Materials and Methods

Candida albicansGene Deletion with a Transient CRISPR-Cas9 System

MATERIALS AND METHODSStrains and culture conditions. C. albicans strains SC5314 (14), BWP17 (ura3Δ::λimm434/ura3Δ::λimm434 his1Δ::hisG/his1Δ::hisG arg4Δ::hisG/arg4Δ::hisG) (15), and a yhb5 mutant derivative of strain SN152 referred to as Noble_ORF_NegativeMutantCollection_Mutant no. 643 (his1Δ/his1Δ leu2Δ/leu2Δ arg4Δ/arg4Δ URA3/ura3Δ::imm434 IRO1/iro1Δ::imm434 yhb5Δ::CdHIS1/yhb5Δ::CmLEU2 contains HIS1 from C. dubliniensis and LEU2 from Candida maltosa) (10) were used as transformation recipients. Fungal strains were grown at 30°C in YPD+uri (2% Bacto peptone, 2% dextrose, 1% yeast extract, and 80 µg/ml uridine) with shaking. C. albicans transformants were selected on YPD+NAT (2% Bacto peptone, 2% dextrose, 1% yeast extract, 80 µg/ml uridine, and 400 µg/ml nourseothricin NAT; Werner BioAgents) for nourseothricin-resistant (NatR) isolates or on synthetic medium (2% dextrose, 1.7% Difco yeast nitrogen base with ammonium sulfate and auxotrophic supplements). All strains were stored as glycerol stocks at −80°C.Plasmids/DNA. To construct the pNAT plasmid, plasmid pCJN542 (16) was cut with SacI and SpeI to remove the TDH3 promoter. The SacI-SpeI fragment containing the nourseothricin resistance cassette (NAT) was blunted and self-ligated (17) to yield plasmid pNAT. The plasmid pV1093 used in this study was a kind gift from Valmik Vyas (3). We cloned the 20-bp guide sequence for ADE2 into the pV1093 vector, yielding pADE2-sgRNA. The CaCAS9 gene was the CAS9 gene that had been codon optimized for expression in C. albicans (3). The CaCAS9 expression cassette containing the ENO1 promoter, CaCAS9 open reading frame (ORF), and CYC1 terminator was PCR amplified from plasmid pV1093 (Fig. 2A). The sgRNA expression cassette containing the SNR52 promoter, guide sequence, and sgRNA scaffold sequence was assembled by the single-joint PCR method (11). In the first step, the SNR52 promoter and sgRNA scaffold components were PCR amplified using both flanking primers and internal chimeric primers (Fig. 3). The chimeric primers overlapped by a 20-base segment that specified the guide sequence. In the second step, both components were joined by primer extension, relying upon annealing of the complementary chimeric primer extensions. In the third step, the joined product was PCR amplified with nested primers to yield the sgRNA cassette (Fig. 3). Gene deletion PCR constructs were synthesized using plasmid pNAT or pRS-ARG4 (15) or the CdARG4 plasmid pSN105 (10), modified slightly, as the template. The primers were designed to include 80 bases with homology to the sequences upstream or downstream from the target gene (Fig. 2B). The oligonucleotides used in this study are listed in Table S1 in the supplemental material. PCR was conducted with Ex Taq in accordance with the manufacturer’s instructions (TaKaRa Bio, Inc.).Table S1 Oligonucleotides used in this study. Download Table S1, DOCX file, 0.1 MB.Copyright © 2016 Min et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.Fungal transformation. PCR products for transformation were purified and concentrated with the GeneJET PCR purification kit (Thermo Fisher Scientific, Inc.). In the original C. albicans CRISPR system, the deletion constructs (8 µg) were cotransformed with the CRISPR expression plasmid (5 µg), which had been linearized by digestion with KpnI and SacI (3). In the transient CRISPR system, the deletion constructs (3 µg) were cotransformed with the CaCAS9 cassette (1 µg) and sgRNA cassette (1 µg), using the lithium acetate transformation method (18). The transformation frequency was calculated as the ratio of the number of cells that form colonies on selective medium divided by the number on nonselective YPD medium.

Article TitleCandida albicansGene Deletion with a Transient CRISPR-Cas9 System

Abstract

C. albicansstrains SC5314 (14), BWP17 (ura3Δ::λimm434/ura3Δ::λimm434 his1Δ::hisG/his1Δ::hisG arg4Δ::hisG/arg4Δ::hisG) (15), and ayhb5mutant derivative of strain SN152 referred to as Noble_ORF_NegativeMutantCollection_Mutant no. 643 (his1Δ/his1Δleu2Δ/leu2Δarg4Δ/arg4ΔURA3/ura3Δ::imm434 IRO1/iro1Δ::imm434 yhb5Δ::CdHIS1/yhb5Δ::CmLEU2containsHIS1fromC. dubliniensisandLEU2fromCandida maltosa) (10) were used as transformation recipients. Fungal strains were grown at 30°C in YPD+uri (2% Bacto peptone, 2% dextrose, 1% yeast extract, and 80 µg/ml uridine) with shaking.C. albicanstransformants were selected on YPD+NAT (2% Bacto peptone, 2% dextrose, 1% yeast extract, 80 µg/ml uridine, and 400 µg/ml nourseothricin NAT; Werner BioAgents) for nourseothricin-resistant (NatR) isolates or on synthetic medium (2% dextrose, 1.7% Difco yeast nitrogen base with ammonium sulfate and auxotrophic supplements). All strains were stored as glycerol stocks at −80°C.


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