Breaking news: plants mutate right on target.

F or millennia, early human civilizations observed phenotypic changes in animals and plants and used these for domestication (1). In recent decades, scientists around the world induced random mutations, mainly in crop plants, to widen the mutation spectra to be used for extensive screening for varieties useful for agriculture and science (2). As mutagens, ethyl methanesulfonate, radiation, Agrobacterium tumefaciens-mediated T-DNA transformation, and transposon mutagenesis have been used. Distinction between WT and mutant was dependent on the phenotype, on sequence specificity of the mutagenizing DNA (in case of transposons or T-DNA), or could be accomplished with tilling. Alternatively, gene expression could be suppressed by use of small interfering RNAs. Targeted mutagenesis in plants, however, was only recently developed, and examples of Zinc finger nuclease (ZFN)-mediated targeting of natural genes by homologous recombination have been published recently (3–6). Now, an even more straightforward technique for mutation, the site-specific breaking and error-prone repair of endogenous genes in Arabidopsis thaliana by the plant machinery, is the topic of two reports presented in PNAS (7, 8). A similar approach using a custom-made meganuclease was also reported for maize (9). In recent years, the development and use of ZFNs or meganucleases, especially for animal systems, increased like an explosion. Meganucleases are reengineered homing endonucleases mostly based on I-CreI that is found in chloroplasts of Chlamydomonas reinhardtii (10). ZFNs rely on the combination of a nuclease domain supplied by the enzyme FokI and sequence-specific Zinc-finger domains designed using specialized programs or assays (11). Proof of concept for the successful activity has been provided earlier (12), and the utility of site-specific induction of double-stranded breaks (DSBs) and their repair by nonhomologous joining of the ends or their repair using homologous rescue sequences has been demonstrated in animal and plant systems (Fig. 1). Whereas the pathway using homologous sequence is not as efficient, both in plants and in animals (except for ES cell lines), direct error-prone rejoining of the broken ends by endogenous pathways turned out to yield mutated versions of the targeted gene in relatively high proportions in animal systems (11). This reflects the relatively higher efficiency of the error-prone nonhomologous repair pathway compared with that dependent on homology, both in higher animal and higher plant systems. Osakabe et al. (7) targeted the A. thaliana gene ABI4, as mutations in this gene are expected to show a strong phenotype. The activities of the respective designed ZFNs were assayed in bacterial two-hybrid systems, in vitro and in transgenic plants. The expression of the ZFNs was activated by a temperature-inducible system and candidates for mutated abi4 were screened for, using the Surveyor nuclease assay (this assay makes use of mismatches between WT and mutated sequences using a mismatch-specific endonuclease). Frequencies as high as 3% were obtained in somatic tissue and transmission to the offspring exhibiting the expected phenotype could be demonstrated. Use of ku80 mutant plants as targets for the ZFNs led to mutation frequencies in the same range as with WT plants, but the extent of sequence degradation at the junction sites was increased. Zhang et al. (8) targeted two Arabidopsis genes, one coding for the ADH 1 and the other for the TT4, both of which, in the homozygous mutated version, exhibit strong phenotypes. Activity and specificity of the chosen ZFNs were tested in yeast and Arabidopsis protoplasts. For induction of the ZFN proteins in the transgenic plants, an estrogen-inducible promoter was used. Somatically mutated plants could be recovered at frequencies of 7% and 16%. The efficiency in this publication may be higher than that in the Fig. 1. Target for ZFN, with the two different subunits of the enzyme drawn below and above the target sequence. The sequence in between is being cut, upon dimerization of the enzyme, yielding a 4-bp-long 5′ overhang. This can be repaired by the host-specific NHEJ activity, usually leaving small deletions or insertions behind. Alternatively, a homologous rescue construct, supplied at the time of enzyme activation, is used by the host’s homologous recombination activity, to replace the endogenous sequence.