Table 1.
Protoplast transfection experimental setup and results thereof
| Guide sequence | Promoter | PEG (%) | Transfection time (min) | DNA (µg) | Protoplasts (/mL) | Number regenerated mutated lines (number analysed) | Frequency mutated lines (%) |
|---|---|---|---|---|---|---|---|
| GT1 | A.t.U6 | 12.5 | 3 | 10 | 80 000 | 8 (161) | 5.0 |
| GT1 | A.t.U6 | 25 | 3 | 5 | 80 000 | 7 (85) | 8.2 |
| GT1 + Cas9a | A.t.U6 | 25 | 3 | 5 + 5 | 80 000 | 4 (130) | 3.1 |
| GT1 | A.t.U6 | 40 | 30 | 15 | 50 000 | 5 (43) | 11.6 |
| GT2 | A.t.U6 | 12.5 | 3 | 10 | 80 000 | 12 (286) | 4.2 |
| GT2 | A.tU6 | 25 | 3 | 5 | 80 000 | 5 (108) | 4.6 |
| GT2 | A.t.U6 | 40 | 30 | 15 | 50 000 | 2 (79) | 2.5 |
| GT4 | S.t.U6 | 12.5 | 3 | 10 | 80 000 | 18 (188) | 9.6 |
| GT4 | A.tU6 | 25 | 3 | 5 | 80 000 | 21 (407) | 5.2 |
| GT4 | S.tU6 | 25 | 3 | 5 | 80 000 | 44 (426) | 10.3 |
| GT4 | S.t.U6 | 40 | 30 | 15 | 50 000 | 3 (138) | 2.2 |
Mutation frequency (%) calculated for each experiment based on mutated lines detected using high-resolution fragment analysis by the total number of regenerated lines analysed
aGT1 and Cas9 expressed from separate vectors