TABLE 1 

RFP mutagenesis with PADH-tRNA-gRFP-HDV

TransformationaTotal no. of transformantsb% of colonies:% of transformants:
RedWhiteSectoredHis+cArg+d
(−) Cas9 (EPC1), (+) vector (−gRFP)~1,000100001000
(−) Cas9 (EPC1), (+) gRFP~1,00010000990
(−) Cas9 (EPC1), (+) gRFP, (+) healing fragment~1,000100001000
(+) Cas9 (EPC2), (+) vector (−gRFP)~1,0001000010080
(+) Cas9 (EPC2), (+) gRFP~1,00089023080
(+) Cas9 (EPC2), (+) gRFP, (+) healing fragment~1,00089209773
  • a Transformations were performed with URA3-marked PADH-tRNA-gRFP-HDV plasmid pND482 (+gRFP) or vector lacking gRFP (−gRFP). (−), donor repair fragment absent; (+), donor repair fragment present. The results shown represent an average of 5 separate experiments, in which the percentage of white colonies ranged from 90 to 98%.

  • b Number of uracil prototrophic transformants.

  • c Percentage of uracil prototrophic transformants that were histidine prototrophs.

  • d Percentage of uracil prototrophic transformants that were arginine prototrophs.