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Imprint
Enabling technologies for RNP-mediated genome editing (ENTIRE)
Project
Project code: 031B0552
Contract period: 01.07.2018
- 30.06.2020
Budget: 499,912 Euro
Purpose of research: Basic research
The aim of the project is to study and optimize the targeted insertion of DNA sequences into the plant genome with the help of genome editing methods. The procedures proposed rely on RNA-protein-complexes (RNPs) which are directed towards defined DNA target sequences to cut the DNA. The double-strand break (DSB) induced by RNP-mediated cleavage can either be removed by an error-prone repair mechanism, which leads to short insertions or deletions, or trigger the insertion of a repair DNA fragment by homologous recombination. While error-prone repair is successfully employed to knock-out distinct genes, the targeted insertion of DNA fragments, e.g. to regulate gene expression, is currently not possible with high efficiency. The project employs different methods for enhancing genome editing efficiency in plants based on RNP delivery. A special focus of the project lies on methods for targeted knock-in of short cis-regulatory sequences and on reducing the screening effort generally linked to any GE experiment. Specifically, the project addresses the following: (i) optimization of the RNP transfection process; (ii) delivery of DNA repair templates in spatial proximity to RNP-induced double-strand breaks for increased knock-in frequency, (iii) evaluation of double-strand breaks mediated by different nucleases in knock-in experiments, and (iv) single-cell detection of successfully edited cells already at the protoplast level prior to callus formation or plant regeneration. As a proof of principle, specific insertions or deletions of cis-regulatory elements will be done in the promoters of target genes of the transcription factor JUNGBRUNNEN1 (JUB1) which has been shown by our lab to be an important regulator of the tolerance to various abiotic stresses, including drought, heat and salinity. By fine-tuning the expression of different target genes of JUB1 it will be possible to enhance the tolerance to environmental stresses without compromising growth, which is an important breeding goal.
Section overview
Subjects
- Process engineering
