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Legends to figures:
Figure 1: Schematic representation of plasmid pCFYF. Elements shown not to scale. The plasmid contains origins and selection markers forE. coli : ori and AmpR, as well as for yeast: ARS-CEN andURA3 . The Cas9 gene is under the control of the inducibleMET3 promoter. The gRNA gene can be cloned under the control of the constitutive SNR52 promoter.
Figure 2: Survival on solid induction medium. Survival is calculated as the ratio of the number of colonies that grow on plates with induction medium (SC-Ind) over the the number of colonies that grow on plates with repression medium (SC-Rep). Black bars: untransformed yeast (with Uracil added to the media); dark gray bars: yeast transformed with the plasmid expressing Cas9 but where no gRNA is cloned; light gray bars: yeast transformed with the plasmid expressing both Cas9 and the gRNA. Error bars represent standard deviation.
Figure 3: Survival to limited induction time and efficiency of induction. Cells are placed in liquid SC-Ind medium for the time, in hours, shown on the graph, followed by plating on repression medium (YPD-Rep). The upper survival curve, starting at 100 %, is the ratio of colonies on YPD-Rep at a given time-point over the number of colonies on YPD-Rep at T0. This is normalized by division with the survival rate in the same conditions, of cells transformed with pCFYF (expressing Cas9 but no gRNA). The lower curve starting at 0 % for C. glabrata and C. nivariensis , and at 40 % for C. bracarensis is the percentage of red colonies on total colonies on SC-Rep medium. Error bars represent standard deviation.
Figure 4: Sequence of NHEJ junction in Ade- cells of the three species. First line shows sequence before the DSB in all three species, with double arrow indicating cut site, and letters in italics, the PAM sequence. Coordinates relative to the first base of coding gene is shown. Following lines show sequences after unfaithful NHEJ events, with letters in bold indicating insertions and dashes indicating deletions, the (-1) indicating deletion of the nt at position 471. The frequency of each repair event is indicated on the right.