Yeast transformation
For selection of transformants of the Cas9 plasmid, and maintenance in SC-Rep. Transformation is done according to a modified one-step lithium acetate transformation protocol from (Gietz et al. , 1995). The duration of the heat shock at 42°C differs according to the species; 25 min for C. glabrata , 20 min for C. bracarensis , 15 min forC. nivariensis .
For induction of DSBs, all species were transformed with the pCFYF plasmid into which is cloned the gRNA specific for each species, but also with the plasmid that does not express any gRNA, to control for medium/plasmid/Cas9 expression effects (see Results).