Green light for gene targeting in plants
- Holger Puchta* and
- Barbara Hohn†,‡
- *Botany II, University of Karlsruhe, D-76128 Karlsruhe, Germany; and †Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland
Precision engineering of eukaryotic organisms requires efficient techniques for homology-based replacement of an endogenous gene by an introduced gene, a process termed gene targeting (GT). Because foreign DNA is preferentially integrated by nonhomologous recombination in plants and animals, special strategies are needed to increase the frequency and facilitate the detection of GT. Developing these strategies has proved particularly difficult in plants, for which GT efficiencies are on the order of 10–3 to 10–4 targeted events per transformed plant (1, 2). This low frequency seems to be the result of a natural barrier to integration of homologous sequences in higher plants. By contrast, efficient GT has been demonstrated in lower plants, such as the moss Physcomitrella patens (3).
The time appears ripe for a breakthrough in this field, and one has now been reported in this issue of PNAS. Avraham Levy and his group (4) describe a jump of between one and two orders of magnitude in GT frequency in Arabidopsis thaliana plants overexpressing the yeast chromatin remodeling protein Rad54. Their innovative approach was facilitated by the development of an ingenious assay that uses fluorescent seeds to identify targeted insertions.
Targeting by Breaking
Previous attempts to improve GT efficiency include the introduction of double-stranded breaks (DSBs) at the target site and modification of proteins involved in homologous recombination (HR). DSBs are obligatory recombination intermediates, and dedicated endonucleases are recruited to introduce them during meiosis. The introduction of a target site for a rare cutter restriction enzyme increased the targeting efficiency to such a site by two orders of magnitude in the somatic tissues in which GT normally takes place (5). However, the DSB has to be introduced at or close to the gene to be changed, which has been achieved in mammalian cells by using reprogrammed endonucleases whose catalytic domain …
