Emerging tools for synthetic biology in plants

Synthetic biology is generally held to be the rational design of biological components to achieve a desired purpose. It attempts to replace the inherent messiness of biology with the ordered precision and predictability of engineering. This is a tall task, and requires an intimate understanding of the biological process that is to be engineered. Not surprisingly, much of the initial work has been done in bacteria where such detailed understanding is a little easier to obtain. Complex multicellular organisms such as plants pose additional problems, but, as this special issue demonstrates, this has not prevented rapid progress. Fortunately, synthetic biology lends itself well to a reductionist approach, as illustrated by the ‘BioBricks’ popularized by the annual iGEM (International Genetically Engineered Machine) competition (Boyle et al., 2012). This allows relatively complex and sophisticated ‘devices’ to be constructed from simple components. In fact, one of the strengths of synthetic biology is that new synthetic tools can be developed that, once obtained, permit the creation of new levels of artificial biological synthesis. Thus synthetic biology is progressing via ‘bootstrapping’, or as Baron von Münchhausen would have put it, it is pulling itself out of the swamp by its own hair. The classic example of synthetic biology is synthetic nucleases. By fusing synthetic domains modeled on the DNA-binding domains of zinc finger (ZNF) proteins or transcription activator-like effectors (TALEs) with the nuclease domain of a natural restriction endonuclease, synthetic enzymes have been developed that enable scientists to induce double-strand breaks (DSBs) in any genomic locus they wish. Holger Puchta and Friedrich Fauser sum up the current knowledge about DSB repair mechanisms in plants and describe the exciting potential of synthetic nucleases in plant genome engineering (Puchta and Fauser, 2014). These new tools had scarcely been tested when a new way to produce an artificial endonuclease was developed and applied to plants, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system. This originates from bacteria and archaea where it serves as an adaptive immune response system that degrades invading foreign plasmid or viral DNA. To create new targeted CRISPR/Cas nucleases requires only a 20-nucleotide RNA sequence to specify the cutting site instead of a redesigned protein, so this system is especially user-friendly. A large number of studies describing the use of this system for inducing mutations in various plant species have already flooded the literature. One does not need to be a fortune teller to see that such tools will be routine for all of us in the near future, making more traditional targeted mutagenesis approaches (e.g. insertion mutagenesis, TILLING) rapidly obsolete. Furthermore, the potential applications of synthetic nucleases go well beyond simply creating mutations. These tools will find uses in various kinds of more comprehensive genome modifications (or even the construction of artificial genomes) and will help us progress into new levels of synthetic biology. The site-directed change of only a few nucleotides in plant genomes deserves special consideration in future biosafety evaluations. Compared with some classical breeding techniques such as irradiation-induced mutation that cause multiple uncharacterized changes in plant genomes, the newly developed technique is as a surgeon's scalpel to a blunderbuss. The use of synthetic nucleases is currently the cleanest way to achieve targeted progress in plant breeding. In their contribution, Frank Hartung and Joachim Schiemann from the German Institute for Biosafety in Plant Biotechnology set this question in a broader context (Hartung and Schiemann, 2014). They sum up the legislation covering genetically modified organisms (GMOs), especially in the European Union, and evaluate the new plant breeding techniques with a special emphasis on artificial nuclease-mediated genome modifications. They suggest that the nature of the plant variety but not the process by which it was obtained should be used as the criterion to classify GMOs. From a practical point of view, plants carrying point mutations, small deletions of a few base pairs or subtle changes in coding sequence induced by the use of synthetic nucleases cannot be discriminated from natural varieties of the same species. Nuclease fusions are just one of many possible application of synthetic DNA-binding domains. In their contribution, Lahaye et al. (2014) describe how the architecture of TALE repeats determines DNA recognition and how arrays of TALE repeats can be fused to various other kinds of protein domains with specific biochemical functions. They discuss how these fusions can be used to regulate plant transcriptomes and even the epigenome. TALEs are currently the most widely used nucleic acid-binding domains in our synthetic toolbox, but competition from the newly emerging CRISPR/Cas technology will be interesting to follow. The TALE proteins are but one example of a whole class of nucleic acid-binding proteins with a modular structure in which each repeat recognizes one nucleotide. It is this modular structure that makes them ideal for synthetic biology. As Yagi et al. (2014) explain, one of the most promising families is the large group of pentatricopeptide repeat (PPR) proteins that, unlike TALEs, bind to single-stranded RNA rather than double-stranded DNA. Applications of PPR proteins are in their infancy, but their tremendous natural diversity suggest that they can be easily tailored to multiple functions. A set of RNA manipulation tools for synthetic biology would be a valuable addition to science. The ultimate goal for synthetic biologists is the creation of an entire synthetic genome. Much excitement surrounded the creation by Craig Venter et al. of ‘Synthia’, a strain of Mycoplasma mycoides constructed by transplanting an entire synthetic genome of M. mycoides into a Mycoplasma capricolum cell (Gibson et al., 2010). Although a technical tour de force, this still falls short of a creating a new, redesigned genome. It is possible that the first true ‘designer’ genomes will be tested in plants; the organelle genomes, and particularly chloroplast genomes, make ideal subjects given their small size and relative lack of essential genes. In a visionary and thought-provoking contribution, Lars Scharff and Ralph Bock give an expert opinion on the possibilities (Scharff and Bock, 2013). The field of synthetic biology is still young, but plant science has already contributed a great deal. Arguably, the wave of small interfering RNA and artificial micro-RNA technology can be considered the first real impact of synthetic biology, and plant scientists were highly influential in its development and uptake. Now plant pathologists and plant molecular biologists have given us TALE and PPR proteins and are developing the next wave of new synthetic biology tools, and their colleagues are at the forefront of genome redesign. It is truly an exciting time to be a plant scientist.