Gene targeting

Targeted insertion of genes in the plant genome

Simple in theory, difficult in practice: Gene targeting can be used to selectively switch off, modify or replace genes. Targeted insertion of genes in the plant genome could further minimise undesirable side effects. It has long been used successfully in bacteria, yeasts and mice. The use of gene targeting in plants, however, has proved very difficult.

Gene targeting is based on homologous recombination, a naturally occurring mechanism in which homologous, i.e. identical or similar, DNA sequences in the genome switch places. This mechanism can be used to integrate a new gene at a particular site in the genome. For this to take place, the gene must be flanked by DNA segments that are identical to the planned integration site (see diagram).

Homologous recombination

Exchanging homologous sequences (left) Two DNA strands with homologous DNA sequences (light blue, dark blue): exchange.

Exchanging a non-homologous DNA segment (below) If a gene (orange) is flanked by sequences that are homologous with another site in the genome, it can be cut out from its original site and inserted at the other.

So much for the theory: in practice however, no efficient methods of gene targeting have yet been developed for use in plants. This is partly due to the fact that homologous recombination does not occur in plants with sufficient frequency or efficiency. One approach then is to boost homologous recombination in the plant cell with the help of transposons and bacterial recombination systems. Another method stimulates the naturally occurring recombination processes in the plant cell to make them take place more frequently and more efficiently.

Gene targeting with transposons and bacterial recombination systems

Transposons and recombination system consist of certain recognition sequences and an enzyme (transposase or recombinase) that can cut DNA segments out of the genome at the site of these recognition sequences and reintegrate them at another site. Unlike transposes, recombinases also need recognition sequences to insert a DNA segment. By contrast, transposes can reintegrate the DNA segment at any site in the genome, but will generally integrate it in regions with high gene expression.

A completed biosafety research project used a recombinase to remove a marker gene and simultaneously integrate another gene in its place. For this to work, both genes have to be flanked by recognition sequences (sites).

A current biosafety research project is inserting a genetically modified transposon that also contains the recognition sequences for a recombinase into the genome of oilseed rape plants. It also contains a reporter gene, which can be used to measure whether the transposon has been integrated in a region with high gene expression. The transposon integration site is also the site intended for gene integration, so it should be as ‘active’ as possible.

Molecular biological methods are used to check that the integration has not triggered any undesirable modifications in the genome, e.g. disabling a plant gene. Plants that meet both criteria are selected as suitable recipient lines for targeted gene insertion.

A recombinase can then be used to insert any chosen transgene into the genome of the recipient line at the transposon integration site.

Gene targeting through stimulation of the cell’s own recombination processes

In this method, a special DNA-cutting enzyme (“I-SceI” restriction enzyme) is used to cut the DNA strand at two sites in the plant genome. It is then possible at these sites to carry out recombination processes and so to achieve a targeted exchange of homologous sequence segments.

Step 1: Producing two different transgenic plant lines

First, two different transgenic plant lines are produced that each contain a target locus (the target site for the integration of a new gene) and a targeting vector. DNA analyses are used to check whether the target locus is at a desired site in the plant genome.

The targeting vector contains a DNA sequence that is homologous with the target locus (green and blue in the diagram), into which the new gene is inserted. The targeting vector is also flanked by two cutting sites for the “I-SceI” restriction enzyme.

Step 2: Crossing the plant lines

The two plant lines – the target locus line and the targeting vector/I-SceI line – are then crossed with each other.

The “I-SceI” restriction enzyme cuts the targeting vector at the I-SceI cutting sites. This triggers homologous recombination between the targeting vector and the target locus.

The cutting site from which the targeting vector was excised is subsequently closed again by homologous or illegitimate recombination. In the process, the cutting sites are destroyed, so the process is irreversible and the targeting vector cannot be reintegrated at its original site.

Step 3: Analysing the plants

DNA analysis is then used to select the plants in which gene targeting has actually worked.

Marker genes are being used initially to establish the gene targeting system in plants. This enables researchers to make the system as efficient as possible. Once this has been achieved, the marker gene can be replaced with a gene of interest. A further long-term goal is to make every naturally occurring sequence in the plant genome available as a target site for gene integration using gene targeting.