Figure 4
Generation of marker-free mutants with the CRISPR/Cas9 technique. (a) Strategies for CRISPR/Cas9-assisted inactivation of pks1. Three protospacers (PS) for inducing DSBs in different regions of pks1 were combined with the sgRNA backbone, cas9, a hygR cassette as well as the AMA1 sequence (Table S1). PS- and PS-adjacent motifs (PAM) are shown. (b) Random mutation of pks1 via non-homologous end joining (NHEJ). WT:A95 protoplasts were transformed with the circular cas9/pks1-sgRNA-containing AMA plasmids (Table S3). 31 mel− mutants were studied. MEA was inoculated with cell suspensions and incubated for 11 days. ∆p/∆p − ∆pks1/∆phd1. Sequencing of PS-spanning regions revealed point mutations and short deletions at the Cas9 sites (green triangles) resulting in truncated proteins. Mutants H2.7 and H2.6 contain in-frame deletions (Fig. S5). (c) Increase of editing efficiency by addition of single-stranded DNA oligonucleotides. WT:A95 protoplasts were co-transformed with cas9-sgRNA-containing AMA plasmids and single-stranded 80-bp-long DNA oligonucleotides (+ O) that covered the Cas9 cutting sites and comprised mutations (Fig. S6). Numbers of differentially pigmented colonies on the transformation plates were quantified from four independent experiments (Table S6). Representative plates for pks1 are shown on the left. The PS-spanning regions of chosen mutants were amplified by PCR and sequenced to detect the mutations at the Cas9 cutting sites (Fig. S6). MEA was inoculated with cell suspensions and incubated for 8 days.
