Fig. 1

Schematic diagram of the seven CRISPR/Cas9 vectors The green arrows, red boxes, and yellow boxes represent the seven promoters, the terminators, and the gRNA sequences, respectively."

Table 1

Primer sequences for real-time PCR"

Table 2

Promoter amplification primers sequences for CRISPR/Cas9 vectors"

Table 3

Primer sequences for positive hair root and target sites"

Fig. 2

Relative expression level of 11 genes in root, stem, trifoliolate leaf, unifoliolate leaf, and shoot apical in heat map Soybean GmActin-1 gene was used as the internal reference, and the relative expression levels of each genes were calculated using 2-ΔΔCt method. The relative expression in heat map was made by GraphPad Prism 7 software, and the relative expression level was represented by the color scale from blue (high) to white (low)."

Fig. 3

Editing efficiency of different promoters on GmSPA1a gene by hair roots transformation. A: U6 and Cas9 sequences were detected by PCR in hair roots using GmRPS5Ab promoter. M: molecular weight marker; blank control: pure water; Negative control: TL1 genomic DNA; positive control: plasmid with Cas9 and U6 sequences. The positive root numbers are marked in red, the U6 sequence is 950 bp and that of Cas9 sequence is 864 bp. B: the number of the total roots, positive roots, and edited roots. The three column charts in the figure are the results of three repetitions, among which the blue column is the number of detected roots, the red column is the number of PCR positive roots, and the green column is the number of edited roots at the GmSPA1a site (the specific number of sampled roots in Table 4)."

Fig. S1

Electrophoresis map of U6 and Cas9 sequence by PCR detection Using CAMV35S, GmHE, ZmUbi, GmRPS5Ac, AtRPS5A, GmYAO promoter, U6 and Cas9 sequences were detected by PCR in hair roots. M: molecular weight marker; Blank control: pure water; Negative control: TL1 genomic DNA; Positive control: plasmid with Cas9 and U6 sequences. The positive root numbers were marked with red, the U6 sequence was 950 bp and that of Cas9 sequence was 864 bp."

Table 4

Number of total hair roots, positive hair roots, and editing efficiency"

Fig. 4

Editing efficiency using different Cas9 promoters A: the editing efficiency of seven promoters on GmSPA1a gene. B: the editing efficiency of GmEID1 gene by three promoters. *: P<0.05; **: P < 0.01; ns: not significant difference."

Table 5

Number of total hair roots, positive hair roots, and editing efficiency"

Fig. 5

Sequencing map of GmSPA1a editing sites A: the example of high peak map; B: the example of low peak map; C: the statistical analysis of the high peak maps and low peak maps ratio of 7 promoters at GmSPA1a edited site. Black column represents the high peak maps, and gray represents the low peak maps in sequenced maps."

Fig. S2

The high peak maps and low peak maps ratio of 7 promoters in GmSPA1a editing sites sequencing maps during the first, second and third root transformation Black column represents the high peak maps, and gray represents the low peak maps in sequenced maps."