Genomic deletions in Aureobasidium pullulans by an AMA1 plasmid for gRNA and CRISPR/Cas9 expression | Fungal Biology and Biotechnology

Strains and cultivation conditions

A. pullulans EXF-150 (CBS 100280) was used as the wild-type and a parent strain for disrupting the URA3 gene [3]. A. pullulans was cultivated at 24 °C on malt extract (MEX) agar plates (1 g/L peptone, 30 g/L malt extract, 15 g agar in tap water). For selection, a minimal medium described by Ueda et al. was used: 0.06% (NH₄)₂SO₄, 0.1% NaCl, 0.5% K₂HPO₄, 0.02% MgSO₄∙7H₂O, 0.4% yeast extract and 2% carbon source (added after autoclaving) [29]. The salts were prepared in a fivefold concentrated stock at pH 6 (adjusted with HCl). Uridine at a final concentration of 5 mM was added to the medium to cultivate the uridine auxotrophic strain. To select for 5-FOA or hygromycin B resistance, these compounds were added at the respective final concentrations of 2 g/L or 100 μg/mL.

Construction of the CRISPR/Cas9 plasmid (adapted from [28])

We aimed for deletion of the gene coding for the orotidine-5-phosphate carboxylase (gene ID: 40743721) using a gRNA target described by Kreuter et al. [19] (no. 14 in Supplementary Table 1). The CRISPR/Cas9 plasmid was assembled in three steps, as displayed in Fig. 1. First, a 248 bp-fragment containing the gRNA was created by multiple overlap extension PCR (MOE-PCR): six primers with overlaps (oligonucleotides no. 1–6 in Supplementary Table 1) were used to generate the fragment in one PCR (Fig. 1A). The 50 µL PCR mix contained 20 µM dNTPs, 0.5 µM of each gRNA primer, 0.25 µM of each structural primer, 1U Q5 DNA polymerase, and Q5 buffer. The reaction was incubated in a thermocycler for 1 min 30 s at 95 °C followed by 35 cycles of 30 s at 95 °C, 20 s at 60 °C and 10 s at 72 °C and followed by the final extension for 5 min at 20 °C. The generated PCR product was analyzed on an agarose gel, and the correct band was cut out and purified [28].

Overview on the CRISPR/Cas9 plasmid assembly. A. Scheme of the multiple overlap PCR to generate the 248 bp fragment. This fragment was assembled by PCR using two primers containing the sequence specific to the gRNA target and four generic primers. The obtained product includes a hammerhead ribozyme, the complementary sequence to the target DNA, the sequence for expression of the gRNA, a hepatitis delta virus (HDV) ribozyme for cutting at the 5’ and 3’ ends of the gRNA and BsaI restriction sites on the 3’ and 5’ ends. B. Overview on the two GGAs to generate the final CRISPR/Cas9 plasmid. The initial GGA using BsaI, leads to the ligation of the 248 bp-fragment and the BB1_L_23_syn_BsaI plasmid. The product of this assembly is then assembled with the plasmid bearing the Cas9 expression cassette via BsbI

Then, this 248 bp-fragment was assembled with the plasmid P1 (Supplementary Table 1) through a GGA with BsaI (Fig. 1B). The resulting plasmid was assembled with the plasmid P2 (Supplementary Table 1) using GGA with BbsI (Fig. 1B). GGA were performed in a final volume of 20 µL containing 2 mM of each construct to assemble, CutSmart Buffer, 100 U of T4 ligase, 2 mM ATP, 30 U BsaI-HF or BbsI (New England Biolabs). The assemblies were incubated in a thermocycler for 45 cycles of 2 min at 37 °C and 5 min at 16 °C followed by a single step of 5 min at 50 °C and 10 min at 80 °C. The resulting products were transformed into E. coli Top10 and selected on LB agar containing the relevant antibiotic.

