CRISPR/Cas targeting of RFP in C. albicans. (A) Yeast colonies that express functional RFP are pink and are fluorescent and can be easily distinguished from colonies that arise through CRISPR-mediated deletion of RFP, which are white and nonfluorescent. Panel B depicts the strategy for quantitating Cas9- and sgRNA-dependent cleavage of RFP. RFP (EPC1) or RFP CaCAS9 (EPC2) hemizygous strains were constructed as described in Materials and Methods. These strains were transformed with or without a donor healing fragment and a series of URA3-marked plasmids that differ in expression of an RFP sgRNA (Fig. 3). The gRNA targets a 20-bp DNA sequence proximal to the RFP PAM site at position 132. The number of white and red colonies in each transformation was counted, and cleavage efficiency was calculated as the percentage of white colonies in the population.
Construction of the RFP donor repair fragment. The donor repair fragment used for repair of the Cas9-induced DSB was constructed by overlap PCR. Two fragments were amplified by PCR, one of which contains a 3′ end that is homologous to the 5′ end of the other. After the two fragments are mixed, these homologous regions anneal to one another, and then a round of DNA replication produces a single fragment with arms of homology to the 5′ and 3′ regions of RFP, but which is deleted for 370 bp of the ORF, including the PAM site at position 132. Homologous recombination of this rfpΔ33-403 fragment with chromosomal RFP can easily be detected by PCR amplification (Fig. 4).
Schematic diagram of sgRNA expression cassettes and the structures of their predicted encoded RNA. The backbone of each of these plasmids is based on the URA3 integrating CIP10 plasmid (35) (see Materials and Methods). RNA secondary structure visualization was performed using VARNY (http://varna.lri.fr/) (41). (A) PSNR52 drives transcription of the sgRNA that consists of the 20-nucleotide gRFP (purple) fused to the 85-nucleotide Cas9 recruiting tracrRNA (green). Note that this sgRNA is identical in all four delivery schemes. The gRNA has the requisite 5′ G and a 3′ poly(U) tail. (B) PADH-HH-HDV. The sgRNA is flanked by the 5′ hammerhead (HH [pink]) and 3′ hepatitis delta virus (HDV [yellow]) ribozymes. HH-sgRNA-HDV transcription is driven by the strong ADH1 promoter. The stem structure formed between the 5′ HH and 5′ gRNA forms the structural motif recognized for autocleavage (depicted by arrowhead). The 3′ cleavage relies on the autonomous pseudoknot structure of HDV (depicted by arrowhead). (C) PADH-tRNA(−HDV). This construct is exactly like that shown in panel B, but the hammerhead sequence is replaced by the 75-bp C. albicans alanine tRNA gene. In this scheme, the mature 5′ end of the sgRNA is generated by endogenous RNase Z, which recognizes the stem structure formed by tRNA 5′ and 3′ sequences. 3′ cleavage relies on the autonomous stem-loop structure of HDV (depicted by arrowhead). Transcription of the tRNA-sgRNA-HDV is driven by the strong ADH1 promoter. (D) PtRNA(−HDV). This construct is exactly like that of panel C, but the PADH1 sequence is deleted. In this cassette, RNA Pol III transcription is driven by the internal A and B box elements of the tDNA promoter, and the mature 5′ end of the sgRNA is generated by endogenous RNase Z, while 3′ cleavage relies on the autonomous pseudoknot of HDV (depicted by arrowhead).
Sectored colony phenotype of RFP mutants. (A) Comparison of red, white, and sectored colony phenotype in visible and red fluorescent light. (B) Examples of various degrees of sectoring that range from mostly red to mostly white. (C) Red and white phenotypes in a sectored colony are genetically stable. A single sectored colony was restreaked on nonselective medium. Below is shown PCR analysis of full-length and rfpΔ33-403 from white “repaired” colonies isolated after transformation in the presence of a donor repair fragment.
RFP CRISPR mutagenesis as a function of sgRNA delivery. RFP Cas9 strain EPC2 was transformed with each of the plasmids depicted in Fig. 3. Shown are the percentages of white mutant (rfpΔ), red (RFP), and sectored colonies that arose after 2 days at 30°C. Transformations were performed in the presence of a donor homologous repair fragment (A) or in its absence (B). The data represent an average from three separate experiments in which all plasmids (plus or minus the repair fragment) were transformed side by side. Approximately 200 colonies per plate were counted and scored as red, white, or sectored. For each experiment, controls were included in which all plasmids were transformed in the isogenic strain that lacks Cas9 (not shown). (C) Light and red fluorescent images of colony phenotype as a function of different sgRNA delivery plasmids depicted in Fig. 3. Note the near absence of sectored colonies when sgRNA is efficiently delivered (PADH-tRNA-gRFP-HDV) compared to when poorly delivered (PtRNA or PSNR52).
