Materials and Methods

New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi

Transformation.

A hybrid lithium acetate/electroporation protocol was used for C. albicans, S. cerevisiae, and C. glabrata. Briefly, a 5-ml yeast extract-peptone-dextrose (YPD) culture of each strain was grown overnight at 30°C on a roller drum to saturation. Cells were pelleted by centrifugation and resuspended in 2 ml of 100 mM lithium acetate–10 mM Tris (pH 8.0)–1 mM EDTA (pH 8.0) to which 50 µl 1 M dithiothreitol (DTT) was added. These cells were incubated on a 30°C roller drum for 1.5 to 15 h. Next, cells were washed twice in 5 ml of ice-cold water, washed once in 5 ml of ice-cold 1 M sorbitol, and resuspended in 500 µl of ice-cold 1 M sorbitol. Cells were kept on ice while transformation mixes were prepared. Cell suspension (40 µl) was added to aliquoted DNA, mixed by pipetting, placed in chilled Bio-Rad gene pulser cuvettes (Bio-Rad catalog no. 1652086), and electroporated on a Bio-Rad Gene pulser with a capacitance of 25 µF and resistance of 200 Ω at 1.8 kV. YPD medium (100 µl) was immediately added to the cuvette, and the cell mixture is incubated for between 1 and 12 h at room temperature before plating onto appropriate selective media.

N. castellii was transformed using a modified version of the lithium acetate method previously described by Gietz and Schiestl (29). A 5-ml YPD culture of N. castellii was grown overnight at 30°C on a roller drum. This preculture was then used to inoculate 50 ml of YPD medium to reach an optical density at 600 nm (OD600) of 0.2. The culture was then grown at 30°C with vigorous shaking in a baffled flask until it reached an OD600 of 0.8. Cells were collected by centrifugation, washed once in 25 ml of water, and resuspended in 1 ml of 100 mM LiAc–100 mM Tris-Cl (pH 7.5). This suspension was transferred to a microcentrifuge tube and centrifuged at top speed for 15 s. The pellet was resuspended in 400 µl of 100 mM LiAc–100 mM Tris-Cl (pH 7.5) and incubated at room temperature for 20 min. For each transformation, 50 µl of cell suspension was added to a microcentrifuge tube. To each transformation tube, the following reagents were added in the following order, with vortex mixing performed after each addition: 240 µl 50% polyethylene glycol (PEG) 3350, 36 µl 1 M LiAc, 12.5 µl boiled single-stranded DNA (ssDNA) (10 mg/ml), and 1.5 µg each plasmid DNA and repair template. Transformation mixes were incubated at 23°C for 30 min with rotation and then heat shocked at 37°C for 20 min. Heat-shocked cells were centrifuged at 8,000 rpm for 15 s, and transformation liquid was removed. Cells were washed with 500 µl of water and resuspended in 200 µl of water. At least 25 µl of this suspension was plated onto selective media.

Strains and growth conditions.

Strains used in this study are listed in Table S2 in the supplemental material. C. albicans strain SC5314, C. glabrata strains BG2 and CLIB138, S. cerevisiae strains BY4741 and FermPro, and N. castellii strains DBP069 and DBP596 were used for experiments. Yeast cells were grown in YPD medium (1% Difco Bacto yeast extract, 2% Difco Bacto peptone, 2% glucose) and selected with nourseothricin (Nat) at between 100 and 200 µg/ml. Flipout of the Natr gene from C. albicans vectors was induced by overnight growth in Difco yeast carbon base supplemented with bovine serum albumin (4%) overnight or by overnight growth in YP maltose medium (1% Difco Bacto yeast extract, 2% Difco Bacto peptone, 2% maltose). Episomal plasmid loss experiments were performed by overnight growth in nonselective liquid YPD medium. Drug-sensitive isolates, which had either flipped out the cassette or lost the plasmid, were identified by plating for single colonies on nonselective media and subsequent identification by replica plating to selective media.

Filamentation experiments were performed with yeast grown overnight in liquid YPD medium, washed twice with RPMI 1640 medium (Thermo Fisher catalog no. 22400-105) supplemented with 10% fetal bovine serum (FBS), and incubated in RPMI medium–10% FBS for the indicated time starting at an optical density of 0.1. CRISPR-mutagenized loci were verified by agarose gel electrophoresis and sequencing of PCR products amplified from target loci.

Plasmids/DNA.

