Editing of the starch branching enzyme gene SBE2 generates high-amylose storage roots in cassava

The production of high-amylose cassava through CRISPR/Cas9-mediated mutagenesis of the starch branching enzyme gene SBE2 was firstly achieved. High-amylose cassava (Manihot esculenta Crantz) is desirable for starch industrial applications and production of healthier processed food for human consumption. In this study, we report the production of high-amylose cassava through CRISPR/Cas9-mediated mutagenesis of the starch branching enzyme 2 (SBE2). Mutations in two targeted exons of SBE2 were identified in all regenerated plants; these mutations, which included nucleotide insertions, and short or long deletions in the SBE2 gene, were classified into eight mutant lines. Three mutants, M6, M7 and M8, with long fragment deletions in the second exon of SBE2 showed no accumulation of SBE2 protein. After harvest from the field, significantly higher amylose (up to 56% in apparent amylose content) and resistant starch (up to 35%) was observed in these mutants compared with the wild type, leading to darker blue coloration of starch granules after quick iodine staining and altered starch viscosity with a higher pasting temperature and peak time. Further 1H-NMR analysis revealed a significant reduction in the degree of starch branching, together with fewer short chains (degree of polymerization [DP] 15-25) and more long chains (DP>25 and especially DP>40) of amylopectin, which indicates that cassava SBE2 catalyzes short chain formation during amylopectin biosynthesis. Transition from A- to B-type crystallinity was also detected in the starches. Our study showed that CRISPR/Cas9-mediated mutagenesis of starch biosynthetic genes in cassava is an effective approach for generating novel varieties with valuable starch properties for food and industrial applications.