Fig. 5.

One-step generation of triple knock-out strains. WT Phaeodactylum cells were simultaneously bombarded with six RNP complexes, each directed against one specific target at either the PtAPT, PtUMPS, or PtAureo1a loci. After two days, cultures were transferred onto 2-FA selective medium supplemented with adenine and uracil. Fourteen 2-FA resistant clones (referred to as strains AUA#1 to #14) were obtained and screened for the presence of mutagenic events at the six target sites. Bar plots present the number of 2-FA resistant colonies harboring mutations at the PtAPT loci only (white bar), at both the PtAPT and PtUMPS loci or the PtAPT and PtAureo1a loci (light gray bars), or at all three loci (dark gray bar). The genotypes of the corresponding clones are presented underneath the graph and written in orange font for PtAPT, blue for PtUMPS and green for PtAureo1a. RC reverse complement insertion. NA not amplifiable, Mix mosaic, ND not determined