Protoplast generation (adapted from [19])

For the generation of protoplasts, a cell suspension of A. pullulans in a sterile solution of 0.8% NaCl/0.05% Tween 80 was prepared by scraping cells from an agar plate. A shake flask containing 20 mL MEX medium was inoculated with the suspension with a target starting OD of 0.05 and incubated overnight (~ 16 h) at 220 rpm and 24 °C. 20 mL of the culture (OD₆₀₀ = 1–2) was centrifuged at 4000 × g for 5 min at room temperature (RT). The supernatant was discarded, and the pellet was resuspended in 20 mL of buffer A (100 mM KH₂PO₄, 1.2 M sorbitol, pH = 5.6, sterilized by autoclaving). The suspension was centrifuged at 4000 × g for 5 min at RT, then the supernatant was removed, and the pellet was resuspended in 15 mL sterile lysing solution (600 mg VinoTaste^® Pro lysing enzymes (Novozymes, catalogue number 2860–689-03–2) dissolved in 15 mL buffer A, sterile filtered). The suspension was placed in a shake flask and incubated in a rotary shaker at 140 rpm and 24 °C for 45–60 min until protoplasts were formed. The protoplast suspension was poured into a 50 mL reaction tube, filled with up to 40 mL with ice-cold 1.2 M sorbitol, and centrifuged at 3000 × g for 10 min at 4 °C. The formed pellet was washed once with 30 mL 1.2 M ice-cold sorbitol and twice with 10 mL ice-cold, sterile buffer B (1 M sorbitol, 10 mM Tris–HCl (pH = 7.5), 25 mM CaCl₂, sterilized by autoclaving), each time centrifuged at 3000 × g for 10 min at 4 °C. The protoplasts were handled gently and always kept on ice. The protoplast suspension and 20% PEG solution (1/3 sterile 60% PEG solution, 2/3 sterile buffer B) were mixed 1:1, and DMSO was added to a final concentration of 1% and mixed carefully. The 60% PEG solution was made of 600 g/L PEG 4000, 10 mM Tris pH = 7.5 and 10 mM CaCl₂. Protoplasts were counted with a Thoma cell counting chamber to verify that the concentration is approximately 10⁶–10⁷ protoplasts per mL. The protoplast suspension was aliquoted (200 µL aliquots) in prechilled reaction tubes and stored on ice (for protoplasts used freshly) or at − 80 °C (for frozen protoplasts). Two transformations were performed for which two different batches of protoplasts were used. The first transformation was made with frozen protoplasts only, and the second transformation was made with fresh and frozen protoplasts from the same batch [19].

Transformation of protoplasts (adapted from [19])

A 200 µL-aliquot of protoplasts, either freshly prepared or frozen and thawed on ice, was used. The aliquot was mixed with 2 or 5 μg undigested plasmid, 2 μL β-mercaptoethanol and 50 μL buffer B. The mix was incubated on ice for 30 min. 50 µL, 200 μL and 500 μL 60% PEG solution were added stepwise to the mix, followed by careful mixing between each step. The mix was incubated at RT for 20 min. Then, 200 μL, 400 μL, 1000 μL and 2500 μL of sterile buffer C (1 M sorbitol, 10 mM Tris–HCl (pH = 7.5)) were added stepwise with careful mixing after each step. The final volume of the transformation solution was 5.2 mL. 20 mL of melted selection medium containing 1 M sucrose and 100 μg/mL hygromycin B was added to the protoplast suspension and poured into plates. Different volumes of the protoplast suspensions were used. The plates were incubated in the dark at 24 °C until colonies were visible (4–14 days) [19].

