Validation of maize codon-optimized Cas9 and three Pol-III promoters driving gRNA expression in maize protoplasts. (A) Sequence of the target site from the ZmHKT1 locus. The PAM, the putative cleavage site (red arrowhead), and the XcmI site (boxed) are indicated. (B,C) Mutation analysis by XcmI digestion of PCR fragments. GFP, 201, 301, 401 (B): PCR fragments amplified from the genomic DNA of maize protoplasts transfected with pUC-GFP (control), pBUN201-ZT1, pBUN301-ZT1, and pBUN401-ZT1, respectively. The three CRISPR/Cas9 vectors have the same gRNA but different Cas9: hCas9-1/2, two types of human-codon-optimized Cas9; zCas9, Zea mays codon-optimized Cas9. GFP, 401, 411, 421 (C): PCR fragments from the pUC-GFP, pBUN401-ZT1, pBUN411-ZT1, and pBUN421-ZT1 transfections, respectively; the three CRISPR/Cas9 vectors have the same zCas9 and gRNA, but the gRNA is driven by three different Pol-III promoters. − and + indicate whether the PCR fragments were digested with XcmI. Mutation efficiency (% indel) calculated based on the percent ratios of residual undigested PCR fragments (+ lanes: 569 bp) to total PCR products (− lanes); the WT indel values should be treated as the background level. (D,E) Alignment of sequences of mutated alleles identified from cloned PCR fragments resistant to XcmI digestion. The mutated alleles include deletions (D) and insertions (E). Dots, deleted bases. Highlighting denotes the degree of homology of the aligned fragments, and only aligned regions of interest are shown. The type of indel and the number of indels of the same type are indicated.