Cas9‐mediated mutagenesis of potato starch‐branching enzymes generates a range of tuber starch phenotypes

Summary We investigated whether Cas9‐mediated mutagenesis of starch‐branching enzymes ( SBE s) in tetraploid potatoes could generate tuber starches with a range of distinct properties. Constructs containing the Cas9 gene and sg RNA s targeting SBE 1 , SBE 2 or both genes were introduced by Agrobacterium ‐mediated transformation or by PEG ‐mediated delivery into protoplasts. Outcomes included lines with mutations in all or only some of the homoeoalleles of SBE genes and lines in which homoeoalleles carried several different mutations. DNA delivery into protoplasts resulted in mutants with no detectable Cas9 gene, suggesting the absence of foreign DNA . Selected mutants with starch granule abnormalities had reductions in tuber SBE 1 and/or SBE 2 protein that were broadly in line with expectations from genotype analysis. Strong reduction in both SBE isoforms created an extreme starch phenotype, as reported previously for low‐ SBE potato tubers. HPLC ‐ SEC and 1 H NMR revealed a decrease in short amylopectin chains, an increase in long chains and a large reduction in branching frequency relative to wild‐type starch. Mutants with strong reductions in SBE 2 protein alone had near‐normal amylopectin chain‐length distributions and only small reductions in branching frequency. However, starch granule initiation was enormously increased: cells contained many granules of <4 μm and granules with multiple hila. Thus, large reductions in both SBE s reduce amylopectin branching during granule growth, whereas reduction in SBE 2 alone primarily affects numbers of starch granule initiations. Our results demonstrate that Cas9‐mediated mutagenesis of SBE genes has the potential to generate new, potentially valuable starch properties without integration of foreign DNA into the genome.