Materials and Methods

Use of CRISPR-Cas9 To Target Homologous Recombination Limits Transformation-Induced Genomic Changes inCandida albicans

MATERIALS AND METHODSStrains and culture conditions. The yeast strains described in the study were constructed starting from C. albicans strain SN148 (His– Arg– Ura– Leu–) (46). Yeast cells were cultured on/in rich YPD medium (1% yeast extract, 2% peptone, 2% dextrose). synthetic defined (SD) medium (0.67% yeast nitrogen base without amino acids, 2% dextrose), and synthetic complete (SC) medium (0.67% yeast nitrogen base without amino acids, 2% dextrose, 0.08% dropout mix with all the essential amino acids), which were used for selection. Solid media were obtained by adding 2% agar.Cloning experiments were conducted using One Shot TOP10 chemically competent Escherichia coli K-12 cells (Thermo Fisher Scientific). E. coli strains were cultured on/in LB (1% Bacto tryptone, 0.5% Bacto yeast extract, 0.5% sodium chloride) or 2YT (1.6% Bacto tryptone, 1% Bacto yeast extract, 0.5% sodium chloride, 0.1% d-glucose) medium, with appropriate antibiotics for selection purposes (50 μg/ml kanamycin, 50 μg/ml ticarcillin). Solid media were obtained by adding 2% agar.All C. albicans strains and E. coli plasmids are listed in Tables S1 and S2 in the supplemental material, respectively.TABLE S2Plasmids used in this study. Download Table S2, XLSX file, 0.01 MB.Copyright © 2020 Marton et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.CRISPR-Cas9-free transformation. Strains constructed using the following CRISPR-Cas9-free protocol underwent four sequential heat shock and lithium acetate/PEG rounds of transformations (47) in order to (i and ii) integrate the BFP/GFP LOH reporter system at a distinct genomic locus, (iii) integrate a unique barcode associated with the URA3 auxotrophic marker at the RPS1 locus, and finally (iv) integrate the LEU2 auxotrophic marker at the RPS1 locus, rendering the strains prototrophic (Fig. 1). The strategy was to integrate the BFP/GFP LOH reporter system into the most telomere-proximal intergenic region of ≥5 kb on each chromosome arm (Table S1). For this purpose, 120-bp primers were designed, each composed of 20 bp complementary to both the PTDH3-GFP-ARG4 and PTDH3-BFP-HIS1 cassettes and 100-bp tails possessing the complementary sequences of the targeted integration locus (Table S3). Each primer pair was used to amplify both the PTDH3-GFP-ARG4 and PTDH3-BFP-HIS1 cassettes from plasmids pCRBluntII-PTDH3-GFP-ARG4 and pCRBluntII-PTDH3-BFP-CdHIS1, respectively. Each cassette was amplified in a total PCR volume of 500 μl, precipitated in 100% ethanol, and resuspended in 100 μl of distilled sterile water. For each transformation, competent cells were transformed with approximately 5 μg of the appropriate DNA cassette. The parental SN148 strain was initially transformed with the PTDH3-GFP-ARG4 cassette and then subjected to a second round of transformation with the PTDH3-BFP-HIS1 cassette. These two transformation steps allowed the integration of the BFP/GFP LOH reporter system at a given intergenic locus (Table S1). The resulting strains, except those possessing the BFP/GFP LOH reporter system on Chr1, were then retransformed with StuI-linearized CIp10-PTET-BC-URA3 plasmids, each containing a unique barcode (BC) sequence and targeting the C. albicans RPS1 locus on Chr1. These plasmids are derived from a private laboratory collection of Cip10-PTET-BC-GTW-URA3 plasmids (unpublished data), each possessing a unique 25-nucleotide barcode sequence. The Gateway (GTW) cassette was removed by HindIII digestion and self-ligation in order to ensure that its presence did not influence the biology of C. albicans. Last, BFP/GFP barcoded strains were rendered prototrophic by a fourth round of transformation involving the integration of the StuI-linearized CIp10-LEU2 plasmid at the RPS1 locus. Conversely, in