Fig. 1. Precise base editing of OsROS1 gene thickens aleurone layer in rice grains. A, Diagram of the target gene OsROS1. Filled black boxes and bold lines represent exons and introns, respectively. TS1 and TS2 indicate the position of the sites targeted by the base-editing guide RNAs. B, Mutations of TS2 in the edited T0 and T1 lines. The putative protospacer-adjacent motifs (PAMs) are shown in green. The number of altered base (coloured in red) is indicated by the letter S followed by a number. S and F represent the amino acid before and after the base-editing in TS2, respectively. C, Plant state of the wild type Zhennuo 19 and the T1 edited line T85-11 at the heading stage. Scale bars, 10 cm. D, Comparison of the appearance of milled rice grains. Scale bars, 5 mm. E, Analysis of the potential off-target sites in the edited T1 line T85-11. Red bases are mismatches to TS2; ‘+’ indicates mutations detected, and ‘-’ indicates mutations not detected. CDS, Coding sequence. F, Transverse sections of dehusked mature grains. Scale bars, 0.5 mm. G-I, Transversally (G) and longitudinally (H) sectioned dehusked mature grains that are stained with Evans blue, and scanning electron microscope images of transverse sections of dehusked mature grains (I). Arrowheads indicate the aleurone. Scale bars, 0.5 mm. ES, Endosperm. J-M, Toluidine blue stained semi-thin sections of aleurone at lateral positions at 9 (J), 12 (K) and 15 DAF (L), and scanning electron microscopy images of the aleurone layer in mature grains (M). The thickness of the aleurone is indicated by the length of yellow lines. Scale bars, 20 μm. ES, Endosperm. DAF, Days after flowering. N, A transversely sectioned dehusked mature grain showing the dorsal, upper lateral, lateral, lower lateral, and ventral positions of the endosperm described in this study. VB, Vascular bundle. O, Average number of aleurone layers at the lateral position of the endosperm. Data are Mean ± SD (n = 10). *, P < 0.05 by the Student’s t-test.

Fig. 2. Nutritional contents of dehusked mature grains in Zhennuo 19 (ZN19) and T85-11 mutant. A, Total protein content. B, Total lipid content. C, Zinc content. D, Calcium content. E, Total phenolic content. F, Vitamin B1 content. G, Vitamin B3 content. H, Vitamin B6 content. I, Vitamin C content. J, Vitamin E content. K, Total starch content. L, Apparent amylose content. Data are Mean ± SD (n = 3). * and ** indicate P < 0.05 and P < 0.01 by the Student’s t-test, respectively.