Genome Editing and Plants
The process by which a plant cell is edited is as follows: a target site for genome editing is designed and screened for potential off-target effects using computer software. The sgRNA representing that target site is synthesized and inserted into a CRISPR-Cas9 expression cassette, containing the gene encoding Cas9 and the sequence of the sgRNA, each under the control of a specific promoter. The cassettes are delivered into plant cells using a variety of methods, ranging from Agrobacterium-mediated to biolistic (gene gun) delivery and even through the use of plant viruses engineered as delivery vectors. Plant cells that have been transformed are then screened for the presence of the desired mutation, either by restriction enzyme analysis or by directly sequencing their genomes (Kumar et al., 2015, Rani et al., 2016).
The various genome-editing systems described above provide a straightforward method for rapid gene targeting within one to two weeks (Shan et al., 2014). Two major advantages are that genome editing is more rapid than both traditional breeding and transgenic approaches, and a selection process using marker sequences or genes is not necessary (Xing et al., 2014). Alterations in the genome can be detected quickly and inexpensively, and selectable markers are not required as they are in marker-assisted selection or transgenesis, respectively (Kim et al., 2016). A single genome editing event can also offer the possibility of simultaneous targeting of multiple (stacked) traits within a single crop; these traits can be carried to all homologues within the plant’s genome, which is no small feat and difficult to control using both traditional breeding and transgenesis (Luo et al., 2016, Raitskin and Patron, 2016)). While humans have a diploid genome (23 pairs of chromosomes), plants can have higher levels of polyploidy (for example, the wheat genome has six copies of each chromosome). It can be challenging for traditional plant breeders and molecular biologists who work with transgenic plants alike to ensure that every chromosome homologue contains the gene of interest and that it is expressed in an optimal fashion (Zhu et al., 2016). As a result of these features, the regulatory path for genome-edited plants into the marketplace is far more straightforward than it is for transgenic crops. Since many of the tools required for genome editing come directly from common bacteria (often harbored within our own GI tracts) and no additional genetic material is added to the genome (unlike the process creating transgenic plants), the promise of global acceptance of genome edited crops by farmers and consumers alike is more likely to be realized. These features provide assurances to scientists that any advances they make to the technology and any forthcoming products are less likely to be left on the shelf or subject to attack; consequently, genome editing has virtually blossomed overnight (Cardi et al., 2016)
At the moment, genome-editing technologies are being specifically optimized for all major crop types. Often a proof of concept is first sought out through the demonstration that a previously well characterized gene can be edited in that crop in such a way that all homologues have been altered in the plant and that the alteration is inherited stably to the next generation (Khatodia et al., 2016). Some of the traits that have been examined include those that are fundamental to crop improvement, such as flower/fruit size, color, grain yield, herbicide tolerance and pest resistance (Barakate et al., 2016). As more and more research groups perfect the conditions for successfully editing a particular crop type, attention will shift to the production of novel traits that will improve vigor, stress tolerance, yield and nutritional content of crop varieties (Basak et al., 2015). Genome editing is also being rapidly incorporated as a tool for scientists to learn even more about how plants cope with abiotic and biotic pressures. The knowledge gleaned from these studies can then be used to generate a second generation of newly genome edited crop varieties that are even better able to manage in a rapidly changing world (Liu et al., 2016, Nangpiur et al., 2016). The next section provides examples of some of the traits that are under examination for economically important crops.
5a. Wheat. Wheat is one of the major food crops in the world but can be difficult to work with due to its large (17 Gb) hexaploid genome. Kumar et al., (2014) used CRISPR-Cas9 to alter genes involved in amino acid and carotenoid biosynthesis in a wheat cell suspension culture as a proof of concept that large complex genomes could undergo genome editing successfully. The same authors were also able to use genome editing to delete a large gene fragment in the wheat genome. Zhang et al, (2016) edited the wheat gene responsible for grain length and weight using particle bombardment. Approximately 16% of the mutants recovered had all six alleles simultaneously knocked out. Both hexaploid bread wheat and tetraploid durum wheat (used predominantly for pasta) were edited in this fashion. Another research group was able to successfully target genes involved in wheat shoot and root development traits (Wang et al., 2014). Simultaneous editing of three homologous alleles of the mlo gene led to a bread wheat variety that is resistant to powdery mildew, a disease that is a threat to food security (Huang et al., 2016).
5b. Maize. CRISPR/Cas9 has been used as a tool to demonstrate that genome editing could have a direct impact on the production of maize crops with new, agronomically helpful attributes (Svitashev et al., 2015, Char et al., 2016). CRISPR-Cas9 was employed to target a number of different genomic regions in maize immature embryos by biolistic transformation. These regions include regulatory elements required for leaf development, male fertility genes and genes involved in amino acid biosynthesis (with the idea of creating herbicide resistant plants, for the latter). Reduction of the antinutrient phytase has also been generated using ZFN technology in maize (Shukla et al., 2009).
