Use of CRISPR systems in plant genome editing: Toward new opportunities in agriculture

Author: Agnès Ricroch (AgroParisTech), Pauline Clairand (AgroParisTech), Wendy Harwood (John Innes Centre)

Abstract

Initially discovered in bacteria and archaea, CRISPR–Cas9 is an adaptive immune system found in prokaryotes. In 2012, scientists found a way to use it as a genome editing tool. In 2013, its application in plants was successfully achieved. This breakthrough has opened up many new opportunities for researchers, including the opportunity to gain a better understanding of plant biological systems more quickly. The present study reviews agricultural applications related to the use of CRISPR systems in plants from 52 peer-reviewed articles published since 2014. Based on this literature review, the main use of CRISPR systems is to achieve improved yield performance, biofortification, biotic and abiotic stress tolerance, with rice (Oryza sativa) being the most studied crop.

Introduction

CRISPR/Cas systems have proved to be important tools for genome editing in model plants and crops. On the one hand, CRISPR/Cas or tools derived from this technology have permitted rapid and straightforward determination of the function of different coding and non-coding DNA sequences in model plants [1–5]. On the other hand, numerous studies describe applications of CRISPR systems for the development of new traits in crops and could be considered as proof-of-concept studies.

The CRISPR/Cas gene-editing system is able to generate heritable, targeted mutations and also to address concerns over the presence of foreign DNA sequences as it can generate transgene-free plants. Therefore, it offers advantages in giving precision that was previously not possible and in allowing the induction of mutations without the presence of transgenes in the final plants. Articles focusing on these areas are considered, together with the analysis of the agricultural opportunities offered by the technology within specific geographic areas. We built up the following bibliographic research quest to gather scientific peer-reviewed articles specifically dealing with trait improvement in crops: ‘CRISPR’ OR ‘clustered regularly interspaced short palindromic’ OR ‘cas9’ OR ‘cas 9’ AND (Plant* OR vegetal OR Spermatophyt* OR algae OR Dicot* OR Monocot* OR Legume* OR Cereal* OR crop*). Then, we submitted this research request in databases such as Infodoc, Sciencedirect, BiblioVie, EBSCO, BergeRicrochGMlibrary and Web of Science to perform this systematic literature review. These search terms may not have captured every relevant article; however, those captured clearly identify key trends.

Induction of heritable targeted mutations and generation of ‘transgene-free’ plants

Regarding economic perspectives and social acceptance of CRISPR/Cas systems, a major concern is the heritability of the gene-induced mutations and the generation of transgene-free plants. Various articles have reported the induction and stable inheritance of single- [6–8] and multiple-targeted mutations [2], studying the T0 plants and T1 and T2 progenies. Generally speaking, the mutants of interest are selected by segregation [9].

Regarding the heritability concern, Pan et al. [8] used a visually interesting tool to demonstrate the inheritance of mutations induced in the PDS gene of Solanum lycopersicum. SlPDS encodes the phytoene desaturase which is a key enzyme in carotenoid biosynthesis. The silencing of this gene causes photobleaching or albino phenotypes. The authors were, thus, able to monitor mutation and inheritance patterns with a visual indicator associated with genotyping and sequencing. They demonstrated that the CRISPR/Cas system can induce heritable mutations in tomato plants (from T0 to T2 generation plants) and that homozygous and biallelic mutants were generated even in the first generation. In addition, a classical study on Arabidopsis thaliana provided a general scheme regarding the heritability of mutations induced by CRISPR/Cas using Agrobacterium-mediated transformation [10].

To generate transgene-free plants, it is necessary to obtain a stable production of CRISPR/Cas-mutated lines without the presence of the CRISPR DNA expression cassettes in the final mutant plants. This can be achieved in many ways. Most studies using Agrobacterium-mediated transformation intend to generate final mutant plants without transferred DNA (T-DNA). As specific primers are used to detect the presence of transgenes encoding CRISPR/Cas components, scientists showed that transgene-free, T2 mutant lines could be obtained by genetic segregation: the targeted mutations were stably passed on in transgene-free plants [Table 1, column ‘Transgene-free plants studied (Yes/No)’]. Other studies show how to generate transgene-free plants using alternative delivery methods [11,12]. These methods include ways of introducing the CRISPR components in a transient fashion such that integration is unlikely, for example using protoplast systems [12]. Alternatively, it is possible to avoid the presence of foreign DNA at any stage of the process, therefore avoiding the possibility of foreign DNA insertion. This can be achieved by the introduction of RNA or a ribonucleoprotein complex [13].

