Team:Guelph/Proposed Implementation
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PROPOSED IMPLEMENTATION
Climate change is a global issue that leaves no one unaffected. Every industry is impacted by climate change, but the one facing the greatest risk is plant agriculture. Abnormal temperature fluctuations along with periods of drought and flooding can kill crops. It also makes plants weaker, and therefore more susceptible to pathogens.
This is a widespread and rapidly developing problem, but iGEM Guelph’s 2021 project can bring us one step closer to the solution. Our project is a ‘proof of concept’, which means it shows that this type of work is possible, but it does not actually implement it. Here, we describe the processes through which our technology might be used, and the considerations that have to be made in order to make that happen.
Using the AlcR promoter we are able to use ethanol to regulate the change within our plants. Ethanol is miscible with water making it an easy substance to irrigate plants with. Irrigation methods are a key player in upscaling our project as the delivery of ethanol needs to be measured. Given how important water is to agriculture, it is safe to assume the farmers have some method of irrigation.
Weather forecasting is a vital tool in farming because to produce successful crops farmers need to be aware of moisture, temperature and light conditions. Farming costs can be greatly reduced by combining irrigation schedule with weather forecasting. As weather patterns become more erratic with the on going climate change crisis weather forecasting tools like the farmer’s almanac or localized weather stations farmers can accurately monitor when and how much to water and fertilize plants.
Forecasting tools can also provide farmers with information on extended droughts and arid conditions. With this knowledge farmers have the ability to alter their plants to help them grow successfully.
In modern day farming, large scale irrigation is done by pipe systems or some form of sprinkler setup. These irrigation systems also have the capability to be automated. Automation of irrigation requires past, present and future data on the weather which can be found in an almanac, from regional weather stations or even gathered from local news networks or government stations for your region. Using this data and an automated irrigation schedule can ensure that farmers plants are also in their peak fitness for their daily climate.
Safety
When planning how to implement our technology as a usable product, it is important to consider the various risks that crop plants such as maize or soy with our genetic modifications might pose to humans and to the general environment. Potential risks which may be posed to the environment by genetically modified (GM) crops include the potential of seeds or pollen escaping into the wild or spreading transgenes into other plant varieties (Weebadde & Maredia, 2011). One danger is that GM or wild type-GM hybrid plants in the wild may outcompete other plants due to the advantage conferred on them by their genetic modifications, thus acting as invasive species and disrupting local ecosystems, or possibly becoming weeds and overrunning non-GM crop fields. The other environmental danger from escape is that the spread of GM plants with pesticidal properties into the wild may lead to pests rapidly evolving resistance to the pesticide, thus rendering the pest-resistant properties of the commercial GM strain ineffectual (Weebadde & Maredia, 2011).
One advantage that our proposed system has over traditional GM crops is the fact that whereas traditional GM crops constitutively express their advantageous trait, our plants’ gene expression only differs from the wild type when stimulated by a chemical inducer, namely ethanol. Since ethanol is not commonly found in nature, if offspring of our plants were to escape they should in theory be phenotypically identical to non-GM plants and thus would pose less danger of becoming invasive. Additionally, our plants have no transgenic resistance to pests or herbicides and thus do not pose a risk for spreading resistance in the environment.
Nonetheless, as with all GM crops it is good to attempt to limit escape if feasible, for instance by growing them at a distance from other crops and from wild plants (Weebadde & Maredia, 2011). Various biological solutions have been proposed to contain the spread of transgenic organisms, known as gene use restriction therapies or GURTs (Hancock & Halsey, 2011). These can include using male-sterile strains to prevent pollen release, or using a site-specific recombinase controlled by an organ- or season-specific promoter to excise transgenes from reproductive cells at the start of the flowering process (Hancock & Halsey, 2011). However, GURTs such as the latter method which entirely prevent the propagation of the GM strain or transgene raise significant ethical concerns, because it prevents farmers from saving seeds and places them in a state of continued dependence towards the company producing the GM seeds. Thus, such exploitative practices ought to be avoided, and have been banned by multiple national and international regulation agencies (Lombardo, 2014). Given the current regulatory climate, it may thus be preferable to control spread through responsible distancing of crops from other plants, and rely on the alcR promoter to inhibit expression of the CRISPRa complex in the wild. Experiments would need to be done to examine whether the alcR promoter is a sufficient inhibitor in the absence of ethanol.
