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Probing gene function in Candida albicans wild-type strains by Cas9-facilitated one-step integration of two dominant selection markers: a systematic analysis of recombination events at the target locus
Infections by the pathogenic yeast Candida albicans are commonly treated with fluconazole, a well-tolerated drug that inhibits ergosterol biosynthesis and thereby growth of the fungus. C. albicans can develop fluconazole resistance by various mechanisms, which often involve the overexpression of genes encoding multidrug efflux pumps, the ATP-binding cassette transporters Cdr1 and Cdr2 and the major facilitator Mdr1 (1, 2). The constitutive overexpression of these genes in fluconazole-resistant isolates is caused by gain-of-function (GOF) mutations in their main transcriptional regulators, Tac1, which controls CDR1 and CDR2 expression, and Mrr1, which regulates MDR1 expression (3–11). Of note, the upregulation of other target genes of these transcription factors also contributes to Tac1- and Mrr1-mediated drug resistance. For example, the deletion of PDR16, which encodes a phosphatidylinositol transfer protein, in a strain with a Tac1 GOF mutation decreased its resistance, and forced PDR16 overexpression in a susceptible strain resulted in increased fluconazole resistance (12). Likewise, an activated form of Mrr1 still conferred increased fluconazole resistance, albeit at a reduced level, when MDR1 was deleted, demonstrating that other Mrr1 target genes are involved in the resistance phenotype (13). Transcriptional profiling of strains with Mrr1 GOF mutations identified genes that are regulated by Mrr1 (9, 13, 14). However, which of these genes contribute to fluconazole resistance has not been revealed so far. The deletion of orf19.7306, which encodes a putative aldo-keto reductase that was originally found by a proteomic analysis to be upregulated in MDR1-overexpressing strains, did not increase fluconazole susceptibility (15). Similarly, the deletion of ALS1 and orf19.7042 (a gene of unknown function) did not reduce fluconazole resistance in a strain with a hyperactive Mrr1 (16). To our knowledge, no further efforts have been undertaken to establish which genes besides MDR1 promote Mrr1-mediated fluconazole resistance.
The development of gene deletion methods based on the bacterial CRISPR-Cas9 system has facilitated the generation of specific knock-out mutants in C. albicans and other Candida species (17–29). The Cas9-mediated double-strand break at the target locus strongly increases the specific integration of selection markers in several non-albicans Candida species and other organisms. In C. albicans, homologous recombination is very efficient, and the vast majority of transformants exhibit a designed gene replacement without the help of Cas9. However, transformation efficiencies normally are too low to enable the simultaneous replacement of both alleles of a target gene in a single step in this diploid yeast, and the generation of homozygous deletion mutants traditionally required the sequential replacement of the two alleles with a recyclable marker or two different selection markers. This has changed with CRISPR-Cas9-based methods, since the double-strand breaks introduced by Cas9 must be repaired on both chromosomes, resulting in the inactivation of both alleles in many transformants. Furthermore, the requirement of a single instead of two sequential transformations also reduces the risk of stress-induced unspecific genomic alterations. A potential caveat that is not usually considered is that the deletion of both target gene alleles in some homozygous mutants may not have occurred by independent insertions of the selection marker but involved a single insertion into one chromosome, which then served as a repair template for the double-strand break on the homologous chromosome. This can result in loss of heterozygosity (LOH) for extended regions on this chromosome and, consequently, effects on the phenotype of the mutants that are not caused by the deletion of the target gene but by loss of allelic variants of other genes, especially when only one allele is functional (4, 30–48). In fact, it has recently been shown that CRISPR-Cas9-based gene editing strongly increased the frequency of LOH in Candida parapsilosis, especially on the target chromosome (49).
The ability to generate homozygous mutants in a single step enables a more rapid testing of candidate genes for their involvement in a specific phenotype. Most CRISPR-Cas9-based gene deletion methods use vectors containing the genes for the Cas9 endonuclease and a gene-specific single guide RNA, which are introduced into C. albicans cells together with the gene deletion cassette containing the selection marker. Instead of generating these expression constructs, one can also use a ribonucleoprotein (RNP) complex consisting of purified Cas9 enzyme and a custom-synthesized guide RNA (gRNA, a complex of a gene-specific crRNA and a universal tracrRNA) together with the gene deletion cassette containing the selection marker. The latter strategy was established for several genetically less tractable haploid Candida species and has recently also been used in C. albicans (17, 18, 20). In our present study, we used this approach to assess the contribution of previously identified Mrr1 target genes to fluconazole resistance of strains containing GOF mutations in this transcription factor. We hypothesized that the use of two different selection markers for Cas9-facilitated, separate replacement of the target gene alleles by the deletion cassettes reduces the occurrence of undesired LOH events on the target chromosome. For this purpose, we used gene deletion cassettes containing the caSAT1 or the HygB marker, which confer resistance to nourseothricin and hygromycin, respectively, and are useful for the genetic manipulation of C. albicans wild-type strains (50, 51). We undertook a detailed analysis of the different gene deletion mutants and compared integration events in transformants after selection for resistance against one or both antibiotics.
