Strains and media.
C. lusitaniae strains RSY284 (haploid a, ura3), RSY286 (haploid α, leu2), RSY147 (haploid a, chx_r), RSY432 (diploid a/α, _ARG4/arg4::FRT ADE2/ade2::FRT URA3/ura3 chx_S/_chx_r) and RSY411 (haploid α, _arg4::FRT ade2::FRT chx_r) were used in this study (see Data Set S1 in the supplemental material). Strains RSY284 and RSY286 were mated to produce CAY5019 (diploid a/α, _URA3/ura3 LEU2/leu2). All strains were grown in YPD (2% Bacto peptone, 2% glucose, 1% yeast extract, 25 µg/ml uridine) at 30°C with shaking, and transformants were selected on YPD supplemented with 200 µg/ml nourseothricin (YPD plus NAT).
NHEJ plasmids and strain construction.
C. lusitaniae strain RSY147 was used to generate a SAT1-recycled leu2− strain (RSY281, haploid a, leu2), using homologous recombination of a leu2 deletion plasmid (pRB310) linearized with ApaI and SacII restriction enzymes (see Data Set S1 for primer sequences). KU70 and LIG4 deletion constructs were generated by PCR amplifying and cloning ~950-bp C. lusitaniae 5′ and 3′ homologous flanks into the pSFS2a plasmid (26) containing the nourseothricin resistance marker (plasmids pRB254 KU70 and pRB247 LIG4; see Data Set S1 for primer sequences). Plasmids were linearized with KpnI and SacII restriction enzymes prior to transformation. C. lusitaniae strain RSY281 was used in electroporation transformations to delete KU70 (strain RSY376) and subsequently delete LIG4 (strain RSY426).
CRISPR plasmids and DNA constructs.
The CTG clade codon-optimized Cas9 (CaCas9) and guide RNA were PCR amplified from plasmid pV1093 (23) (see primers in Data Set S1). To optimize Cas9 and guide RNA expression in C. lusitaniae, the C. albicans promoters were replaced with C. lusitaniae constitutive TDH3 and SNR52 promoters, respectively (plasmids pRB732 to -734). The TDH3 locus for C. lusitaniae, CLUG_03499, is annotated in the Candida Genome Database (CGD) (35). The C. lusitaniae SNR52 locus was identified using the BLASTN tool on CGD to obtain the region of the C. lusitaniae genome with the greatest homology to the C. albicans SNR52 gene. For TDH3 and SNR52, the upstream ~1-kb regions were PCR amplified from C. lusitaniae genomic DNA, and fusion PCR was used to attach these fragments to the Cas9 and sgRNA scaffolds (see primers in Data Set S1). sgRNAs were designed to target C. lusitaniae ADE2, UME6, MTLalpha1, and REC8 genes by using 20-bp protospacers immediately followed by a PAM sequence of the structure NGG (or preceded by CCN if located on the reverse strand). Protospacers were identified based on previously established criteria (23), but we considered only candidates located within the first half of the genes so that mutations would more likely generate nonfunctional proteins. We also selected sgRNAs that had minimal off-target effects identified by cross-referencing the 12 nucleotides (nt) proximal to the PAM sequence against the C. lusitaniae genome using NCBI download Clavispora lusitaniae ATCC 42720 (assembly ASM383v1). By using oligonucleotide primers containing the unique 20-bp protospacer sequence plus a 20- to 30-bp overlap with the upstream or downstream sequence, we amplified the sgRNA in two fragments; these fragments were then stitched together by fusion PCR to integrate the new protospacer (see primers in Data Set S1). Deletion constructs were generated with either long (~1-kb) or short (80-bp) homologous flanks. Long-flank deletion constructs were generated using fusion PCR of 1-kb upstream and downstream flanking regions plus ~1.8 kb of the SAT1 marker, which were then cloned into a pCR-Blunt II-TOPO vector (ADE2, pRB620; UME6, pRB744; MTLalpha1, pRB768; REC8, pRB748). Short-flank deletion constructs were generated by PCR amplifying the ~4.3-kb SAT1 marker and flipper cassette with long oligonucleotides that contained ~80 bp of the upstream and downstream flanking regions of the target gene. PCRs (50-µl mixtures) were conducted with Phusion enzyme (Thermo Scientific) to generate all DNA constructs (1× Phusion HF buffer, 200 µM deoxynucleoside triphosphates dNTPs, 0.2 µM each primer, ~20 to 50 ng template DNA, and 1 unit Phusion DNA polymerase; run according to the manufacturer’s recommendations). To stitch constructs together by fusion PCR, the products from prior Phusion PCRs were combined and diluted 10-fold, and then 1 µl was used as a template in the fusion PCR.
C. lusitaniae transformations.
An electroporation transformation protocol used for both haploid and diploid strains was adapted from reference 21. (i) Overnight cultures were diluted to an optical density at 600 nm (OD600) of 0.4 in liquid YPD and then grown until they reached a final OD600 of 1.5 to 1.7. (ii) Cells were pelleted and resuspended in 10 ml of transformation buffer composed of 0.1 M lithium acetate (LiOAc), Tris-EDTA, pH 8.0, and 0.01 M dithiothreitol. (iii) Cells were incubated on a shaker at 22°C for 1 h, washed with ice-cold water, and resuspended in ice-cold 1 M sorbitol to acquire a concentration of 150 OD units/ml. (iv) Forty microliters of cells was combined with 3 µg deletion construct, 1 µg Cas9 construct, and/or 1 µg sgRNA construct (Table 1) in 0.2-cm electroporation cuvettes (Bio-Rad, Hercules, CA). (v) Cells were electroporated at 1.8 kV, 200 Ω, and 25 µF for 4.5 to 5 ms (Bio-Rad MicroPulser); immediately resuspended in 1 ml YPD; and allowed to recover overnight at 30°C. (vi) Cells were plated onto selective medium (YPD plus NAT) to identify successful transformants.
The use of CRISPR-Cas9 has transformed our ability to edit genome sequences from bacteria to humans. Here, we describe a species-specific version of CRISPR-Cas9 that has been modified from that developed for the relatedCandidaclade speciesCandida albicans(23,24). Our results highlight the necessity of modifying this system and the use of species-specific promoters to drive expression of CRISPR-Cas9 components for achieving high transformation efficiencies inC. lusitaniae. We show that CRISPR can be used to target multiple genes and that transformation efficiencies for template-directed repair are further increased upon deletion of the NHEJ pathway inC. lusitaniae. As an alternative approach, Hogan and colleagues have utilized the transformation of RNA-protein complexes that allow species-independent CRISPR-Cas9 editing ofCandidagenomes (see accompanying paper by Grahl et al. 34). There are therefore now two distinct CRISPR-Cas9 methodologies that support efficient genome editing inC. lusitaniae.