Another risk associated with GM plants which must be considered is the possibility of health risks to humans, particularly since we envision using this technology in food plants. There is a possibility that our modified crops could cause allergic reactions or have toxic effects when consumed, and tests in this regard would need to be conducted before the technology could be transformed into a marketable product. To test for allergenicity, we can conduct in vitro tests using immunoblotting to see if exposed animal or human test subjects produce antibodies specific to the dCas9 protein, similar to what has been done with Bt corn (Bawa & Anilakumar, 2013). For toxicity tests, it is a simple matter of testing first on animal and then human test subjects (Weebadde and Maredia, 2011). The only transgenic protein in our crops is the dCas9-VP64 fusion protein. Since it is a version of Cas9 whose endonuclease activity has been inactivated, the protein is unlikely to cause any gene edits to the plant other than the edits caused by the initial insertion of the transgenic material. Other research has shown that Cas9 is easily digested and readily denatured at high temperatures, and that its only resemblance to known allergens is one peptide sequence which is similar to serine carboxypeptidase 2, an occasionally allergenic protein found in wheat (Nakajima et al., 2016). There is thus not a high prior likelihood of GM plants with dCas9 causing problems, although experimental trials would have to be conducted to be sure.
Conclusion
In conclusion, the use of crops which have been genetically modified to have inducible CRISPR-mediated up-regulation of target genes could have an increased survival rate of extreme weather effects, if coupled with good weather prediction systems. Farmers would apply ethanol to their plants in advance of cold snaps or drought to increase the expression of the appropriate survival genes. From a safety perspective, our technology would have an advantage relative to other GM crops in that the only transgenic protein would be an inactivated Cas9 repurposed as a specific gene transcription activator, so most phenotypic changes would be the result of the upregulation of the plant’s own genes. Since the CRISPR activation system is under the control of an ethanol-inducible promoter, the effects would only manifest in the presence of ethanol and thus pose a lower risk in the event of seeds escaping into the wild.
References
AgriTech Tomorrow. Weather Forecasting for the Farmer. <a href="https://www.agritechtomorrow.com/article/2020/02/weather-forecasting-for-the-farmer/11981">https://www.agritechtomorrow.com/article/2020/02/weather-forecasting-for-the-farmer/11981</a>. Accessed Oct 5, 2021.
Bawa, S., & Anilakumar, K.R. (2013). Genetically modified foods: Safety, risks and public concerns - A review. Journal of Food Science Technology 50(6). doi: <a href="https://dx.doi.org/10.1007%2Fs13197-012-0899-1">10.1007/s13197-012-0899-1</a>
Hancock, J., & Halsey, M. (2011). Control and Monitoring of Gene Flow from Genetically Engineered Crops. In R. Grumet, J. Hancock, K.M. Maredia & C. Weebadde. (Eds.), Environmental Safety of Genetically Engineered Crops (pp. 75-86). Michigan State University.
Lombardo, L. (2014). Genetic use restriction technologies: A review. Plant Biotechnology Journal 12(8). doi:<a href="https://doi.org/10.1111/pbi.12242">https://doi.org/10.1111/pbi.12242</a>
Nakajima, O., Nishimaki-Mogami, T., & Kondo, K. (2016). Cas9 in genetically modified food is unlikely to cause food allergy. Biological and Pharmaceutical Bulletin 39, 1876-1880.
The Old Farmer’s Almanac. Long-Range Weather Forecast for 2021. <a href="https://www.almanac.com/weather/longrange">https://www.almanac.com/weather/longrange</a>. Accessed Oct 5, 2021.
Weebadde, C., & Maredia, K. M. (2011). Environmental Biosafety Issues Associated with Genetically Modified Crops. In R. Grumet, J. Hancock, K.M. Maredia & C. Weebadde. (Eds.), Environmental Safety of Genetically Engineered Crops (pp. 21-29). Michigan State University.v