Genome editing is a type of genetic engineering where the DNA in an organism is inserted, deleted or replaced with another DNA using molecular scissors or genetically modified nucleases (Gaj, Gersbach & Barbas, 2013). The process can be done in plants or in animals to either increase the quantity of production or to improve the quality of production. The organism can also be modified to be an insect, pest or drought resistant which allows it to breed even in those areas with harsh climatic conditions. Genome editing is very important as it helps reduce food shortages in those countries that have embraced the technology of gene editing. Application of new technologies of gene editing like clustered regularly interspaced short palindromic repeats (CRISPR), has proven to be very efficient in editing genes. This is especially of foods (Shan et al., 2013). In this discussion, we shall focus on the applications of CRISPR, its importance and some of the challenges that come with in as far as gene editing in foods is concerned.
CRISPR makes it easier for scientists to precisely alter target genes in order to produce the desired traits in crops with respect to the reason for editing. For instance, it can be applied either to improve the nutrient content in crops or to make them disease and drought resistant. The application of this technology is unique in that it leaves no traces of foreign DNA after the crop or the plant’s genome has been modified. The process is very simple and efficient to use as it requires only three components: Cas9, crRNA (CRISPR RNA) and trRNA (Transactivating RNA).These makes the technology very different from the previously applied technologies like RNA interference and homologous recombination (Shan et al., 2013). With CRISPR, short RNAs can be used to guide nuclease proteins to specific targets within the plant’s genomes.
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CRISPR can be applied in crop gene editing to help produce the desired traits. It can be applied for gene silencing. In this particular case, the use of crRNA and trRNA or the single guide RNA, locations within complex plant genomes can be targeted by cas9 protein(endonuclease) for a specific double stranded break (Gaj et al., 2013). The breaks can be repaired by an endogenous mechanism and when this happens these genes are silenced permanently. This is important when creating disease or drought resistant crops. For instance, tomatoes can be altered to be disease resistant if specific genes responsible for attractions of insects are silenced using CRISPR. The technique can also be applied to create a knocking effect. The double-stranded breaks can be used as an opportunity in inserting a new gene with desired traits (Gaj et al., 2013). That means that a new gene can be inserted into the plant's genome to either help increase yield production or increase its resistance to pests. For instance, crops with low protein content can be modified through the insertion of new genes in order to increase their protein percentages (Cong et al., 2013).
The application or the use of CRISPR is more important as compared to other methods of gene editing. First, it involves no protein engineering. That, therefore, makes it very easy to identify guide RNA for specific targets, making the process cost-effective as well as fast. For instance, if there is a need to alter the genes in order for a guide RNA to match the precise target, only 20 nucleotides need to be changed. This means that the processes of assembling large guide RNA libraries are relatively inexpensive. The other importance is the ability and ease of multiplexing when using CRISPR. Several genes can be edited at the same time making it important for eliminating or knocking out redundant genes. That can also be useful when performing modifications of large genomic deletions or insertions. In addition multiplex editing using cas9 requires only monomeric cas9 proteins while other techniques like zinc finger nuclease require separate dimeric proteins specific for every specific target sites (Cong, Ran, Cox, Lin, Barretto, Habib & Zhang, 2013).
The use of gene knockouts in plants through the use of CRISPR technology has proven to be very useful in eliminating genes that lower crop yields or those genes that affect food quality in a negative manner. For instance, Wang used the technology in modifying genes of the mildew resistance locus in wheat, which successfully generated plants that were resistant to powdery mildew disease (Shan, Wang, Zhang, Chen, Liang & Gao, 2013). Also, the application of precise nucleotide exchange through the use of oligonucleotide donor sequence may be helpful in modifying regulatory sequences which can be useful in improving crop yields (Gaj et al., 2013).
The technique, however, arouses a lot of criticism, the major concern being the targeting of specificity given that cas9 can allow the occurrences of some mismatches between the guide RNA and the complementary target DNA. This usually results from off-site targeting. Since there is not much engineering of genes in plants when using the technique, that means that the plants or foods that have been modified using this technology may not be classified as genetically modified food. This is because the plants that are created using CRISPR are transgene -free mutated plants. That makes them be not classified as transgenic as is the case when other methods of genetic modifications like RNAi are used (Shan et al., 2013).
In conclusion, as seen above, despite the fact that CRISPR is simple and easy to use it faces one major challenge of offsite targeting which if solved in future may leave the technology as the best for gene editing. Therefore, there is a need for structural analysis of CRISPR RNA and its interactions with the other components (trRNA and guide RNA) for improved developments of engineered nucleases with more accurate or high specificity and efficiency.
References
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., ... & Zhang, F. (2013). Multiplexgenome engineering using CRISPR/Cas systems. Science , 339 (6121), 819-823.
Shan, Q., Wang, Y., Li, J., Zhang, Y., Chen, K., Liang, Z., ... & Gao, C. (2013). Targeted genome modification of crop plants using a CRISPR-Cas system. Nature biotechnology , 31 (8), 686-688.
Gaj, T., Gersbach, C. A., & Barbas, C. F. (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in biotechnology , 31 (7), 397-405.
