Figure 5. SpEDIT allows simultaneous editing at two non-homologous genes at distinct locations in the S. pombe genome.

A. Schematic of experiment to simultaneously generate targeted point mutations in clr5 ⁺ and meu27 ⁺. Two sgRNA-loaded pLSB plasmids (with different selection markers) were co-transformed with two HR templates that create the desired point mutations and disable the corresponding PAM (NGG) sequence. Transformed cells were then selected on selective plates containing both cloNAT and hygromycin. B. sgRNA and HR template sequences for clr5 (left) and meu27 (right) are shown, along with Sanger sequencing chromatograms for a successfully edited clone. Full HR template sequences can be found in Table 1. C. Percentage of cloNAT- and hygromycin-resistant transformants harbouring the targeted mutations in clr5 and meu27 as revealed by Sanger sequencing. sgRNA, single guide RNA. HR, homologous recombination donor template.