Use of RNA-Protein Complexes for Genome Editing in Non- albicans Candida Species

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome modification systems have greatly facilitated the genetic analysis of fungal pathogens. In CRISPR-Cas9 genome editing methods designed for use in Candida albicans, DNAs that encode the necessary components are expressed in the target cells. Unfortunately, expression constructs that work efficiently in C. albicans are not necessarily expressed well in other pathogenic species within the genus Candida or the related genus Clavispora. To circumvent the need for species-specific expression constructs, we implemented an expression-free CRISPR genome editing system and demonstrated its successful use in three different non-albicans Candida species: Candida (Clavispora) lusitaniae, Candida glabrata, and Candida auris. In CRISPR-Cas9-mediated genome editing methods, a targeted double-stranded DNA break can be repaired by homologous recombination to a template designed by the investigator. In this protocol, the DNA cleavage is induced upon transformation of purified Cas9 protein in complex with gene-specific and scaffold RNAs, referred to as RNA-protein complexes (RNPs). In all three species, the use of RNPs increased both the number of transformants and the percentage of transformants in which the target gene was successfully replaced with a selectable marker. We constructed mutants defective in known or putative catalase genes in C. lusitaniae, C. glabrata, and C. auris and demonstrated that, in all three species, mutants were more susceptible to hydrogen peroxide than the parental strain. This method, which circumvents the need for expression of CRISPR-Cas9 components, may be broadly useful in the study of diverse Candida species and emergent pathogens for which there are limited genetic tools. IMPORTANCE Existing CRISPR-Cas9 genome modification systems for use in Candida albicans, which rely on constructs to endogenously express the Cas9 protein and guide RNA, do not work efficiently in other Candida species due to inefficient promoter activity. Here, we present an expression-free method that uses RNA-protein complexes and demonstrate its use in three Candida species known for their drug resistance profiles. We propose that this system will aid the genetic analysis of fungi that lack established genetic systems.

Keywords: CRISPR; Candida; auris; genome editing; glabrata; lusitaniae; molecular methods.

FIG 1

Scheme for creating the gene deletion constructs and gene-specific crRNAs. (A) The components needed to create the gene deletion cassettes were generated in three PCRs. The locus or ORF to be deleted is shown in red, and the nourseothricin resistance gene (NAT1) is shown in orange. Regions of 500 to 1,000 bp flanking the target ORF (left flank [LF] and right flank [RF]) were amplified using the primers shown in PCRs 1 and 3. The NAT1 cassette was amplified in PCR 2 with primers 5 and 6. Primers 2 and 3 contained reverse-complemented sequences to primers 5 and 6 that were used to fuse the NAT cassette to the LF and RF amplicons in the PCR 4 stitching reaction with nested primers 7 and 8. The resulting gene deletion construct was used for transformation. (B) The gene-specific part of the crRNA is a 20-bp sequence (underlined) that ends with a CRISPR-Cas9 PAM site and is directly adjacent to an additional PAM site (PAM sites shown in bold).

FIG 2

PCR genotype analysis to determine RNP-mediated knockout efficiency in C. lusitaniae. (A) Schematic indicating the locations of primers used to detect transformants with the C. lusitaniae CLUG_04072Δ::NAT1 genotype. Amplification of the left and right flanking regions (LF and RF, respectively) was performed using sets of primers that included one annealing within the NAT1 locus and another annealing to the genome immediately outside the deletion construct flank (depicted in blue or green). The NAT1 gene is shown in orange. The predicted sizes for the LF and RF amplicons are shown. (B) Amplicons from reactions using primers 1 and 9 (LF) and primers 4 and 10 (RF) are shown. Genomic DNA isolated from the parental strain (P) and 10 randomly selected NAT^r colonies (1 to 10) from transformation reactions that included either the gene deletion construct and CRISPR RNPs (+RNPs) or the gene deletion construct alone (−RNP) was used as the template. Transformants for which there is a band present in both the LF and RF reactions were considered positive transformants with the NAT1 gene properly integrated at the CLUG_04072 locus. Black arrows indicate the location of the correct band size for each amplicon.

FIG 3

PCR genotype analysis to determine RNP-mediated knockout efficiency in C. glabrata and C. auris. (A) Schematic indicating the locations of primers used to detect C. glabrata and C. auris transformants with cta1Δ::NAT1 and QG_05842_05843Δ::NAT1 genotypes, respectively. Amplification of the left and right flanking regions (LF and RF, respectively) was performed using sets of primers that included one annealing within the NAT1 locus and another annealing to the genome immediately outside the deletion construct flank (depicted in blue or green). The NAT1 gene is shown in orange. The predicted sizes for the LF and RF amplicons are shown. (B) Amplicons from reactions using primers 1 and 9 (LF) and primers 4 and 10 (RF) are shown. Genomic DNA isolated from the parental strain (P) and 10 randomly selected NAT^r colonies (1 to 10) from transformation reactions that included either the gene deletion construct and CRISPR RNPs (+RNPs) or the gene deletion construct alone (−RNP) was used as the template. Transformants for which there is a band present in both the LF and RF reactions were considered positive transformants with the NAT1 gene properly integrated at either the CTA1 or QG_05842_05843 locus in C. glabrata and C. auris, respectively. Black arrows indicate the location of the correct band size for each amplicon.

FIG 4

Comparison of parental strains and transformants lacking catalase genes in C. lusitaniae, C. auris, and C. glabrata. Comparison of serially diluted parental strain (P) and four confirmed catalase knockouts (Δ-a to Δ-d), verified by the method demonstrated in Fig. 2 and 3. Images were captured after 24 h of growth on either YPD or YPD plus 3 mM H₂O₂ as indicated.