Table 1: Agricultural applications of the use of CRISPR systems in the 52 articles studied (2014–2017) (For detailed information about Table 1, please check in the original pdf file.)

The heritability and the transgene-free character of the generated plants were demonstrated in several studies, confirming that these areas should no longer be a concern for agricultural applications. This opens up many opportunities for different agricultural and industrial applications of CRISPR systems and below we focus on those that were developed in proof-of-concept studies.


Agricultural and industrial proof-of-concept studies

To study the agricultural and industrial applications of CRISPR/Cas systems in plants, 52 articles dealing with trait improvement of crops were selected to assess how scientists are directing their use. The use of CRISPR/Cas systems covers various applications, from biotic stress tolerance to abiotic stress tolerance, and also includes the achievements of improved yield performance, biofortification and enhancement of plant quality (Table 1 and Figure 1). Table 1 summarizes the main information found in these applied research articles, with a view to

• - understanding the main applications of CRISPR/Cas systems in plant genome editing;
• - looking at whether the production of transgene-free plants was addressed in the studies and
• - detailing the main strategy used and method of delivery of the CRISPR components.

First of all, the application of CRISPR/Cas systems is mainly achieved directly in crops: 42 out of 52 articles studying 15 crops (Figure 1). Few studies use model plants for transient assays before studying the stable and heritable patterns of CRISPR-induced mutations in the target crop(s). Several trends can be observed with regard to the scope of applications of CRISPR/Cas systems in plant genome editing. The most important group of target applications relates to yield traits followed by the achievement of biotic or abiotic stress tolerance (Figure 2). Biotic stress tolerance includes induced tolerance to viral, fungal and bacterial diseases with a higher number of articles exploring plant tolerances to viral disease (Figure 2). As for abiotic stress tolerance, the two main objectives are to achieve herbicide and natural environmental stress tolerances (Figure 2). Environmental stress includes cold, salt, drought and nitrogen stress. All of these trait improvements are related to economic and agronomic challenges faced by farmers as pathogens, and environmental conditions are important threats that need to be dealt with in agriculture. Furthermore, plant breeders are continually trying to increase yield performances. The most studied crop is rice (Oryza sativa) (Figure 1) followed by other major crops: maize (Zea mays), tomato (S. lycopersicum), potato (Solanum tuberosum), barley (Hordeum vulgare) and wheat (Triticum aestivum) (Figure 1). Finally, the emergence of biofortification in the list of applications can be related to that of metabolic engineering in the 1990s.


For the achievement of viral disease resistance, two main strategies are observed:

• - the integration of CRISPR-coding sequence in the host plant genome that targets and interferes with the virus genome once it is incorporated in the plant: the aim is to establish a CRISPR-like immune system in the host genome [7,14] and
• - the induction of a CRISPR-mediated targeted mutation in the host plant genome that will confer improved virus resistance traits [9].

As an example, Ji et al. [7] demonstrated resistance to the Geminivirus, beet severe curly top virus using a CRISPR/Cas-based approach in the model plants Arabidopsis and Nicotiana benthamiana. The resulting plants were highly resistant to the virus. An extensive knowledge of plant biology and gene functionalities is required before using CRISPR/Cas systems in a specific species for a particular application. The application of CRISPR/Cas gene editing requires the precise definition of the target DNA sequence and the availability of good genome sequence data of the studied species in order to allow design of single-guide RNAs (sgRNA). The presence of a PAM sequence (protospacer-adjacent motif) upstream of the sequence complementary to the sgRNA is also required and it is necessary to search for putative off-target sites.

Once the decision is taken to employ CRISPR/Cas systems for a given application, scientists need to choose adapted delivery methods and strategies to fulfill their objectives. Table 1 lists a selection of articles with agricultural applications that could be considered as proof-of-concept studies for future commercial application of CRISPR/Cas systems in plants. It shows that conventional Agrobacterium-mediated transformation using plasmid vectors containing, for example, Cas9/sgRNA expression cassettes is mainly used to deliver the system to plants. However, additional delivery methods have also been implemented such as

• - protoplast transfection in Linum usitatissimum and S. tuberosum [12,15];
• - biolistic delivery in T. aestivum, O. sativa and Z. mays [6,16,17], and
• - use of reconstituted viral replicons in N. benthamiana, S. tuberosum and Papaver somniferum [14,18,19,59,60].

Table 1 also describes how CRISPR/Cas systems can be used not only for site-directed mutagenesis (gene knockout) but also for gene insertion or replacement and multiplex genome editing (column ‘Delivery method//Main strategy’).

