Different Golden Gate cloning systems use different overhangs to assemble DNA parts. It is often not possible to use parts from systems in the same reaction, so converting clones from one system to another is needed. To facilitate this conversion, we developed 'oligo stitching' to readily convert and modify existing DNA parts. Oligo stitching relies on the use of ssDNA oligos in a Gibson assembly reaction. Hence, the method is PCR-free. We reported this method in ACS Synthetic Biology in De Saeger et al., 2022. You can see a short video here where first-author Jonas describes the method.

High-throughput experiments require readily-available donor material for transfection or transformation. In many plant systems, nothing is more convenient than protoplast systems. Using tens to dozens of plants, enough protoplasts can be recovered to transfection 1 to 2 96-well plates. We use this approach in both wheat and maize in numerous applications. Most commonly, we use it to optimize our gene editing reagents. In Gaillochet et al., 2023 we reported the development of LbCas12a base editors in wheat and maize using a series of iterative transfections (over months) of wheat protoplasts. In this video you can see how we prepare protoplasts from etiolated maize seedlings. 

Following the preparation of protoplasts, it is usually time to transfect with plasmids or RNPs. This video shows how we perform protoplast transfections in the lab on a variety of species (maize, wheat, physco). It can easily be adapted for high-throughput multi-well assays.