Materials and Methods

Dramatic Improvement of CRISPR/Cas9 Editing inCandida albicansby Increased Single Guide RNA Expression

MATERIALS AND METHODSPlasmid construction. The plasmids and their relevant features are listed in Table 3, and the sequence of each relevant cassette and the oligonucleotides used for the construction are listed in Table S1 in the supplemental material. All DNA generated by PCR was verified by DNA sequence analysis.TABLE 3 Plasmids used in this studyPlasmid no.NameRelevant feature(s)Reference/sourcepND294CIp10CaURA3 RPS1 integrative plasmid42pND500CIp-His1CaHIS1 RPS1 integrative plasmid16pND383CIp-Arg4CaArg4 RPS1 integrative plasmidThis studypND442YPB1-ADHpCaADH1 promoter/URA3/C. albicans ARS35pND354CIpHis1-PADH1 yEmRFPyEmRFP driven by CaADH1 promoter in CIp-His16pND425CIpArg4-PTEF CaCas9Codon-optimized Cas9 driven by AgTEF1 promoter in CIp-ArgThis studypND459YPB-PADH1 HH gRFP HDVPADH1HH gRFP HDV in YPBThis studypND465YPB-PADH1 HH gLeu2 HDVPADH1HH gLeu2 HDV in YPBThis studypND468YPB-PADH1 tA gRFP HDVPADH1tRNA gRFP HDV in YPBThis studypND479YPB-PtRNA gRFP HDVPtRNA gRFP HDV in YPBThis studypND474pV1090PSNR52-gRNA/SATR integrative plasmid1pND476pV1090-gRFPPSNR52-gRFP in pV1090This studypND489pV1025CaCas9/SAT flipper ENO1 integrative plasmid1pND486CIp10-PADH1 HH gRFP HDVPADH1HH sgRFP HDV in CIp10This studypND482CIp10-PADH1 tA gRFP HDVPADH1tRNA sgRFP HDV in CIp10This studypND483CIp10-PtRNA gRFP HDVPtRNA sgRFP HDV in CIp10This studypND484CIp10-PSNR52 gRFPPSNR52 sgRFP in CIp10This studypND494CIp10-PADH1 tA SapI HDVCIP10-based cloning vector for ligation of gRNA PADH1tA-SapI2× HDV in CIp10This studypND499CIp10-PADH1 tA gLEU2 HDVPADH1tRNA sgLeu2 HDV in CIp10This studypND501CIp-dpl-PADH1 tA SapI HDVpND494 but contains ura3-dpl200 instead of URA3This studyOpen in a separate windowTABLE S1 DNA sequence of plasmids, gRNA, and expression cassettes used in this study. sgRNA and promoters are color coded according to Fig. 3. Download TABLE S1, PDF file, 0.1 MB.Copyright © 2017 Ng and Dean.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.CIp-His1-PADH1 yEmRFP (pND354) is a HIS1 integration plasmid that contains the C. albicans codon-optimized yEmRFP (15). CIp-ARG4 (pND383) was constructed by replacing the URA3 gene in CIp10 (35) with a 1-kb BamHI/SacI fragment containing C. albicans ARG4. Unless otherwise noted, CIp plasmids were linearized with StuI to target integration at RPS1.A C. albicans codon-optimized CAS9 gene, encoding CaCas9 endonuclease with a hemagglutinin (HA) epitope tag and nuclear localization signal at the C terminus and driven by the strong Ashbya gossypii TEF1 promoter, was synthesized (Genescript, NJ) and cloned into CIp-ARG4 as a KpnI/SacI fragment to produce CIp-ARG4-PTEF CaCAS9 (pND425). YPB1-ADHp (pND442) is a 2µ CaURA3 CaARS plasmid that contains the ADH1 promoter and terminator (35).YPB-PADH1 HH gRFP HDV (pND459) contains an sgRNA that targets the RFP PAM at position +132. sgRNA expression is driven by the RNA polymerase II (Pol II) ADH1 promoter (PADH1); the 5′ and 3′ ends are flanked by the self-cleaving hammerhead (HH) (36) and hepatitis delta virus (HDV) (37) ribozymes, respectively. A 229-bp BglII/MluI fragment (Table S1) containing the HH-gRFP-HDV sequence was synthesized and cloned into the BglII/MluI fragment of YPB1-ADHp, downstream of PADH1.YPB-PADH1 HH gLeu2 HDV (pND465) is identical to YPB-PADH1 HH gRFP HDV but contains a gRNA that targets the CaLEU2 PAM site at position +123 and an HH sequence whose 6 nucleotides at the 5′ end are complementary to the first 6 nucleotides of gLeu2 (Fig. S1). The HH-gLeu2-HDV cassette