Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.
This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.
Candida albicans is the most prevalent human fungal pathogen1,2,3. Understanding differences between C. albicans and mammalian molecular biology is critical to development of the next generation of antifungal therapeutics. This requires investigators to be able to quickly and accurately genetically manipulate C. albicans.
Genetic manipulation of C. albicans has historically been challenging. C. albicans does not maintain plasmids, thus all constructs must be incorporated into the genome. Furthermore, C. albicans is diploid; therefore, when knocking out a gene or introducing a mutation, it is important to ensure that both copies have been changed4. In addition, some C. albicans loci are heterozygous, further complicating genetic interrogation5. To genetically manipulate C. albicans, it is typical to perform multiple rounds of homologous recombination6. However, the diploid nature of the genome and laborious construct development have made this a potentially tedious process, especially if multiple changes are required. These limitations and the medical importance of C. albicans demand the development of new technologies that enable investigators to more easily manipulate the C. albicans genome.
Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing is a powerful tool that allows researchers to change the sequence of a genome. CRISPR requires three components: 1) the Cas9 nuclease that cleaves the target DNA, 2) a 20 base guide RNA that targets Cas9 to the sequence of interest, and 3) repair template DNA that repairs the cleavage site and incorporates the intended change7,8. Once the guide brings Cas9 to the target genome sequence, Cas9 requires a protospacer adjacent motif (PAM) sequence (NGG) directly upstream of the guide sequence to cleave the DNA9. The requirement for both the 20 base guide and PAM sequences provides a high degree of targeting specificity and limits off-target cleavage.
CRISPR systems have been designed to edit the genomes of a diverse set of organisms and tackle a wide variety of problems10. Described here is a flexible, efficient CRISPR protocol for editing a C. albicans gene of interest. The experiment introduces a stop codon to a gene, causing translation termination. Other edits can be made depending on the repair template that is introduced. A fragment marked with nourseothricin (Natr) containing yeast codon-optimized Cas9 (CaCas9) and a guide RNA is incorporated into the C. albicans genome at a neutral site. Cotransformation with the repair template encoding the desired mutation leads to repair of the cleavage by homologous recombination and efficient genome editing. Described below is the editing of TPK2, but all C. albicans open reading frames can be targeted multiple times by CRISPR. The CRISPR system is flanked by FRT sites and can be removed from the C. albicans genome by induction of flippase encoded on the CaCas9 expression plasmid. The C. albicans CRISPR system enables investigators to accurately and quickly edit the C. albicans genome11,12.
1. Identification and Cloning of Guide RNA Sequence
2. Designing and Generation of Repair Template
3. Transformation of C. albicans with Repair Template and Plasmid
4. Streaks for Single Colonies
5. Colony PCR
6. Restriction Digestion of Colony PCR
7. Saving Strains
8. Removal of Natr Marker
Sequences of guide RNAs and repair templates that target C. albicans TPK2, a c-AMP kinase catalytic subunit, were designed according to the guidelines suggested above. Sequences are shown in (Table 1, Figure 1). Guide RNAs were cloned into CaCas9 expression vectors and cotransformed with repair template in wild-type C. albicans. An EcoRI restriction digestion site and stop codons in the repair template disrupt the PAM site and facilitate screening for correct mutants (Figure 1). Transformants were streaked for single colonies and screened by colony PCR and restriction digestion for incorporation of the repair template (Figure 2). Restriction digestion quickly distinguishes wild-type from mutant sequences.
Oligonucleotide Name | Oligonucleotide Sequence |
Forward Guide Primer_3 | ATTTGgggtgaactatttgttcgccG |
Reverse Guide Primer_3 | AAAACggcgaacaaatagttcacccC |
Repair Template Forward_3 | tcagcaatatcagcaacaatttcaacaaccgcagcaacaactttatTAAGAATTCggcga |
Repair Template Reverse_3 | attttgtccagtttgggctgcagcagggtgaactatttgttcgccGAATTCTTAataaag |
Forward Check Primer | ttaaagaaacttcacatcaccaag |
Reverse Check Primer | actttgatagcataatatctaccat |
Sequencing Primer | ggcatagctgaaacttcggccc |
Table 1: List of oligo nucleotides used for this study. Sites added for cloning purposes are capitalized and bolded in the guide primer sequences. Sequences in the repair template that mutate the genomic DNA are capitalized and bolded.
