# Modifying the potato tuber storage protein patatin targeting improved thermal stability

## TL;DR

Researchers used gene editing to modify patatin proteins in potatoes to improve their stability under heat and acidic conditions.

## Contribution

A CRISPR/Cas9-based approach was used to edit patatin genes, achieving over 10% mutation efficiency and designing variants with altered stability.

## Key findings

- CRISPR/Cas9 targeting with a single sgRNA achieved over 10% mutation frequency in patatin genes.
- Four patatin variants were designed and expressed, but none showed improved thermostability compared to wild-type.
- One modified patatin showed reduced sensitivity to pH changes, suggesting potential for further optimization.

## Abstract

Gene editing of the patatin gene cluster using a single-guide RNA sequence consistently modifies over 10% of the targeted genes in modified individuals.

Patatins have gained recent attention, as a group of highly nutritious proteins with excellent functional properties. Some techniques have been suggested for industrial-scale patatin purification, mostly as a by-product from potato starch processing. The purification process has proved to be a challenge due to the low thermostability of patatins, especially under acidic conditions. One strategy to make patatin more accessible for extraction would be to stabilize the protein structure through the introduction of point mutations. Here, we show that the tuber expression of patatin genes is dominated by a few genes from the extended gene family, most of which were predicted to be catalytically inactive. We have further evaluated the suitability of the patatin gene cluster as a target for clustered regularly interspaced repeat (CRISPR)/Cas9-based mutagenesis. In the mutation study, we show that targeting using a single single-stranded guide RNA (sgRNA) can lead to mutations in over 10% of all alleles. Finally, four patatin variants with amino acid substitutions were designed based on in silico analysis of patatin protein structure. These modified patatins were then heterologously expressed in bacteria and evaluated for increased thermostability. While none of the mutant proteins performed better than a wild-type variant, with regard to their thermal properties, one candidate proved to be less sensitive to shifting pH, making it an interesting candidate for further optimizations.

The online version contains supplementary material available at 10.1007/s00425-025-04766-2.