was synthesized (Genescript, NJ), and cloned into YPB1-ADHp vector as a BglII/MluI fragment.YPB-PADH1 tA gRFP HDV (pND468) contains the 75-bp C. albicans tRNAAla gene between the ADH1 promoter and the sgRNA in YPB1-Adhp. The tRNA gene was amplified by PCR from genomic DNA. Gibson assembly (38) was used to assemble the tRNA-sgRNA-HDV fragments and the YPB-ADHp vector.YPB-PtRNA gRFP HDV (pND479) was generated by deleting the ADH1 promoter of YPB-PADH1 tA gRFP HDV (pND468) by digestion with NotI and BglII, filling in overhangs with Klenow DNA polymerase and ligating.To construct the CIp10 series of sgRNA delivery plasmids, each of the sgRNA cassettes described above (PADH1 HH gRFP-HDV 1,113 bp, PADH1 tA gRFP-HDV 1,147 bp, and PtRNA gRFP HDV 546 bp) was cloned into CIp10 as SalI/MluI fragments to produce pND486, pND482, and pND483, respectively. To construct CIp10-PSNR52 gRFP, the gRFP gRNA was first cloned into pV1025 as described previously (1). The 1,112-bp PSNR52 gRFP-tracr fragment was then amplified by PCR using primers with SalI and MluI sites and inserted into CIp10 to produce CIp10-PSNR52 gRFP (pND484).CIp10-PADH1 tA SapI HDV (pND494) is a vector that allows cloning and expression of any gRNA such that the sgRNA transcript is flanked with 5′ tRNA and 3′ HDV and transcribed by PADH1. It was constructed by using site-directed mutagenesis to replace the RFP gRNA segment in CIp10-PADH1 tA gRFP HDV with a cassette containing two SapI sites and 22 bp of intervening sequence, including a ClaI site (Fig. S2). In addition, site-directed mutagenesis replaced the unique SapI site at position 4381 of CIp10-PADH1 tA gRFP HDV with an NsiI site (Fig. S2). It should be noted that while constructing this plasmid, we discovered that the CIp10 sequence (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF181970","term_id":"5923859","term_text":"AF181970"}}AF181970) between KpnI and SacI (containing pBluescript KS+ sequence) was incorrectly annotated and actually flipped, which places the T7 promoter adjacent to RPS1 and the T3 promoter adjacent to CaURA3. To allow sequential introduction of additional gRNAs into the same strain, we also constructed a plasmid that is identical to pND494 but contains the recyclable ura3-dpl200 allele (26). This URA3 is flanked by 200-bp repeats that facilitate its recycling by FOA selection.Strains and growth conditions. C. albicans strains were grown in standard rich YPAD medium (2% Bacto-peptone, 2% dextrose, 1% yeast extract, 20 mg/liter adenine) or synthetic dropout (SD) (2% dextrose, 2% Difco yeast nitrogen base with ammonium sulfate) supplemented with the appropriate nutritional requirements. Uridine (75 mg/liter) was added to all media except SD(−Ura).The C. albicans strains used in this study are listed in Table 4 and were derived from BWP17. EPC1 contains a single integrated copy of the RFP gene (16) and was constructed by targeting StuI-linearized CIp-HIS-PADH-RFP to RPS1. This integration results in a duplication of RPS1 flanking PADH1-RFP, HIS1 and the intervening plasmid sequence. Single integration of PADH1-RFP at RPS1 was confirmed by Southern blotting (not shown). EPC2, which expresses both RFP and CaCAS9, was constructed by targeting CIp-ARG-PTEF1-CaCas9 (see below) in a second round of integration into the second RPS1 allele in EPC1. An isogenic strain, RJY54 that expresses CaCAS9 but not RFP, was constructed by targeting CIp-ARG-PTEF1-CaCas9 (see below) to RPS1. HNY30 (eno1Δ::Cas9) was constructed by targeting integration of the KpnI/SacI Cas9/SAT-flipper cassette of pV1025 (1) in BWP17 and then plating SATR colonies on YPAD