Tailor-Made Plants Using Next-Generation Molecular Scissors

When people hear the words “mutation” or “mutant,” they usually associate them with something negative (e.g., a disease) or even evil (as in a science fiction movie). But mutations are simply changes or variations, which are not inherently positive or negative. As a matter of fact, mutations are the basis of evolution and biodiversity and are often favorable. And mutations are the essence of agriculture.

The variety of traits found in nature reflects random genetic changes that are often the result of mistakes in the repair mechanism initiated after DNA damage. Mutations effected through genome editing can confer resistance to pests, increase biomass, remove allergens, and enhance adaptability to changing environmental conditions such as drought. Such mutations can help improve product quality and yield, reduce costs, and protect the environment.

Background

Since the practice of agriculture began about 10,000 years ago, humans have tried to adapt plants to their convenience, identifying and crossing varieties with traits that were beneficial or advantageous. For a long time, adaptations were achieved only by observing and selecting plants that were naturally available, collecting the seeds of those that looked best and propagating them over generations of seedlings. This practice came to be called plant breeding, and in this paper is referred to as conventional breeding.

In the late 1920s it was discovered that the occurrence of “natural” variability could be increased by exposing plants to radiation or chemicals that cause extensive DNA damage and thus mutations (Stadler 1928). This process was called mutation breeding. The findings both considerably expanded the range of useful traits available for breeding and accelerated the establishment and release of new, improved plant varieties, many of which now show up on the dinner table every day.

However, with mutation breeding many genetic changes are induced randomly, which means that it is still necessary to screen a large number of individual plants to identify those (if any) carrying the mutation in the gene of interest. Furthermore, it may not be clear what alterations might result from all the other randomly induced mutations.

The long-sought solution to these problems came about in the 1990s with the discovery of site-specific nucleases (SSNs), a sort of sophisticated “molecular scissors” that can be programmed to cut DNA precisely at a predetermined site (Kim et al. 1996), enabling the directed introduction of changes (mutations) in the target gene. This technology is called genome (or gene) editing.

CRISPR/Cas9: First the Gene Is Cut…

The first programmable molecular scissors had limited applicability because protein engineering was required to “program” them, a capacity available in only a few specialized laboratories. The real breakthrough came in 2012 with the development of a particularly efficient SSN based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system (for a review of this technology, see Bortesi and Fischer 2015).

The principle behind the CRISPR/Cas9 system is quite simple. The system works like a vaccination for bacteria: when a bacterium is infected by a virus, it retains a short piece of the viral sequence and uses it to recognize and destroy the virus by efficiently and quickly cleaving its DNA.

What makes the CRISPR/Cas9 system such an exceptional and interesting tool for genome editing is that no complex protein engineering is necessary to change the specificity: all one needs is the Cas9 protein, which is activated upon interaction and binding with a short piece of RNA called the single guide RNA (sgRNA). The activated Cas9/sgRNA complex locates the target site by scanning genomic DNA for sequences complementary to a short stretch in the sgRNA. With changes to just 20 nucleotides (or bases) in the sgRNA, the system can be “programmed” to cleave any sequence of choice and only that sequence (figure 1). In other words, scientists now have a simple instrument that makes it possible to identify and cut precisely 20 base pairs among, for example, the approximately 840 million that make up the potato genome.

Since nowadays one can order 20-base-long nucleic acids for a few dollars and get them the next day, it becomes clear what a simple yet potent tool the CRISPR/Cas9 system represents. Part of its power comes from the fact that, because it’s so easy and cheap to change the specificity, one can test several sequences at the same time and select only the most efficient one for study. This is why researchers worldwide jumped on this technology and many even approached genome editing for the first time.

Figure 1

A further advantage of the CRISPR/Cas9 system over other SSNs is that it allows researchers to easily target multiple loci at the same time, a concept called multiplexing, by using multiple sgRNAs with different sequences for each target (figure 1). This feature is especially important for crop improvement, because most of the relevant agronomic traits, such as yield, quality, and stress response, are jointly regulated by many genes.

…Then It’s Repaired

Once the DNA is cut it has to be repaired, and the different possible outcomes of the repair process can be exploited for various applications (figure 2).

Figure 2

Plants mostly repair a DNA break by “stitching” the loose ends together via an error-prone process called nonhomologous end joining, which frequently results in the modification (mutation) of one or several random base pairs at the cut site (figure 2a). The nature of these modifications cannot be predicted and often destroys the function of the gene, generating a gene knockout (KO).

When DNA is cut in the presence of a small piece of exogenously supplied (“donor”) DNA that matches the sequence around the break but for a few mutations, the plant can (but doesn’t necessarily) use it as a patch to repair the break. Through this process, called gene conversion, researchers can determine not only the position but also the nature of the resulting mutation (figure 2b). Such homology-dependent repair is technically more difficult, but it affords control of the outcome and can be useful to, for example, change or fine-tune the expression of a gene instead of completely destroying it.

