The DNA changes that the researchers specifically induce in the plants are produced at the cellular level. One prerequisite for further research and application is that the cells modified in this way have to grow into robust plants. This is made possible by a special feature that distinguishes plants from humans and many other living beings: their cells are totipotent. For instance, in principle a complete plant can regenerate from each cell again. Nonetheless, in practical terms, this means a high level of knowledge, care, diligence, and time. "With carrots we need about twelve weeks from the single cell to the plant and about six more weeks until they reach the greenhouse. With potatoes, the first step already takes four to five months until the plants are in the greenhouse, and it takes another eight to twelve weeks," explains Unkel.
Here you can have a look into the laboratory of AG Sprink:
Sensitive plant tissue cultures
Depending on the culture and growth phase, different nutrients and phytohormones have to be provided via nutrient media and the plants and tissues have to be regularly transferred to new media. This is where care comes into play: when technical employees Annett Beyer and Jana Goldschmidt work to transfer the plants and tissue with tweezers and a scalpel at one of the cleanbenches in the laboratory, a constant enemy lurks, namely contaminants such as bacteria or fungi. They can quickly overgrow the approaches and ruin weeks and months of work. "Nothing works here without sterile working," says Goldschmidt. "Especially in summer, when temperatures are warm, it is sometimes a challenge to keep the cultures free of contamination," adds Beyer. All materials and media used are therefore autoclaved, the two TAs flambé tools such as tweezers and scalpel before each operation with alcohol and a Bunsen burner and they never allow themselves to reach the cleanbench without having first thoroughly disinfected their hands.
The Path of the Genome Editing Reagent into the Cell Nucleus
However, this work, which the two colleagues are conscientiously planning and carrying out, is already the second step, whereby first the plant cells have to be transformed, i.e. the genome editing reagent has to be introduced into the plant cells so that it can carry out its work on the DNA there. "Another issue that runs through all our projects, in addition to the off-targets, is that we try to do genome editing DNA-free," explains Sprink. Normally, the genome editing reagents are introduced into the plant cells via a DNA construct using agrobacteria, the coding sequences are integrated into the plant-genome, and expressed in the cells by gene expression. However, this does not always work with all plants, and it potentially produces more off-targets.
DNA-free genome editing aims to circumvent this step by, introducing for example Cas9 and single guide RNA directly into the plant cells. "We produce Cas9 recombinant in E. coli and purify it. We do this ourselves, because Cas9 is not available on the market for our applications in plants, or is not available in the quality we need. We also produce our own guide RNA in the laboratory." Post-Doc Janina Metje-Sprink carries out liquid chromatography over several columns as required, on average once a month. "From about two liters of culture, I can clean up about 20 to 30mg of pure Cas9. All in all, this takes five days," explains the scientist.
A complex of Cas9 and single guide RNA is then introduced into the plant in different ways. "At present, we use protoplasts, i.e. plant cells without cell walls, and polyethylene glycol," explains Sprink. However, the researchers also want to establish other DNA-free transformation methods, e.g. via Gene Gun, microinjection or RNA viruses.
Once in the cell nucleus, the CRISPR/Cas9 reagent can then do its work on the DNA and is degraded again after approximately 48 to 72 hours. "In this way, one obtains a genetically-modified organism, but not a transgenic one," states Sprink, explaining the difference compared with agrobacteria transformation. In order to compare off-target rates and efficiencies, employees usually work with both methods in parallel in their projects. One side effect of DNA-free GE is that the selection markers commonly used in classical transformation techniques, such as antibiotic resistance or herbicide tolerance genes, which serve to distinguish transformed from non-transformed cells, are omitted. As an alternative, the AG has developed a Cas9-GFP construct that allows the visual control of DNA-free transformations via fluorescence microscopy.
What sounds rather technical and simple here is anything but trivial. Depending on the culture, functioning transformation protocols are almost worth their weight in gold. For example, Enikö Lörincz-Besenyei brought a protocol for the potato from her previous working group with her to Quedlinburg, whereby it also took 18 months alone to re-establish this protocol there. This may also relativize a few "omnipotence theories" that are sometimes already attributed to genome editing. Only once the plant cells have finally been transformed does the equally demanding cell and tissue culture begin. Therefore, it requires a lot of work and is impossible without functioning cooperation.
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