Trichoderma reesei—T. reesei strain QM6a (ATCC13631) (Li et al., 2017) was used in this study. The targeted gene is the ura5 auxotrophy gene.

Sphaerulina musiva—S. musiva isolate MN14 was used in this study to establish the CRISPR-Cas9 RNP-mediated transformation. This species was isolated from canker of an interspecific hybrid poplar (P. nigra x P. maximowiczii) tree in Minnesota, United States (Tabima et al., 2020). The gene targeted in this transformation is the pyrG encoding for Orotidine 5′-phosphate decarboxylase that confers prototrophy to uracil and uridine.

Trichoderma reesei—To ensure the successful development of the CRISPR-Cas9 RNP-mediated transformation system, we tested the gRNA (5ʹ-GGA​TGC​CGA​AAT​CAT​GGC​CGt​gg-3ʹ, PAM shown in lowercase) designed and confirmed in the study by Liu et al. (2015) that targets the ura5 gene in the model fungus T. reesei.

Sphaerulina musiva—In addition to the Cas9 nuclease, the CRISPR-Cas9 gene editing requires a custom single guide RNA (sgRNA) that is made up of two parts: i) the Crispr RNA (crRNA) that comprises 20 nucleotide sequence complementary to a region in the target gene that will direct the Cas9 nuclease activity and ii) the trans-activating crRNA (tracrRNA) that serves as a binding scaffold for the Cas9 nuclease. First, we run a Blast search using the pyrG gene from A. nidulans FGSC A4 (XM_658,669.1) against the S. musiva strain SO2202 genome on NCBI. A local blastn search against the genome of strain MN14 was later performed to locate the coordinates of pyrG in the isolate of interest. For the design of the crRNA, the web tool CRISPOR (http://crispor.tefor.net/) is used. This tool lists possible crRNA sequences within a specific DNA region and provides scores such as specificity, efficiency prediction, out of frame, and off targets. To start generating crRNA sequences, copy and paste the sequence of the target gene, and select the genome of interest from their list. If the genome does not exist in the list, the GCF_/GCA_ ID from NCBI can be sent to the CRISPOR support team. Lastly, we selected a Protospacer Adjacent Motif (PAM). For our analysis, the gene of interest was the Orotidine 5′-phosphate decarboxylase encoding gene (pyrG) from Sphaerulina musiva strain MN14 and the selected PAM is -NGG specific for Streptococcus pyogenes (Sp)-derived Cas9 (SpCas9). Below are the different criteria we followed in the selection of the crRNA sequence.

⁃ It is recommended to select from the list gRNAs that target conserved coding domains of the gene of interest.

After selecting the crRNA sequence with the best location, high predicted activity score and low off-target scores, we used the RNAfold webserver (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) to ensure that the RNA molecule will fold into the correct 3D structure for binding to the Cas9 enzyme. We generated a combined sequence including the protospacer and the Cas9-specific scaffold sequence and proceed to secondary structure prediction on RNAfold. For our analysis the gRNA scaffold sequence used is: GTT​TTA​GAG​CTA​GAA​ATA​GCA​AGT​TAA​AAT​AAG​GCT​AGT​CCG​TTA​TCA​ACT​TGA​AAA​AGT​GGC​ACC​GAG​TCG​GTG​C (Liu et al., 2015).

Lastly, we evaluated the RNAfold results and chose a final crRNA candidate from those that passed the different filtering steps. In this regard, it is recommended that the crRNA and tracrRNA exhibit interactions without affecting the essential stem-loop structures of the sgRNA. The secondary structure of the sgRNA should contain three stem loops, interrupted by five ring structures. High binding probabilities should also be displayed through the entire structure, except for the protospacer region.

The designed crRNA, tracrRNA (Cat No. 1072533) and the Cas9 nuclease (Cat No. 1081059) were synthesized by Integrated DNA Technologies (IDT). First, we diluted the crRNA and tracrRNA to a final concentration of 200 µM in IDT duplex buffer (Cat No. 1072570). To prepare the sgRNA, we mixed 5 µl of 200 µM crRNA and 5 µl of 200 µM tracrRNA and completed the volume to 20 µl with IDT duplex buffer. The sgRNA mixture was later heated to 95°C for 5 min and allowed to cool down at room temperature for at least 20 min. Lastly, to assemble the Cas9/sgRNA ribonucleoprotein (RNP) complex, we mixed 6 µl of sgRNA and 8.3 µl of the Cas9 nuclease (60 µM stock) and completed the volume to 25 µl using RNase-free water.