to screen for SATS strains that lost the SAT-flipper cassette. HNY25 was constructed by knocking out LEU2 in RJY54 using the URA3-marked CRISPR/Cas gLEU2 plasmid p465. After confirming the homozygous leu2 mutation by PCR, uracil auxotrophs were selected on plates containing 5-fluoroorotic acid (FOA) and further screened for spontaneous loss of CIp-ARG-PTEF1-CaCas9 by arginine auxotrophy. HNY31 (leu2Δ/leu2Δ eno1Δ::Cas9) was constructed by targeting integration of the KpnI/SacI Cas9/SAT-flipper cassette of pV1025 in HNY25 and screening for SAT sensitivity.TABLE 4 Strains used in this studyStrainGenotypeReferenceBWP17ura3Δ::λ imm434/ura3Δ::λ imm434 his1Δ::hisG/his1Δ::hisG arg4Δ::hisG/arg4Δ::hisG27EPC1BWP17 RPS1::PADH1 RFP-HIS1 RPS1This studyEPC2BWP17 RPS1::PADH1 RFP-HIS1-RPS1::PTEF1 CaCas9-HA-ARG4-RPS1This studyRJY54BWP17 RPS1::PTEF1 CaCas9-HA-ARG4-RPS1This studyHNY25BWP17 and leu2Δ/leu2ΔThis studyHNY30BWP17 eno1Δ::CaCas9This studyHNY31BWP17 eno1Δ::CaCas9 leu2Δ/leu2ΔThis studyOpen in a separate windowYeast transformation and quantitation of RFP cleavage. Yeast transformations were performed by the lithium acetate protocol (39) with the following modifications. Fresh overnight cultures (12 to 16 h) were diluted 1:20 and incubated for ~5 h (optical density at 600 nm OD600 of 5). Five milliliters was harvested, washed once with H2O and once with 100 mM lithium acetate (LiOAc), and resuspended in 500 µl 100 mM LiOAc. The concentration of plasmid DNA was titrated to yield ~200 colonies per plate. Typically, transformations included 50 µl of the cell suspension (2.5 OD600 units), ~2 to 4 µg of linearized sgRNA plasmid (1 pmol), 1 to 2 µg of RFP donor repair DNA fragment (15 pmol), or annealed, filled-in repair oligonucleotides (200 pmol). After transformation, plates were incubated at 30°C for 2 days before being viewed by fluorescence microscopy with a Zeiss microscope equipped with low-power magnification (1.5 to ×10). Visual detection of pink (RFP) and white (rfp) colonies required 3 days of incubation. The number of red, white, and sectored colonies per plate was counted to determine the efficiency of Cas9-mediated cleavage. Cleavage efficiency was calculated as the no. of white colonies/total no. of colonies per plate.PCR analysis of Cas9-mediated cleavage. For analyses of RFP mutagenesis, white colonies were patched and replica plated on selective media to test for prototrophy of selective markers (URA3, HIS1, and ARG4). Genomic DNA from 20 white colonies per experiment was prepared and used as the template for PCR amplification using primers specific for RFP (Fig. S1). Colonies were inoculated into 0.5 ml medium and incubated at 30°C with shaking for 2 to 3 h. After harvesting of cells, pellets were suspended in 30 µl 0.2% SDS, heated for 3 min at 95°C, and centrifuged for 1 min at 14,000 × g. Three microliters of the supernatant was used as genomic DNA template in a standard 25-µl PCR.For the analyses of LEU2 deletions, after transformation with various sgLEU2 gRNA plasmids or control vectors, and with or without donor repair fragments, colonies were patched onto to SD(−Ura) and replica plated onto SD(−Ura) and SD(−Leu) plates. PCR amplification of Leu2+ and Leu2− colonies was performed, followed by digestion with EcoRI to determine the percentage of colonies that were heterozygous or homozygous for the leu2Δ allele.Construction of donor healing fragments. The rfpΔ repair fragment targeted deletion of nucleotides 55 to 402 of the RFP ORF, including the PAM site located at +132. It was generated by fusion PCR (40). PCR was used to amplify two fragments: one homologous to