Gene editing in agriculture
Gene editing involves making distinct changes in the DNA sequence of a plant/animal cell creating a desired genetic make-up. Gene editing is a powerful tool for genome editing which requires high specificity and uses enzymes known as nucleases which cut the DNA at the desired site while another sequence is inserted to replace the cut sequence (Fridovich-Keil, 2019). The development of gene editing techniques was challenging; however, the breakthrough of gene editing was made successful by the ability to make breaks in the target area of double strand DNA and make repairs with a replacement sequence. DNA double strand breaks (DSBs) are made using nuclease enzyme which is followed by initiation of DNA repair mechanisms. DSBs repairs can be made in two ways, through homologous (HR) and non-homologous end joining (NHEJ). Non-homologous end joining causes random insertions or deletions resulting in gene knockouts. Alternatively, a DSB can cause an overhang allowing for the introduction of a template sequence with compatible overhangs. Homologous recombination is achieved when a template with homology to region surrounding the DSB is used in repairing the DNA damage (Bortesi & Fischer, 2015).
Origin of CRISPR/Cas9
Clustered DNA repeats were first identified by a Japanese scientist, Yoshizumi Ishino, in the 1980s who found strange repeats in bacterial DNA. The term clustered regularly interspaced short palindromic repeats (CRISPR) was coined in 2001 by Francisco Mojica and Ruud Jansen. Later, independent scientists revealed that the clustered repeats in bacteria DNA came from viruses that had previously attacked the bacteria which was a smart way of defence against the attacking viruses (Richardson, 2019). CRISPR/Cas9 gene editing technology was first described by Jennifer Doudna and her colleagues in the US in 2012 (Jinek et al., 2012) and modified in 2013 (Ran et al., 2013). Cas9 (CRISPR associated systems 9) is the protein that performs the cleavage to effect site specific nicks on both sides of double stranded DNA using RuvC and HNH sites (Gasiunas et al., 2012).
Use of CRISPR/Cas9 system in eukaryotic gene editing
CRISPR is a RNA guided system that can be used to edit genomes by using a lead RNA with a specific 20-nucleotide sequence (Ran et al., 2013). The CRISPR/Cas9 technology involves the design of DNA-RNA heteroduplex which is bound to a Cas9 nuclease enzyme. The CRISPR part which is made up of small interfering sequences with identical repeats known as spacers that target the genome sequence acts as a guide to the desired target. The Cas9 enzyme cuts the target DNA at the intersection of target-specific sequence and the palindromic repeat sequence of the guide RNA (Jinek et al., 2012).
Application of gene editing technology in tackling challenges in agriculture
CRISPR/Cas9 gene editing technology applications have been demonstrated to have a huge potential in improving crops such as rice. With several studies focusing on the potential for improvement of biotic and abiotic stress tolerance (Jaganathan et al., 2018). Precision genome editing using CRISPR technology has been used successfully in crops such as wheat, rice and maize. In wheat, TaMLO gene knock out using CRISPR has been identified to confer resistance to powdery mildew disease (Wang et al., 2014). Kim et al. (2018) reported on successful editing of abiotic response genes namely TaDREB2-which is responsible for wheat dehydration responsive element binding protein 2 and TaERF3 which controls ethylene responsive factor 3. In maize, CRISPR technology is being used to improve nutritional composition by knocking off phytic acid biosynthesis genes (Liang et al., 2014). CRISPR/Cas9 is also being used to improve horticultural crops in order to enhance shelf life, increase disease resistance and yield (Karkute et al., 2017). There are several other crops that have undergone improvement using CRISPR/Cas9 technique and will soon be available for cultivation. They include, a browning resistant mushroom, a waxy corn with enriched amylopectin, late flowering green brittle grass, high oil content camelina and drought tolerant soybean (Jaganathan et al., 2018).
CRISPR/Cas9 system can be used to introduce genes controlling traits that are hard to introduce through other methods of conventional breeding such as traits controlled by multiple genes (quantitative traits). Gene editing tools that can introduce multiple genes of interest can accelerate breeding of agricultural crops.
