Table 2.
Comparison of transformants and mutation frequency obtained using different sgRNAs targeting fcy1 or pyrG
| Plasmid with sgRNA | Hygromycin-resistant strains | 5-FC- or 5-FOA-resistant strains | fcy1 or pyrG mutants by PCRa | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Rep 1 | Rep 2 | Rep 3 | Rep 1 | Rep 2 | Rep 3 | Rep 1 | Rep 2 | Rep 3 | |
| Empty vector | 2 | 4 | 8 | 0b | 0b | 0b | – | – | – |
| fcy1 sgRNA No. 1 | 5 | 11 | 18 | 1 (20%)c | 0 | 0 | 1/1 | – | – |
| fcy1 sgRNA No. 2 | 28 | 23 | 34 | 22 (78.6%)c | 15 (65.2%)c | 30 (88.2%)c | 5/5 | 4/5 | 4/5 |
| pyrG sgRNA No. 1 | 17 | 19 | – | 8 (47.1%)c | 18 (94.7%)c | – | 7/8 | 10/10 | – |
| pyrG sgRNA No. 2 | 7 | 7 | – | 2 (28.6%)c | 2 (28.6%)c | – | 2/2 | 2/2 | – |
aThe number of mutants which can be identified by difference size, or no amplified, bands compared to wild-type control were observed in the genomic PCR experiment (Additional file 1: Fig. S2A and B)
bThe number of 5-FC- or 5-FOA-resistant strains
cPercentage of 5-FC- or 5-FOA-resistant strains from the total number of hygromycin-resistant strains