Targeted insertion of genes at specific points in the plant genome

Topic

In plant transformation , the DNA is usually integrated at random into the genome at any point (illegitimate recombination ). Targeted insertion (homologous recombination) would make it possible to carry out genetic modifications at specific points in the plant genome. Individual genes could then be switched off or modified.

The aim of this project was to develop a method with which DNA can be inserted at specific points with the help of targeted double-strand breaks in the plant itself. At the same time, illegitimate recombination should be suppressed.

Further information on homologous recombination

• Recombination - New arrangements of genes

Summary

It was possible to establish a novel technique for eliminating transgene sequences from the plant genome.

Transgenic plants were produced in which the expression of certain proteins involved in illegitimate recombination was suppressed. Unfortunately, however, it was not possible by this means to assist the targeted insertion of new DNA sequences at specific points in the genome. Other approaches must be tried to achieve this objective.

Experiment description

The method used in the project consists of two complementary approaches. In the case of a successful trial, the plan is to combine them.

1. A rare-cutter restriction enzyme called "I-SceI" is used to cause a break in the DNA strand (double-strand break) at a particular point in the plant genome. This increases the probability that the gene sequence being transferred will be inserted at precisely this point. The effectiveness of this process is checked using a test system (involving repairing a defective marker gene )

2. Certain factors are known (from Arabidopsis ) to play a role in illegitimate recombination. These can be inhibited by introducing certain RNA fragments (gene  silencing ). The aim is to tip the scales in favour of targeted integration .

The idea is that the procedure should be applicable for different plant species, although initially it is being developed for tobacco and Arabidopsis.

Results

for 1. The usability of the rare-cutter restriction enzyme I-SceI for the targeted modification of the plant genome was demonstrated spectacularly in a test system. It was possible in the process to develop a novel technique for the elimination of transgene sequences from the plant genome. By using cutting sites of the I-SceI enzyme in the transgene construct before and after the marker gene, it is possible to induce double-strand breaks either side of the marker gene following expression of I-SceI in the plant. The marker gene can then be removed (see diagram).

for 2. It was possible to isolate sequences of several proteins involved in illegitimate recombination from tobacco. Transgenic plants were produced in which the function of these proteins was suppressed.

However, it was not possible by this means to assist the targeted insertion of new DNA sequences at specific points in the genome, which indicates that other, as yet unknown, factors are involved in illegitimate recombination in plants. Future approaches to establish the technique in plants must therefore take new directions. There will be a follow-up project for this purpose.

Transgene with selection marker (top). By using a special cutting system, the marker gene is removed and the DNA strand is repaired. (Diagram: Prof. H. Puchta, University of Karlsruhe)