Fig. 6

Ribozymes flanked gRNA driven deletion of SeILV6 in S. pastorianus IMX1187. a Representation of the gRNA expression cassette in pUDP010. The gRNA _SeILV6 was flanked on its 5′ by a hammerhead ribozyme (HH represented in orange) and on its 3′ by a hepatitis delta virus (HDV represented in bronze) ribozyme. This construct was under the control of the RNA polymerase II promoter ScTDH3 and the ScCYC1 terminator. Upon ribozyme self-cleavage, a mature gRNA comprising the SeILV6 guiding spacer (in yellow) and the constant structural gRNA fragment (in green) is released. b Schematic representation of the SeILV6 editing upon transformation of IMX1187 with pUDP010. The primers for the validation of transformants are indicated. c Validation of transformants of the S. pastorianus IMX1187 strain with pUDP010 in presence of a 120 bp repair DNA. The lanes (1–5) corresponding to the transformants obtained with repair DNA showed a PCR product of 640 bp corresponding to the deleted allele. One of the transformants exhibiting an SeILV6 deletion was renamed IMK1205. The control lane labelled CBS1483 and lane 6 corresponding to one transformant obtained without repair DNA showed a PCR product corresponding to the wild type fragment at 1616 bp. The lane labelled with L designated the position of the DNA ladder [Gene ruler DNA ladder Mix (ThermoFischer Scientific #SM0332)]