Multiplex genome editing with CRISPR/Cas9. (A) Schematic depiction for simultaneously targeting of multiple genes. Five sgRNAs were assembled using Golden Gate system and inserted into the multiplex expression vector. Blue arrows indicate tRNA and orange boxes indicate each sgRNA. (B) Identification of mutations in the targeting locus of PI3K genes. Clones revealing low amplification efficiency of the PCR products compared to the control were candidate mutants. Clone numbers presented in red were used for further sequence analysis. Parental strain AX2 was used as a control. The sizes of the PCR products were 217 bp, 167 bp, 184 bp, 126 bp and 102 bp, respectively. Gel images were cropped, but no other bands were present. (C) Summary of candidate mutants mediated by multiplex CRISPR/Cas9.