CRISPR/Cas9 Vs previous gene editing technology
Transcription activator like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs) were used for gene editing prior to the discovery of CRISPR/Cas9. Both TALENs and ZFNs combines endonucleases FokI and DNA binding domains to give rise to chimeric proteins. Their deployment in gene editing is limited because ZFNs binding domain are not easy to construct while the tandem repeat units of TALENs are complex in nature, which may affect the stability of the vector in plant and animal genomes. While TALENs are easier to construct than ZFNs when longer recognition sequences are involved, the process of vector construction is difficult and complex because ZFNs and TALENs functions as dimers which have weak bonds (Ma et al., 2015). ZFNs have less target specificity than TALENS, have low number of target sites and also produce many off target cuts (Chen & Gao, 2013). Construction of TALENs is complex as they require a thymidine base at the onset and are repetitive (Jaganathan et al., 2018). Not all TALENS function effectively to deliver the expected mutations, thus they require validation to confirm their success (Bortesi & Fischer, 2015). CRISPR/Cas9 employ RNA-DNA binding as opposed to protein-DNA binding in the previous methods to lead the nuclease activity. This unique characteristic makes the design simple and versatile for application in a range of eukaryote sequences (Fridovich-Keil, 2019).
The heteroduplex construct in CRISP/Cas9 is stable as its RNA is designed to bind only to the target sequence based on DNA base pairing (Jinek et al., 2012). CRISPR/Cas9 has yielded more successful mutations in both plants and animals than ZFNs and TALENs (Bortesi & Fischer, 2015). CRISPR/Cas9 does not require cloning and can reach targets that are beyond other nucleases as it can cut methylated human DNA (Bortesi & Fischer, 2015). CRISPR/Cas9 main advantage over ZFNs and TALENs is that it can be used to edit multiple target sites simultaneously (Bortesi & Fischer, 2015).
ZFNs have been used in the modification of genes in a number of plant cells and organisms. It has been used in Arabidopsis for targeted gene inactivation. The ABI4 gene was deleted and inactivation giving rise to ABA and glucose insensitive phenotypes (Chen & Gao, 2013).
TALENs have been used to modify a number of organism’s genomes including in Drosophila. TALENs were designed to modify the yellow gene on the sex chromosome in Drosophila and introduced to the embryos. Inheritable modifications were detectable with a duration of four weeks (Chen & Gao, 2013).
Random mutagenesis has been used in breeding programs to introduce new genetic alleles that are not present in nature. Mutation breeding involves the use of radiation (x-rays or gamma radiation), chemical mutants and site directed mutagenesis. New varieties of crops bred through mutagenesis are in cultivation in many countries. Some of the crops include, wheat, rice, leguminous crops, soybean and vegetatively propagated crops. However, this method of plant breeding requires laborious screening to select mutants which have the desired traits and mutation confirmation (Bortesi & Fischer, 2015). Genome editing can hasten breeding by introducing desirable traits to an elite background. The off- target mutations caused by CRISPR/Cas9 gene editing is way below those caused physical chemical mutagenesis (Bortesi & Fischer, 2015).
Regulation and ethics relating to the application of gene editing
Gene editing technology has been successfully applied for editing genomes to correct genetic disorders in humans and confer resistance to pest and diseases in plants. However, the CRISPR/Cas9 gene editing technology has not been without controversy over its application in editing human embryos. The debate has been going on whether the editing of genes in human embryos is ethical. The scientists behind the discovery of this technology have sought to limit the application of CRISPR/Cas9 gene editing technology in human embryos (Fridovich-Keil, 2019).
Another concern on the application of CRISPR/Cas9 gene editing technology is due to off target mutations which were reported in early studies. These off-target mutations could have long term effects on patients including malignancy. Although some improvements have been made to overcome this short coming, including considered design of guide RNA and biolistic delivery of CRISPR/Cas9 ribonucleoproteins, more studies are required to minimise/eliminate off-target effects and long lasting safety for any treatments (Jaganathan et al., 2018). Any gene editing that involves germline is permanent and it may have long term consequences that are not clear for now. There is a strong opposition on germline modifications as they may lead to genetic enhancement that is not therapeutic. The ethical confines of CRISPR/Cas9 technology have to be clearly defined.