Models for DSB repair at the hemizygous RFP locus by homology-mediated repair and NHEJ. (A) Schematic diagram of homology-mediated and NHEJ pathways of DSB repair in RFP in the absence of donor repair fragment. A variety of homology-directed repair pathways between duplicated RPS1 loci are possible. Both flip-out and single-strand annealing (SSA) are consistent with the observed simultaneous loss of both RFP and HIS1 when a DSB is introduced in RFP. (B) Alignment of the wild-type RFP sequence with that of RFP amplified from a white colony isolated after CRISPR mutagenesis in the absence of a donor repair fragment. The sequence targeted by the RFP gRNA is underlined in red and precedes the PAM site (AGG, denoted with blue asterisks) at n = 132. Note that the deletion begins 3 bases from the PAM, precisely at the predicted Cas9 cleavage site.
Markerless deletion of LEU2. (A) Schematic diagram of the LEU2 locus, showing the PAM site at position 123. Sequences homologous to the repair fragment are colored in green and red. Each 60-mer oligonucleotide ends with a 20-bp sequence of complementarity, including a restriction site that is absent in LEU2 (EcoRI), shown in blue. When annealed and extended, this repair fragment contains 47 bp of homology to sequence flanking the DSB. Homologous recombination (HR) results in a 434-bp deletion of LEU2, which is replaced by a unique EcoRI site. (B) PCR analysis of DNA from LEU2 and leu2Δ mutants isolated by CRISPR (see text). EcoRI digestion of PCR products verified the leu2 deletion genotype. MW, molecular weight.
↵a Transformations were performed with URA3-marked PADH-tRNA-gRFP-HDV plasmid pND482 (+gRFP) or vector lacking gRFP (−gRFP). (−), donor repair fragment absent; (+), donor repair fragment present. The results shown represent an average of 5 separate experiments, in which the percentage of white colonies ranged from 90 to 98%.
↵a Transformations were performed with BWP17 (Cas9−) or HNY30 (Cas9+) with URA3-marked LEU2 gRNA plasmid, p499 (+gLEU2), or the vector that lacks gRNA (p494). (−), donor repair fragment absent; (+), donor repair fragment present. The results shown represent the average from 3 experiments.
↵b The number of Ura+ transformants that were leucine auxotrophs.
↵c The percentage of LEU2/leu2Δ heterozygotes was based on PCR analysis of LEU2 using genomic DNA isolated from 40 random Ura+/Leu+ transformants.
↵d The percentage of leu2Δ/leu2Δ homozygotes was based on PCR analysis of LEU2 amplified from genomic DNA isolated from 60 random Ura+/Leu− transformants.
Plasmids used in this study
CaURA3 RPS1 integrative plasmid
CaHIS1 RPS1 integrative plasmid
CaArg4 RPS1 integrative plasmid
CaADH1 promoter/URA3/C. albicans ARS
yEmRFP driven by CaADH1 promoter in CIp-His
Codon-optimized Cas9 driven by AgTEF1 promoter in CIp-Arg
YPB-PADH1 HH gRFP HDV
PADH1HH gRFP HDV in YPB
YPB-PADH1 HH gLeu2 HDV
PADH1HH gLeu2 HDV in YPB
YPB-PADH1 tA gRFP HDV
PADH1tRNA gRFP HDV in YPB
YPB-PtRNA gRFP HDV
PtRNA gRFP HDV in YPB
PSNR52-gRNA/SATR integrative plasmid
PSNR52-gRFP in pV1090
CaCas9/SAT flipper ENO1 integrative plasmid
CIp10-PADH1 HH gRFP HDV
PADH1HH sgRFP HDV in CIp10
CIp10-PADH1 tA gRFP HDV
PADH1tRNA sgRFP HDV in CIp10
CIp10-PtRNA gRFP HDV
PtRNA sgRFP HDV in CIp10
PSNR52 sgRFP in CIp10
CIp10-PADH1 tA SapI HDV
CIP10-based cloning vector for ligation of gRNA PADH1tA-SapI2× HDV in CIp10
pADH/tRNA plasmids used for high-efficiency gRNA expression. (A) Schematic diagram of the CIp-PADH/tRNA vector that contains a cloning cassette containing two nonpalindromic SapI sites, separated by a unique ClaI, at the tRNA-tracrRNA junction. These RPS1 integration plasmids are marked with URA3 (p494) or ura3-dpl200 (p501). (B) The sequence of the SapI cassette (blue) at the tRNA (green)-tracrRNA (purple) junction. The positions of ClaI and SapI sites within the cassette are underlined, and arrows depict the SapI cleavage sites. Beneath the SapI cassette is an example of how to design two gRNA oligonucleotides (boxed, using LEU2 gRNA). Note the gRNA (red) oligonucleotides contain CAA overhangs that create sticky ends when annealed, which allow ligation of these oligonucleotides into SapI-digested vector. Ligation results in recreation of the RNase Z tRNA recognition cleavage site, as well as the correct fusion of the gRNA with the tracrRNA. Correct ligation products are screened by the loss of the ClaI site. Download FIG S1, PDF file, 1.8 MB.
NHEJ DNA sequence trace. Shown is a partial DNA trace of an rfp mutant allele whose sequence is consistent with an NHEJ event. A schematic diagram of a portion of the sequence aligned to RFP is depicted, showing deletion endpoints. (See the sequence alignment in Fig. 6B.) Download FIG S2, PDF file, 0.2 MB.