Figure 2.

CRISPR/Cas9-targeted mutagenesis of GmIPK1 in soybean protoplasts. (A) Schematic diagram of GmIPK1 gene structure and four guide RNA target sites used in this study (red arrows). Based on a comparison with GmIPK1 cDNA, the coding regions (4158 bp) of the GmIPK1 gene are divided into seven exons interrupted by six introns. (B) The vector pJY_GmU6-10_SpCas9_ Bar was used for CRISPR/Cas9-mediated soybean gene editing with guide RNAs: AtRPS5A^P, Arabidopsis AtRPS5A promotor; GmU6-10^P, soybean U6-10 promotor; sgRNA, single guide RNA; Nos^P, nopaline synthase promoter; Nos^T, nopaline synthase terminator; SpCas9, human codon-optimized S. pyogenes Cas9; NLS, nuclear location signal; Bar, selective marker gene. (C) Four sgRNA sequences of GmIPK1 are shown. Mutation rates tested in soybean protoplasts were determined by indel frequencies (%) through deep sequencing at target regions of the GmIPK1 gene.