Validation of the toolkit by targeted mutation of a maize gene. (A) Sequence of a region of maize ZmHKT1 with two target sites indicated. (B) Physical map of T-DNA carrying two-gRNA expression cassettes. The alignment of target of gRNA with its target gene is shown. Only aligned regions of interest are displayed. -rc, reverse complement. (C) Mutation analysis of 20 T0 transgenic lines by XcmI or SphI digestion of PCR fragments. The lines used for sequencing analysis are indicated with boxes. (D) Alignment of sequences of mutated alleles identified from cloned PCR fragments from two representative T0 transgenic lines. Highlighting denotes the degree of homology of the aligned fragments, and only aligned regions of interest are displayed. The number of indels of the same type is indicated.