Cas Endonuclease Technology for Plant Research and Breeding
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1. Historical View of Genetic Modification in Crop Plants
For more than 10,000 years, plants have been cultivated to feed humans and their livestock, as a source of raw materials and to generate energy. Over time, the driving forces of evolution, domestication, and breeding of cultivated plants have changed according to environmental conditions and societal demands. To cope with the growth of the world’s population and the consequent increase in demand for food, the total production needs to be substantially increased during the next decades [1,2]. Not only is the growing world population a challenge, but also the expected climatic changes [3,4]. Thus, the plants of the future also have to be better adapted to harsh weather conditions [5,6]. In addition, the demands of modern society are increasing; food should be of better and consistent quality, healthier, and more diverse. At the same time, society calls for a substantially reduced application of fertilizers and pesticides to render agriculture more sustainable .
In nature, mutations occur spontaneously and alterations that provide an advantage under the given conditions are likely to prevail over time. For modern plant breeding, however, the rate of spontaneous mutations is too low to keep up with the demands for crop improvements. Moreover, it is in the nature of spontaneous mutations that they are unpredictable in terms of both position and resultant nucleobase sequence. In the 1930s, mutation breeding emerged, in which mutations are deliberately induced with chemicals such as ethylmethanesulfonate (EMS) or with ionizing radiation. Induced mutagenesis breeding has so far produced over 3000 approved varieties  and for many further cultivars it has not been documented which induced mutations they have inherited from germplasms they derive from. Using this technology, however, thousands of mutations occur at different sites in a single plant’s genome at a time. Therefore, a vast number of undesirable mutations have to be eliminated via cumbersome and time-consuming back-crossing procedures . The method of Targeted Induced Local Lesions in Genomes (TILLING) represented a significant progress in the detection of mutations. With TILLING or Eco-TILLING, respectively, it is possible to readily identify induced and spontaneous mutations in known genes of interest [8,9].
The method of gene transfer using Agrobacterium tumefaciens was introduced in the 1980s . Ever since, genes derived from unrelated organisms or genetically modified gene variants can be introduced into plant genomes. This technique makes it possible to modify or introduce performance-determining genes into cultivated plants more quickly and in a more targeted manner. However, there are some limitations, such as the fact that the transferred gene is integrated at a random position in the genome and that changes to the plant’s own genes are only possible to a very limited extent. The insertion site in the genome then also determines whether and how effectively the introduced gene is expressed, and thus to what extent the respective trait will be modified. In addition, not all plants can be transformed equally well by the use of Agrobacterium, with cereal species being a particular challenge in this respect .
With the new methods for targeted genome modification that are based upon customizable endonucleases, it is possible to modify DNA sequences at a previously defined site of choice in the host genome. The standard application of this technology is site-directed mutagenesis, in which the location of the mutation can be precisely determined, whereas the resulting nucleobase sequence is random . However, the precision can be increased by a variety of approaches. The most sophisticated one involves the use of an artificial DNA repair template implemented via homology-directed DNA repair. By this still very challenging principle, any sequence of choice can be created at any predefined genomic locus.
2. Platforms of Customizable Endonucleases
In plant research and biotechnology, four platforms of customizable endonucleases have been used so far; meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the RNA-guided, clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated (Cas) endonucleases.
Meganucleases are naturally occurring endonucleases that recognize and cut comparatively long (>12 bp) DNA sequences. The most commonly used meganuclease is the I-SceI from Saccharomyces cerevisiae [13,14]. The ability of this endonuclease to induce double-strand breaks in plants was first demonstrated by Puchta et al.  using Nicotiana protoplasts. Meganucleases are very specific and efficient. It is, however, very difficult to reengineer these enzymes for target sequences other than their native ones . Hence, their use is fairly restricted as compared to the alternative endonuclease platforms [17,18]. As a consequence, meganucleases have been used in plants almost exclusively for basic research, in particular for DNA repair mechanisms .
Zinc-finger nucleases are hybrid proteins with a DNA recognition domain consisting of at least three zinc-fingers combined with a FokI restriction endonuclease domain . Each zinc-finger specifically interacts with three base pairs (bp) of the genomic target sequence and several zinc-fingers can be consecutively assembled to recognize and bind a total of 9 to 12 bp of DNA . Zinc-finger nucleases must always be used in pairs, since their FokI endonuclease domain is only catalytically active if it is present as a dimer . The target motifs on the DNA are selected in such a way that the two zinc-finger nuclease monomers bind to the target DNA in antiparallel orientation and at a suitable distance to each other. A DNA double-strand break (DSB) is then catalyzed in the interspace between the two binding sites [22,23]. In addition to the particularly complex production process of zinc-finger nucleases, there are limitations in the selection of possible binding sites, as well as unpredictable neighbor effects between adjacent zinc-fingers on the DNA binding specificity .
In 1989, it was shown for the first time how transcription activator-like effector (TALE) proteins are transferred from a pathogenic bacterium of the genus Xanthomonas into a host plant . The TALE-based nucleases (TALENs) act, similarly to zinc-finger nucleases, as dimers . The DNA-binding domains consist of up to 30 copies of highly conserved repeats of typically 34 amino acids. Only the amino acid positions 12 and 13 (called repeat-variable di-residues) of each repeat are not uniform, because they specify the different DNA base pairs to bind . A FokI endonuclease linked with such a TALE binding domain induces the double-strand break in the target motif . Since each repeat recognizes just one nucleobase of the DNA target, the design and assembly of the binding domain is more straightforward and versatile than with zinc-finger nucleases .
The platform of RNA-guided Cas endonucleases is derived from the CRISPR/Cas adaptive immune system of microbes. The most commonly used Cas endonuclease is from Streptococcus pyogenes (Sp). The two-component system consists of the Cas restriction enzyme and an artificial guide RNA (gRNA) that navigates the Cas protein to a cognate DNA sequence motif . The gRNA consists of a structurally functional and a variable part. The 3′-tail forms a spatial structure required for binding with Cas to form a ribonucleoprotein complex. The variable part of the gRNA located at the 5′-end usually comprises 20 nucleotides and defines the DNA-binding specificity of the gRNA according to the principle of complementary base pairing. The genomic target motif of such ribonucleoprotein complex includes the same nucleotide sequence as the variable part of the gRNA, which enables the gRNA to specifically bind the opposite DNA strand. This part of the target DNA is often referred to as the protospacer, a term adopted from the microbial immune system these molecules originate from. In addition, this major part of the target motif is complemented by the so-called protospacer-adjacent motif (PAM), which is recognized and bound by the Cas protein. In the case of SpCas9, the nucleobase triplet NGG, in which N stands for any of the four nucleobases, represents the PAM site . The high versatility of the gRNA 5′-end allows a wide variety of target sequences of choice to be addressed. The comparatively simple application and the efficiency and reliability achievable in higher organisms have rendered this platform the most popular and frequently used tool for site-directed genome modification today [27, 28].
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