB media and culture for ~3 h at 37 °C, 220 rpm.
    2. Spin down cells and resuspend in 30 μl of 0.2% SDS and boil for 4 min.
    3. After centrifugation of cells, 3 μl of the supernatant (containing genomic DNA) is added to 18.25 μl water, 2.5 μl PCR buffer, 20 μl 2.5 mM dNTP, 1 μl of each primer (10 mM stock) and 0.25 μl Taq polymerase. The PCR conditions are 30 cycles of 0.5 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C.
    1. PCR products can be analyzed by agarose gel electrophoresis. It is strongly recommended that prior to this PCR analysis, streak for single colonies to avoid the possibility of mixing colonies of differing genotypes. For an example of these analyses, see Ng and Dean, 2017, Figure 7.
    2. The design of oligonucleotides used for verification (see Procedure C above) should consider the need to detect a size mobility shift if deletion or alteration produces a smaller PCR product than the wild type allele. As described in Procedure B, susceptibility to restriction digest with a unique enzyme introduced into the donor repair fragment is very useful for mutant verification. In cases where this is not possible, PCR products can be screened directly by DNA sequencing. In either case, genomic sequencing of the deletion alleles should be performed for any mutant strain that will be used for subsequent phenotypic analyses.
    3. CRISPR-mediated mutation frequency varies depending on the locus and the nature of the mutation, but this procedure generally results in frequencies ranging from 30-95%. Once mutant strains have been obtained and verified, stock 2-3 strains from single colonies in 15% glycerol at -80 °C.

  7. Regeneration of ura3 auxotrophic strains
    1. When using strains transformed with pND501, ura3 mutants that lose the ura3dpl200 allele can be isolated by selection on 5-FOA containing plates (http://cshprotocols.cshlp.org/content/2016/6/pdb.rec086637.short). Prior to 5-FOA selection, streak out the mutant strain on fresh media for single colonies.
    2. Pick 3-4 colonies and streak them on 5-FOA containing plates. Alternatively, use fresh liquid culture, dilute, and plate out (frequency of looping out is ~1 in 105).
    3. Dilute overnight culture 1:100, 1:10,000, 1:100,000. Plate 100 µl of each dilution on FOA-containing plate. Screen FOA-resistant colonies on SD-URA plates to confirm the loss of URA3 gene.

Data analysis

During yeast transformation, it is important to include the following controls: No plasmid DNA (to ensure that transformants are uracil prototrophs and that SD (Ura) plates or transformation solutions are not contaminated); no gRNA plasmid (to ensure that transformants arise from gRNA vector uptake), no donor repair fragment. This last control provides an estimate of the number of colonies that are likely to arise from homologous recombination of the repair fragment at the target locus. The fold stimulation of colonies that arise in the presence versus absence of healing fragment may provide an estimate of mutation frequency. Generally, we have found that a high mutation frequency results in a large stimulation of colonies that arise in the presence versus absence of repair fragment. The frequency of mutation and repair by this method is about 30-90%. It should be noted that several other CRISPR-mediated systems for introducing mutations in C. albicans have been described (Vyas et al., 2015; Min et al., 2016; Nguyen et al., 2017), each having its own strength and weakness. It is hoped that as these methods are used more frequently, facile genetic analyses of C. albicans will become the norm.


This protocol was adapted primarily from Ng and Dean, 2017. Authors have no conflicts of interest or competing interests. HN was supported in part by a Stony Brook University URECA-Biology Alumni Research award. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.


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  2. Gasiunas, G., Barrangou, R., Horvath, P. and Siksnys, V. (2012). Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A 109(39): E2579-2586.
  3. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A. and Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096): 816-821.
  4. Labun, K., Montague, T. G., Gagnon, J. A., Thyme, S. B. and Valen, E. (2016). CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res 44(W1): W272-276.
  5. Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E. and Church, G. M. (2013). RNA-guided human genome engineering via Cas9. Science 339(6121): 823-826.
  6. Min, K., Ichikawa, Y., Woolford, C. A. and Mitchell, A. P. (2016). Candida albicans gene deletion with a transient CRISPR-Cas9 system. mSphere 1(3).
  7. Ng, H. and Dean, N. (2017). Dramatic improvement of CRISPR/Cas9 editing in Candida albicans by increased single guide RNA expression. mSphere 2(2).
  8. Nguyen, N., Quail, M. M. F. and Hernday, A. D. (2017). An efficient, rapid, and recyclable system for CRISPR-mediated genome editing in Candida albicans. mSphere 2(2).
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  10. Vyas, V. K., Barrasa, M. I. and Fink, G. R. (2015). A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families. Sci Adv 1(3): e1500248.
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白色念珠菌是最普遍和最重要的人类真菌病原体。 CRISPR作为基因编辑手段的出现极大地促进了 C中的遗传分析。白色假丝酵母。 在这里,我们描述一个详细的分步过程来构建和分析 C。 白色念珠菌缺失突变体。 该协议使用质粒,允许合成的双链体23mer引导寡聚脱氧核苷酸在高拷贝gRNA表达的简单连接。 表达密码子优化的Cas9的白色念珠菌菌株。 该协议允许在9天内分离和鉴定缺失菌株。