Shi et al., 2016, used CRISPR/Cas9 to generate novel variants of the ethylene response gene ARGOS8. Overexpression of ARGOS8 has been shown to improve grain yield under drought stress conditions. Several mutants generated using CRISPR/Cas9 were able to increase grain yield by five bushels per acre under stress conditions. The same plants experienced no yield loss under well-watered conditions, showing that genome editing can generate novel types of drought resistant crops. Along the same lines, Qi et al., 2016, were able to change storage protein content in maize using CRISPR-Cas9.
TALENs have also been used as genome editing tools in maize. As a proof of concept, Char et al., (2015), have shown that mutations can be generated at the maize glossy2 (gl2) locus, responsible for the waxy layer on leaves. Furthermore, scientists at Dupont Pioneer have edited the Wx1 gene that creates ‘waxy corn’ used for producing specialty starch for processed foods, adhesives and high-gloss paper.
Genome editing can also be used to directly alter maize pathogens, and thus identify what specific interactions cause infection, so that plants can be modified to become resistant to those interactions. For example, Schuster et al., (2016), used the CRISPR/Cas9 system to alter genes in the fungal maize pathogen Ustilago maydis. The fungal mutants can then be tested for their ability to infect maize plants, and using this reverse genetics approach, the virulence genes of the pathogen can be identified and their function during infection determined. With this knowledge, new maize crops edited to resist fungal infection can be designed and generated.
5c. Rice. Genome editing has been extensively used to modify rice for a number of purposes (Li et al., 2016a, b, Xu et al., 2017). The authors Blanvillain-Baufumé et al., (2016), used TALEN as a genome editing tool to examine bacterial leaf blight infection in rice. Targeted mutations in the plant gene involved in leaf blight infection were generated and the ability of proteins from a variety of different bacterial strains to bind to these rice mutants and promote infection was examined. A number of the genome edited rice plants showed resistance to several of these bacterial strains, demonstrating that while new plants that are resistant to Xanthomonas infection could be developed, the nature of that resistance could also be studied in detail via direct plant pathogen interactions.
Rice resistant to rice blast, a fungal pathogen, has been developed by using CRISPR-Cas9 to alter a gene that is involved in the plant stress response (Wang et al., (2016a, b). By creating a variety of mutations in this gene, the selected plants were demonstrated to resist rice blast but displayed no difference when compared to wild type plants with respect to agronomic traits such as plant height, leaf length, grain weight and number. Another research group located in China used the CRISPR/Cas9 system to alter genes in rice responsible for enhanced grain number density and larger size, simultaneously. The results showed that CRISPR/Cas9 can modify stacked, multiple traits in a single cultivar (Li et al., 2016).
5d. Soybean. Genome editing technologies have also been employed for soybean. Du et al., (2016), used CRISPR/ Cas9 to alter soy flower size and color. The genome editing technique for soybean has been further optimized through the development of an online web tool that quickly identifies a high number of potential CRISPR/Cas9 target sites (Michno et al., 2015). Another research group used CRISPR/Cas9 to develop herbicide tolerance in soy (Li et al., 2015). Other examples of genome editing in soybean can be found in Sun et al., (2015), Jacobs et al., (2015) and Cai et al., (2015).
5e. Citrus. Citrus is an economically important slow-growing tree crop found worldwide. Over half of citrus grown commercially in the world is sweet orange. The genome of sweet orange has been successfully modified using CRISPR/Cas9 (Jia and Wang, 2014). More recently, Duncan grapefruit has been edited by CRISPR/Cas9 for resistance to Citrus canker, one of the worst pathogens of citrus. The bacteria which produced citrus canker injects a protein into infected citrus plant cells that suppresses plant defense and promotes bacterial growth and canker development. This bacterial effector protein can turn on genes in the cell of the citrus plant that aid in tumor development and bacterial infection by binding directly to the promoter region of the plant DNA. By altering the sequence of this promoter region using genome editing, grapefruit plants were developed that were resistant to this disease (Jia et al., 2016).
5f. Tomato. Tomato, another economically important crop, has been studied for its nutritional enhancement properties through alteration of the carotenoid pathway (Brooks et al., 2014). Recently, Pan et al., (2016) used the CRISPR/Cas9 system to target two genes responsible for altering the color of tomato fruit. The frequency of mutation was high and albino phenotypes were observed in tomato for two generations, indicating that the mutations were stably inherited and exhibited no off target effects. Another study conducted by Cermak et al., (2015), examined the use of CRISPR/Cas9 delivered by a geminivirus vector to overexpress anthocyanin in tomato, which turns the fruit a deep purple colour. Anthocyanin, a compound found in blueberries, is associated with reduced cardiovascular and cancer risks. Tomatoes are less expensive, globally available and easier to grow than blueberries, and thus providing similar nutritional benefits is desirable.