RESULTS
Deletion of GRP2 using PCR-amplified selection markers with short homology regions
We chose GRP2, which is strongly upregulated in C. albicans strains containing GOF mutations in MRR1 (13), as the first target for gene deletion with two different selection markers. The quickest way to obtain a gene deletion cassette is the amplification of a selection marker by PCR with oligonucleotides in which upstream and downstream sequences of the target gene are incorporated. Since the promoter (from the ACT1 gene) and downstream sequences (from the URA3 gene) of the caSAT1 and HygB selection markers are identical, both selection markers could be amplified with the same pair of oligonucleotides. Equal amounts of the so-generated GRP2 deletion cassettes were mixed with Cas9 enzyme and GRP2-specific gRNA and used to transform two independently generated derivatives of the C. albicans wild-type strain SC5314 that contained a GOF mutation in both MRR1 alleles (see Materials and Methods for details). We selected 16 nourseothricin- and hygromycin-resistant transformants (eight from parent strain A and eight from parent strain B) from plates containing both antibiotics for Southern hybridization analysis. The expected outcome after the integration of the caSAT1 and HygB selection markers into either of the two GRP2 alleles is illustrated in Fig. 1A. Hybridization of EcoRI-digested genomic DNA with a probe from the GRP2 upstream region demonstrated that 12 transformants had lost both GRP2 alleles, whereas four transformants (clones A1, A4, A5, and B3) had retained one of the wild-type GRP2 alleles (Fig. 1B, top panel). In addition to the wild-type fragment, these clones showed new bands that were larger than expected and corresponded to a tandem integration of both selection markers into one of the two GRP2 alleles, which was confirmed by rehybridization of the blot with probes from the GRP2 downstream region and with caSAT1- and HygB-specific probes (Fig. 1B, middle panels). The two GRP2 alleles in strain SC5314 can be distinguished by an upstream NdeI restriction site polymorphism, and hybridization of NdeI-digested DNA showed that clones A1, A4, and A5 had integrated HygB, followed by caSAT1, in GRP2 allele 2, while clone B3 contained caSAT1, followed by HygB, in GRP2 allele 1 (Fig. 1B, bottom panel, see also Fig. 2 for an illustration of all integration events).
Fig 1
Deletion of GRP2 using the caSAT1 and HygB selection markers. (A) Structure of the GRP2 locus in the wild type and mutants containing the caSAT1 and HygB selection markers in either of the two GRP2 alleles. Arrows on the lines representing the chromosomes point toward the telomere. The locations of diagnostic NdeI (N) and EcoRI (E) sites and the sizes of corresponding fragments are shown. (B) Southern hybridizations of EcoRI- or NdeI-digested genomic DNA of the wild-type strain SC5314 (WT), the parental strains SCMRR1R34A (PA) and SCMRR1R34B (PB), and nourseothricin- and hygromycin-resistant transformants A1 to A8 and B1 to B8, respectively, with the probes specified on the left. The identities of the hybridizing fragments are indicated on the right side of the blots. M, size markers (in kb).
Target gene
Selection
Δ/Δ frequencya
Δ/Δ with LOH
GRP2
Nou
8/24
3/8
Hyg
10/24
1/10
Nou + Hyg
24/24
10/24
GLX3
Nou
3/24
1/3
Hyg
3/24
2/3
Nou + Hyg
24/24
15/24
OYE23
Nou
3/24
3/3
Hyg
3/24
1/3
Nou + Hyg
22/24
4/22
OYE32
Nou
1/22
0/1
Hyg
7/24
3/7
Nou + Hyg
18/24
7/18
a
Not including homozygous deletion mutants with tandem integrations.