【背景】℃。白色念珠菌是一种难以处理遗传的有机体。由于它通常作为不容易进行有性生殖的二倍体存在,所以纯合隐性突变需要对每个基因座进行连续修饰。克隆间规则间隔短回文重复(CRISPR)突变的发展和应用。白色念珠菌促进遗传操作,因为它允许两个等位基因同时突变(Vyas et al。,2015; Min et al。,2016; Ng and Dean,2017 )。 CRISPR基因编辑涉及将RNA引导的核酸酶募集至邻近NGG原型间隔子邻接基序(PAM)的互补靶位点(Jinek等人,2012; Cong等人, 2013年;马里等人,2013年)。 CRISPR相关(Cas)核酸酶通过与结合Cas9的激活CRISPR RNA(tracrRNA)相关的指导RNA之间的互补碱基配对以高特异性进行靶向(Gasiunas等人, em>,2012)。由于染色体靶序列仅约20个核苷酸,与〜80核苷酸tracrRNA融合的单个指导RNA(sgRNA)的表达与Cas9一起足以用于靶向双链DNA切割。由于染色体断裂是致命的,因此双链断裂修复具有很强的选择压力。在 C中。白色念珠菌共同表达供体修复片段,其含有与断裂侧翼区域的同源性,允许通过同源重组来修复该断裂。因此,供体修复片段的适当设计允许引入序列缺失,置换或其他染色体改变。

我们以前的研究表明,促进高效率CRISPR突变的关键因素是C。白色念珠菌基因依赖于最佳的gRNA表达(Ng和Dean,2017)。为此,我们创建了允许高水平的gRNA表达的gRNA表达载体。这种高水平表达的基础部分是由于存在强的RNA聚合酶II启动子(ADH1 / em>)。该启动子驱动由5'tRNA和3'肝炎三角洲病毒(HDV)核酶RNA侧接的sgRNA的表达。这些5'和3'侧翼RNA序列的存在用于促进高效的转录后加工以产生具有精确末端的成熟sgRNA。在存在适当的供体修复片段的情况下,这种增加的sgRNA表达显着改善了C中的CRISPR / Cas诱变。白色假丝酵母。在实践中,诱变的执行非常简单。 gRNA表达质粒克隆依赖于两个编码短gRNA的寡核苷酸退火和连接到预切的载体中。诱变包括将sgRNA质粒和供体修复片段共转化到C.表达密码子优化的Cas9核酸酶的白色念珠菌菌株。以下描述了设计,合成和克隆gRNA寡核苷酸,愈合片段构建,酵母转化和突变验证的详细方案。

关键字白色念珠菌, Cas9, CRISPR, 真菌遗传学, gRNA, 酵母


  1. 1.5 ml微量离心管(可由任何科学分销商销售)
  2. 用于培养2-5毫升酵母和细菌培养物的玻璃或一次性圆底无菌试管(可由任何科学经销商出售)
  3. 塑料培养皿(可由任何科学分销商出售)
  4. PCR管
  5. 牙签
  6. 酵母菌株(表1)


  7. 质粒(表2)


