CRISPR-Cas9 for Fungal Genome Editing

Transformation of M. thermophila and M. heterothallica protoplasts was performed according to a previously described procedure [44 (link)]. For amdS mutagenesis, the promoter Ptef1 (MYCTH_2298136) and the full-length acetamidase-encoding gene amdS (GenBank number: M16371.1) were amplified from genomic DNA of M. thermophila and the plasmid p3SR2 using the paired primers Ptef1-F2/R2 and amdS-F/R, respectively. The amdS gene was fused with Ptef1 by overlapping PCR using the primer pair Ptef1-F2/amdS-R. The Ptef1-amdS cassette was then ligated into the EcoRI and HindIII sites of a pCAMBIA-0380 plasmid [56 (link)], thereby generating the expression plasmid p0380-Ptef1-amdS. This constructed plasmid was transformed into the WT strain via Agrobacterium-mediated transformation. Colonies grown for 5 days at 35 °C were selected in medium containing 10 mM acetamide as the sole nitrogen source. Positive transformants were identified by PCR with paired primers (Additional file 7: Table S1). From these transformants, the amdS expression strain M1 was chosen and used for further CRISPR/Cas9 manipulation. Briefly, 10 µg of the Cas9-expression PCR cassette bar-Ptef1-Cas9-TtprC and the gRNA expression PCR product U6p-amdS-sgRNA at a molar concentration ratio of 1:1 was co-transformed into protoplasts of the recipient strain M1. After transformation, amdS mutants were inoculated onto MM agar plates supplemented with 2 mg mL⁻¹ FAA and 100 µg mL⁻¹ phosphinothricin. After 3 days incubation at 35 °C, FAA-resistant mutants were isolated and tested for growth on acetamide medium, followed by identification and sequencing via PCR with paired primers (Additional file 7: Table S1).
For single-gene editing, 10 µg of the Cas9-expression PCR cassette bar-Ptef1-Cas9-TtprC, gRNA expression PCR cassette U6p-cre1-sgRNA, and donor-cre1 was mixed at a molar concentration ratio of 1:1:1 and added to the fungal protoplasts. Control experiments were performed by adding 10 µg of donor-cre1 alone, or only the Cas9 cassette and donor-cre1, or only U6p-cre1-sgRNA and donor-cre1 to the fungal protoplasts. Transformants were screened for bar resistance with phosphinothricin (100 µg mL⁻¹) and neo resistance with G418 (40 µg mL⁻¹), followed by PCR identification with paired primers (Additional file 7: Table S1).
For multiple genomic edits, the generated amdS mutant M2 containing a Cas9 expression chassis was used as a host. Multiple genomic modification involving the cre-1, res-1, gh1-1, and alp-1 loci was performed in M2 protoplasts through co-transformation of two, three, or four sets of sgRNA expression cassettes and donor DNA fragments at the same molar concentration. The putative transformants were selected on MM supplemented with 100 µg mL⁻¹ phosphinothricin and 40 µg mL⁻¹ G418, followed by sequential identification via PCR with paired primers (Additional file 7: Table S1).