Team:KU Leuven/Proof Of Concept
BLADEN
Proof of concept
EvolvR used in continuous directed evolution (CDE) in plants
BLADEN strives to accelerate the engineering of plants using EvolvR for continuous directed evolution (CDE). EvolvR has previously never been applied in plants and neither has CDE. EvolvR has so far been tested successfully in Escherichia coli and Saccharomyces cerevisiae [1][2]. We hope to make this technology available in plants as well. As a proof-of-concept for EvolvR in planta, we first applied the technology to a simplified system, opting for a Tobacco Bright Yellow 2 (BY-2) suspension culture, allowing us to work at cellular level as opposed to a whole plant, saving us a lot of time. 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].
In order to demonstrate that our own EvolvR construct is functional in plant cells, we decided to first test EvolvR in a cell-free system based on a BY-2 cell lysate. Since the transcriptional and translational machinery is preserved in the lysate, we can quickly determine which of our EvolvR variants would be most suitable in a more complex BY-2 environment [5]. Next, we wanted to verify EvolvR activity in BY-2 cells by guiding EvolvR to mutate the essential glutamine synthetase gene targeted by the herbicide BASTA [6]. The presence of EvolvR can be confirmed by visualizing the transformed cells under a fluorescence confocal microscope since it is co-expressed with mCherry as a transformation marker. By applying selective pressure to transformed BY-2 cells, we strive to create a BASTA-resistant BY-2 cell line using CDE, followed by sequencing of the evolved BY-2 glutamine synthetase gene. Transformation conditions were optimized and initial results can pave the way for further research and development of the BLADEN platform for CDE in plants.
EvolvR activity in a cell-free BY-2 environment
Testing EvolvR in a cell-free BY-2 environment has many advantages. Firstly, by using an in vitro system, we avoid any potential toxicity of the protein to the cell related to the expression of EvolvR. Secondly, we avoid any potentially disruptive interactions with other proteins in the plant cell that may affect the function of EvolvR. Once we establish a functional EvolvR system in a eukaryotic environment derived from plant cells, we can start testing in plant cells.
While cell-free systems are often used for protein synthesis applications, we sought to use it to show that EvolvR can fold correctly in this specific eukaryotic environment and is still able to function as expected in a CDE experiment. Thus, we decided to adapt the established protocol by adding each component of interest (EvolvR, gRNA scaffold with protospacer, target, and positive control) on separate plasmids into the system. We have described our plasmid design and assembly process in detail on the Experiments page. In order to test whether EvolvR is functional in the cell-free system, we would insert a target plasmid carrying a ΔsfGFP(Y93X) gene that is not being expressed in the system but can be mutated by EvolvR. As this variant of superfolder GFP contains a nonsense mutation, if we target EvolvR to this mutation, we expect it to mutate this region and revert this mutation back to the wildtype. We can then confirm that EvolvR has induced mutations in the target gene via next-generation sequencing, isolating the target plasmid after EvolvR has been expressed in the cell-free system for 48 hours.
EvolvR in BY-2 plants
Ultimately, we strived to test all our plant EvolvR constructs in Tobacco BY-2 cells. Firstly, we wanted to optimize transformation conditions for the BY-2 EvolvR experiments. We accomplished this by first testing different concentrations and volumes of BY-2 and Agrobacterium tumefaciens, to determine the best transformation conditions. Additionally, we tested the transformation protocol both on solid and in liquid media. The common procedure during transformation of a BY-2 suspension cell culture is to incubate BY-2 cells with A. tumefaciens carrying a plasmid of interest for up to two days. They are then plated on solid medium to let the transformed BY-2 cells grow into calli, which can take up to three weeks to become visible. To accelerate this process, we also tested BY-2 transformed cell growth in liquid medium (more on this can be found on the Engineering Success page). We also tested different antibiotics and herbicide concentrations on wild-type (WT) cells. We assessed the effect of different antibiotics (timentin, carbenicillin, vancomycin, kanamycin and hygromycin) on WT BY-2 cells and Agrobacterium carrying our EvolvR constructs to determine what combinations and concentrations were most effective against the bacteria, but minimally affected the BY-2 cells. Next, the best sublethal (and lethal) BASTA concentrations were determined both on plates and in liquid media to optimize the selection pressure applied during later CDE experiments with BY-2 cells. More detailed explanations and results can be found on the “Experiment” and “Results” pages.
Secondly, we confirmed that we can successfully transform BY-2 cells with a plant destination vector carrying EvolvR. This is an enCas9-PolI5M-unspecific construct (pGGK_35SP_NLS-N7_enCas9-PolI5M_P2A-mCherry-NLS_35ST_unspecific-gRNA) which would be used as a control in CDE experiments and does not target the glutamine synthetase gene. This vector includes an mCherry reporter, which localizes to the nucleus as it is fused to a nuclear localization signal (NLS). We visualized mCherry using a fluorescence confocal microscope and were able to determine that we successfully transformed BY-2 cells with this EvolvR construct and created a transgenic BY-2 cell line containing EvolvR.
Finally, we wanted to validate whether EvolvR is mutating the user-defined target region of the BY-2 genome, which we chose to be the glutamine synthetase gene encoding for the target of BASTA herbicide. As the BY-2 genome is not fully characterized, we sequenced the target glutamine synthetase BY-2 gene so as to choose the best protospacers for the EvolvR experiments in BY-2. These protospacers were then inserted into our own plant EvolvR constructs, which is described on the Experiments page. Next, we transformed our constructs into A. tumefaciens strain C58C1 Rif (pMP90). These bacteria could then be used to transform BY-2 cell suspension cultures via Agrobacterium-mediated transformation (AMT). Our plan was to transform a WT BY-2 liquid cell suspension culture with our EvolvR construct carrying the BASTA target protospacer. After 48 hours, an array of antibiotics is added at specific previously determined concentrations. These would kill off the Agrobacterium while leaving the transformed BY-2 cells unharmed. 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 AMT. 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. Initial conclusions regarding EvolvR activity in plant cells can be drawn by sequencing the BY-2 genes targeted by EvolvR. If we can demonstrate that the targeted mutation rate in the target gene is higher than the global mutation rate, we can infer that EvolvR is active in BY-2 plant cells. Additionally, at 3 days post transformation, we would add a sublethal concentration of BASTA as described in the transformation optimization experiments to start the CDE experiment. This selection pressure would cause the beneficial mutations generated by EvolvR to become more abundant as they would have a higher fitness compared to the less BASTA-resistant variants. By harvesting the BY-2 cells again and sequencing the target gene, we can further validate that EvolvR is indeed functional in plant cells. Thus, providing a tunable technology for evolution in plants. While we were unable to finish BY-2 experiments with EvolvR, we have shown that EvolvR can be transformed into plant cells and we have built a large toolkit to be used for CDE in BY-2 cells. Our plan is to validate the activity of EvolvR in living plant cells as we believe that EvolvR has a large potential for the accelerated evolution of plants. Additionally, hardware designs such as CellED can further advance the application of our product in plant research.
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
[5] Buntru, M., Vogel, S., Spiegel, H., & Schillberg, S. (2014). Tobacco by-2 cell-free lysate: An alternative and highly-productive plant-based in vitro translation system. BMC Biotechnology, 14(1). https://doi.org/10.1186/1472-6750-14-37[6] Liaw, S.-H., Kuo, I., & Eisenberg, D. (1995). Discovery of the ammonium substrate site on glutamine synthetase, a third cation binding site. Protein Science, 4(11), 2358–2365. https://doi.org/10.1002/pro.5560041114