  8. 电子。大肠杆菌感受态细胞(DH5α)(如所述制备,例如, https://www.neb.com/protocols/2012/06/21/making-your-own-chemically-competent-cells 或可以从新英格兰生物实验室购买)
    注意:我们使用CaCl 2 处理过的DH5α,但可以购买。
  9. 甘油(任何来源)(在H 2 O中50%,高压灭菌)
  10. 鲑鱼精子DNA(Sigma-Aldrich,目录号:D1626; 10mg / ml,超声处理并煮沸)
  11. Sap I(新英格兰生物实验室,目录号:R0569S)
  12. Cla(I)(New England Biolabs,目录号:R0197S)
  13. 小牛肠磷酸酶(CIP)(新英格兰生物实验室,目录号:M0290L)
  14. 定制的寡核苷酸(长度在18-60个核苷酸之间,Eurofins MWG Operon,Alabama)
  15. T4多核苷酸激酶(New England Biolabs,目录号:M0201S)
  16. T4连接酶(New England Biolabs,目录号:M0202L)
  17. 2x YT(请参阅 https://www.elabprotocols.com/protocols/#!protocol=5436配方) (+100μg/ ml氨苄青霉素)液体培养基和培养板(细菌选择)
  18. 氨苄青霉素(任何来源)
  19. Taq 聚合酶(任何来源)
  20. 聚乙二醇(PEG)3350(Sigma-Aldrich,目录号:P4338)
  21. 醋酸锂(任何来源)
  22. Nco I或 Stu I
  23. 尿苷(任何来源)
  24. 0.2%SDS
  25. 琼脂糖(任何来源)
  26. Qiaquick凝胶提取试剂盒(QIAGEN,目录号:28706)
    1. 酵母提取物(BD,Bacto TM,目录号:212750)
    2. 胰蛋白胨(BD,Bacto TM TM,目录号:211705)
    3. 氯化钠(NaCl,认证ACS等级≥99.0%)(Fisher Scientific,目录号:S271)
  27. YPD
    1. 酵母提取物(BD,Bacto TM,目录号:212750)
    2. 蛋白胨(BD,Bacto TM,目录号:211677)
    3. 葡萄糖(Fisher Scientific,目录号:D16)
  28. 缺乏尿嘧啶的合成葡萄糖(SD-URA)液体培养基和平板(尿嘧啶原养型酵母选择)(参见 http://cshprotocols.cshlp.org/content/2015/2/pdb.rec085639.short
    1. 细菌琼脂(用于盘子)
    2. 葡萄糖(Fisher Scientific,目录号:D16)
    3. 酵母氮碱无W / O氨基酸W /硫酸铵(BD,目录号:291940)
    4. 所有氨基酸,嘌呤和嘧啶(任何来源)
  29. SD-complete + 5'FOA( http://cshprotocols.cshlp.org/content/2016/6/pdb.rec086637.short )。
    1. 细菌琼脂
    2. 葡萄糖(Fisher Scientific,目录号:D16)
    3. 尿嘧啶
    4. 酵母氮碱无W / O氨基酸W /硫酸铵(BD,目录号:291940)
    5. 5-氟乳清酸(5-FOA)(奥克伍德产品,目录号:003234)


  1. 移液器(能从1μl-10 ml准确移液)(任何来源)
  2. 热循环仪(MJ Research,型号:PTC-200)
  3. 涡(任何来源)
  4. 微型离心机能够14kG(Eppendorf)
  5. 加热块(37°C,44°C)
  6. 培养箱(30°C,37°C)
  7. DNA凝胶电泳仪
  8. 紫外透射仪(或手持式紫外灯)
  9. 摇床培养箱(或滚筒)(30°C,37°C)
  10. 介质灭菌设备(高压灭菌器或压力锅)



  1. 3对不同的gRNA靶标(如果可能的话,靶向起始ATG上游150个碱基的启动子区域中的一个PAM)(〜23个碱基/寡核苷酸)。
  2. 2个用于供体修复片段的寡核苷酸(〜60个碱基/引物)。
  3. 2个寡核苷酸(正向和反向)用于基因分型。 

  1. 退火并填写供体修复片段。
  2. 退火并将编码gRNA的寡核苷酸连接到SapI消化的和去磷酸化的载体中。
  3. 转换成 E。大肠杆菌感受态细胞。 

确认质粒插入 (见程序B)
  1. 培养细菌并制备gRNA质粒(37°C孵育6-8小时)。
  2. 检查gRNA质粒是否存在ClaI位点的缺失。
  3. 消化用于酵母转化的StuI。
  4. 为第二天转化放置过夜酵母培养物。

第6-7天。 C。白色念珠菌转化(见程序E)

