6. Conclusions and final considerations

the entire genome in question. ZFNs act at two sites of the DNA sequence at the cellular level, on the forward and reverse strand, respectively. Since cleavages of specific regions within the genome can be observed, ZFNs are capable to recognize two adjacent sequences, and once the corresponding cleavage occurs, the FokI restriction enzyme domains produce a dimerization

[31, 34, 35]. As listed in Table 1, some examples of experiments related to transient transfection

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

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

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

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


prior to the cleavage of the corresponding DNA loci. Thus, DSBs with 5<sup>0</sup>

based on CRISPR/Cas9-mediated NHEJ may be cited.

48 Transgenic Crops - Emerging Trends and Future Perspectives

of specific plant genes for basic research purposes [43].

exhaustiveness unattainable.

plant diseases [56].

normal conditions [61].

In recent years, the progress in the development of new tools for molecular genetic research has been evident since their application by simple, versatile, and efficient experimental techniques. From all the genome edition systems based on the nucleases application, CRISPR/Cas9 is the most friendly and simple method. It is now clear that the utility of this technology for the modification of specific loci is limited only by the interest of the researcher. In coming years, debates are expected about the best use of organic and conventional agriculture, sustainable farming, and all that coming from biotechnology. Thus, CRISPR/Cas9 technology has changed the way we see the future of agriculture. On the other hand, the implementation and easy accessibility to the CRISPR/Cas9 technology has allowed the generation of diverse molecular methodologies that constitute significant advances in the genome edition and its subsequent exploitation for agrarian and health purposes. Therefore, this technique is considered a revolutionary tool.

Nevertheless, despite the great potential of the CRISPR/Cas9 technique, it is also common to find evident limitations, since other alternatives have been proposed to improve genome editing with biotechnological processes [81]. Molecularly, the components of the CRISPR/ Cas9 system are too large to be introduced into a viral genome (e.g.), and thus, they are most commonly used in gene therapy to transport foreign genetic material into human and plant cells. One solution for this problem is to use a smaller type of a Cas9 enzyme, obtained from S. aureus [82]. This enzyme is small enough to fit in the virus. The mini-Cas9 complex has been used in mice to correct the gene responsible for Duchenne muscular

Understanding CRISPR/Cas9: A Magnificent Tool for Plant Genome Editing

http://dx.doi.org/10.5772/intechopen.81080

51

The Cas9 enzyme not always cleavages where it is intended within the genome (a certain DNA sequence must be nearby for that to happen). This is easily accomplished in many genomes, but it may be a limitation in some experiments. Researchers are constantly looking for microbes to obtain enzymes with different genome characteristics to expand the number of sequences that can be modified. One of these enzymes is called Cpf1, and it can be an interesting alternative, since it is smaller than Cas9 and has different characteristics in its sequence that make it highly specific [84, 85]. Another enzyme called C2c2 is able to target RNA instead of DNA, which is why it has a great potential to study RNA and to fight plant

Several laboratories apply CRISPR/Cas9 technology in order to eliminate specific regions in a gene sequence, thus repressing its function. Those who want to exchange a sequence for another face a more difficult task. When Cas9 cleavages the DNA, the cell makes mistakes by regrouping the loose ends, thus obtaining desired deletions. Researchers who want to rewrite a DNA sequence rely on different repair pathways that can insert new sequences (a process that occurs at a much lower frequency). Cas9 directs the sequence dictated by its guiding RNA, but does not cut it; instead, the bound enzyme changes the letters of DNA, ultimately producing a

In contrast to the above, recent new gene editing systems have been released using a protein called NgAgo to cleavage DNA at a predetermined site without the need for an RNA guide or a genome-specific neighbor sequence. Instead, the protein (of bacterial origin) is programmed using a short DNA sequence corresponding to the target sequence. However, laboratories have failed to reproduce the results so far, so the effectiveness of this technique cannot be affirmed [88]. There are also other genetic editing systems, some of which have existed for years. For example, scientists rely heavily on a system called lambda Red, which can be programmed to

In light of the above considerations, we can finally conclude that the biotechnological tools that belong to cas9 toolbox that in synergism with new bioinformatic algorithms increase their potential in a specific and powerful way and help position this technology as a magnificent last generation method for genomic edition, which is considered a revolutionary scientific discovery for both basic and applied research including the field of plant biotechnology, even when there are inconclusive details of its application in the laboratory and very probably a

dystrophy [83].

viruses with this type of genomes [86].

