Materials and Methods

Implementation of a CRISPR-Based System for Gene Regulation inCandida albicans

MATERIALS AND METHODSStrains and growth conditions. The strains used are listed in Table S1 in the supplemental material. Cells were grown at 37°C in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) or complete SD medium (2% glucose, 0.5% ammonium sulfate, 0.17% yeast nitrogen base and amino acids) unless otherwise stated. The analysis of susceptibility/resistance to different compounds was performed using the standard drop test as follows. Cultures grown at 37°C from stationary-phase cells were adjusted to 2 × 107 cells/ml, serially 10-fold diluted, and deposited (5 µl) onto solid SD plates supplemented (or not) with hydrogen peroxide at different concentrations. Plates were incubated at 37°C for 24 h and were then scanned. When necessary, doxycycline was added to either liquid or solid medium at 5 to 10 or 20 mg/liter, respectively. For flow cytometry (FC) analysis, cells were recovered 24 h after growth at 37°C in SD complete medium (stationary phase) or at the indicated times after dilution in fresh SD medium (log phase) from stationary-phase cultures. Cells were fixed in 1% phosphate-buffered saline (PBS)-formaldehyde and washed twice with PBS previous to fluorescence-activated cell sorter (FACS) analysis performed using a Guava cytometer (Millipore).TABLE S1List of strains used in the study. We include the name referred in the manuscript, the lineage or the laboratory where the strain was originated, and the genotype. Download Table S1, XLSX file, 0.01 MB.Copyright © 2019 Román et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.Molecular biology procedures and construction of plasmids. All plasmids and oligonucleotides are listed in Table S2 and Table S3, respectively. C. albicans CAS9 (CaCAS9) was amplified with primers Up-Cas9 and Rev-Cas9 (Table S3) using pV1093 (kindly provided by Vyas et al.) (13) as the template. CadCAS9 was obtained by overlapping PCR to introduce the corresponding mutations using primer pair dCas9-o1 and dCas9-o2 and primer pair dCas9-o3 and dCas9-o4. Both the CaCAS9 and CadCAS9 amplicons were introduced in the intermediate pGEM-T vector (Promega), digested with SalI/NotI, and then inserted in the pNRU expression vector, with URA3 used as a selection marker (41), to generate pNRU-CAS9 and pNRU-dCas9 plasmids. Both plasmids were finally digested with KpnI/SacII to force homologous recombination at the ADH1 locus. pNIM1RX-dCas9 vectors, with SAT1 used as a selection marker, were generated by the insertion of the SalI/NotI dCas9 fragment into the construct that had been digested previously with the same pair of enzymes, and pNIM1RX-RFP vector was generated by replacing the 5´ ADH1 XbaI/SacI fragment from pNIM1R-RFP (42) with a 1.630-bp XbaI/SacI fragment from the pNIMX vector (43), containing the 5´ ADH1 and the TDH3 promoter. Homologous recombination at the ADH1 locus was forced after enzymatic digestion with KpnI/SacI. Transformants were selected in SD Ura− or YPD nourseothricin (200 mg/liter) media, and the levels of expression of the corresponding Cas9 and dCas9 proteins were detected by Western blotting using Cas9 polyclonal (Clontech) and anti-Flag clone M2 (Sigma) antibodies. GAL4 and NRG1 transcriptional modulators were amplified by PCR with primer pairs Up-GAL4/Rev-GAL4 and Up-NRG1/Rev-NRG1, subcloned into intermediary vector pGEM-T, digested with XhoI/NotI, and finally inserted into pNRU-dCas9 or pNIM1RX-dCas9 vectors that had been previously digested with XhoI/NotI, generating corresponding vectors pNRU-dCas9-Gal4, pNIM1RX-dCas9-Gal4, pNRU-dCas9-Nrg1, and pNIM1RX-dCas9-Nrg1.TABLE S2List of plasmids used in the work. The main characteristics of the plasmid are indicated, such as the name of the plasmid, the promoter and gene regulated, the parental vector, the marker used, and the integration region in the Candida albicans genome. Download Table S2, XLSX file, 0.01 MB.Copyright © 2019 Román et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.TABLE S3List of oligonucleotides used in the work. In addition to the sequences, their uses are also indicated in the notes. Colors in the sequence indicate the base changes that introduce a restriction recognition site or a mutation. Download Table S3, XLSX file, 0.01 MB.Copyright © 2019 Román et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.The gene reporter vector containing a CAT1 promoter fused to GFP strain pCAT1-GFP was obtained as follows. pDU1-GFP_myc was constructed as follows. ACT1 promoter was amplified by PCR with Up-pACT1 and Rev-pACT1, digested with SalI/NheI, and cloned into pDU0-L, which had been digested previously with the same pair of enzymes, to generate pDU1-L (44). The click beetle luciferase (CbLUC) open reading frame (ORF) was removed from pDU1-L by cutting with SalI and BglII and replaced by GFP_Myc obtained from SalI/BglII digestion of plasmid pNIM1_MoGFP_carboxi_ca_myc (45). We amplified 1,600 bp containing the coding region of HIS1 with primers o-up-HIS1-SpeI and o-rev-HIS1-SacII and cloned the result into pDU1-GFP_myc, previously digested with SpeI and SacII, to generate pDH0-GFP_myc. To generate a cloning site, we replaced the 5´ ARD1 region of pDH0-GFP_myc with the amplicon generated by PCR using primers Up-5 ARD1-KpnI and Low-5 ARD1-BamHI BswiI and SalI (Table S3) containing the same 5´ ARD1 region (455 bp) by digestion with KpnI and SalI, generating vector pDH0MGFP_myc. A 1,000-bp fragment containing the putative CAT1 regulatory region was amplified by PCR with Up-1kbp Pr CAT1 BamHI and Low-1kbp Pr CAT1 BsiWI (Table S3), digested with the indicated enzymes, inserted into plasmid pDH0MGFP_myc, previously digested with BamHI and BsiWI, and treated with alkaline phosphatase (New England Biolabs NEB) to generate the final vector pDH8M-GFP_myc. KpnI and SalI digestion was used to force homologous recombination in the ARD1 locus to generate the pCAT1-GFP tester strain. Transformants were selected in SD His− media and confirmed for GFP expression by Western blotting using anti-GFP antibody