Team:KU Leuven/Plant
BLADEN
Awards: Best plant-
based project
Plant Synthetic Biology
Due to climate change, local conditions around the world are changing rapidly. The weather is becoming more unpredictable with large and sudden deviations from stable local conditions. The effects of climate change have a particularly large impact on agriculture and farmers must adapt their crops to these new conditions. Plants are versatile organisms with an extensive secondary metabolism that allows them to typically respond rapidly to biotic and abiotic stresses, but they cannot adapt and evolve at the same pace as the climate is changing, at least not through classical breeding techniques or on their own.
With our project BLADEN we strive to accelerate the evolution of plants using EvolvR, a CRISPR-based method, for continuous directed evolution (CDE). EvolvR has never before been applied in plants and as far as we are aware, CDE has never been performed in plants either. EvolvR has so far been tested in Escherichia coli and Saccharomyces cerevisiae [1][2]. Therefore, our hypothesis is that it would work in planta as well. As a proof-of-concept for EvolvR in planta, we first chose to test it in a simpler plant system instead of doing so in a whole plant directly. We opted for a Tobacco Bright Yellow 2 (BY-2) suspension culture, allowing us to work at the single cell level and to test EvolvR much faster than in a whole plant. Tobacco BY-2 cells are a plant cell line established from Nicotiana tabacum cultivar Bright Yellow-2 [3]. This cell line exhibits homogeneity and rapid growth rates and is therefore often used as a model organism for plant research [4]. The cells form small clusters in liquid culture, but each cell is still exposed to changes in the culture medium. We also confirmed these characteristics in our own culture under a microscope. Since it has even been referred to as the “HeLa” system of higher plants, we chose this plant cell line to demonstrate the activity of EvolvR in plant cells [4]. We also explored an opportunity to use cambial meristematic cells (CMC) of Catharanthus roseus as they would create a more homogenous culture of separate single cells and allow for an easier selection of higher-fitness mutants.
We built vectors carrying 3 different variants of EvolvR. We used a linker that can be easily replaced by a custom-designed gRNA expression cassette in the construct, so that these vectors can be used to express EvolvR in any kind of CDE experiment in plants. The vectors we built express EvolvR fused with an NLS that transfers it into the nucleus where it can access the genomic DNA. It also contains the fluorescent marker mCherry for easy detection of a successful transformation, which makes for an easy control method. One of the vectors was tested in BY-2 cell culture and showed successful transformation and expression of the fluorescent marker.
As mentioned on the proof-of-concept page, our plan was to transform a WT BY-2 cell suspension culture with an EvolvR construct carrying the BASTA target protospacer. After 48 hours, we would add an array of antibiotics at specific concentrations determined in the optimization experiments. These antibiotics would kill off the Agrobacterium without harming the transformed BY-2 cells. The WT BY-2 cells will be affected by supplemented kanamycin, while the transformed BY-2 cells will contain a kanamycin resistance cassette after successful Agrobacterium-mediated transformation. The selection of transformed BY-2 cells out of a liquid transformation mixture with WT BY-2 cells and Agrobacterium would be continued throughout the whole experiment to ensure only transformed BY-2 cells survive. We also wrote a guide about possible use of EvolvR which can be applied to any plant system.
EvolvR is a very promising tool for CDE of plants, which has never been described before. We have shown that a large construct containing EvolvR can effectively be transformed into BY-2 cells and our next steps are to validate the activity of EvolvR in these cells to confirm that it is able to mutate a target genomic region in BY-2 cells. The toolkit, protocols, and hardware design we have built in our project will certainly advance the field of plant biotechnology towards a future in which plants can rapidly be evolved towards novel phenotypes. Examples of such phenotypes are higher resilience and resistance to drought, floods, or high salinity and sodicity, as well as any other kind of external stress factor. EvolvR can also be applied to create plants that produce higher yields of compounds such as nutrients of pharmaceutically active compounds.
More detailed explanations and results can be found on the Experiment, Proof of concept, Results and in the Lab books.
References
[1] Halperin, S. O., Tou, C. J., Wong, E. B., Modavi, C., Schaffer, D. V., & Dueber, J. E. (2018). CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. Nature, 560(7717), 248–252.https://doi.org/10.1038/s41586-018-0384-8
[2] Tou, C. J., Schaffer, D. V., & Dueber, J. E. (2020). Targeted diversification in the S. cerevisiae genome with CRISPR-guided DNA polymerase I. ACS Synthetic Biology, 9(7), 1911–1916.https://doi.org/10.1021/acssynbio.0c00149
[3] Experimental Plant Division, R. B. R. C. (2021, September 7). RPC00001: Nicotiana tabacum by-2 cell suspension culture. rpc00001: Nicotiana tabacum BY-2 cell suspension culture -BRC plant cell line documentation. Retrieved October 19, 2021, from https://plant.rtc.riken.jp/resource/cell_line/web_documents/cell_lines/rpc00001.html
[4] Nagata, T., Inzé, D., & Matsuoka, K. (2006). Tobacco BY-2 Cells: From Cellular Dynamics to Omics (1. Aufl., Vol. 58). Springer-Verlag. https://link.springer.com/content/pdf/10.1007%2F3-540-32674-X.pdf