Fig. 1. Targeted adenine base editing (ABE) in rice genome.A, Schematic diagrams of rice codon-optimized tools pHUN411-ABE and pHUN411-ABE-sg2.0. B, Schematic illustration of the target site in the Wx gene. The protospacer adjacent motif (PAM) and sgRNA sequences are underlined. The desired point mutation of Wx-mq is indicated in red. C, Representative Sanger sequencing chromatograms of the ABE-edited Wx allele. Arrows mark the T to C substitutions. The protospacer sequence is underlined. D, The base editing efficiency of the different sgRNAs and different targets. E, Schematic illustration of the target site in the GL2 and OsGRF2 genes by the GL2-sg protospacer. The PAM and sgRNA sequences are underlined. The binding sequence of the miR396 is indicated in red. The asterisks show the mutation sites in the wild-type GL2 allele with large grain size. F, Representative Sanger sequencing chromatograms of the ABE-edited GL2 allele. RB, Right border; LB, Left border; NTL, Number of transgenic line; NEL, Number of edited line; BEE, Base editing efficiency.

Fig. 1. Sequence of rice codon-optimized ABE7.10.The sequence of wild type ecTadA, linker, TadA*7.10, nCas9(D10A) and NLS was labeled by blue, orange, red, black and cyan respectively.

Fig. 2 Genotyping of some targeted wx mutants.The mutations in the target region are indicated in red. The genotype of the wx targeted mutant was analyzed by PCR sequencing. The numbers located far right indicate the ratio of the number of clones with the corresponding genotype to the number of total sequenced clones. WT, wild-type; Tx to C, the T at position x is mutated to C.

Fig. 3 The sequence of the extended sgRNA scaffold.The structures of the sgRNA-scaffold-2.0 sequence were generated with RNAfold and visualized using VARNA (Darty et al., 2009). The adapted and extended RNA is shown in red.

Fig. 4 Sanger sequencing chromatograms of the OsGRF3 target region of representative ABE-Gl2-sg edited lines.The targeted T to C mutation(s) is indicated by arrow(s). The target sequence is underlined.

Fig. 5 Genotyping of some transgenic plants targeted by ABE-GL2-sg.The mutations within the target region are indicated in red. The genotype of the GL2 and OsGRF3 target regions of the mutant was analyzed by PCR sequencing.

Fig. 6 Alignment of the miR396 binding region in the rice growth-regulating factor family.The GL2-sg shows 100% sequence identity with GL2 and OsGRF3, whereas there is one base mismatch with that of OsGRF5.

Fig. 7 The mutation of protein sequence resulting from the adenine base editor-induced editing.The amino acid of the target region of Wx (A) and GL2/OsGRF3 (B) was indicated. The PAM and protospacer sequence was labeled by blue and orange, respectively.