and by FC.Bioinformatic analysis of CAT1 upstream region. The 1,594-bp upstream CAT1 ORF (orf19.6229) (see Fig. S1D in the supplemental material) was analyzed by the use of different software programs for determination of potential regulatory regions. In the text that follows, the numbering refers to this specific region; therefore, “1595” represents the “A” in the ATG starting codon. Neural Network Promoter Prediction (http://www.fruitfly.org/seq_tools/promoter.html) predicted the following stretches of regulatory DNA sequences: positions 124 to 174 (score of 0.91, potential transcriptional start site TSS at position 164), 804 to 854 (score of 0.93, potential TSS at 844), and 1389 to 1439 (score of 0.96, potential TSS at 1429). Potential TATA boxes were analyzed with Comet Software (https://zlab.bu.edu/~mfrith/comet/form.html) with a threshold E value of lower than 8, which gave the following results: TATA1 (positions 1398 to 1412, positive strand, score of 0.939, E value of 4.08), TATA2 (positions 131 to 145, positive strand, score of 0.376, E value of 7.53), TATA3 (positions 1111 to 1125, negative strand, score of 0.332, E value of 7.9), and TATA4 (positions 1294 to 1308, negative strand, score of 0.272, E value of 8.44). Combining the two analyses and considering the associated probabilities, the most probable promoter regulatory region would be between positions 1389 and 1398 or between positions 1412 and 1439. Guides were selected in this 1,594-bp region using CHOCHOP software (http://chopchop.cbu.uib.no/index.php).sgRNA design, generation and cloning. We use different approaches for sgRNA design and cloning depending on the strategy used for the sgRNA-expressing method. In one approach, sgRNAs were inserted into a BsmBI cloning site in the corresponding vector, pV1090 or pV1093 (kindly supplied by Vyas et al. 13). sgRNAs were generated by phosphorylation and annealing of complementary single-stranded DNA (ssDNA) oligonucleotides (see Table S3) and then inserted into the corresponding vector, previously digested with BsmBI, and treated with alkaline phosphatase, calf intestinal (CIP) (NEB). In some cases, we used an NEB Builder HiFi master assembly commercial kit, which allows direct cloning (in one step and with high efficiency) of the sgRNAs whose sequences harbor the desired target sequence (20 nt in length) flanked with a 25-nt sequence that hybridized with the cloning vector. Insertion of the correct sgRNAs was confirmed by sequencing using o-seq-pV1093, 200 bp downstream sgRNA cloning site (Table S3). The corresponding sgRNAs were integrated into the RP10 locus after KpnI and SacII digestion, and transformants were selected in YPD plus nourseothricin (clonNAT; Werner BioAgents) (200 mg/liter). The correct integration was confirmed by PCR analysis of genomic DNA with 0-to-100 bp of guide cloning site pV1090 up (in pSNR52) and Rev-ORFX-con pv1090 (outside the RP10 integration site). In the second strategy, as previously described (19), ssDNA sequences incorporated a SapI recognition sequence. After the two-step protocol was performed as previously described, sgRNAs were directly cloned into the pND494 vector digested with SapI and treated with alkaline phosphatase (CIP; NEB). The correct insertion was confirmed by analysis of the loss of the ClaI site in the cloning site. The corresponding sgRNAs were integrated into the RPS1 locus after StuI digestion, and transformants were selected in SD Ura−.Yeast scaffold RNA (scRNA) sequence design. scRNA sequences with RNA recruitment hairpins were synthetized for C. albicans codon optimization on the basis of the codon usage of four highly expressed C. albicans genes (HWP1, ENO1, MRPS9, and ACT1) (GenScript, USA) following the sgRNA sequence described previously (23, 32). The XmaI-AvaI fragment with RNA binding protein MCP fused to the transcriptional activator VP64 sequences was removed from the pUC57-MCP-VP64 MS2 plasmid and accommodated in the pV1093 vector previously digested with XmaI/AvaI, generating the pV1093-MCP-VP64 plasmid. Then, the XhoI-SacII fragment with the sgRNA cloning site and recruitment hairpin MS2 sequences from the pUC57-MCP-VP64 MS2 plasmid were exscinded and inserted into pV1093-MCP-VP64 digested with XhoI-SacII to obtain the final vector, pV1093-MS2-MCP-VP64. Finally, the desired sgRNAs were inserted into the BsmBI sgRNA cloning site as previously described. The final plasmids were then digested with KpnI and SacI to force recombination in the ENO1 locus. Transformants were selected in YPD plus nourseothricin (clonNAT; Werner BioAgents) (200 mg/liter), and correct integration was confirmed by PCR with primers Up-int-ENO1 and Rev-int-pV1093-MCP-VP64-MS2 (Table S3).Protein extracts and immunoblot analysis. All procedures involving cell lysis, protein extraction, gel electrophoresis, and transfer to nitrocellulose membranes were performed as previously described (46, 47). Protein extracts were measured at A280 to equalize the amounts of protein loaded for Western blot analysis, and the blots were probed with anti-GFP, anti-Flag clone M2 (Sigma), or anti-Cas9 (Clontech). Western blots were developed according to the instructions of the manufacturer (Amersham Pharmacia Biotech) using a Hybond ECL kit.Flow cytometry analysis. Epifluorescence microscopy images were obtained by the use of an Eclipse TE2000-U inverted microscope (Nikon) coupled with an Orca C4742-95-12 ER charge-coupled-device camera (Hamamatsu). Image capture and processing were performed with AquaCosmos Imaging System 1.3 software. A Guava EasyCyte cytometer and InCyte software (Millipore) were used for flow cytometry and qualitative and quantitative analysis of GFP fluorescence. Data processing and analysis were done by using FlowJo software.Statistical analysis. Statistical differences between two groups were calculated using Student’s two-tailed unpaired t tests, correcting for multiple comparisons using the Holm-Sidak method, with α= 0.05. Computations were performed with the assumption that all rows were sampled from populations with the same standard deviation.