Figure 1: Diagram of guide RNA and repair template design. (A) Labeling of all the PAM sequences in the first 100 nucleotides of TPK2. PAM sequence 3 (PAM_3) is highlighted, as that is the sequence used in this study. (B) Guide RNA design using PAM_3. 20 base primers designed using SnapGene are lowercase and blue. Additional bases required for cloning are uppercase and green shown offset. (C) Repair template primers that insert a TAA stop codon and EcoRI site are inserted into the TPK2 reading frame. DNA that differs from the wild-type sequence is red and uppercase. (D) Example of how a guide is designed on the positive strand of DNA using PAM_4. (E) Schematic diagram of pV1524 after cloning of the guide RNA and digestion with KpnI and SacI. Neut5L-5' and Neut5L-3' target the vector to the Neut5L site in the C. albicans genome. CaENO1p is the promotor that drives expression of the yeast-optimized CaCas9. Natr is the nourseothricin resistance cassette. CaSNR52p is the promotor driving guide RNA expression (sgRNA). FRT sites are cleaved and recombined by flippase (FLP) removing the CRISPR cassette upon flippase expression. A schematic similar to (E) was published by Vyas et al.11. Please click here to view a larger version of this figure.
Figure 2: Introduction and confirmation of a stop codon and EcoRI restriction site to TPK2. Primers used for amplification are listed in Table 1. Wild-type and mutant sequences are shown below the gel. This figure has been modified from Vyas et al.11. Please click here to view a larger version of this figure.
Figure 3: Cartoon description of repair templates that will generate (A) deletions and (B) insertions. Grey dashes in (A) depict intervening sequences not present in the repair template primers. Please click here to view a larger version of this figure.
C. albicans CRISPR efficiently edits the C. albicans genome. pV1524 encodes a yeast codon-optimized Cas9 and is designed such that investigators can easily clone guide RNA sequences downstream of the CaSNR52 promoter (Figure 1)11. It must be ensured that only a single copy of the guide sequence has been cloned into CaCas9 expression vectors by sequencing, as extra copies will impede genome editing. If multiple copies of the guide are introduced consistently, one should lower the concentration of the annealed guide used in ligation. The vector and protocol described allow targeting of any C. albicans gene. Although C. albicans is diploid, only a single transformation is required to target both alleles of a gene. Furthermore, the processive nature of CRISPR-CaCas9 genome editing enables researchers to target multiple members of gene families. Many gene families such as the secreted aspartyl proteases (SAPS) and agglutinin-like sequence proteins (ALS) are important for C. albicans virulence. CRISPR genome editing will facilitate investigation of these gene families.
The protocols described above introduce a stop codon to an open reading frame, resulting in the phenotypic equivalent of a null (Figure 2). A wide variety of genetic alternations can be made by varying the repair template. Nonsense, missense, and silent mutations can be inserted via recombination with an appropriate repair template. Incorporation of a restriction site streamlines transformant screening, as those without must be screened by sequencing12,13. In addition, C. albicans CRISPR enables researchers to generate insertions and deletions, making it an ideal system to insert affinity tags, perform promotor swaps, and generate knockouts (Figure 3). Screening for correct transformants for these mutations is more laborious, as it is necessary to sequence the edits to confirm correct incorporation of the repair templates. Furthermore, Southern blot may be necessary to ensure additional copies of a gene have not been inserted at additional locations in the genome. The requirement of the NGG PAM site places slight limitations on the regions of the genome that can be targeted. The development of alternative CRISPR systems that use alternative nucleases such as Cpf1 or variations on the Cas9 system have/will alleviate many of these limitations14. To the investigators' knowledge at this time, these systems have not yet been applied to C. albicans.
The CRISPR system described in the above protocol has been developed such that it can be applied in a wide variety of species including Saccharomyces cerevisiae, Naumouozyma castellii, and the human pathogen Candida glabrata11. Transformation and efficient editing of these yeast requires slight changes to the described protocol, but the framework for editing these alternate genomes is remarkably similar to that described for C. albicans12. Furthermore, yeast provide an excellent mechanism to develop genome editing procedures. In yeast, when ADE2 is mutated, a precursor to the adenine biosynthesis pathway accumulates, turning the cells red. This easily observable phenotype allows investigators to identify edited cells and quickly troubleshoot genome editing protocols. Combined with the extensive molecular biology toolbox available for fungi, protocols for editing numerous yeast species have been developed15,16. Such a broad application of genome editing technology in fungi has the potential to significantly impact a wide variety of scientific disciplines.