strains bearing the BFP/GFP LOH reporter system on Chr1, the BC-URA3 and LEU2 cassettes were integrated on Chr4 at the CDR3-tG(GCC)2 locus to avoid loss of the latter upon LOH. For these strains, the BC-URA3 and LEU2 cassettes were generated using the same PCR amplification protocol described above for the PTDH3-GFP-ARG4 and PTDH3-BFP-HIS1 cassettes, where long-tailed primers (Table S3) were used for amplification from plasmids CIp10-PTET-BC-URA3 and CIp10-LEU2, respectively. Throughout the strain construction process, selective pressure was always maintained in order to ensure the selection of transformants carrying all integration cassettes. In addition, at each transformation step, junction PCRs were conducted to ensure the proper integration of cassettes using the primers listed in Table S3.TABLE S3List of primers. Download Table S3, XLSX file, 0.02 MB.Copyright © 2020 Marton et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.CRISPR-Cas9-dependent transformation. In contrast to the CRISPR-Cas9-free transformation method, both homologs may be simultaneously targeted for cassette integration with the CRISPR-Cas9-dependent transformation protocol. Thus, by directing a DNA DSB with a locus-specific sgRNA, the BFP/GFP LOH reporter system can be introduced with only one exposure to heat shock and lithium acetate/PEG, rather than two treatments. Hence, only two transformation rounds were required for strain construction, where (i) the BFP/GFP LOH reporter system was integrated at distinct genomic loci and (ii) strains were simultaneously barcoded and rendered prototrophic using both URA3 and LEU2 auxotrophic markers (Fig. 1). A total of 11 unique 20-bp sgRNAs were designed using CHOPCHOP (48), targeting the same integration loci as those chosen in the CRISPR-Cas9-free protocol for integration of the BFP/GFP LOH reporter system (Table S3). Because the CIp10-derived BC-URA3-LEU2 plasmids could not be targeted to the RPS1 locus on Chr1 in the strains bearing the BFP/GFP LOH reporter system on Chr1, an additional sgRNA was designed to target the BC-URA3 and LEU2 markers on Chr4 at the CDR3-tG(GCC)2 locus (Table S3) in these strains.We used a transient CRISPR-Cas9 system (21), which does not necessitate the genomic integration of either Cas9 or sgRNAs. The construction of sgRNAs and the amplification of Cas9 cassettes from the pV1093 plasmid were conducted as described by Min et al. (21), while BFP-HIS1-, GFP-ARG4-, BC-URA3-, and LEU2-bearing cassettes were constructed as described above. SN148 cells were cotransformed with 3 μg of the PTDH3-GFP-ARG4 cassette, 3 μg of the PTDH3-BFP-HIS1 cassette, 1 μg of the Cas9 cassette, and 1 μg of sgRNA using the lithium acetate/PEG transformation protocol. Transformants were then selected on SC-Arg-His medium, and junction PCRs were performed in order to ensure proper integration of both cassettes at the targeted locus. Strains bearing the BFP/GFP LOH reporter system on Chr1 were then transformed using the transient CRISPR-Cas9 system targeting BC-URA3 and LEU2 cassette integration at the CDR3-tG(GCC)2 locus on Chr4. For the remaining strains, both BC-URA3 and LEU2 markers were integrated in one transformation step. We did this using the Gateway recombination system, where the LEU2 gene was transferred from a pDONR-LEU2 plasmid into Cip10-PTET-BC-GTW-URA3 plasmids (unpublished data) by an LR reaction. The unique Cip10-PTET-BC-LEU2-URA3 plasmids were then linearized by StuI and integrated at the RPS1 locus. These transformants were selected on SD medium, and junction PCRs were performed.Strain phenotyping. All selected strains underwent basic phenotypic characterization upon validation of cassette integration at targeted loci by junction PCRs. The functionality of the auxotrophic markers was evaluated by drop tests on SC medium with the appropriate dropout amino acid mix, based on the marker