TABLE 1 .
Transformation outcomes
| Construct | Transformation no.a | Recipient strainb | Introduced gene |
No. of transformants recovered |
||||
|---|---|---|---|---|---|---|---|---|
| sgRNA1 | sgRNA2 | Split-marker repair template | Total | His− | Leu− | |||
| UME6 deletion | 1 | SN152 | 0 | 0 | 0 | 0 | ||
| 2 | SN152 | 0 | 0 | ume6Δ::r1HIS1r1 | 39 | |||
| 3 | SN152 | UME6 (1 µg) | 0 | ume6Δ::r1HIS1r1 | 333 | |||
| BRG1 deletion and HIS1 excision in ume6Δ::r1HIS1r1 | 4 | MH101 | 0 | 0 | 0 | 0 | ||
| 5 | MH101 | 0 | 0 | brg1Δ::r2LEU2r2 | 12 | |||
| 6 | MH101 | BRG1 (1 µg) | 0 | brg1Δ::r2LEU2r2 | 1,120 | 0 | ||
| 7 | MH101 | BRG1 (1 µg) | HIS1 (1 µg) | brg1Δ::r2LEU2r2 | 564 | 22 | ||
| 8 | MH101 | BRG1 (1 µg) | HIS1 (3 µg) | brg1Δ::r2LEU2r2 | 276 | 42 | ||
| 9 | MH101 | BRG1 (1 µg) | HIS1 (9 µg) | brg1Δ::r2LEU2r2 | 156 | 30 | ||
| BCR1 deletion and LEU2 excision in ume6Δ::r1 brg1Δ::r2LEU2r2 | 10 | MH110 | 0 | 0 | 0 | 0 | ||
| 11 | MH110 | 0 | 0 | bcr1Δ::r1HIS1r1 | 26 | |||
| 12 | MH110 | BCR1 (1 µg) | 0 | bcr1Δ::r1HIS1r1 | 134 | 0 | ||
| 13 | MH110 | BCR1 (1 µg) | LEU2 (1 µg) | bcr1Δ::r1HIS1r1 | 47 | 22 | ||
a
All transformations included a CAS9 gene, following the method of Min et al. (10). Approximate amounts of each PCR product in a typical transformation were the following (unless otherwise stated within the table): CAS9, 1 µg; sgRNA1, 1 µg; sgRNA2, 1 µg; split-marker cassette A, 1.5 µg; split marker cassette B, 1.5 µg.
b
All strains are of genotype his1Δ/his1Δ leu2Δ/leu2Δ arg4Δ/arg4Δ. MH101 has the additional genotype ume6Δ::r1HIS1r1/ume6Δ::r1HIS1r1. MH110 has the additional genotype ume6Δ::r1/ume6Δ::r1 brg1Δ::r2LEU2r2/brg1Δ::r2LEU2r2, in which the ume6Δ::r1 allele is marked only with one copy of the flanking repeat from the r1HIS1r1 marker cassette.