Development of a CRISPR-Cas9 System for Efficient Genome Editing of Candida lusitaniae

Abstract

Candida lusitaniae is a member of the Candida clade that includes a diverse group of fungal species relevant to both human health and biotechnology. This species exhibits a full sexual cycle to undergo interconversion between haploid and diploid forms. C. lusitaniae is also an emerging opportunistic pathogen that can cause serious bloodstream infections in the clinic and yet has often proven to be refractory to facile genetic manipulations. In this work, we develop a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (Cas9) system to enable genome editing of C. lusitaniae. We demonstrate that expression of CRISPR-Cas9 components under species-specific promoters is necessary for efficient gene targeting and can be successfully applied to multiple genes in both haploid and diploid isolates. Gene deletion efficiencies with CRISPR-Cas9 were further enhanced in C. lusitaniae strains lacking the established nonhomologous end joining (NHEJ) factors Ku70 and DNA ligase 4. These results indicate that NHEJ plays an important role in directing the repair of DNA double-strand breaks (DSBs) in C. lusitaniae and that removal of this pathway increases integration of gene deletion templates by homologous recombination. The described approaches significantly enhance the ability to perform genetic studies in, and promote understanding of, this emerging human pathogen and model sexual species. IMPORTANCE The ability to perform efficient genome editing is a key development for detailed mechanistic studies of a species. Candida lusitaniae is an important member of the Candida clade and is relevant both as an emerging human pathogen and as a model for understanding mechanisms of sexual reproduction. We highlight the development of a CRISPR-Cas9 system for efficient genome manipulation in C. lusitaniae and demonstrate the importance of species-specific promoters for expression of CRISPR components. We also demonstrate that the NHEJ pathway contributes to non-template-mediated repair of DNA DSBs and that removal of this pathway enhances efficiencies of gene targeting by CRISPR-Cas9. These results therefore establish important genetic tools for further exploration of C. lusitaniae biology.

Keywords: CRISPR; DNA double-strand break; homology-directed repair; nonhomologous end joining.

FIG 1

CRISPR components and targeting construct were optimized for transient CRISPR-Cas9 transformations in C. lusitaniae. Primers used to generate these constructs are shown, and their sequences are listed in Data Set S1 in the supplemental material. Cas9 was previously codon optimized for the Candida clade, CaCas9 (23), and the constitutive C. lusitaniae TDH3 promoter ensured maximal expression. The single guide RNA (sgRNA), which enables Cas9 to identify the target gene, was composed of the C. lusitaniae constitutive SNR52 promoter, a 20-bp protospacer sequence specific to the target gene, and the guide RNA backbone structure; terminator regions were included on both CRISPR components to help ensure efficient expression. Deletion constructs were also included in the transformation reaction to promote homology-directed repair of double-strand breaks created by Cas9. Two types of deletion constructs were generated by flanking a nourseothricin resistance marker (caSAT1) by either long (~1-kb) or short (~80-bp) regions of homology to the target gene. PCR construction of the long-flank deletion construct is shown in the figure; the short-flank deletion construct was generated using primers with ~80-bp homology to the target locus.

FIG 2

Genotypic analysis of red colonies arising from CRISPR-Cas9 targeting of ADE2 in a C. lusitaniae haploid strain. (A) A C. lusitaniae-optimized CRISPR-Cas9 system was used to target the ADE2 locus, and a large percentage of red-colony (ade⁻) phenotypes was observed (gamma of image adjusted to emphasize red/white color differences). (B) PCR assays for identifying ade2 genotypes, with arrows indicating the relative positions of the primers used (see Data Set S1 for sequences). WT, wild type. (C) For all 12 red colonies shown here, the 5′ and 3′ junction checks were positive and the ORF checks were negative, indicating successful replacement of the ADE2 target gene with the nourseothricin resistance marker. The parent strain (RYS284) was used as a wild-type control (P), and template DNA was omitted in the negative control (N).

FIG 3

Genotypic analyses and sequencing of C. lusitaniae diploid transformants. (A) PCR assays for identifying ade2 or wild-type (WT) genotypes, with arrows indicating the relative positions of the primers used (see Data Set S1 for sequences). (B) Results of genotypic analyses for red and white diploid colonies, indicating that most red colonies exhibit replacement of ADE2 with the SAT1 gene. However, two red colonies (colonies 1 and 8) had positive SAT1 junction checks but still contained the ADE2 ORF, indicating that one allele was repaired via homologous recombination and the other was repaired via NHEJ. P, parent strain (CAY5019); N, negative control (no DNA). (C) Sequencing of the ADE2 ORF in colonies 1 and 8 shows that mutagenesis via NHEJ resulted in a 1- or 2-nucleotide deletion within the protospacer preceding the PAM sequence (red box).