CRISPR/Cas9 and The Future of Global Health

BY AKHIL UPNEJA

The discovery of CRISPR/Cas9 has revolutionized the field of genetic engineering in countless ways. From targeting genes conferring antibiotic resistance to creating disease models in animals, the technique offers scientists a fast, cheap, and accurate alternative to every other gene-editing system on the market. While its applications in human disease continue to take massive strides, including a recent human trial, CRISPR has entered the global health realm as scientists attempt to solve some of the most important crises in the field. UC Berkeley and UC San Francisco plan to invest $125 million over the next five years to create solutions to some of the world’s most pressing problems, and CRISPR co-discoverer Jennifer Doudna, who received an honorary degree from Yale in 2016, has publicly said that the technology can and will be used to promote global health in the agriculture and microbial worlds.¹ In this piece, I will expand on how CRISPR is being used, and will continue to be used, in the fight against insect-borne diseases and the global food shortage.

To understand how the technique is being used, it is first important to understand how CRISPR works at a fundamental level. The system, which takes the form of a double-stranded circular piece of DNA called a plasmid, contains DNA sequences for dozens of elements. The two most important elements for this discussion are a destroyer protein called Cas9 and a 20-nucleotide guide RNA sequence that can be designed by scientists to target any gene in the genome. The guide RNA sequence attaches to Cas9 and acts as a homing signal that takes the protein to the specified location and destroys the gene of interest. CRISPR/Cas9 can be used not only for deletion, but also for modification and insertion, which will be critically important for the applications discussed below.

This technology is already having a profound impact on issues relating to the global food crisis in three ways: decreasing flowering time, preventing disease, and increasing yield in drought conditions. First, scientists at the Cold Spring Harbor Laboratory have been experimenting with flowering times for the domesticated tomatoes that are sold in supermarkets all across North America. They found that while domesticated tomatoes are not sensitive to the amount of daylight they get per day, their ancestors — the wild equatorial species — are particularly sensitive to the Sun’s rays.² The researchers were able to trace this back to SP5G, an anti-florigen gene that regulates flowering periods depending on the amount of daylight present. Expression of SP5G spiked in the equatorial tomatoes when they grew in greenhouses in the US, and domestic tomatoes also contained residual amounts of SP5G expression. The researchers decided to eliminate SP5G from the tomato genome, letting the tomato plant flower regardless of daylight. Using CRISPR/Cas9, SP5G was deleted, and they found a rapid increase in flowering time, increasing yield and making the process of production much more rapid.² Increasing the productivity of each individual crop could potentially yield vast dividends for the world’s food supply.

Second, crops resistant to diseases represent a new wave in holding crop yields steady. Powdery mildew is a disease that manifests itself as a white fungus on the surface of plant parts.³ It is a ruthless killer that has developed resistance to many traditional fungicides, and thus it exemplifies exactly the type of case that scientists are looking to eliminate with CRISPR. Before CRISPR/Cas9, it took years of selectively cross-breeding specific crops to pass down favorable genes. With this technique, however, farmers also inevitably get unfavorable genes that are a part of the genomes of the “favorable” plants, thus making this process inefficient and only partially successful. In 2014, scientists at the Chinese Academy of Sciences in Beijing identified three genes that were repressing the ability of wheat to protect itself from powdery mildew.⁴ The scientists used CRISPR/Cas9 to introduce loss-of-function mutations to each of these three genes. Through their experiments, the bread wheat showed heritable resistance to powdery mildew, a huge breakthrough in the field of disease-resistant crops.⁴

Third, drought conditions are a huge crop killer. An analysis published in Nature in 2016 showed that droughts have reduced crop yields by 10 percent over the past 50 years.⁵ The authors warned that drought conditions continue to get worse with climate change, and that yields will continue to deteriorate as global temperatures rise.⁵ Maize in particular has been severely affected by drought. The UN’s Food and Agriculture Organization declared that maize production had declined in Malawi by 27 percent due to extreme heat and low rainfall.⁶ To combat this, scientists continue to search for ways to encourage plants to adapt to low-water conditions. A great example of where CRISPR has aided with this discovery is in maize, which has a natural ethylene response that regulates stress in drought conditions. A study published in 2016 found that in maize, a gene named ARGOS8 was a negative regulator of ethylene responses.⁷ It was hypothesized that by increasing expression of ARGOS8, crop yields in drought conditions could potentially increase. CRISPR was used to insert a promoter before the ARGOS8 gene to increase expression, and modified crops were tested for their yield against unchanged crops in both drought conditions and normal conditions. In overexpressing ARGOS8, the scientists found that maize production substantially increased in the changed crops with less water, and that with normal amounts of precipitation, yield remained unchanged.⁷ As drought becomes an increasingly important issue, CRISPR/Cas9 modifications represent game-changers in the fight against the global food shortage.

Perhaps the most controversial application of CRISPR/Cas9 involves the use of gene drives as a weapon against insect-borne diseases. Normally, genes have two copies in a genome, and the likelihood that any given copy is passed down to offspring is 50 percent. However, if present, a gene drive is a molecular mechanism that makes the transmission of a particular allele almost 100 percent. Scientists at the Wyss Institute engineered a gene drive utilizing CRISPR-Cas9 that can be incorporated into fly genomes to ensure that a specific allele is passed down from generation to generation. Suppose that a fly carrying a blue coloring allele mates with one that carries a black coloring allele. Their offspring will subsequently have one copy of each allele. If we want the blue allele to be passed down, we would engineer two sequences into the germ-line cells of the fly: a sequence coding for Cas9 and another sequence coding for guide RNAs against the black allele. When the blue allele is then transcribed and translated, Cas9 and guide RNA are created with it. Cas9 subsequently attaches to the guide RNA and combs the genome to find the black allele. When the black allele is identified, Cas9 creates a break in the DNA. At this point, there are two mechanisms by which the cell can repair its DNA. The first is the non-homologous end-joining pathway, in which the DNA simply tries to stick the two ends back together. This is an error-prone pathway because a part of the DNA was cut out, so the DNA will not be the same when it is ligated back together. The second pathway is the homologous recombination pathway. In this pathway, the DNA searches for the analogous place where the DNA was cut on the complementary strand. In this case, that would be where the blue allele is. A copy of the allele, including the gene drive sequence, is then made and inserted in the cut strand. Thus, the fly now has two copies of the same allele with the gene drive in place, meaning that any fly that it mates with will become blue in the same way, regardless of the allele pairing of the other parent.⁸

