Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple Candida Species

Abstract

Many Candida species that cause infection have diploid genomes and do not undergo classical meiosis. The application of clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) gene editing systems has therefore greatly facilitated the generation of gene disruptions and the introduction of specific polymorphisms. However, CRISPR methods are not yet available for all Candida species. We describe here an adaption of a previously developed CRISPR system in Candida parapsilosis that uses an autonomously replicating plasmid. Guide RNAs can be introduced in a single cloning step and are released by cleavage between a tRNA and a ribozyme. The plasmid also contains CAS9 and a selectable nourseothricin SAT1 marker. It can be used for markerless editing in C. parapsilosis, C. orthopsilosis, and C. metapsilosis We also show that CRISPR can easily be used to introduce molecular barcodes and to reintroduce wild-type sequences into edited strains. Heterozygous mutations can be generated, either by careful selection of the distance between the polymorphism and the Cas9 cut site or by providing two different repair templates at the same time. In addition, we have constructed a different autonomously replicating plasmid for CRISPR-Cas9 editing in Candida tropicalis We show that editing can easily be carried out in multiple C. tropicalis isolates. Nonhomologous end joining (NHEJ) repair occurs at a high level in C. metapsilosis and C. tropicalisIMPORTANCECandida species are a major cause of infection worldwide. The species associated with infection vary with geographical location and with patient population. Infection with Candida tropicalis is particularly common in South America and Asia, and Candida parapsilosis infections are more common in the very young. Molecular methods for manipulating the genomes of these species are still lacking. We describe a simple and efficient CRISPR-based gene editing system that can be applied in the C. parapsilosis species group, including the sister species Candida orthopsilosis and Candida metapsilosis We have also constructed a separate system for gene editing in C. tropicalis.

Keywords: CRISPR; Candida; genome editing.

FIG 1

The pCP-tRNA plasmid system for gene editing in C. parapsilosis. (A) The plasmid shares the main features of the pRIBO system (29), namely, the SAT1 gene (nourseothricin resistance), autonomously replicating sequence 7 (ARS7) from C. parapsilosis, and the CAS9 gene expressed from the C. parapsilosis TEF1 promoter. (B) The pRIBO and pCP-tRNA systems differ in the cassette used to express the sgRNA. In pCP-tRNA, the RNA pol II GAPDH promoter is followed by the tRNA^Ala sequence (in pink), two SapI restriction sites (in yellow), the scaffold RNA (in blue), and the hepatitis delta virus (HDV) sequence (in orange). (C) Like pRIBO, pCP-tRNA is easily lost. Transformed cells were patched to YPD plates without nourseothricin (NTC) for 48 h and were then streaked on YPD and YPD plus NTC. Colonies from YPD were repatched after 48 h. All transformants lost NTC resistance after just two passages. (D) The target guide (in green, representing ADE2-B in panel E) was generated by annealing two 20-bp oligonucleotides carrying overhang ends (in pink and blue) and cloned into SapI-digested pCP-tRNA. The guide RNA is released by cleavage after the tRNA^Ala and before the HDV ribozyme. (E) Editing of ADE2 using the pCP-tRNA system. Transformation of C. parapsilosis CLIB214 with pCP–tRNA–ADE2-B and a repair template (RT-B [29]) resulted in the introduction of two stop codons that disrupted the gene function, producing pink colonies that failed to grow in the absence of adenine (SC-ade). A white Ade-positive (Ade⁺) wild-type colony is shown as a control. The transformants were screened by PCR using the mutADE2B-F primer derived for the edited site and the downstream ADE2_REV primer, which generates a product only when the mutation is present as described in reference . WT (wild type), CLIB214 strain; NC, no DNA.

FIG 2

Editing and reconstitution of CPAR2_101060. (A) The plasmid pCP-tRNA-CP101060 was generated to target CPAR2_101060. The guide sequence recognized by Cas9 is boxed in black, and the PAM is shown in bold. The Cas9 cut site is indicated by red scissors. C. parapsilosis CLIB214 cells were transformed with pCP-tRNA-CP101060 and a repair template (RT30_101060) generated by overlapping PCR using RT30_101060_TOP and RT30_101060_BOT oligonucleotides. The repair template contains two 30-bp homology arms (HA) that flank an 11-bp sequence containing coding stop codons in all three possible reading frames (in red, with all reading frames indicated below the sequence) and a 20-bp unique barcode (in orange). The gel shows results of screening of 15 transformants by PCR using primer CP101060_TAG_F, which anneals to the barcode, together with the CP101060_WT_R downstream primer. Sequencing confirmed that stop codons were introduced into both alleles of CPAR2_101060. (B) To replace the cpar2_101060* edited alleles with wild-type sequences, a PAM site (bold) upstream from the edited site (red) was selected. The guide RNA is boxed in light blue. Transformation with pCP-rec-tRNA-Cp101060a containing this guide resulted in Cas9 cleavage 36 bp upstream from the mutated region (indicated by blue scissors). The repair template (rec-RT101060a) generated by overlapping PCR with primers rec-RT-101060aTOP and rec-RT-101060aBOT was designed to replace the edited site and barcode with wild-type sequences. It also included a single G1420C synonymous SNP so that the reconstituted and wild-type alleles could be distinguished. The wild-type sequence was successfully reintroduced in 2/9 transformants tested. The scheme is not drawn to scale.

