TABLE 2 

LEU2 mutagenesis

TransformationaNo. of transformants:% of:
Ura+LeubLEU2/leu2 heterozygotescleu2Δ/leu2Δ homozygotesd
(−) Cas9, (+) vector620000
(−) Cas9, (+) gLEU2700000
(−) Cas9, (+) gLEU2, (+) repair fragment650000
(+) Cas9, (+) gLEU228000
(+) Cas9, (+) gLEU2, (+) repair fragment760608080
  • a Transformations were performed with BWP17 (Cas9) or HNY30 (Cas9+) with URA3-marked LEU2 gRNA plasmid, p499 (+gLEU2), or the vector that lacks gRNA (p494). (−), donor repair fragment absent; (+), donor repair fragment present. The results shown represent the average from 3 experiments.

  • b The number of Ura+ transformants that were leucine auxotrophs.

  • c The percentage of LEU2/leu2Δ heterozygotes was based on PCR analysis of LEU2 using genomic DNA isolated from 40 random Ura+/Leu+ transformants.

  • d The percentage of leu2Δ/leu2Δ homozygotes was based on PCR analysis of LEU2 amplified from genomic DNA isolated from 60 random Ura+/Leu transformants.