Our main motivation to simultaneously use two different selection markers for Cas9-facilitated, single-step generation of homozygous mutants was the expectation that independent integrations of caSAT1 and HygB in the two alleles of a target gene would decrease the risk of an undesired LOH at the target locus, which may occur when a marker is integrated into only one target allele, and the altered chromosome then serves as a template to repair the Cas9-mediated double-strand break on the homologous chromosome. LOH, which was rarely observed in heterozygous mutants containing only one allelic replacement, was indeed a significant problem when using Cas9 for the generation of homozygous mutants. Surprisingly, this was not ameliorated by the simultaneous use of caSAT1 and HygB. A possible scenario that could explain this finding is that a Cas9-mediated double-strand break in one allele of the target locus is first repaired using the homologous chromosome as a template and results in LOH before subsequent insertion of the selection markers into both chromosomes. Such LOH events can involve large regions on the same chromosome arm after break-induced replication and cause altered phenotypes that are unrelated to the deletion of the target gene. We always tested for LOH events between the target site and the telomere, which is the region that is most likely to be affected by break-induced replication, but it has been demonstrated that LOH can also extend for longer regions from the break site toward the centromere (52). Indeed, many glx3Δ mutants exhibited an LOH event at the centromere-proximal SpeI site, which is located 1 kb upstream of GLX3 allele 1, in some cases without a concomitant LOH at the telomere-proximal PstI site in the downstream region (see Fig. S4; Table S2). Therefore, the observed LOH frequency is probably an underestimation of undesired LOH events on the target chromosome.
Interestingly, we also obtained mixed clones consisting of subpopulations containing either the wild-type gene, the caSAT1 marker, or the HygB marker on the same chromosome. This indicates that marker integration events also occur during colony growth in some, but not other cells, especially as long as the Cas9 enzyme is still present in the cells (subpopulations may also contain aneuploid cells with chromosome duplications). This was observed because we analyzed the primary colonies appearing on the selection plates. Consequently, LOH events were also observed in subpopulations of these clones. Such mixed clones would be resolved after further restreaking for single colonies.
While the frequency of Cas9-induced LOH may depend on the transformation method (e.g., electroporation versus other protocols and use of expression cassettes for Cas9 and gRNA versus purified Cas9/gRNA complexes) and the genetic background (note that we used strains containing a hyperactive Mrr1, in which all tested target genes were constitutively overexpressed), and alterations in other genomic regions can be induced by the transformation stress, the findings of our study indicate that checking mutants for LOH at the target locus is especially important when their construction involved Cas9-mediated chromosome breakage.
MATERIALS AND METHODS
Strains and growth conditions
The C. albicans wild-type reference strain SC5314 (53) and its derivatives SCMRR1R34A and SCMRR1R34B, which contain the P683S GOF mutation in both MRR1 alleles (MRR1P683S-FRT/MRR1P683S-FRT) (13), were used for the experiments described in this study. Strains were routinely grown in YPD liquid medium (10 g yeast extract, 20 g peptone, and 20 g glucose per liter) at 30°C in a shaking incubator. For the selection of transformants, 200 µg/mL nourseothricin (Werner Bioagents) and/or 1 mg/mL hygromycin B was added to YPD agar plates containing 15 g agar per liter.
Plasmid constructions
To generate a GRP2 deletion cassette, ca. 0.5 kb of GRP2 upstream and downstream sequences was amplified by PCR with primers GRP2.13/GRP2.14 and GRP2.15/GRP2.16, respectively (all oligonucleotide primers used in this study are listed in Table S5). The PCR products were digested with ApaI/XhoI (upstream sequence) and PstI/SacII (downstream sequence), gel-purified, and ligated together with an XhoI-PstI fragment from plasmid pNIM1 (54) containing the caSAT1 selection marker in the ApaI/SacII-digested pNIM1 to generate pGRP2M1. An XhoI-PstI fragment containing the HygB selection marker from pSNF1ex3 (55) was then substituted for the caSAT1 marker to obtain pGRP2M2. Deletion cassettes containing the caSAT1 and HygB selection markers for other Mrr1 target genes were generated in the same way using the gene-specific primers listed in Table S5.
C. albicans transformation
Strains were transformed by electroporation (51) with the gene deletion cassettes described above. GRP2 deletion was also performed with PCR-amplified selection markers obtained with primers GRP2.11 and GRP2.12 (Table S5), which bind to the upstream and downstream sequences of both caSAT1 and HygB and contain 90 and 91 additional nucleotides corresponding to sequences at the start and end, respectively, of the GRP2 coding sequence.
RNP complexes were generated using the CRISPR-Cas9 system from Integrated DNA Technologies, Inc., basically as reported by Grahl et al. (17). The gRNA was generated by mixing equimolar concentrations (2 µM final) of the gene-specific crRNA and the universal tracrRNA, with a final volume of 3.6 µL per transformation. This mixture was incubated at 95°C for 5 minutes and allowed to cool down at room temperature. To assemble the RNP complex, 3.6 µL of the gRNA was mixed with 3 µL of 4 µM Alt-R S.p. Cas9 nuclease V3 and incubated at room temperature for 5 minutes. For each transformation, 40 µL of competent cells was mixed with 6.6 µL of the RNP complex plus 100 ng of each repair template and transferred to an electroporation cuvette (0.2 cm). Electroporation was performed with a 1,800-V pulse. After electroporation, the cells were resuspended in 1 mL YPD and incubated at 30°C for 4 h with shaking. Appropriate dilutions of the recovered cells were spread on YPD plates supplemented with 200 µg/mL nourseothricin, 1 mg/mL hygromycin B, or both antibiotics. After 2 days of incubation at 30°C, single colonies were picked for further analysis.
