Genome editing via engineered designer nucleases has revolutionized the possibilities for plant breeding. According techniques bring the development of crops with optimized characteristics into target reach. A key remaining obstacle for the success of engineered genome editing is the efficient transfer of molecular tools into plant cells. In particular, a successful delivery method should (1) be broadly applicable to a diversity of plant species, (2) enable a vector-free and DNA-free editing, and (3) produce uniformly edited individual plants. In the biomedical field, laser-based techniques are efficient and safe tools for the delivery of molecular tools to mammalian cells, but the potential of this technique has so far not been explored for genome editing in plant cells. Compared to other technologies, like electroporation, laser based techniques could provide a combination of a high-throughput approach with a single cell resolution, allowing for clonal selectivity. In this project, we aim to develop an innovative laser-based optoporation method for delivery of CRISPR/Cas9 ribonucleoprotein-complexes into potato protoplasts with the functional goal to reduce glycoalkaloids in potato breeds. This cooperative project combines the expertise in laser-based techniques and genome editing of teams at the Laser Zentrum Hannover e.V. and the Leibniz Universität Hannover, respectively. The workflow for protoplast optoporation includes (1) enzymatic digestion of plant material to produce protoplasts, (2) co-incubation of the protoplasts with a gold nanoparticle containing buffer, (3) addition of the CRISPR/Cas9 riboprotein complex, (4) scanning laser irradiation of the sample, (5) washing and further processing of the modified protoplasts. The optoporation method utilizes gold nanoparticles as absorbers to focus the laser energy to a confined volume in close proximity to the protoplast membrane. The mode of action of this technique can be described as follows: The sample is irradiated with a wavelength matched to the plasmon resonance of the gold nanoparticles (532 nm). This results in a strong absorption of the laser energy by the gold nanoparticles yielding a strong thermal heating. Utilizing short laser pulses (< 1 ns) prevents excessive heat diffusion to the surrounding medium. However, the surface temperature of the particle rises above the critical temperature of water causing evaporation of the medium. The resulting cavitation can induce transient opening of the cellular membrane, allowing volume exchange between the intra- and extracellular space. At the applied intensities and wavelength, the direct absorption of light is minimal and has no effect on the protoplast. At Laser Zentrum Hannover e.V., a technical demonstrator for high-throughput protoplast optoporation has been developed, integrating the laser process in a laser-safe and easy to use benchtop device. Currently, the graphical user interface and the final automation are under development. Protocols for isolation and transportation of the protoplasts between the partner institutes have been developed and optimized. The laser transfection standard operating procedure from mammalian cell cultures have been adapted with respect to the protoplast culture conditions. Osmolality of the gold-nanoparticle solution and the transfection buffer were identified as crucial parameters for the viability of the protoplast. First experiments to test for the parameter space for gold nanoparticle mediated optoporation are ongoing. At Leibniz Universität Hannover, protocols for potato protoplast generation and plant regeneration from protoplasts have been established. CRISPR/Cas9 ribonucleocomplexes genome editing in protoplasts have been generated. To easily detect and quantify successful genome editing in potato protoplasts, a reporter system has been developed and will be transformed into potato to generate stable transgenic reporter lines. These lines can then be used to optimize the laser-based delivery process. Using genome editing, we will address an agriculturally relevant trait in potato. Glycoalkaloids (a-solanine and a-chaconine) are compounds that are toxic for human and animal nutrition and can cause severe food poisoning. Using optoporation mediated genome editing, we will reduce the concentration of glycoalkaloids in potato. To reduce or block glycoalkaloid synthesis, different potato genes encoding biosynthetic enzymes or key regulators are targeted by a collection of sgRNAs. The overarching goal is to establish this innovative genome editing delivery technology by optimizing the optoporation procedure for low toxicity, different cargo molecules, miniaturization, and automated delivery to a large sample size of individual clonal cells. Future experiments will focus on optimizing of the optoporation for protoplast modification and apply it to CRISPR/Cas9 complex delivery for genome editing Transformed protoplasts will be developed into callus, be screened for successful editing events, and be regenerated into plants.