tested. Overnight-saturated cultures of selected strains in liquid YPD medium were spotted onto solid YPD, SC-His, SC-Arg, SC-Ura, and SC-Leu media and were placed at 30°C for 24 h to monitor growth. Furthermore, the functionality/intensity of both fluorescent proteins (BFP and GFP) was validated by flow cytometry (MACSQuant analyzer Miltenyi Biotec) and fluorescence microscopy (Olympus IX83). The colony morphology of all strains was also assessed on both solid YPD and SD media at 30°C. Finally, doubling times were evaluated in liquid YPD medium at 30°C by measuring the optical density with a Tecan Infinite system over a 24-h period.DNA extraction and whole-genome sequencing of strains. Prototrophic strains were cultured in 5 ml of liquid SD medium overnight at 30°C, and DNA was extracted by following the manufacturer’s protocol using the Qiagen QIAamp DNA minikit. The DNA was eluted in a total volume of 100 μl. The genomes were sequenced at the P2M Platform of Institut Pasteur by using the Illumina sequencing technology. Libraries were prepared with the Nextera XT sequencing kit, and NextSeq500 platforms were used to generate 151-bp paired-end reads.Identification of gross chromosomal rearrangements. Sequences and genomic variations were analyzed as described in references 49 and 50. Each set of paired-end reads was mapped against the C. albicans reference genome, SC5314 haplotype A and haplotype B (version A22-s07-m01-r57), using Minimap2 (51). SAMtools, version 1.9, and Picard tools, version 2.8.1 (http://broadinstitute.github.io/picard), were then used to filter, sort, and convert SAM files. SNPs were called using the Genome Analysis Toolkit (GATK), version 3.6, according to GATK best practices. SNPs were filtered using the following parameters: VariantFiltration, QD < 2.0, LowQD, ReadPosRankSum < –8.0, LowRankSum, FS > 60.0, HightFS, MQRankSum < –12.5, MQRankSum, MQ < 40.0, LowMQ, HaplotypeScore > 13.0. Sequencing depths were also calculated using the Genome Analysis Toolkit (Table S4). The GATK variant filtration walker (VariantAnnotator) was used to add allele balance information to VCF files. The value of allele balance at heterozygous sites (ABHet) is a number that varies between 0 and 1. ABHet is calculated as the number of reference reads from individuals with heterozygous genotypes divided by the total number of reads from such individuals. Thus, a diploid genome will be defined by an ABHet value of 0.5. In contrast, while a triploid strain may contain either three identical alleles (an allelic frequency of 1) or two identical alleles and one different allele (frequencies of 0.66 and 0.33), a tetraploid strain may have allelic frequencies of either 0.5 (2 × 2 identical alleles), 1 (4 identical alleles), or 0.25 and 0.75 (3 identical alleles and 1 different allele). In order to obtain an average ABHet value per chromosome, we evaluated the ABHet and allele balance at homozygous positions (ABHom) with the AlleleBalance annotation GATK module (Table S5). Histograms were built based on the number of SNPs with ABHet values with the matplotlib 2D graphics package (52), with blue and red representing ABHet and ABHom values, respectively.Data availability. Genome sequences of the 57 engineered C. albicans isolates described in this study have been deposited in the NCBI Sequence Read Archive under BioProject ID PRJNA659611. All other relevant data are available from the corresponding author upon request.

Article TitleUse of CRISPR-Cas9 To Target Homologous Recombination Limits Transformation-Induced Genomic Changes inCandida albicans

Abstract

Genome sequences of the 57 engineeredC. albicansisolates described in this study have been deposited in the NCBI Sequence Read Archive under BioProject IDPRJNA659611. All other relevant data are available from the corresponding author upon request.


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