Under certain conditions one can even induce a plant to use a much larger piece of DNA as a patch to repair the break, thereby introducing a new gene (or a whole new metabolic pathway) at a precise location (figure 2c). This offers an unprecedented way of controlling the insertion of foreign DNA, in contrast to the usual way of generating transgenic plants, which, relying on random integration in one or multiple genomic locations, risks compromising some essential functions of the plant or altering its metabolism in unpredictable ways.

Why Are Programmable Molecular Scissors Needed?

Conventional breeding is limited by the fact that it relies on existing genetic variation and requires lengthy backcrossing to introduce selected traits into elite lines (these are the best lines, with many positive features—especially, among agronomic traits, high yield). Mutation breeding using radiation or chemicals increases genetic variation but requires extensive screening and, again, lengthy backcrossing.

Using the SSN approach, both drawbacks can be overcome by expanding and explicitly controlling the genetic variation. The outstanding advantages of this technology are not only high efficiency and relative simplicity but also the remarkable precision and speed with which desired mutations can be achieved compared to conventional or mutation breeding (table 1).

Table 1

Genome editing for crop improvement mostly aims to increase biomass, boost uptake of soil nutrients such as phosphorus and nitrogen to reduce both the costs (of purchase quantities and application) and environmental pollution associated with the use of fertilizers, or confer resistance to pests to improve product quality and yield while limiting the use of toxic pesticides.

Knockouts are the simplest and most frequent outcome of genome editing in plants. They are very useful for studying the function of a gene and can also have important practical applications. For instance, by simply knocking out a plant gene essential to a particular viral infection it has been possible to generate a resistant cucumber variety (Chandrasekaran et al. 2016), and a significant increase in rice yield was achieved by the simultaneous KO of three genes that restricted grain weight (Xu et al. 2016). The latter is an example in which speed of mutation and multiplexing are striking advantages, because several genes had to be modified to obtain an appreciable trait improvement.

The use of SSNs such as the CRISPR/Cas9 system also enables targeted molecular trait stacking, the combination of multiple, otherwise segregating traits at a specific position in the plant genome. For example, maize genes that confer greater drought tolerance, disease resistance, and yield could be stacked in an elite variety either simultaneously or in subsequent rounds of targeted integration. And the entire array of genes could thus be mobilized into another germplasm by simple crossing because it would behave as a single locus. This would eliminate the need to introduce one trait at a time via conventional breeding and would avoid excessively lengthy timescales and severe downstream breeding challenges (e.g., from extended backcrossing to eliminate unwanted genomes while ensuring inheritance of desired trait[s]).

Besides conventional agriculture for food, feed, or fuel, plants can be used for molecular farming (as production platforms for biopharmaceuticals, technical proteins, or small molecules; Arora and Narula 2017; Tschofen et al. 2016) or as a source of bio-based materials, such as rubber, starch, cellulose, and lignin. In this context, SSN-mediated genome editing is a valuable tool to rapidly optimize plants and plant cell cultures by, for example, eliminating potentially immunogenic plant-specific glycans for the production of pharmaceuticals (Hanania et al. 2017; Mercx et al. 2016), improving the composition of starch for industrial applications (Andersson et al. 2017), or diverting a metabolic pathway to boost the accumulation of valuable natural compounds (Alagoz et al. 2016; Li et al. 2017).

Last but not least, the ability to control integration of new genetic material in a so-called “safe harbor” (a site in the genome where a transgene is consistently and stably expressed with no adverse effects on the plant’s fitness) via SSN-mediated targeted insertion may accelerate the development and possibly the approval of new transgenic lines for recombinant protein production.

Off-Target Effects

The use of SSNs and especially the CRISPR/Cas9 system may be accompanied by off-target effects, mutations that occur in sequences other than those intended to be modified. They are basically the only, albeit major, criticism of this technology.

While it is true that off-target mutations can happen and measures have to be taken to eliminate or reduce as much as possible their occurrence, it is also true that since the first use of CRISPR/Cas9, knowledge of the system has increased so much that in many cases it is possible to predict and prevent such mutations (Zischewski et al. 2017). Improvements in experimental design and protein engineering, for example, have substantially increased the fidelity of the CRISPR/Cas9 system. And recently identified anti-CRISPR proteins of viral origin can be used to switch off the system after it has done its job, to prevent random off-target cutting (Shin et al. 2017). In addition, plants as a whole have the following advantages:

1. Off-target effects are very rare in plants (Bortesi et al. 2016). Even without the application of high-fidelity approaches such as the use of improved Cas9 versions (Kleinstiver et al. 2016; Slaymaker et al. 2016), in all studies reported in the literature it was always possible to identify lines bearing the desired mutation(s) and no off-target effects.
2. Researchers can evaluate the outcome of genome editing in several plant lines and select only those that do not show unintended modifications.
3. Off-target effects can be eliminated by backcrossing, as is regularly done in mutation breeding with radiation or chemicals. The difference is that off--target effects are substantially fewer (if any) when the CRISPR/Cas9 system is used.

