Multiplex CRISPR-Cas9 Gene-Editing Can Deliver Potato Cultivars with Reduced Browning and Acrylamide.

Storing potato tubers at cold temperatures, either for transport or continuity of supply, is associated with the conversion of sucrose to reducing sugars. When cold-stored cut tubers are processed at high temperatures, with endogenous asparagine, acrylamide is formed. Acrylamide is classified as a carcinogen. Potato processors prefer cultivars which accumulate fewer reducing sugars and thus less acrylamide on processing, and suitable processing cultivars may not be available. We used CRISPR-Cas9 to disrupt the genes encoding vacuolar invertase () and asparagine synthetase 1 () of cultivars Atlantic and Desiree to reduce the accumulation of reducing sugars and the production of asparagine after cold storage. Three of the four guide RNAs employed induced mutation frequencies of 17-98%, which resulted in deletions, insertions and substitutions at the targeted gene sites. Eight of ten edited events had mutations in at least one allele of both genes; for two, only the was edited. No wild-type allele was detected in both genes of events DSpco7, DSpFN4 and DSpco12, suggesting full allelic mutations. Tubers of two Atlantic and two Desiree events had reduced fructose and glucose concentrations after cold storage. Crisps from these and four other Desiree events were lighter in colour and included those with 85% less acrylamide. These results demonstrate that multiplex CRISPR-Cas9 technology can generate improved potato cultivars for healthier processed potato products.