  1. 稀释酵母培养物1:20进行转化(在30°C孵育5小时)。
  2. 转换成 C。白色的。

  1. 通过PCR分析Ura <+>转化体
  2. 划出单个菌落以避免混合突变体,通过PCR重新检查,然后储存2-3个突变株。

  1. 设计和订购gRNA,敲除验证寡核苷酸
    1. 确定您想要进行诱变的基因或DNA区域,并找到合适的gRNA靶序列。对于Cas9切割的要求是紧邻约20个碱基对目标下游的PAM序列(5'-NGG-3')的存在。这个5'-NGG-3'可以在任一DNA链上。这个序列在 C中必须是唯一的。白色念珠菌基因组以防止脱靶切割。一个有用的网站用于识别和排列合适的gRNA,是CHOP CHOPv2( http://chopchop.cbu.uib.no / )(Labun等人,2016年)。一般而言,我们坚持使用本网站获得的PAM网站的排名来指导目标网站的选择。
    2. 需要对应于20bp双链序列的两个互补寡核苷酸。每个寡核苷酸的长度为23个碱基,并且在其5'末端还必须包含适当的序列以提供粘性末端用于将退火的dsDNA直接连接到用SapI切割的pND494或pND501中(图1)。选择使用哪种载体是基于后续实验是否需要尿嘧啶原养型或营养缺陷型菌株。如果需要尿嘧啶营养缺陷型,pND501允许回收 Ura - 基因型(见程序H)。将这些退火的寡核苷酸连接到其中的克隆盒(参见下面的图1)已被设计为允许与上游tRNA序列和下游tracrRNA两者的精确融合(Ng和Dean,2017)。该整个tRNA-sgRNA-HDV序列由pND494和pND501载体中的ADH1启动子驱动(图1A)。最终的结果是表达侧翼与tRNA和核酶的sgRNA用于精确的转录后加工以产生合适的5'和3'末端。
    3. 图1描绘了gRNA表达载体图谱(A)和两个互补的gRNA寡核苷酸(B)的设计实例。图1B中的顶部序列描述了pND494 / 501表达载体中存在的含Cla I的Sap I盒。下面的序列描述了退火和连接到该盒中后编码gRNA的寡核苷酸。此实例显示双链体编码LEU2基因特异性gRNA的连接,盒装序列指示合成的每种寡核苷酸。&nbsp;

      图1.如何设计用于表达克隆的编码gRNA的寡核苷酸pND494和pND501克隆载体(图A)含有设计用于退火直接连接的SapI I克隆盒gRNA寡核苷酸(图B)。上部DNA双链体(B)中显示了SapI I克隆盒的DNA序列。下面显示的是连接编码靶向gRNA的LEU2靶标的双链退火寡核苷酸的实例(来自Ng和Dean,2017)。区域中的方框表示每个寡核苷酸的序列。注意每个23个碱基的寡核苷酸包含突出端序列,该突出端序列与用SapI消化的载体的粘附端结合。由于 Sap I的切割是非回文的,载体的重新连接是不可能的,因此大大减少了假阳性E的背景。大肠杆菌转化子(C4)。

    4. 当订购gRNA寡核苷酸时,我们通常靶向3个不同的PAM位点(其需要6种不同的gRNA寡核苷酸),因为gRNA依赖性CRISPR诱变的效率以序列依赖性方式变化。针对3个不同的PAM网站最大限度地获得正确的突变体的可能性。实验证据表明,靶向转录起始位点附近的5'近侧区域导致更高的诱变频率(Radzisheuskaya等人,2016)。因此如果可能的话,选择第一个起始ATG甲硫氨酸密码子上游150个碱基(5')内的一个靶标作为目标位点之一。
      注意:请勿在gRNA寡核苷酸中包含PAM SITE。
    5. 设计引物用于验证正确的染色体突变

  2. 创建一个合适的修复模板
    用于产生缺失等位基因的最简单的修复模板使用一对60bp的寡核苷酸,其具有〜47bp的同源性(在5'末端)与侧翼于染色体断裂位点的序列,3'末端〜20bp重叠,以及独特的限制位点(例如,见Vyas等人,2015; Ng和Dean,2017)的重叠确认。显示修复策略删除 LEU2 基因(来自Ng和Dean,2017)和修复寡核苷酸序列的示例如图2所示。