CRISPR has greatly improved the efficiency of genome engineering in C. albicans, but to date CRISPR has not been used to perform genome wide screens in C. albicans. Current protocols require a repair template to introduce mutations, as the nonhomologous end joining pathway in C. albicans is inefficient12. The generation of repair template oligos for every gene is a significant barrier to the execution of genome-wide screens. The confluence of decreased costs of DNA synthesis and advances to CRISPR technologies will make development of deletion libraries more feasible. For instance, expression of a repair template from the CaCas9 vector paves the way for the development of sustainable plasmid libraries that target every gene11. Furthermore, transient Candida CRISPR protocols that do not require CaCas9 expression vector incorporate into the C. albicans genome have been developed17. In addition, increased guide expression increases genome editing efficiency18. These, and other advances to CRISPR technologies, are crucial to the development of genome-wide screens in C. albicans19,20,21,22.
The C. albicans genome is diploid, but A and B alleles are not always identical5. Such heterozygosity provides both challenges and opportunities. If one aims to target both alleles, a PAM site, guide sequence, and repair template that will act on both copies of the gene must be used. However, depending upon single nucleotide polymorphisms present in a gene, the C.albicans CRISPR system enables investigators to target a single allele. Such precision has the potential to allow investigators to examine functional differences between alleles. Targeting specific alleles must be done carefully, as loss of heterozygosity (LOH) at an allele or of an entire chromosome has been observed. When editing single C. albicans alleles, one must examine adjacent DNA sequences to determine if a clone has maintained a diploid SNP profile. In addition, off-target effects are quite low for C. albicans CRISPR, but whole genome sequencing can be considered for key strains.
The authors have nothing to disclose.
The authors thank Dr. Gennifer Mager for reading and helpful comments on the manuscript. This work was supported by Ball State University laboratory startup funds and NIH-1R15AI130950-01 to D.A.B.
Chemicals | |||
Agar | BD Bacto | 214010 | |
agarose | amresco | 0710-500G | |
Ampicillan | Sigma-Aldrich | A9518 | |
Bacto Peptone | BD Bacto | 211677 | |
Bsmb1 | New England Biolabs | R0580L | |
Calf intestinal phosphatase (CIP) | New England Biolabs | M0290L | |
Cut Smart Buffer | New England Biolabs | B7204S | |
Dimethyl Sulfoxide (DMSO) | Sigma-Aldrich | D8418 | |
dNTPs | New England Biolabs | N0447L | |
Ethylenediaminetetraacetic acid (EDTA) | Sigma-Aldrich | 3609 | |
Glacial Acetic Acid | Sigma-Aldrich | 2810000ACS | |
Glucose | BDH VWR analytical | BDH9230 | |
Glycerol | Sigma-Aldrich | 49767 | |
Kpn1 | New England Biolabs | R3142L | |
LB-Medium | MP | 3002-032 | |
Lithium Acetate | Sigma-Aldrich | 517992 | |
L-Tryptophan | Sigma-Aldrich | T0254 | |
Maltose | Sigma-Aldrich | M5885 | |
Molecular Biology Water | Sigma-Aldrich | W4502 | |
NEB3.1 Buffer | New England Biolabs | B7203S | |
Nourseothricin | Werner Bioagents | 74667 | |
Poly(ethylene glycol) PEG 3350 | Sigma-Aldrich | P4338 | |
Sac1 | New England Biolabs | R3156L | |
Salmon Sperm DNA | Invitrogen | AM9680 | |
T4 Polynucleotide kinase | New England Biolabs | M0201S | |
T4 DNA ligase | New England Biolabs | M0202L | |
Taq polymerase | New England Biolabs | M0267X | |
Tris HCl | Sigma-Aldrich | T3253 | |
uridine | Sigma-Aldrich | U3750 | |
Yeast Extract | BD Bacto | 212750 | |
Equipment | |||
Electrophoresis Appartus | |||
Incubator | |||
Microcentifuge | |||
PCR machine | |||
Replica Plating Apparatus | |||
Rollerdrum or shaker | |||
Spectrophotometer | |||
Waterbath |