5. CRISPR/Cas9 applications in crop genetic improvement: brief overview

The relevance of agriculture for human survival is hard to underestimate. Crops provide food, fiber, and raw materials for a growing human population that faces an increasing amount of challenges, including loss and degradation of arable land as well as climate change. In this context, the rational use of all the available biotechnological tools is of paramount importance to attain a worthy life quality both in developed countries and the third world. Crop genome editing is among the most promising techniques to cope with the aforementioned agricultural challenges. However, it is worth noting that the development of those methodologies is useful not only for genetic improvement of agricultural crops but also to functional characterization of specific plant genes for basic research purposes [43].

Three short reports, published in 2013, demonstrated the feasibility of CRISPR/Cas9 system for genetic engineering of crops, based on the pioneer works of Li et al. (using A. thaliana), Nekrasov et al. (with N. benthamiana), and Shan et al. (O. sativa and T. aestivum), respectively [36, 40, 44]. After that, a plethora of research on crop genome editing has been published. The works listed below include some of the most representative studies on the matter, according to our best knowledge. However, the amazing dynamism of the field makes the presumption of exhaustiveness unattainable.

Given its undeniable worldwide relevance as a staple food, it is not surprising that rice has been one of the most studied crops in terms of CRISPR/Cas9 mediated genetic edition [14, 38, 45–48]. Among the main modifications proposed to this crop, it can be found herbicide resistance [49, 50], improved nitrogen use efficiency [51], and resistance against the rice blast disease [52].

Wheat (both durum and bread wheat) has also been subjected to extensive research in order to optimize the effectiveness of the genome editing process [53–55] as well as the acquisition of novel attributes including heritable broad-spectrum resistance to powdery mildew and other plant diseases [56].

Other crops widely used for the validation of technical improvements in CRISPR/Cas9 system are soybean [12, 57, 58] and maize [59, 60]. The latter cereal has also been modified in order to exhibit advantageous traits. For instance, this technology has generated novel variants of the ARGOS8 gene on maize. The ARGOS8 edited variants significantly increased grain yield under drought stress conditions, compared to wild-type maize, and had no yield loss under normal conditions [61].

The CRISPR/Cas9 system was also used to investigate the influence of specific genes on the phenotype development in tomato plants [51, 62, 63], as well as to achieve features of agronomic importance, such as delayed ripening of tomato fruit [64] or parthenocarpy [65]. Other members of the Solanaceae family reported to have undergone genetic editing via CRISPR/ Cas9, include tobacco (Nicotiana tabacum) [38, 66], potato (Solanum tuberosum) [67], and petunia (Petunia hybrida) [68].

The CRISPR/Cas9 technology has also been used to confer molecular immunity against tomato yellow leaf curl virus (TYLCV), using N. benthamiana as host [69], as well as inducing complete resistance to Turnip mosaic virus (TuMV) [70], and improve the stress response in the model plant A. thaliana [71].

In the case of the emerging oil seed plant, Camelina sativa, the CRISPR/Cas9-targeted genetic edition has improved its fatty acid composition, obtaining a seed oil of superior quality on multiple levels, which besides being healthier, was more stable to oxidation and better suited for biofuel production [72, 73].

In addition to that, this technology has been used to obtain a nontransgenic cucumber strain (Cucumis sativus L.), resistant to cucumber vein yellowing disease, papaya ringspot mosaic virus-W, and zucchini yellow mosaic virus [74], as well as to successfully induce targeted mutagenesis in the Chardonnay grape cultivar that enhanced its endurance to powdery mildew, and to increase the golden delicious apple cultivar resistance to fire blight disease [75].

Besides the aforementioned, other crops in which the CRISPR/Cas9 technology has been optimized include barley (Hordeum vulgare) and Brassica oleracea [76], watermelon (Citrullus lanatus) [77], as well as the nonherbaceous sweet orange (C. sinensis cultivar Valencia) [42] and poplar (Populus tomentosa) [78].

Finally, we would like to stress that there is no scientific evidence whatsoever to assume that genetic modifications produced by modern biotechnological tools, such as CRISPR/Cas9, represent a higher health or environmental risk than conventional breeding techniques. However, public distrust caused by genetically modified crops has led to many countries to implement highly strict and costly regulations that make very difficult to successfully commercialize such products. Interestingly, since CRISPR/Cas9 genetic editing does not necessarily implies the incorporation of foreign DNA, according to some interpretations, the existing legislation might not be applicable to this technology. Therefore, the scientifically informed public discussion of such legal framework is imperative [43, 75, 79].