    图2.用于基因缺失的修复模板的设计用于删除LEU2基因座的供体修复片段合成示意图,靶向在位置137的PAM位点处的断裂(图A)使用图1B中描绘的寡核苷酸。与修复模板同源的染色体序列以红色和绿色着色。每个60聚体寡核苷酸以互补性的3'20bp序列结束,包括在LEU2(Eco EcoRI RI)中不存在的限制性位点,如蓝色所示。当退火和延伸时,该修复片段与DSB侧翼序列具有47bp的同源性。在本例中,取自Ng和Dean,2017,同源重组导致LEU2 的434缺失和独特的EcoRI位点的插入,这简化了假定突变体的基因分型。图B显示每个“修复片段”寡核苷酸的序列及其比对。序列用颜色编码为红色,蓝色和绿色,以指示每个序列的位置,如面板A所示。

  3. 将gRNA寡核苷酸克隆到gRNA载体中
    pND494或pND501均可用于任何ura3ΔCAS9白色念珠菌菌株中的gRNA表达。如果进一步的表型分析需要URA3 的损失,那么这两个载体的唯一区别是pND501具有可回收的URA3等位基因(Wilson等人,2000) / (见下文)。
    1. 消化2微克载体(pND494或pND501)和 Sap I
      SAP I
      H 2 O

      1. 。在37°C孵育5-15分钟以获得快速消化/高保真酶。
      2. 旋转。
      3. 加入1μl小牛肠磷酸酶(CIP)(或南极磷酸酶)。

      4. 在37°C孵育1小时
      5. 用QIAquick Gel Column纯化(不需要在凝胶上运行);用30μl洗脱缓冲液或TE,pH8.0洗脱。
    2. 磷酸化和退火sgRNA寡核苷酸(来自Vyas等人,2015年)。
      1. 加入PCR管中:100μMOligo 1(上图)
        100μMOligo 2(底部)

        10x T4连接酶缓冲液 5微升
        H 2 O
      2. 在热循环仪中孵育:
        37°C 30分钟
        95°C 5分钟
        以最慢的升温速率冷却至16°C,机器可以完成(我们的速度为0.1°C /秒)。
    3. 退火的sgRNA寡核苷酸的连接
      1. 在PCR管中组装:10x T4连接酶缓冲液&nbsp;
        20-40 ng
        H 2 O
      2. 在热循环仪或冷/热块中孵育管:
        16°C 30分钟
        65°C 10分钟
    4. 将5μl转化到50-100μl感受态细菌DH5α细胞中。
      在2xYT(或LB)+氨苄青霉素平板上铺板细胞。 37°C孵育过夜;使用任何公开的协议制备质粒DNA。
    5. 筛选菌落
      这个协议通常导致〜10-20个菌落,没有单独的切割载体的背景。通过限制性分析来鉴定正确的克隆,筛选通过用gRNA寡核苷酸置换在SapI盒中预测的ClaI位点的缺失(注意,pND494含有单一的 I site,在盒式磁带中,而pND501包含两个 Cla I站点)。任何可能正确的克隆都应通过DNA序列分析进行验证。以下引物对于测序gRNA连接是有用的,特别是如果这些载体将用于多基因敲除。这些引物(参见下面的序列)与侧翼5'-ADH1启动子和3'HDV核酶序列同源。

      5'位于tRNA上游的ADH1 下方(-247相对于+1)

      3'(在HDV的3'端包括 Mlu I站点)

  4. 修理模板的准备
    使用〜60点碱基的寡核苷酸与在〜3’ 端的20bp重叠创建修复模板,对象混合寡核苷酸退火和DNA复制,以产生〜100bp的(参见图2)的产物。
    Taq 聚合酶
    H 2 O

  5. ℃。白色念珠菌转化
    gRNA矢量标有 URA3 进行选择,和 RPS1 以允许整合在染色体 RPS1 与斯图我。质粒线性化的斯图 I(或的Nco 我如果gRNA序列包含一个斯图 I位点),变成了一个Cas9表达的酵母菌株(即,表1中的那些),并在SD( - Ura)板上选择。

    使用改进后的醋酸锂方案(Walther and Wendlund,2003)进行转化
    1 M LiOAc(无菌)

    1. 将新鲜菌落接种至YPD +75μg尿苷/ ml。
    2. 将100μl从过夜稀释到2ml YPD +尿苷中,在30℃振荡4-6小时。
    3. 旋转1ml细胞(约5OD单位),用H 2 O洗涤,然后用1ml 0.1M LiOAc洗涤。