T where once there was a C [87].

gross ignorance of its nature, for now.

alter DNA sequences without the need for a RNA guide.

The major challenges for CRISPR/Cas9 technology will focus on two underlying aspects. First, the corresponding ethical or bioethical discussion, in order to demarcate what should or should not be done with this tool considering the risks that we could face by using promising technology, an even more when this is accessible and cheap. On the other hand, the legal consequences in terms of intellectual property that today literally generate wars between law firms and universities for the patents generated by thousands of investigations must be considered. Although many scientists consider CRISPR/Cas9 system as a "Holy Grail of genetic engineering," we must not lose sight of the objectivity and rationality when interpreting the consequences of its use. Additionally, demanding compliance with all the necessary safety steps before this technology becomes a trivial routine, especially if this tool is used for genome editing, and the genetic improvement of living beings must be imperative.

The features of the CRISPR/Cas9 system have allowed opening the possibility of using it to perform gene and cell therapy, in addition to its application in plant genetic improvement. In general, this technology has been used as a tool to perform point mutations, homologous recombination by HDR, and silencing and activation or repression of gene transcription. Thanks to these properties, its application has been possible for genetic monitoring, analysis of metabolic pathways, functional genomic research, generation of animal models, discovery of possible targets for disease treatments, and, even, correction of phenotypes. Another application of great importance that should continue to be developed is the generation of more precise and representative plant lines for the study of phytopathogenic diseases. Knockdown, knockout, and knocking models show the advantage of being able to be quickly and efficiently generated with this system. Also, CRISPR/Cas9 is considered a great biotechnological tool in the field of human therapies since its capacity to perform genetic level corrections/deletions, which is traduced in the possibility of regulating transcription or translation pathways.

In relation to the introduction of CRISPR/Cas9 in agricultural and environmental sciences, several studies recognize the possibilities of this technique to improve crop varieties [25, 80]. Uncertainty about safety and efficacy of genome editing requires evaluating its potential and utility by applying the precautionary principle. Research on this technology also unchains important legal and social debates among genetic engineering and genomic editing, in order to establish whether new mechanisms are needed to regulate research, confined use, voluntary release and if it is necessary to evaluate the possible impact on the environment just like in consumers' health, among other aspects. The application of the precautionary principle in any case must be done considering the available scientific evidence and raising the necessary social and economic considerations, in order to avoid a dogmatic interpretation that could undermine or stop scientific progress.

Nevertheless, despite the great potential of the CRISPR/Cas9 technique, it is also common to find evident limitations, since other alternatives have been proposed to improve genome editing with biotechnological processes [81]. Molecularly, the components of the CRISPR/ Cas9 system are too large to be introduced into a viral genome (e.g.), and thus, they are most commonly used in gene therapy to transport foreign genetic material into human and plant cells. One solution for this problem is to use a smaller type of a Cas9 enzyme, obtained from S. aureus [82]. This enzyme is small enough to fit in the virus. The mini-Cas9 complex has been used in mice to correct the gene responsible for Duchenne muscular dystrophy [83].

debates are expected about the best use of organic and conventional agriculture, sustainable farming, and all that coming from biotechnology. Thus, CRISPR/Cas9 technology has changed the way we see the future of agriculture. On the other hand, the implementation and easy accessibility to the CRISPR/Cas9 technology has allowed the generation of diverse molecular methodologies that constitute significant advances in the genome edition and its subsequent exploitation for agrarian and health purposes. Therefore, this technique is considered a revo-

The major challenges for CRISPR/Cas9 technology will focus on two underlying aspects. First, the corresponding ethical or bioethical discussion, in order to demarcate what should or should not be done with this tool considering the risks that we could face by using promising technology, an even more when this is accessible and cheap. On the other hand, the legal consequences in terms of intellectual property that today literally generate wars between law firms and universities for the patents generated by thousands of investigations must be considered. Although many scientists consider CRISPR/Cas9 system as a "Holy Grail of genetic engineering," we must not lose sight of the objectivity and rationality when interpreting the consequences of its use. Additionally, demanding compliance with all the necessary safety steps before this technology becomes a trivial routine, especially if this tool is used for genome

