The BS‑Talen project set out to develop a CRISPR/Cas9 cassette system for systematic and targeted gene activation and inactivation in potato and rapeseed, with the aim of increasing resistance to the pathogens Verticillium longisporum in rapeseed and Streptomyces scabies in potato. Solana Research worked closely with the partner university Christian‑Albrechts‑Universität (CAU) to advance the potato work. Because the CRISPR/Cas9 system proved markedly more efficient than the originally planned TALEN constructs, the TALEN approach was abandoned early in the project. CAU quickly established an effective vector system for CRISPR/Cas9 delivery.

The first target in potato was the susceptibility factor TXR1, which is thought to render the plant vulnerable to Streptomyces scabies. Despite extensive transformation and screening efforts, no mutations were obtained in this gene. In December 2016 the project was expanded to include two additional susceptibility genes for the late blight pathogen Phytophthora infestans. Mutations were successfully introduced into the CESA3 gene in two different potato cultivars. Initially the edited lines were simplex for the mutation, but after a second round of tissue‑culture cycles, homozygous lines carrying the mutation on all four alleles of the tetraploid genome were generated. Phenotypic tests, however, did not reveal an enhanced resistance to P. infestans, possibly because one of the mutations was a 9‑bp deletion that removed three amino acids without causing a frameshift, leaving the gene functionally active.

A total of 200 transgenic lines were produced using seven CRISPR/Cas9 constructs (K210–K216). Transformation efficiencies were above 50 % for kanamycin‑resistant constructs and only about 10 % for hygromycin‑resistant constructs. CAPS‑PCR analysis identified 15 mutant lines, representing 9 % of the PCR‑positive lines. Sequencing of five of these lines revealed deletions of 1, 9, or 15 base pairs; the 9‑bp deletion in line SR696‑5 removed three amino acids. None of the mutant lines showed improved disease resistance, underscoring the need for complete knockout of all alleles in the tetraploid genome.

To avoid stable integration of the Cas9 cassette and thereby circumvent the regulatory requirements for genetically modified organisms, the project pursued transient expression of the CRISPR/Cas9 system. This required a robust protoplast preparation and regeneration protocol, which was subcontracted to Saaten‑Union Biotec GmbH. The subcontract achieved a genotype‑independent protocol for protoplast isolation, regeneration, and transient expression of the green fluorescent protein reporter. The European Court of Justice ruling of 25 July 2018, however, limited the use of transiently edited plants in the European Union, complicating the translation of these technical advances into field‑ready varieties.

The collaboration involved Solana Research, CAU, and Saaten‑Union Biotec, with the project funded by German research agencies. The timeline spanned from the initial design of the CRISPR/Cas9 system through the generation of mutant lines and the establishment of a protoplast system, with ongoing efforts to produce fully knocked‑out lines and to evaluate their resistance phenotypes. The project demonstrates the feasibility of CRISPR/Cas9‑mediated editing in polyploid crops and highlights the technical challenges of achieving complete gene knockout and regulatory compliance in potato and rapeseed.