Plasmids for CaCas9 system vectors are listed in Table S1. All key components were verified by sequencing and restriction analysis. Vectors and sequences will be available on Addgene. Transformations were performed with between 0.3 and 5 µg of plasmid DNA (smaller amounts were used for episomal plasmids and larger amounts for linear vectors for integration) and with between 1 and 5 µg of a repair template (where applicable). Repair templates were generated with 60-bp oligonucleotide primers containing 20-bp overlaps at their 3′ ends centered at the mutation point, which consisted of either stop codons or an open reading frame (ORF) deletion. N. castellii repair templates were 500 bp in length. Oligonucleotide sequences are listed in Table S3. Primers were extended by thermocycling performed with Ex Taq (TaKaRa catalog no. RR001A), and double-stranded products were purified directly from the PCR using a QIAquick gel extraction kit with isopropanol. Phosphorylated and annealed guide sequence-containing primers were ligated into calf intestinal phosphatase (CIP)-treated, BsmBI-digested parent vectors as described previously (9). The correct clones were identified by sequencing.

TABLE S1 

Plasmids used in this study. Download TABLE S1, PDF file, 0.1 MB.

Copyright © 2018 Vyas et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

TABLE S2 

Strains used in this study. Download TABLE S2, PDF file, 0.1 MB.

Copyright © 2018 Vyas et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

TABLE S3 

Oligonucleotides used for repair template generation. Download TABLE S3, PDF file, 0.05 MB.

Copyright © 2018 Vyas et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Computational analysis for guide design.

We designed guide sequences corresponding to each annotated gene in the genomes of C. albicans, C. glabrata, S. cerevisiae, and N. castellii (see the supplemental guide tables DOI https://doi.org/10.17605/OSF.IO/ARDTX available at http://osf.io/ARDTX/), using updated guide selection and scoring methods (21) (summarized in Table 1).

For C. glabrata, S. cerevisiae, and N. castellii, we listed all 20 nucleotides (nt) followed by NGG (or CCN plus 20 nt) in the genome. We removed any targets that had 6 instances of "T" in the 20 nt before the NGG because their presence would result in premature termination from polymerase (Pol) III promoters. We kept only the guides that were hitting the coding regions of genes. To find putative off-targets, we took the 14 nt closest to the protospacer-adjacent-motif (PAM) sequence, constructed the four possible sequences of the PAM, and mapped these with Bowtie, allowing up to 3 mismatches. For each original guide, we calculated the on-target score using rule set 2 for sgRNA on-target activity script (described in reference 21) as well as all the possible off-target scores for all the places in the genome that the 14-nt NGG hit with ≤3 mismatches (cutting frequency determination CFD scoring) (21). We defined guides as having no off-targets if the off-target scores at other locations were lower than 0.2. To find guides that targeted more than one gene, we followed the requirements that the off-target score for the other genes hit was 0.5 or higher and that there were no other hits with off-target scores higher than 0.2 and lower than 0.5. We also removed guides with off-targets in intergenic regions.

For C. albicans, using rule set 2 for the sgRNA on-target activity script (21), we rescored the guides uniquely targeting the two alleles that we had previously designed (9). Additionally, to assess whether those guides could be used on the Unified Solo vector, we checked that the guide was not targeting the vector and the absence of BsmBI sites in the guide.

Sequences (FASTA files) and annotations (GFF files) for C. albicans and C. glabrata were downloaded from the Candida Genome Database (30). C. albicans sequences from strain SC5314 (assembly 22, version A22-s05-m01-r03) and C. glabrata sequences from strain CBS138 (version s02-m07-r06) were used for guide selection. N. castellii guide design was based on sequences (FASTA files) and annotations (GFF files) for strain CBS4309 (assembly ASM23724v1.31) obtained from Ensembl (31). S. cerevisiae guide design was based on sequences (FASTA files) and annotations (GFF files) downloaded from the Saccharomyces Genome Database (32). The sequences were from strain S288C (version sacCer3/SGD_2010).

Data availability.

Species-specific guide sequence tables (for single-gene, multigene, and/or allele-specific guides) are available at the Open Science Frameworks repository (DOI https://doi.org/10.17605/OSF.IO/ARDTX; https://OSF.IO/ARDTX).

Article TitleNew CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi

Abstract

We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans, Saccharomyces cerevisiae, Candida glabrata, and Naumovozyma castellii. These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both C. albicans and S. cerevisiae rely heavily on homology-directed repair, whereas C. glabrata and N. castellii use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms.

IMPORTANCE CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens C. albicans and C. glabrata, as well as in the related yeasts S. cerevisiae and N. castellii. The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species.


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