Gene editing technologies including CRISPR/Cas9, ZFNs and TALENs are not classified as genetically modified organisms (GMOs) in many countries and may face lesser regulations than GMOs. In the US CRISPR/Cas9 edited plants may be sold freely without regulatory monitoring. This will reduce the time taken and costs to release plants improved this technology. Regulatory restrictions have hindered the adoption of GMOs especially in Europe and other countries outside the US (Jaganathan et al., 2018).
The outlook for CRISPR/Cas9 technology is bright as it will have wide applications in agriculture going in to the future. It’s application in plant and animal improvement will help in solving some of the challenges that conventional plant breeding has not been able to overcome. Breeding plants that are high yielding, are pest and disease resistant, tolerance to abiotic stress, enhance aesthetic value, increased shelf life and nutritional value are some of the areas this technology will be applied in the future. The ability to edit multiple genes is a huge advantage of CRISPR/Cas9 technology over other breeding methods. Virus resistance in plants is one of the areas where gene editing technology will be useful. Conferring specific and broad-spectrum resistance will be important in combating viral diseases in cultivated crops. Another area of interest is elimination/reduction of anti-nutritional factors in legumes and leafy vegetables through modification of biosynthesis pathways.
It will be important to create regulatory framework to govern the production and commercialisation of gene edited plants. The technology will also have to gain wide acceptance among consumers for it to be successfully deployed commercially in agriculture.
- Bortesi, L., & Fischer, R. (2015). The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnology Advances. https://doi.org/10.1016/j.biotechadv.2014.12.006
- Chen, K., & Gao, C. (2013). TALENs: Customizable Molecular DNA Scissors for Genome Engineering of Plants. Journal of Genetics and Genomics, 40(6), 271–279. https://doi.org/https://doi.org/10.1016/j.jgg.2013.03.009
- Fridovich-Keil, J. L. (2019). Gene editing. Retrieved December 19, 2019, from Encylopaedia Britannica website: https://www.britannica.com/science/gene-editing
- Gasiunas, G., Barrangou, R., Horvath, P., & Siksnys, V. (2012). Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America, 109(39). https://doi.org/10.1073/pnas.1208507109
- Jaganathan, D., Ramasamy, K., Sellamuthu, G., Jayabalan, S., & Venkataraman, G. (2018). CRISPR for Crop Improvement: An Update Review. Frontiers in Plant Science, 9, 985. https://doi.org/10.3389/fpls.2018.00985
- Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 337(6096), 816–821. https://doi.org/10.1126/science.1225829
- Karkute, S. G., Singh, A. K., Gupta, O. P., Singh, P. M., & Singh, B. (2017). CRISPR/Cas9 Mediated Genome Engineering for Improvement of Horticultural Crops. Frontiers in Plant Science, 8. https://doi.org/10.3389/fpls.2017.01635
- Kim, D., Alptekin, B., & Budak, H. (2018). CRISPR/Cas9 genome editing in wheat. Functional and Integrative Genomics, 18(1), 31–41. https://doi.org/10.1007/s10142-017-0572-x
- Liang, Z., Zhang, K., Chen, K., & Gao, C. (2014). Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. Journal of Genetics and Genomics, 41(2), 63–68. https://doi.org/10.1016/j.jgg.2013.12.001
- Ma, X., Zhang, Q., Zhu, Q., Liu, W., Chen, Y., Qiu, R., … Liu, Y.-G. (2015). A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Molecular Plant, 8(8), 1274–1284. https://doi.org/10.1016/J.MOLP.2015.04.007
- Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nature Protocols, 8(11), 2281–2308. https://doi.org/10.1038/nprot.2013.143
- Richardson, M. W. (2019). CRISPR Explained. Retrieved December 19, 2019, from BrainFacts website: https://www.youtube.com/watch?v=UKbrwPL3wXE
- Wang, Y., Cheng, X., Shan, Q., Zhang, Y., Liu, J., Gao, C., & Qiu, J. L. (2014). Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology, 32(9), 947–951. https://doi.org/10.1038/nbt.2969