This step is optional but recommended to test the ability of the sgRNA to cleave in vitro. First, we designed a primer set (Smusiva_pyrG F/R) that will amplify a fragment harboring the chosen sgRNA target site. Genomic DNA from S. musiva strain MN14 was extracted following the protocol from Tannous et al. (2018). The targeted DNA fragment was amplified using the designed primer set and a standard DNA polymerase following the manufacturer instructions. We later prepared the sgRNA by combing 0.5 µl of the 200 µM crRNA and 0.5 µl of the 200 µM tracrRNA from the step above into a final volume of 10 µl duplex buffer. The mixture was incubated at 95°C for 5 min, then allow it to cool at room temperature. To form the RNP complex, we combined the 10 µl of sgRNA with 1.6 µl of Cas9 (60 µM stock) and completed the volume to 100 µl with Cas9 dilution buffer (30 mM HEPES, 150 mM KCI, pH 7.5). The mixture was incubated at room temperature for 10 min for optimal formation of the RNP complex. The in vitro digestion reaction was performed by combining 1 µl of the Cas9 dilution buffer, 1 µl of the Cas9-RNP complex, 3 µl of the purified PCR product into a final volume of 10 µl with nuclease free water. The reaction was incubated at 37°C for 60 min. To release the DNA substrate from the Cas9 nuclease, we added 2 μl of Proteinase K (10 mg/mL) to the reaction, then incubated the mixture at 56°C for 10 min. Lastly, the digestion reaction and the PCR product were analyzed on a 2% agarose gel to visualize the cleaved products.

First, the cryo-preserved spores of T. reesei and S. musiva were activated on PDA and KV8 agar medium (180 ml V8 vegetable juice, 2 g CaCO₃, 20 g agar in 1 L distilled water, pH = 7–7.2) supplemented with streptomycin (100 μg/ml), respectively. The plates were incubated for at least 5 days at 25°C. To isolate protoplasts from fungal cells, we inoculated 10⁸ fresh spores of each of the fungal species collected using sterile water. The morphology of spores collected from both species are shown in Figures 1A, B Depending on the fungal species, the incubation time prior to protoplasting can vary. For T. reesei, 24 h growth were required to get enough fungal biomass, whereas for Sphaerulina musiva which is a slow grower species, we allowed 72 h of growth prior to protoplasting. To harvest the germlings (or germinated spores), we transferred the liquid culture into 50 mL falcon tubes and centrifuged at 4,000 g for 5 min. The supernatant was discarded, the germlings were washed with sterile water to eliminate all traces of media and centrifuged again to collect the germling pellet. To lyse the cell wall of the young germlings and release protoplasts, the germling pellet was resuspended in 10 ml of protoplasting solution (100 mg of lysing enzyme from T. harzianum and 50 mg of yatalase in 10 ml osmotic medium (1.2 M MgSO₄ and 10 mM Sodium Phosphate Buffer). The protoplasting solution/germlings preparation was incubated at 30°C with shaking at 80 rpm for 3–4 h. The preparation should be checked every hour for protoplast release. Protoplasts will appear as shown in Figure 1C, d after complete cell wall digestion. Protoplasts are cells without cell walls and are thus very sensitive to mechanical disruption. Make sure to handle them gently when pipetting. To collect protoplasts, we overlayed the preparation gently with 10 ml of cold Trapping buffer (0.6 M Sorbitol and 0.1 M Tris-HCl, pH 7), and centrifuged at 5,000 g and 4°C for 15 min. The protoplasts were collected from the interface into a new 15 ml centrifuge tube and overlayed with an equal volume of cold STC buffer (1.2 M Sorbitol, 10 mM CaCl₂, 10 mM Tris-HCl, pH 7.5). The protoplasts were later pelleted by centrifuge at 3,000 g and 4°C for 10 min. To initiate the transformation, we combined 100 µL of isolated protoplasts with 25 µl of the RNP complex mixture and completed the volume to 200 µL using STC buffer. We vortexed the solution gently to ensure it is thoroughly mixed. A negative control should be prepared by mixing 100 µL of isolated protoplasts with 100 µL of STC buffer. The transformation mixture and the negative control were incubated on ice for 50 min. Later, 1.2 mL of PEG were added to both mixtures and incubated for an additional 20 min at room temperature. Then, we added 5 mL of STC buffer to both mixtures and gently homogenized them. After incubation, we combined the transformation mixture with 45 mL of molten Sorbitol Minimal Medium (SMM) top agar medium (SMM with reduced amount of agar by half) (Tannous et al., 2023) supplemented with uracil (0.56 g/L) and uridine (1.26 g/L) and poured 5 mL of the mixture on top of plates containing 20 mL of SMM supplemented with the same amino acids. For the negative control, we combined 1 mL of the mixture with 5 mL of Top agar medium and poured on top of SMM medium plates supplemented with the same amino acids. The first layer of top agar was allowed to set by incubating the cultures overnight at 21°C (for S. musiva) and 25°C (for T. reesei). The next day, we added an additional layer of SMM top agar supplemented with 5- Fluoroorotic acid (5-FOA) at a final concentration of 1.5 mg/ml of medium. The cultures were incubated again until single transformants were observed growing through both layers of agar. To check the stability of the protoplasts through the transformation process, we recommend having a positive control plate not supplemented with 5-FOA.