FIG 3

Editing of ADE2 in C. metapsilosis. (A) Plasmid pCP-tRNA propagates in C. metapsilosis SZMC8093, and it is lost after two passages on YPD in the absence of nourseothricin (NTC, 200 μg/ml). (B) C. metapsilosis was transformed with plasmid pCP-tRNA-CmADE2b, targeting CmADE2 either without (left side) or with (right side) a repair template (CmRTADE2b, generated by overlapping PCR with primers CmRTADE2b_TOP/CmRTADE2b_BOT) designed to introduce two stop codons. The transformants were replica plated on YPD and Sc-Ade. Almost all colonies were pink and were unable to grow in the absence of adenine. (C) In the presence of the repair template, most transformants contained the inserted stop codons, identified by PCR using primers pCmADE2b_FWD and CmADE2_REV. Results of colony PCR of 15 representative colonies are shown; more are shown in Fig. S1 at https://doi.org/10.6084/m9.figshare.7776761. The wild-type (WT) strain was included as a control. (D) Many pink transformants were obtained even in the absence of the repair template. Sequencing of the region surrounding the Cas9 cut site revealed a variety of repair events, including insertions and deletions (indicated in red), resulting in either frameshift or deletion of His23. These presumably resulted from NHEJ.

FIG 4

The pCT-tRNA plasmid system for gene editing in C. tropicalis. (A) The plasmid shares the main features of the pCP-tRNA system, except that different regulatory elements and a different autonomously replicating sequence are used. The SAT1 gene (nourseothricin resistance) is flanked by a CdTEF1 promoter and a MgPGK1 terminator, and the CAS9 gene is expressed from the M. guilliermondii TEF1 promoter. Autonomously replicating sequence 2 (ARS2) was derived from C. albicans (43, 44). The cassette for the expression of the sgRNA is highlighted in purple on the plasmid map and is represented in more detail in the scheme on the right side. The color coding is the same as in Fig. 1. The only differences from the cassette in Fig. 1B are the promoter (AgTEF1p) and the terminator (ScCYC1t). (B) Editing of CtADE2 using the pCT-tRNA system. The guide gCtADE2.1 was generated by annealing CtAde2.1_gTOP and CtAde2.1_gBOT oligonucleotides (see Table S2 at https://doi.org/10.6084/m9.figshare.7776842) and was cloned into SapI-digested plasmid pCT-tRNA to generate pCT-tRNA-CtADE2.1. Transformation of C. tropicalis Ct46 with pCT-tRNA-CtADE2.1 and the R60-CtADE2-b repair template resulted in the introduction of a stop codon that disrupted the gene function, producing pink auxotrophs. Pink colonies were also observed when cells were transformed with pCT-tRNA-CtADE2.1 without any repair template, presumably due to NHEJ-like repair events (see also Fig. S3 at https://doi.org/10.6084/m9.figshare.7776824). (C) pCT-tRNA-CtADE2.1 is easily lost. Representative pink colonies were patched to YPD plates without nourseothricin (NTC) for 48 h and were then streaked on YPD and YPD plus NTC. Colonies from YPD were repatched after 48 h. All transformants lost NTC resistance after just two passages. (D) The transformants were screened by PCR using the s2CtAde2.1fw primer derived from the edited site and the downstream pCtADE2.1_REV primer, which generates a product only when the mutation is present. (E) Result of PCR screening of 5 representative transformants. WT, Ct46 strain; NC, no DNA.

FIG 5

Generation of heterozygous mutations with the CRISPR-Cas9 system. (A) Varying the distance between the Cas9 cut site and introduced DNA change. (B) Using a mixture of repair templates. (A) C. parapsilosis CLIB214 cells were transformed with pCP-tRNA-CP101060 and either the Het_LL1 repair template or the Het_LL2 repair template. Both Het_LL1 and Het_LL2 carry three nucleotide changes that introduce a codon change and disrupt the PAM (GGG to ATC, resulting in a Gly-to-Ile amino acid change) (yellow). The two templates also carry an additional SNP which introduces a silent mutation at either 10 bp (C>G) or 19 bp (T>C) upstream from the Cas9 cut site. Sixteen transformants obtained with Het_LL1 and Het_LL16 transformed with Het_LL2 yielded a PCR product using primer HetLLd, which anneals at the ATC codon change, and the CP101060_WT_R downstream primer. HetLLd does not anneal perfectly to the sequence introduced by Het_LL1, because of the C>G SNP. The cartoons show the sequencing results. With repair template Het_LL1, all 7 transformants contained the amino acid change at both alleles; in 3 transformants, G was introduced at both alleles 10 bp upstream of the cut site, and four retained the wild-type C base. With repair template Het_LL2, all 6 transformants again contained the amino acid change at both alleles; 5 transformants contained either the wild-type base or the mutated base at the additional site, but one was heterozygous for a T>C SNP 19 bp from the cut site. The combination of alleles in this strain is referred to as CP101060-ATC/CP101060-ATC-SNP. (B) C. parapsilosis CLIB214 was transformed with pCP-tRNA-CP101060 and a mixture of two repair templates, mixHet_1 and mixHet_2. MixHet_2 is designed to introduce two silent mutations that do not change the coding sequence but that do disrupt the PAM site, preventing Cas9 from cutting again at the edited site. mixHet_1 also contains two mutations which change the protospacer preventing the gRNA binding and which introduce a stop codon. Among the 16 transformants, 2 (indicated by the red arrows) generated PCR products when amplified with primers annealing to either of the targeted editing sites (HetLLe or HetLLf) and the CP101060_WT_R downstream primer. Sequencing of these colonies confirmed that different repair templates had been incorporated at each allele. The chromatogram shows one example. Genotype: CP101060-Stop/CP101060-SNP.