Geographic distribution of studied articles

As there are distinct research, economic and regulatory contexts in the world, it was interesting to focus on the importance of the use of CRISPR/Cas systems in plant genome editing depending on the country where studies were carried out. Regarding articles with agricultural applications, Figure 3 shows that China and the U.S.A. are ranked first with 22 (42%) and second with 10 articles (19%), respectively. Europe, which includes the U.K., Sweden, France, Hungary, Germany, Austria and Belgium, had 9 articles (17%). Four studies were carried out in Japan and two in Israel. Five studies were carried out in each of the following countries: Saudi Arabia, Turkey, Korea, Philippines and India. This figure is consistent with the globalized economic, regulatory and research contexts and can be partly explained by the uncertain regulatory framework in Europe that may be holding back work towards commercial application (Figure 3).

Figure 3. CRISPR studies by country. Number of articles studying the use of CRISPR systems in plant genome editing with agricultural applications according to the country of the research team (2014–2017).

Regarding the plant species studied according to the country of the research team (Figure 4), the dominance of rice (O. sativa) is again observed, and mainly in China, which is in accordance with the Chinese research and economic contexts. Additionally, the application of CRISPR/Cas systems in maize (Z. mays) seems to be mainly studied in the U.S.A. Efficient systems for genome editing in soybean have also been reported for example [61]. Other crops that were studied include vegetables and industrial plants:

- Cucumis sativus, Citrus paradisi and Citrus sinensis,
- L. usitatissimum, P. somniferum, Taraxacum kok-saghyz, Salvia miltiorrhiza and Dendrobium officinale and
- the model plant Lotus japonicus.

Figure 4. Plant species studied by country. Plant species studied in articles using CRISPR systems in plant genome editing with agricultural applications according to the country of the research team (2014–2017).

In terms of methods, the generation of transgene-free plants (that is to say plants in which the Cas9/sgRNA-expressing sequence was not integrated) is important to examine in relation to the geographic location of the research team describing the specific agricultural application. Although the number of reviewed articles is low, one trend worth noting is that only one of the studies carried out in the U.S.A. addressed the generation of transgene-free plants (Figure 5). In contrast, Chinese and European studies paid particular attention to the generation of transgene-free plants. This is likely to be linked to GMO regulatory requirements and intellectual property considerations that differ from country to country.

Figure 5. Generation of transgene-free plants by country. Sorting of the 52 articles according to the country of the research teams showing whether the generation of transgene-free plants was studied (green) or not (red).

Conclusion

Since 2013, considerable progress has been made in plant genome editing thanks to CRISPR/Cas systems. This technology has allowed straightforward, cost-effective and efficient gene editing compared with previous technologies, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), making it accessible to many researchers. However, this emerging method is still developing and scientific efforts must continue to be made in order to obtain a mature technology and to realize the full potential of the technology. CRISPR/Cas-based technologies are, however, advancing at a rapid pace with the description of many new technological advances. Such advances are often first described in animal systems and then transferred to plants. A recent example is the application of ‘base editing’ in a crop where a specific base change was achieved in wheat rather than the usual mutation at a specific site involving a small insertion or deletion [62].

Concerns have been raised over the relationships that may exist between the use of this method and GMOs, and the many studies related to the generation of transgene-free plants [63] show that scientists aim to demonstrate that this technology is distinct from GM technology. In the U.S.A., the legal status of CRISPR/Cas-induced mutations is that they are exempt from GMO laws. In Europe, in October 2016, the French Council of State asked the European Court of Justice whether CRISPR/Cas and other site-directed mutagenesis tools should fall under the EU GMO legislation. The European Court of Justice has 18 months to reply. Moreover, ethical concerns could also emerge regarding the impact on public health and the environment of using CRISPR/Cas in plants. However, the use of this system already represents an emerging market, with CRISPR/Cas applications spanning a wide range of industries including research, agricultural and biomedical [64]. The agricultural applications described in this literature review represent only the very first, initial uses of this exciting technology, and we can expect many more valuable opportunities for agriculture in the near future.

Summary

• A systematic review of 52 scientific articles from 2014 to mid-2017 regarding the use of CRISPR systems for agricultural applications.
• The principal species studied is rice. The main applications are yield performance, biofortification and tolerance to abiotic and biotic stress (virus, fungi and bacteria). China published most articles in this area followed by the U.S.A. and Europe.
• The heritability of the induced mutations and the development of transgene-free plants are the most studied areas.

[1-64]: For detailed information of references, please check in the originial pdf file.