Fig. 5

Deletion of ScURA3 in IMX1187 and IMX585 using RNA III polymerase dependent (SNR52p) gRNA expression. a Representation of the native and deleted ScURA3. The plasmid pUDR107 carried a gRNA under the control of the SNR52p. Primers used for validation of the deletion are indicated. b Validation of transformants of the S. pastorianus IMX1187 strain with pUDR107 in presence or not of a 120 bp repair DNA. The PCR reactions were performed with the primers 9314 and 4728. All lanes (1–20) showed a PCR product of 1698 bp corresponding to the wildtype allele. The lane labelled with L designated the position of the DNA ladder [Gene ruler DNA ladder Mix (ThermoFischer Scientific #SM0332)]. c Sanger sequencing results of purified PCR fragments of ten transformants derived from the transformation of IMX1187 with pUD107 (gRNA _URA3 ). The gRNA spacer used to direct Cas9 is indicated in bold and the PAM sequence is underlined. d Validation of transformants of the S. cerevisiae IMX585 strain with pUDR107 in presence or not of a 120 bp repair DNA. The PCR reactions were performed with the primers 4727 and 4728. The lanes (1–10) corresponding to transformants obtained with repair DNA showed a PCR product of 1440 bp corresponding to the deleted allele. The control lane labelled CEN.PK113-7D showed the wild type fragment at 2244 bp. The lane labelled with L designated the position of the DNA ladder