Article TitleImplementation of a CRISPR-Based System for Gene Regulation inCandida albicans

Abstract

The 1,594-bp upstreamCAT1ORF (orf19.6229) (seeFig. S1Din the supplemental material) was analyzed by the use of different software programs for determination of potential regulatory regions. In the text that follows, the numbering refers to this specific region; therefore, “1595” represents the “A” in the ATG starting codon. Neural Network Promoter Prediction (http://www.fruitfly.org/seq_tools/promoter.html) predicted the following stretches of regulatory DNA sequences: positions 124 to 174 (score of 0.91, potential transcriptional start site TSS at position 164), 804 to 854 (score of 0.93, potential TSS at 844), and 1389 to 1439 (score of 0.96, potential TSS at 1429). Potential TATA boxes were analyzed with Comet Software (https://zlab.bu.edu/~mfrith/comet/form.html) with a threshold E value of lower than 8, which gave the following results: TATA1 (positions 1398 to 1412, positive strand, score of 0.939, E value of 4.08), TATA2 (positions 131 to 145, positive strand, score of 0.376, E value of 7.53), TATA3 (positions 1111 to 1125, negative strand, score of 0.332, E value of 7.9), and TATA4 (positions 1294 to 1308, negative strand, score of 0.272, E value of 8.44). Combining the two analyses and considering the associated probabilities, the most probable promoter regulatory region would be between positions 1389 and 1398 or between positions 1412 and 1439. Guides were selected in this 1,594-bp region using CHOCHOP software (http://chopchop.cbu.uib.no/index.php).


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