Genetic analysis of mutants
Genomic DNA from C. albicans strains was isolated as described previously (51). The DNA was digested with appropriate restriction enzymes, separated on an agarose gel (0.8%–1.0%), transferred by vacuum blotting onto a nylon membrane, and fixed by UV crosslinking. Southern hybridization with enhanced chemiluminescence-labeled probes (including a labeled size marker) was performed with the Amersham ECL Direct Nucleic Acid Labelling and Detection System (Cytiva) according to the instructions of the manufacturer. The upstream and downstream sequences of the deletion cassettes were used as gene-specific probes. An additional probe specific for the OYE32 coding sequence (to distinguish the band corresponding to OYE32-1 from that corresponding to oye32-2∆::HygB, see Fig. S6A and D) was amplified with primers OYE32.05/OYE32.06. Marker-specific probes were amplified with primers SAT1.01/SAT1.04 and HygB-3/HygB-7, respectively, thereby excluding sequences that are contained in both caSAT1 and HygB. The sequences of tandem integrations of the selection markers with short homology regions in the GRP2 locus were determined by amplifying the fusion between the HygB and caSAT1 markers with primers HygB-5 and SAT8, followed by sequencing. The order of the markers in clone B2 was determined by PCR with the primer pairs HygB-5/GRP2.16 and SAT1.03/GRP2.16.
The fluconazole susceptibilities of the strains were determined by a previously described broth microdilution method (56), with slight modifications. A 2-day-old colony from a YPD agar plate was suspended in 2 mL of a 0.9% NaCl solution, and 4 µL of the suspension was mixed with 2 mL 2× SD-CSM medium [13.4 g yeast nitrogen base without amino acids (MP Biomedicals), 40 g glucose, and 1.58 g complete supplement medium (MP Biomedicals) per liter]. A twofold dilution series of fluconazole (Sigma) was prepared in water, starting from an initial concentration of 512 µg/mL. One hundred microliters of each fluconazole solution was then mixed with 100 µL of the cell suspension in a 96-well microtiter plate, and the plates were incubated for 24 h at 37°C. The MIC of fluconazole was defined as the drug concentration that abolished or drastically reduced visible growth compared to a drug-free control.
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The adaptation of gene deletion methods based on the CRISPR-Cas9 system has facilitated the genetic manipulation of the pathogenic yeast Candida albicans, because homozygous mutants of this diploid fungus can now be generated in a single step, allowing the rapid screening of candidate genes for their involvement in a phenotype of interest. However, the Cas9-mediated double-strand breaks at the target site may result in an undesired loss of heterozygosity (LOH) on the affected chromosome and cause phenotypic alterations that are not related to the function of the investigated gene. In our present study, we harnessed Cas9-facilitated gene deletion to probe a set of genes that are constitutively overexpressed in strains containing hyperactive forms of the transcription factor Mrr1 for a possible contribution to the fluconazole resistance of such strains. To this aim, we used gene deletion cassettes containing two different dominant selection markers, caSAT1 and HygB, which confer resistance to nourseothricin and hygromycin, respectively, for simultaneous genomic integration in a single step, hypothesizing that this would minimize undesired LOH events at the target locus. We found that selection for resistance to both nourseothricin and hygromycin strongly increased the proportion of homozygous deletion mutants among the transformants compared with selection on media containing only one of the antibiotics, but it did not avoid undesired LOH events. Our results demonstrate that LOH on the target chromosome is a significant problem when using Cas9 for the generation of C. albicans gene deletion mutants, which demands a thorough examination of recombination events at the target site.
IMPORTANCE
Candida albicans is one of the medically most important fungi and a model organism to study fungal pathogenicity. Investigating gene function in this diploid yeast has been facilitated by the adaptation of gene deletion methods based on the bacterial CRISPR-Cas9 system, because they enable the generation of homozygous mutants in a single step. We found that, in addition to increasing the efficiency of gene replacement by selection markers, the Cas9-mediated double-strand breaks also result in frequent loss of heterozygosity on the same chromosome, even when two different selection markers were independently integrated into the two alleles of the target gene. Since loss of heterozygosity for other genes can result in phenotypic alterations that are not caused by the absence of the target gene, these findings show that it is important to thoroughly analyze recombination events at the target locus when using Cas9 to generate gene deletion mutants in C. albicans.
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Title
Probing gene function in Candida albicans wild-type strains by Cas9-facilitated one-step integration of two dominant selection markers: a systematic analysis of recombination events at the target locus
