Creation of a potato mutant lacking the starch branching enzyme gene stsbe3 that was generated by genome editing using the CRISPR/dMac3-Cas9 system

Abstract

The potato tuber starch trait is changed depending on the composition of amylose and amylopectin. The amount of amylopectin is determined by the activity of the starch branching enzymes SBE1, SBE2, and SBE3 in potato. SBE3, a homolog of rice BEI, is a major gene that is abundant in tubers. In this study, we created mutants of the potato SBE3 gene using CRISPR/Cas9 attached to the translation enhancer dMac3. Potato has a tetraploid genome, and a four-allele mutant of the SBE3 gene is desired. Mutations in the SBE3 gene were found in 89 of 126 transformants of potato plants. Among these mutants, 10 lines contained four mutant SBE3 genes, indicating that 8% efficiency of target mutagenesis was achieved. These mutants grew normally, similar to the wild-type plant, and yielded sufficient amounts of tubers. The potato starch in these tubers was similar to that of the rice BEI mutant. Western blot analysis revealed the defective production of SBE3 in the mutant tubers, suggesting that these transformants were loss-of-function mutants of SBE3.