    4. 在100μl0.1 M LiOAc中重悬(应该显得浓稠)。
    5. 将TF混合物,50μl酵母细胞和30μl线性化质粒DNA(〜30μg)或15μlPCR反应物(1-10μg/μl)混合,使总体积为360μl。

    6. 在30°C孵育过夜(最多24小时)。

    7. 在44°C热冲击15分钟
    8. 将整个混合物铺在SD-URA板上。在30°C孵育2-3天。

  6. 突变验证
    1. 在〜400μlLB培养基中接种一个含有单个菌落的细胞的牙签并在37℃,220rpm下培养约3小时。
    2. 旋转细胞并重悬于30μl0.2%SDS并煮沸4分钟。
    3. 细胞离心后,将3μl上清液(含有基因组DNA)加入18.25μl水,2.5μlPCR缓冲液,20μl2.5mM dNTP,1μl各引物(10mM原液)和0.25μlTaq 聚合酶。 PCR条件是94℃0.5分钟,55℃1分钟和72℃1分钟的30个循环。
    1. PCR产物可以通过琼脂糖凝胶电泳进行分析。强烈建议在进行PCR分析之前,对单菌落划线以避免混合不同基因型菌落的可能性。有关这些分析的示例,请参阅Ng和Dean,2017,图7.
    2. 用于验证的寡核苷酸的设计(参见上面的 程序C )应该考虑需要检测大小迁移移位改变比野生型等位基因产生更小的PCR产物。如 程序B 中所述,使用引入供体修复片段的独特酶进行限制性消化的易感性对突变验证非常有用。在不可能的情况下,可以通过DNA测序直接筛选PCR产物。在任何一种情况下,应对任何将用于随后的表型分析的突变株进行缺失等位基因的基因组测序。
    3. CRISPR介导的突变频率取决于基因座和突变的性质而变化,但是该过程通常导致30-95%的频率范围。一旦获得并验证了突变菌株,在-80℃下在15%甘油中储存来自单菌落的2-3个菌株。

  7. ura3 营养缺陷型菌株的再生
    1. 当使用用pND501转化的菌株时,可以通过在含有5-FOA的平板上进行选择来分离丢失了ura3dp1121等位基因的突变体ura3突变体( http://cshprotocols.cshlp.org/content/2016/6/pdb.rec086637.short )。在选择5-FOA之前,将新鲜培养基上的突变菌株划出单菌落。
    2. 挑取3-4个菌落,并在包含5-FOA的平板上划线。或者,使用新鲜的液体培养物,稀释并消除(循环出现的频率在10 -5中为〜1)。
    3. 稀释过夜培养物1:100,1:10,000,1:100,000。在含有FOA的平板上每板稀释100μl。在SD-URA平板上筛选FOA抗性菌落以确认URA3基因的丢失。


在酵母转化过程中,重要的是包括以下对照:无质粒DNA(以确保转化体是尿嘧啶原养型,并且SD( - Ura)平板或转化溶液未被污染)。没有gRNA质粒(为了确保转化子来自gRNA载体摄取),没有供体修复片段。最后一个对照提供了可能由修复片段在靶基因座处的同源重组引起的菌落数量的估计。在存在与不存在愈合片段的情况下产生的集落的倍增刺激可以提供对突变频率的估计。一般而言,我们发现高突变频率导致在存在与不存在修复片段的情况下产生的集落的大的刺激。用这种方法进行突变和修复的频率约为30-90%。应该指出的是,其他几种CRISPR介导的在 C中引入突变的系统。已经描述了白色念珠菌(Vyas等人,2015; Min等人,2016; Nguyen等人,,2017年)。 ),每个人都有自己的优势和弱点。希望随着这些方法被更频繁地使用,对C进行简单的遗传分析。白种人将成为常态。


该协议主要适用于Ng和Dean,2017年。作者没有利益冲突或利益冲突。 HN得到石溪大学URECA-生物学校友研究奖的部分支持。这项研究没有得到公共,商业或非营利部门的任何资助机构的特别资助。


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引用:Dean, N. and Ng, H. (2018). Method for CRISPR/Cas9 Mutagenesis in Candida albicans. Bio-protocol 8(8): e2814. DOI: 10.21769/BioProtoc.2814.