Individual isolates were later picked and screened on new SMM plates supplemented with 5-FOA and uracil and uridine. Lastly, we extracted gDNA from all selected T. reesei and S. musiva transformants using the method cited above.

To screen for the genome editing events in T. reesei, we designed the primer set (Treesei_Ura5 F/R) that will amplify fragment of the ura5 gene harboring the sgRNAs target sites. For S. musiva, we used the same set of primer designed for the in vitro validation of the gRNA cleavage to amplify the fragment harboring the gRNA cut site. The primer sequences are presented in Table 1. We amplified the two fragments using a standard DNA polymerase and purified the PCR products prior to sequencing using the QIAquick PCR Purification kit (Qiagen, Cat. No. 28104). The sequences from the different transformants were lastly aligned with the WT sequence of the gene for INDEL detection at the gRNA cut site.

The approach described in this study employed an RNP version of the CRISPR-Cas9 genome editing system and the critical step in this protocol is the design and synthesis of a high quality sgRNA. The location of the designed crRNA in the pyrG sequence of S. musiva strain MN14 is shown on Figure 2A. Figure 2B displays the scores of the crRNA sequence provided by CRISPOR. This molecule was selected in such a way that it: i) specifically targets the pyrG gene (with an MIT specificity score of 100), ii) shows high interaction score with the target sequence (with a CFD specificity score of 100), iii) is capable of effectively cleave the target region (with a Doench score of 58), iv) shows slight similarity to other regions in the genome (with 1 off target predicted), and v) folds correctly in order to bind with the Cas9 protein. The ability of this sgRNA to target and cleave the target region was later confirmed in vitro by showing two products of the expected sizes (Figure 2C).

For both species, one successful transformation was performed. Ten random transformants were selected from the ura5-targeted transformation in T. reesei. All 10 selected transformants presented random deletions at the gRNA cut site as shown in the alignment with the WT sequence of this gene in Figure 3B. For this transformation, we used the same gRNA designed by Liu et al. (2015). The difference between the transformation methods was the format of the CRISPR components. In the previous study they were introduced to the cells as mRNA (in vitro transcribed) format, whereas here the Cas9 and the sgRNA were delivered as RNP complex. Regardless of the delivery format, we recorded a similar transformation efficiency of 100%.

For S. musiva, since this is the first CRISPR-cas9 editing protocol developed on this species, 20 transformants were randomly picked and subjected to a second screening on new SMM plates supplemented with 5-fluoroortic acid (5-FOA) and uracil and uridine. The disruption of the pyrG gene allows an easy selection on media supplemented with 5-FOA, a chemical that can be converted into the toxic intermediate 5-fluorodeoxyuridine through pyrG activity, thus generating S. musiva △pyrG mutant strains that display auxotrophy for uracil and uridine but insensitivity to 5-FOA.Of the 20 transformants, 19 were able to grow on the screening plates. An amplification and sequencing of the pyrG region harboring the gRNA cut site from these transformants and their alignment with the WT pyrG sequence showed various indels at the gRNA cut site in 17 isolates (Figure 3B). Two isolates S. musiva T17 and T18 showed no differences in the sequence while aligned with the WT copy of the pyrG gene. To summarize, a 100% editing efficiency was observed in T. reesei, while 90% editing efficiency was observed following S. musiva’s transformation.