Genome Editing with Site-specific Nucleases

© Reprinted from Zhu, C., Bortesi, L., Baysal, C., Twyman, R.M., Fischer, R., Capell, T., Schillberg, S., Christou, P., n.d. Characteristics of Genome Editing Mutations in Cereal Crops. Trends in Plant Science., with permission from Elsevier
The four most important nucleases used for genome editing are a) meganucleases, b) Zinc finger nucleases, c) TALENs, and d) the CRISPR / Cas9 system

Site-specific nucleases introduce double-strand breaks at pre-determined DNA sequences, allowing precise and efficient genome engineering. Various targeted modifications can be generated by site-specific nucleases, including simple gene knockouts, complex gene conversions that require precise nucleotide exchange, transgene insertion, and molecular trait stacking. Genome editing can therefore accelerate plant breeding by facilitating the precise and predictable modification of endogenous genes directly in an elite background as well as the introduction of completely new traits, even complete biosynthesis pathways, in a controlled manner.

We use both zinc finger nucleases and the CRISPR/Cas9 system in plants and plant cell cultures to perform single and multiple gene knockouts, and to understand and address the challenges raised by the targeted integration of large transgene fragments.

Selected Publications

Baysal, C., Bortesi, L., Zhu, C., Farré, G., Schillberg, S., Christou, P. (2016). CRISPR/Cas9 activity in the rice OsBEIIb gene does not induce off-target effects in the closely related paralog OsBEIIa. Molecular Breeding 36: 108.

Bortesi, L., and Fischer, R. (2015). The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnology Advances 33 (1).

Jansing, J., Schiermeyer, A., Schillberg, S., Fischer, R., Bortesi, L. (2019)
Genome editing in agriculture: Technical and practical considerations International Journal of Molecular Sciences, 20 (12), 2888.

Kirchhoff, J., Schiermeyer, A., Schneider, K., Fischer, R., Ainley, W.M., Webb, S.R., Schinkel, H, Schillberg, S.
Gene expression variability between randomly and targeted transgene integration events in tobacco suspension cell lines (2020) Plant Biotechnol Rep

Schiermeyer, A., Schneider, K, Kirchhoff, J., Schmelter, T., Koch, N, Ke, J., Herwartz, D., Blue, R., Marri, P., Samuel, P., Corbin, D.R., Webb, S.R., Gonzalez, D.O., Folkerts, O., Fischer, R., Schinkel, H., Ainley, W. M., Schillberg, S. (2019)
Targeted insertion of large DNA sequences by homology‐directed repair or non‐homologous end joining in engineered tobacco BY‐2 cells using designed zinc finger nucleases. Plant direct 3, No.7, Art. e00153.

Schneider, K., Schiermeyer, A., Dolls, A., Koch, N., Herwartz, D., Kirchhoff, J., Fischer, R. et al. (2016). Targeted gene exchange in plant cells mediated by a zinc finger nuclease double cut. Plant Biotechnology Journal 14 (4).