the 5′ region of RFP and the other homologous to the 3′ region. The 5′ fragment also contained a 3′ 20-bp tail that was homologous to the 5′ end of the second fragment (Fig. 2A). These two fragments were mixed, annealed, and then extended. After extension, the full-length “fused” fragment was amplified by PCR. The resulting 593-bp fragment contains a 285-bp arm of upstream sequence homology and a 308-bp arm of downstream sequence homology to regions flanking the DSB. Approximately 1 to 2 µg of this DNA (~5 pmol) was used for transformation of yeast. Recombination with RFP results in deletion of an internal 370 bp within the RFP ORF, including the PAM site to produce the rfpΔ33-403 allele.The LEU2 donor repair fragment was made by annealing and filling in two 60-mer nucleotides that contained 20 bases of sequence complementarity at their 3′ ends. This complementary region included an EcoRI recognition sequence (highlighted in blue in Fig. 7A). The resulting fragment contained 47 bp of homology to sequences flanking the DSB. Three microliters of each oligonucleotide (300 pmol) was annealed and then extended in a 25-µl reaction mixture containing 0.2 mM deoxynucleoside triphosphate (dNTP), buffer, and Taq DNA polymerase (Denville Scientific, Inc., United States) and subjected to 25 to 30 cycles of PCR with an extension time of 30 s. Twenty microliters of this reaction mixture was used per yeast transformation as a repair donor fragment (~240 pmol of repair fragment). Recombination with LEU2 results in replacement of an internal 434-bp fragment within the LEU2 ORF, including the PAM site, with an EcoRI site to produce the leu2Δ71-505 allele.

Article TitleDramatic Improvement of CRISPR/Cas9 Editing inCandida albicansby Increased Single Guide RNA Expression

Abstract

Genetic analysis ofC. albicanshas been complicated because it is a diploid that does not readily undergo sexual reproduction. Without CRISPR, genetic modifications, including knockouts, must be applied to both chromosomes, requiring sequential modification of each locus. The application of the CRISPR/Cas system as described by Vyas et al. (1) has enormous potential because it can circumvent these problems. However, as has been found in other systems, the efficiency of CRISPR/Cas can be frustratingly variable. Studies from other systems suggest that several factors influence efficacy of CRISPR/Cas, including (i) location and accessibility of gRNA target site, (ii) gRNA sequence, and (iii) sgRNA intracellular levels. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas inC. albicans. Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis inC. albicans. Large-scale analyses of the sgRNA target site effects, chromatin structure, and gRNA sequence preferences have led to an increasing knowledge base, as well as online tools that help design gRNAs. Features of a “good” gRNA include guanines at the −1 and −2 positions (i.e., a 3′ GG) and cytosine at the −3 DNA cleavage site and at +1 relative to the N0G0G0PAM site (29,–32). Features of a good target position appear to be nucleosome-free locations upstream of transcriptional start sites (29,–31,33,34). It is notable that neither the gRFP nor its target location used in the present study conforms to any of these predictive guides, yet when optimized for expression, it nevertheless resulted in an almost 100% mutation frequency. These results suggest that inC. albicans, sgRNA levels may in part compensate for a less than optimal gRNA design. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing inC. albicans.


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