Table 1.
Characteristics and efficiency of the CRISPR-Cas9 mediated NHEJ and HDR experiments in the tested M. circinelloides strains.
| Host strain | Target | Elements used for transformationa | Repair mechanism | Selection method | Transformation frequency (per 105 protoplasts) | Targeting efficiencyb |
|---|---|---|---|---|---|---|
| M. circinelloides CBS277.49 | carB | Cas9 (50 µM), gRNA (50 µM) | — | white phenotype | 0 | — |
| M. circinelloides CBS277.49 | carB | Cas9 (100 µM), gRNA (100 µM) | NHEJ | white phenotype | 2 × 104 | 100% |
| M. circinelloides CBS277.49 | carB | Cas9 (200 µM), gRNA (200 µM) | NHEJ | white phenotype | 1.5 × 104 | 100% |
| M. circinelloides MS12 | carB | Cas9 (50 µM), gRNA (50 µM) | — | white phenotype | 0 | — |
| M. circinelloides MS12 | carB | Cas9 (100 µM), gRNA (100 µM) | NHEJ | white phenotype | 1.25 × 104 | 100% |
| M. circinelloides MS12 | carB | Cas9 (200 µM), gRNA (200 µM) | NHEJ | white phenotype | 1.2 × 104 | 100% |
| M. circinelloides MS12 | carB | Cas9 (100 µM), gRNA (100 µM), template DNA (5 µg) | HDR | white phenotype, complementation of uracil auxotrophy | 2 | 100% |
| M. circinelloides MS12 | carB | template DNA (5, 10 or 15 µg) | — | white phenotype, complementation of uracil auxotrophy | 0 | 0 |
| M. circinelloides MS12 | carB | Cas9 (100 µM), gRNA (100 µM) | — | white phenotype, complementation of uracil auxotrophy | 0 | 0 |
| M. circinelloides MS12 | hmgR2 | Cas9 (100 µM), gRNA (100 µM), template DNA (5 µg) | HDR | complementation of uracil auxotrophy | 1 | 100% |
| M. circinelloides MS12 | hmgR2 | template DNA (5, 10 or 15 µg) | — | complementation of uracil auxotrophy | 0 | 0 |
| M. circinelloides MS12 | hmgR2 | Cas9 (100 µM), gRNA (100 µM) | — | complementation of uracil auxotrophy | 0 | 0 |
aApplied concentrations of the elements used are presented in parentheses. bTargeting efficiency means the percentage of the isolated transformants, in which the mutation occurred in the targeted site.