GMO Regulation

As highlighted above, SSNs such as the CRISPR/Cas9 system can be used for a variety of applications. Some are clearly transgenic, such as the targeted introduction of a new gene to produce a pharmaceutical protein. In other cases, the resulting plant may carry only mutations that are indistinguishable from naturally occurring ones or from changes induced through mutation breeding. These plants may or may not be regulated as genetically modified organisms (GMOs), depending on the country.

When GMO regulations were first conceived, the types of genetic modifications that researchers were able to perform were limited and transgenic plants constituted a clear and defined class of GMOs involving the stable introduction of foreign DNA. Now, with the advent of SSNs and the rapid evolution of genome editing technologies, it is possible not only to perform a great variety of modifications but to do so in multiple ways, transcending a single definition. For example, one can perform genome editing via an intermediate transgenic approach (i.e., introducing the transgenes to produce SSNs in a plant and then removing them through crossing once the desired modification has been achieved), or even do the whole procedure without introducing DNA at all (i.e., using the SSNs as proteins or RNA/protein complexes; Liang et al. 2017; Svitashev et al. 2016).

Every jurisdiction that has considered the regulation of genetically modified products has had to choose whether to adopt a product- or process-based approach (Marchant and Stevens 2015). For example,

• Canada has adopted a strictly product-based evaluation of plants with novel traits, so it is the characteristics of the product that are scrutinized, not the process.
• The United States has a hybrid system: the regulatory trigger is process-based, but the risk assessment is product-based. Applications involving genome-edited plants are reviewed on a case-by-case basis.
• The European Union uses a process-based approach: products generated using recombinant DNA are subject to burdensome premarket risk assessment and approval, labelling, and traceability requirements (which do not apply to other products such as those created by mutagenesis), even if in the final product there remains no trace of foreign DNA and the modification is indistinguishable from naturally occurring mutations.

Given the importance of the topic and the challenges of applying the existing binary transgenic/-conventional regulatory framework to products modified using these new genome editing techniques, the European Commission is evaluating the legal classification of “new plant breeding techniques,” including SSN-mediated genome editing. Many academic scientists as well as seed and crop companies are urging a shift toward product- -rather than process-based classification of genome-edited plants. This would lower the currently prohibitive costs of regulatory approval and could bring the commercialization of improved plant -varieties within the reach of entities other than big multi-national corporations.

Conclusions and Outlook

Given worldwide dependence on agriculture for food, feed, and fuel, and the increasingly urgent need to reduce dependence on petroleum, plant genome editing represents an invaluable tool to improve selected traits in elite cultivars, offering a rapid and effective means to address sustainability challenges. With the global population expected to top 9 billion by 2050, it is projected that the world’s food production will need to increase by 60–110 percent. Worryingly, most of the land used to grow wheat, maize, and rice is endangered by the effects of climate change (Pugh et al. 2016).

The SSN-mediated genome editing technology is already mature and safe enough to be used in plants, with the possibility of selecting and backcrossing to eliminate unwanted mutations if needed. As a matter of fact, the CRISPR/Cas9 system has been successfully used for genome editing of single or multiple genes in a wide range of plant species, including not only model plants used for research but also important crops such as rice, maize, and wheat (Bortesi and Fischer 2015).

Since CRISPR/Cas9 was first used for genome editing, variants of the system have been discovered with features that may be desirable for different applications (Steinert et al. 2015). Recently developed hybrids use the flexibility and ability of the CRISPR/Cas9 system to guide an enzyme that can modify a nucleotide without causing a break in the DNA (Gaudelli et al. 2017).

While research is rapidly moving forward to optimize the technology and its implementation in plants, the main limitations to its widespread application beyond the lab seem to lie elsewhere (Brinegar et al. 2017). Inadequate regulatory policies in some countries may limit commercialization of genome-edited crops to the few large corporations profitable enough to afford the costs for regulatory approval. Negative public perceptions of genetic engineering may influence policymakers and pressure governments to ban cultivation of even approved crops, as happens in Europe. Last, uncertain intellectual property protection, due to a long-running legal battle over patents for CRISPR/Cas9 genome editing (Ledford 2017), may discourage companies to invest in this technology.

Given the ease and speed of creating new mutations, SSN-mediated genome editing has the potential to effectively and rapidly generate a quantum leap in crop yields. Importantly, the use of genome editing to improve the adaptability of plants to new environmental conditions could help to maintain the biosphere and even prevent the extinction of certain plant species.

