Construction and Verification of CRISPR/Cas9 Gene Editing Vector for Cassava MeSSIII Gene

Starch glucan chain structure of cassava root is the key factor to determine starch quality. Soluble starch synthase III (SSIII) is the key enzyme to regulate the synthesis of long chain in plant amylopectin glucan. Cassava has two MeSSIII homologous genes MeSSIII-1 and MeSSIII-2. To study the effect of cassava MeSSIII on the quality formation of cassava root starch, a double gene editing vector for MeSSIII-1 and MeSSIII-2 was constructed. The sgRNA target for MeSSIII-1 and MeSSIII-2 was designed simultaneously by online software CRISPR-Pv2.0 based on the conserved segments, and the recombinant pCAMBIAP1301-Cas9MeSSIII-gRNA plasmid was constructed by digestion and ligation. The gene editing vector was transformed into LBA4404 Agrobacterium competent cells and used to infect the friable embryogenic callus of cassava, and the their DNA was extracted. The target segments of MeSSIII-1 and MeSSIII-2 were amplified by PCR for Sanger sequencing, and analyzed the editing of target position. The results showed that the target sites of MeSSIII-1 and MeSSIII-2 were successfully edited. This study helps to further obtain mutants of theMeSSIII gene to analyze the role of this gene in the cassava starch synthesis pathway.