Legume Genomics and Genetics 2025, Vol.16, No.2, 72-80 http://cropscipublisher.com/index.php/lgg 77 problems-like high regulatory costs and long approval cycles-often become the "roadblocks" for its promotion and application. Figure 2 The scheme of binary vector used for CRISPR/Cas9 mediated gene editing on KTI1/KTI3, and transgenes and gene editing have been detected in the leaves of four T0 soybean plants. (A) The CRISPR/Cas9 construct harbors three necessary elements exhibited as below: the selection cassette consists of MAS promoter, Bar gene (soybean transformation selection marker), and MAS terminator; the Cas9 cassette consists of U10 promoter, Cas9 gene, and OCS terminator; three guide RNA cassettes and each of them consists of a U6 promoter, and one sgRNA. (B) The sequences of three sgRNAs is shown here. Two sgRNAs were designed, synthesized, and assembled to the plasmid to target on KTI1, while one sgRNA was designed, synthesized, assembled to the plasmid to target on KTI3. The fragments of two transgenes, (C) Cas9 and (D) Bar, have both been detected in lines #2, #5, #11, and #17 by PCR, but not lines #4 and #7. The WM82 gDNA serves as the template for negative control, while the plasmid DNA serves as the template for positive control. (E) The gene editing on KTI1 and KTI3 has also been observed in the leaf tissues of plants at T0 generation. The double peak sequence around the sgRNA region indicates the gene editing was ongoing but not completed (Adopted from Wang et al., 2023)
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