Selection of transformants

Hygromycin B-resistant transformants were streaked on plates containing 5-FOA. Some positive transformants on 5-FOA were randomly selected and underwent two isolation passages on MEX plates supplemented with uridine. Transformants were considered resistant to 5-FOA when colonies grew better than the negative control (wild-type strain EXF-150) and similarly to the positive control (strain #6 (∆URA3 mutant, generated with the RNP method [19])). The colony forming units per µg DNA (CFU) were estimated as follows:

Colony PCR and sequencing

After the first transformation assay, 2 transformants resistant to 5-FOA and hygromycin B were selected for colony PCR with primers 7 and 8 (Supplementary Table 1). The colony PCR was performed following the protocol of Wu et al. for rapid screening of yeast colonies [30]. 2 μL of the fungal cell suspension was used as a template in a 50 μL colony PCR using Q5 DNA polymerase (NEB) and the wild-type strain as a control. The molecular mass of the PCR products was checked on an 0.8% agarose gel. For visualization, a Bio-Rad ChemiDoc Imaging System was used. PCR products of the correct molecular mass were purified with the Thermo Scientific GeneJET PCR Purification Kit and sent for sequencing to Microsynth AG with the same primers as for PCR, primers 7 and 8.

Extraction of genomic DNA, PCR and sequencing

After the second transformation assay, no colony PCR was performed. Isolated transformants were directly used for DNA extraction and PCR on the genomic material. DNA extraction was performed on 33 transformants from the second transformation assay and one transformant from the first transformation assay. The selected transformants were cultivated overnight in liquid MEX medium, then centrifuged and about 50 mg of the cell biomass was used as starting material for the DNA extraction. 50 mg of cells were placed in a 2 mL tube with glass beads (0.37 g of 0.1 mm diameter beads, 0.25 g diameter of 1 mm diameter beads and one bead of 5 mm diameter) in 1 mL CTAB buffer (1.4 M NaCl, 100 mM TrisCl pH 8.0, 10 mM EDTA, 2% CTAB and 1% polyvinylpyrrolidone) and mechanically disrupted using a FastPrep high-speed homogenizer (MP Biomedicals) at 4 m/s for 30 s. The tube was then incubated for 20 min at 65 °C. The supernatant and foam were transferred to a 2 mL tube and mixed with 400 μL phenol and 400 μL chloroform by manual horizontal shaking. Then an incubation step of 5–10 min at room temperature was performed followed by centrifugation at 12,000 g at 4 °C for 10 min. After centrifugation, 650 μL of the top phase were transferred to a 1.5 mL tube, 650 μL chloroform were added and the tube mixed by manual horizontal shaking for 30 s, followed by centrifugation at 12000 g at 4 °C for 10 min. 500 μL of the top phase were transferred to a new 1.5 mL tube and 4 μL of RNAse A at 10 mg/mL added to the tube, the tube was mixed by inversion 6 times and incubated 30 min at 37 °C. After incubation 350 μL of isopropanol were added to the tube, the tube was mixed by inversion 6 times and incubated 10 min at room temperature. Then DNA was precipitated by centrifugation at 21000 g 4 °C for 30 min. The supernatant was removed and 1 mL of ice cold 70% ethanol added, followed by centrifugation at 21000 g 4 °C for 10 min. The supernatant was removed and the remaining ethanol evaporated by incubation of the tube at 50 °C. The obtained pellet was dissolved in 50 μL ultrapure water or TE buffer. The concentration and purity were checked using NanoDropOne^C (Thermo Fisher Scientific) and agarose gel electrophoresis. A PCR was performed using Q5 DNA polymerase (NEB) according to manufacturer protocol with the primers 9 and 10 or 11 and 12 (Supplementary Table 1). Sequencing was performed by Microsynth AG using primers 10, 11 and 12 (Supplementary Table 1).

Phenotype analysis

After one passage on plates with uridine and 5-FOA and two isolation passages on MEX plates, the transformants selected for DNA extraction were grown on MEX plates supplemented with uridine, MEX plates supplemented with uridine and hygromycin B and MEX plates supplemented with uridine and 5-FOA. The wild-type and a mutant bearing a genomic hygromycin B resistance were used as controls. Plates were incubated at 25 °C for 2–3 days.