Figure 3. SpEDIT can generate targeted ade6 and ura4 point mutants in asynchronous cells with 100% mutagenesis efficiency.

A. Schematic of experiment to generate targeted ade6 and ura4 point mutants. A sgRNA-loaded pLSB or pMZ379 plasmid was co-transformed with an HR template that creates a premature STOP codon by disabling the PAM (NGG) sequence. sgRNA and HR template sequences for ade6 and ura4 are shown. Full HR template sequences can be found in Table 1. B. After transformation, cloNAT-resistant colonies were picked and re-streaked to non-selective YES plates. Cells were then replica-plated to indicated media to assess their phenotype. Representative plates from two independent experiments are shown. Quantification is shown in C– D. C– D. Percentage of cloNAT-resistant transformants displaying a mutant phenotype (pink cells, ade6; uracil auxotrophy and FOA resistance, ura4) after asynchronous ( C) or G1-synchronized ( D) wild-type cells were transformed with a sgRNA-loaded SpEDIT/pLSB (developed here, Figure 2) or pMZ379 ( Rodríguez-López et al., 2016) plasmid targeting ade6 ⁺ or ura4 ⁺ (or no sgRNA plasmid as control). An HR template targeting the same or a different gene was co-transformed as indicated. n = number of cloNAT-resistant colonies assayed. Note that when an HR template targeting a different gene or no HR template was co-transformed into asynchronous cells, the number of cloNAT-resistant colonies obtained was drastically reduced. Experiment was repeated twice with similar results. E. For each condition in C– D, five colonies displaying the mutant phenotype (or 5 cloNAT-resistant colonies for no sgRNA plasmids) were taken and the gene targeted by the sgRNA was sequenced to confirm changes in its DNA sequence. Both ade6 and ura4 were sequenced when no sgRNA was used. Edited clones harbour the change contained in the corresponding HR template. Other mutations at PAM disrupt the PAM (NGG) sequence and the corresponding gene coding sequence. For asynchronous cells transformed with pLSB- ura4 (no HR template) and pMZ379- ade6 (no HR template) only two and one colonies were respectively obtained and analysed. * No colonies were obtained for these conditions. sgRNA, single guide RNA. PAM, proto-spacer adjacent motif. HR, homologous recombination donor template. N/S, non-selective medium. FOA, 5-fluoroorotic acid.