Dramatic Improvement of CRISPR/Cas9 Editing in Candida albicans by Increased Single Guide RNA Expression

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) P_SNR52 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) P_ADH-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) P_ADH-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) P_tRNA(−HDV). This construct is exactly like that of panel C, but the P_ADH1 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 (P_ADH-tRNA-gRFP-HDV) compared to when poorly delivered (P_tRNA or P_SNR52).

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.