The question is not whether the use of programmable molecular scissors and other methods of genome editing to modify plants will have an impact on society, but when and where.

References

Alagoz Y, Gurkok T, Zhang B, Unver T. 2016. Manipulating the biosynthesis of bioactive compound alkaloids for next-generation metabolic engineering in opium poppy using CRISPR-Cas 9 genome editing technology. Scientific Reports 6: 30910.

Andersson M, Turesson H, Nicolia A, Falt AS, -Samuelsson M, Hofvander P. 2017. Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by -transient CRISPR-Cas9 expression in protoplasts. Plant Cell Reports 36(1):117–128.

Arora L, Narula A. 2017. Gene editing and crop improvement using CRISPR-Cas9 system. Frontiers in Plant -Science 8:1932.

Bortesi L, Fischer R. 2015. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnology -Advances 33(1):41–52.

Bortesi L, Zhu C, Zischewski J, Perez L, Bassié L, Nadi R, -Forni G, Lade SB, Soto E, Jin X, and 9 others. 2016. -Patterns of CRISPR/Cas9 activity in plants, animals and microbes. Plant Biotechnology Journal 14(12):2203–2216.

Brinegar K, Yetisen A, Choi S, Vallillo E, Ruiz-Esparza GU, Prabhakar AM, Khademhosseini A, Yun SH. 2017. The commercialization of genome-editing technologies. -Critical Reviews in Biotechnology 37(7):924–932.

Chandrasekaran J, Brumin M, Wolf D, Leibman D, Klap C, Pearlsman M, Sherman A, Arazi T, Gal-On A. 2016. Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology. Molecular Plant Pathology 17(7):1140–1153.

Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR. 2017. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551(7681):464–471.

Hanania U, Ariel T, Tekoah Y, Fux L, Sheva M, Gubbay Y, Weiss M, Oz D, Azulay Y, Turbovski A, and 2 others. 2017. Establishment of a tobacco BY2 cell line devoid of plant-specific xylose and fucose as a platform for the production of biotherapeutic proteins. Plant Biotechnology Journal 15(9):1120–1129.

Kim YG, Cha J, Chandrasegaran S. 1996. Hybrid restriction enzymes: Zinc finger fusions to Fok I cleavage domain. Proceedings of the National Academy of Sciences 93(3):1156–1160.

Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales AP, Li Z, Peterson RT, Yeh JJ, and 2 others. 2016. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523(7561):481–485.

Ledford H. 2017. Bitter CRISPR patent war intensifies. Nature, October 26. doi:10.1038/nature.2017.22892.

Li B, Cui G, Shen G, Zhan Z, Huang L, Chen J, Qi X. 2017. Targeted mutagenesis in the medicinal plant Salvia miltiorrhiza. Scientific Reports 7:43320.

Liang Z, Chen K, Li T, Zhang Y, Wang Y, Zhao Q, Liu J, Zhang H, Liu C, Ran Y, Gao C. 2017. Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nature Communications 8:14261.

Marchant GE, Stevens YA. 2015. A new window of opportunity to reject process-based biotechnology regulation. GM Crops Food 6(4):233–242.

Mercx S, Tollet J, Magy B, Navarre C, Boutry M. 2016. Gene inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 suspension cells. Frontiers in Plant Science 7:40.

Pugh TAM, Mueller C, Elliot J, Deryng D, Folberth C, Olin S, Schmid E, Arneth A. 2016. Climate analogues suggest limited potential for intensification of production on current croplands under climate change. Nature Communications 7:12608.

Shin J, Jiang F, Liu JJ, Bray NL, Rauch BJ, Baik SH, Nogales E, Bondy-Denomy J, Corn JE, Doudna JA. 2017. Disabling Cas9 by an anti-CRISPR DNA mimic. Science Advances 3(7):e1701620.

Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. 2016. Rationally engineered Cas9 nucleases with improved specificity. Science 351(6268):84–88.

Stadler LJ. 1928. Mutations in barley induced by X-rays and radium. Science 68(1756):186–187.

Steinert J, Schiml S, Fauser F, Puchta H. 2015. Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus. Plant Journal 84(6):1295–1305.

Svitashev S, Schwartz C, Lenderts B, Young JK, Cigan AM. 2016. Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nature Communications 7:13274.

Tschofen M, Knopp D, Hood E, Stoger E. 2016. Plant molecular farming: Much more than medicines. Annual Review of Analytical Chemistry (Palo Alto Calif) 9(1):271–294.

Xu R, Yang Y, Qin R, Li H, Qiu C, Li L, Wei P, Yang J. 2016. Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice. Journal of Genetics and Genomics 43(8):529–532.

Zischewski J, Fischer R, Bortesi L. 2017. Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases. Biotechnology Advances 35(1):95–104.