The features of the CRISPR/Cas9 system have allowed opening the possibility of using it to perform gene and cell therapy, in addition to its application in plant genetic improvement. In general, this technology has been used as a tool to perform point mutations, homologous recombination by HDR, and silencing and activation or repression of gene transcription. Thanks to these properties, its application has been possible for genetic monitoring, analysis of metabolic pathways, functional genomic research, generation of animal models, discovery of possible targets for disease treatments, and, even, correction of phenotypes. Another application of great importance that should continue to be developed is the generation of more precise and representative plant lines for the study of phytopathogenic diseases. Knockdown, knockout, and knocking models show the advantage of being able to be quickly and efficiently generated with this system. Also, CRISPR/Cas9 is considered a great biotechnological tool in the field of human therapies since its capacity to perform genetic level corrections/deletions, which is traduced in the possibility of regulating transcription or translation pathways.

In relation to the introduction of CRISPR/Cas9 in agricultural and environmental sciences, several studies recognize the possibilities of this technique to improve crop varieties [25, 80]. Uncertainty about safety and efficacy of genome editing requires evaluating its potential and utility by applying the precautionary principle. Research on this technology also unchains important legal and social debates among genetic engineering and genomic editing, in order to establish whether new mechanisms are needed to regulate research, confined use, voluntary release and if it is necessary to evaluate the possible impact on the environment just like in consumers' health, among other aspects. The application of the precautionary principle in any case must be done considering the available scientific evidence and raising the necessary social and economic considerations, in order to avoid a dogmatic interpretation that could under-

editing, and the genetic improvement of living beings must be imperative.

lutionary tool.

50 Transgenic Crops - Emerging Trends and Future Perspectives

mine or stop scientific progress.

The Cas9 enzyme not always cleavages where it is intended within the genome (a certain DNA sequence must be nearby for that to happen). This is easily accomplished in many genomes, but it may be a limitation in some experiments. Researchers are constantly looking for microbes to obtain enzymes with different genome characteristics to expand the number of sequences that can be modified. One of these enzymes is called Cpf1, and it can be an interesting alternative, since it is smaller than Cas9 and has different characteristics in its sequence that make it highly specific [84, 85]. Another enzyme called C2c2 is able to target RNA instead of DNA, which is why it has a great potential to study RNA and to fight plant viruses with this type of genomes [86].

Several laboratories apply CRISPR/Cas9 technology in order to eliminate specific regions in a gene sequence, thus repressing its function. Those who want to exchange a sequence for another face a more difficult task. When Cas9 cleavages the DNA, the cell makes mistakes by regrouping the loose ends, thus obtaining desired deletions. Researchers who want to rewrite a DNA sequence rely on different repair pathways that can insert new sequences (a process that occurs at a much lower frequency). Cas9 directs the sequence dictated by its guiding RNA, but does not cut it; instead, the bound enzyme changes the letters of DNA, ultimately producing a T where once there was a C [87].

In contrast to the above, recent new gene editing systems have been released using a protein called NgAgo to cleavage DNA at a predetermined site without the need for an RNA guide or a genome-specific neighbor sequence. Instead, the protein (of bacterial origin) is programmed using a short DNA sequence corresponding to the target sequence. However, laboratories have failed to reproduce the results so far, so the effectiveness of this technique cannot be affirmed [88]. There are also other genetic editing systems, some of which have existed for years. For example, scientists rely heavily on a system called lambda Red, which can be programmed to alter DNA sequences without the need for a RNA guide.

In light of the above considerations, we can finally conclude that the biotechnological tools that belong to cas9 toolbox that in synergism with new bioinformatic algorithms increase their potential in a specific and powerful way and help position this technology as a magnificent last generation method for genomic edition, which is considered a revolutionary scientific discovery for both basic and applied research including the field of plant biotechnology, even when there are inconclusive details of its application in the laboratory and very probably a gross ignorance of its nature, for now.
