Fig. 1. Generation of lines with decreased apparent amylose content via CRISPR/Cas9-mediated base editing of Waxy gene in elite rice varieties.A, Schematic diagram of the targeted site in the Waxy gene. Black boxes and line indicate the exon and intron, respectively. The sequence of the targeted site is shown with the protospacer adjacent motif (PAM) sequences labeled in red color. B, Mutation type of different Wx edited lines. The Wx gene sequence at the targeted sites is shown. Wxb and Wxmp are used as controls. PAM sequences are in red. The mutated bases in different lines are marked as blue color with bold, and ‘-’ indicates nucleotides deletion. The mutated amino acid type and position is indicated in the brackets. C, Brown rice phenotypes of edited lines and their corresponding wild types. The Wxm4 mutant is milky white compared with other lines. SJ2, Songjing 2; LQD3, Longqingdao 3; SJ18, Suijing 18. Scale bars, 5 mm. D, Apparent amylose content in different mutation types compared with their corresponding wild type. Values are Mean ± SE (n = 3). P values were calculated by the Student’s t test (*, P < 0.05; **, P < 0.01).

Supplemental Fig. 1. Identification of transgenic plants in T0 generation through PCR assay with hygromysin gene specific primer. M indicates DL2000 DNA Ladder Mix; Lines 1-19 are transgenic plants; Line 20 is WT, Line 21 is water used as a negative control.

Supplemental Fig. 2. Identification of edited Wx mutants.Sequencing chromatograms and sequence of Wx genes in WT and different edited mutants are shown. The PAM (CCA) site is underlined and the edited bases are highlighted in red. ‘- ’ indicates nucleotides deletion.

Supplemental Fig. 3. Detection of GBSSI in parents and edited lines. SDS-PAGE assay of starch granule-bound GBSSI (top) and total seed proteins (bottom) from mature seeds. M indicates protein marker; Line 1 is SJ2, Line 2 is Wxm1, Line 3 is LQD3, Line 4 is Wxm2, Line 5 is SJ18, Line 6 is Wxm3, Line 7 is Wxm4, Line 8 is Wxmq.

Supplemental Fig. 4. Grain morphology in parents and edited lines.(A and B) Quantification of grain length (A) and grain width (B) in WT and edited lines, respectively. Data are means±SE (n = 20).(C) The grain picture of WT and edited lines.

Supplemental Fig. 5. Amylopectin structure analysis in parents and edited lines.(A-C) indicate difference in chain length distribution of amylopectin in WT and edited lines. (D-G) show chain length fraction differences in WT and edited lines.

Supplemental Fig. 6. Agronomic traits analysis in parents and edited lines.After collected the plant, the above agronomic traits were investigated. For each mutation type, at least 10 individual plants were used for analysis. Data are means ± SE (n = 10).