Fig 5. Telomere vector-mediated editing for introduction of marker-free CRISPR/Cas edits into B. cinerea.

(A) Experimental setup. (B) Applications of non-selected editing performed in this study. pTEL-Fen can be propagated in E. coli with selection for ampicillin (Amp^R) and kanamycin (Kan^R). After transformation into B. cinerea it is linearized to a minichromosome with telomere ends. (C-F) Generation and characterization of a Sod1-GFP knock-in mutant. (C) Transformation efficiency and percentage of fluorescent transformants (below; n = 3). (D) Cytoplasmic and putative peroxisomal localization of Sod1-GFP fluorescence, as indicated by similarity to the fluorescence pattern of a mutant expressing GFP fused to a SKL peroxisomal targeting motif, and strain Bcgfp1 expressing GFP only [15]. (E) Immunoblot detection of Sod1-GFP with GFP antibodies. Loaded are B. cinerea WT, two transformants of Sod1-GFP mutant, and a strain (Bcgfp1) expressing cytoplasmic GFP only. Transformant Sod1-GFP #4 was used also in 5D and 5F. (F) Native gel electrophoresis of B. cinerea protein extracts stained for superoxide dismutase activity (inhibition of superoxide-mediated reduction of nitroblue tetrazolium). Lanes showing WT (expressing Sod1) and mutant (expressing Sod1-GFP, arrowheads).