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Imprint
ProTAL (ProTAL)
Project
Project code: 031B0541
Contract period: 01.07.2018
- 30.06.2020
Budget: 368,108 Euro
Purpose of research: Experimental development
Keywords: TALEN, rice, Xanthomonas oryzae bacteria
TALEN are highly efficient, highly specific, and can be positioned in a genomic context with the greatest flexibility of all genome editing tools. Nevertheless, their construction is significantly more cumbersome than the currently favored CRISPR/Cas system. This project aims to alleviate this burden by generating custom TALEN proteins in a high-throughput measure. Several procedures will be tested to increase the solubility and stability of TALEN proteins which is the current bottleneck in the use of TALEN proteins. Besides standard procedures, specific innovative approaches will be used to control the protein solubility by exploiting natural protein variations or novel protein fusions. Currently, TALEN are applied in pairs. Thus, in a second part of the project, highly active single-chain-TALEN (scTALEN) will be established to allow the use of monomeric TALENs which will further simplify the applicability of this genome editing tool. To enhance the activity of such nucleases, the DNA-binding strength and the DNA processivity will be enhanced. The aim is to establish a monomeric TALEN which can be applied as a soluble and stable protein and which shows a high in vivo activity at target sites. In contrast to CRISPR/Cas, the specificity of TALEN in the context of complex genomic DNA has not throughly been assessed. Soluble and stable TALEN proteins will be used to clarify their genome-wide specificity using CIRCLE-seq. Finally, the TALEN protein will be used to edit the rice genome at multiple positions corresponding to virulence targets of natural TALEs from rice-pathogenic Xanthomonas oryzae bacteria. Multiplex editing of such loci will disable the virulence factors and generate rice plants with resistance against these important rice pathogens. The use of TALEN proteins instead of DNA or RNA might allow for a wider acceptance of this technology, because no foreign nucleic acid is transmitted and no transgenic intermediate organism is produced. These optimizations will re-establish TALEN as an alternative to CRISPR/Cas and will make their unique advantages available for a broad usership.
Section overview
Subjects
- Plant Breeding
- Biotechnology