To explain how gene drives have been employed in the fight against malaria, it is important to first understand how malaria is spread in vulnerable populations. First, a parasite called Plasmodium falciparum enters mosquitoes in gametocyte form.⁹ Two gametes then fuse to create a zygote, which develops into an ookinete. These ookinetes travel to mosquito guts, where they make their home and mature into oocysts. These oocysts release sporozoites that invade the circulatory fluid of mosquitoes, settling in their salivary glands. When mosquitoes bite humans, they inject sporozoites into our bloodstream, and these sporozoites cause malaria in our bodies.⁹ Malaria continues to be a deadly killer – the World Health Organization (WHO) estimates that there were 212 million new cases of malaria in 2015 alone, causing 429,000 deaths worldwide.¹⁰ While prevention efforts by the WHO have reduced the disease burden by 20% over the past five years, drastic measures need to be taken, in sub-Saharan Africa and beyond, to stop the spread of this deadly disease.¹⁰

The understanding of how malaria is spread through humans led to a scientific breakthrough in 2015, when scientists from UC Irvine and UC San Diego were able to identify several antibodies that conferred malaria resistance onto mosquitoes. Publishing their results in Proceedings of the National Academy of Sciences, the paper identified IC3 and 2A10 as potential malaria stoppers.¹¹ IC3 blocks an enzyme called chitinase 1, which is critical in oocyst formation. By blocking the spread of malaria at such an early stage, parasites are theoretically unable to ever get into the salivary glands of the mosquitoes, thus stopping malaria’s spread to humans. The other antibody, 2A10, is a later-stage inhibitor that stops sporozoites from successfully infecting cells, essentially acting as a second inhibitor in case IC3 does not work. In 2015, the same lab decided to add these the genes coding for these antibodies, with CRISPR-mediated gene drives attached, into the genome of Anopheles stephensi, an urban Indian mosquito species well-known for its ability to infect humans in the sub-continent with malaria. Within just a few generations, the genes were transferred successfully into over 99 percent of mosquitoes. In those tested mosquitoes that received the genes, no sporozoites were found in their salivary glands, an encouraging sign that the CRISPR-mediated gene-drive works and that this technique can be applied to other insect-borne diseases like West Nile virus and dengue fever.¹¹

Still, controversies abound regarding whether or not these engineered mosquitoes should be released into the wild. The main ethical issue concerns the accuracy of experimental testing – indeed, there is no accurate experimental method for testing how mosquitoes will behave in the wild.¹² Were scientists to release an untested product, the environment would reap the consequences on evolution, not just in the experimental species, but also in non-contact species that rely on the experimental organisms for survival.¹² Additionally, there is the idea that evolution always finds a way. Indeed, studies have found that over time, mosquitoes will start favoring the non-homologous end-joining pathway that will confuse the gene drive system, simply stop mating with mosquitoes carrying the gene-drive, or develop an unknown resistance that computer models cannot identify.¹³

While evolution will always play out over the long-run, it is critically important that we continue attempting to innovate in order to at least delay the rapid onset of diseases killing millions of people each year. Regardless of the ethics behind gene drive, scientists must push forward with CRISPR’s answers to the food shortage problems across the world. The World Bank estimates that the world needs to produce 50 percent more than our current output of food by 2050, while climate change cuts production by 25 percent in the same time period.¹⁴ CRISPR provides a simple, yet efficient mechanism for modifying specific genes of interest, and it remains the best available option for us to continue innovating in order to fulfill the most basic needs of our society today.

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References:

1. Sanders, R. (2017). CRISPR research institute expands into agriculture, microbiology. University of California. Retrieved from http://news.berkeley.edu/2017/01/24/crispr-research-institute-expands-into-agriculture-microbiology/.
2. Soyk, S. et al. (2016). Variation in the flowering gene SELF PRUNING 5G promotes day-neutrality and early yield in tomato. Nature Genetics. Retrieved from http://www.nature.com/ng/journal/v49/n1/abs/ng.3733.html.
3. Floriculture and Ornamental Nurseries: Powdery Mildew. (2009). In Agriculture and Natural Resources. San Francisco, CA: University of California.
4. Wang, Y., Cheng, X., Shan, Q., Zhang, Y., Liu, J., Gao, C., & Qiu, J. (2014). Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology. Retrieved from http://www.nature.com/nbt/journal/v32/n9/full/nbt.2969.html.

5. Lesk, C., Rowhani, P., & Ramankutty, N. (2016). Influence of extreme weather disasters on global crop production. Nature. Retrieved from http://www.nature.com/nature/journal/v529/n7584/full/nature16467.html.
6. Tembo, F. (2015). Malawi confirms maize deficit: Agriculture production down by 27.7 percent. Nyasa Times. Retrieved from http://www.nyasatimes.com/malawi-confirms-maize-deficit-agriculture-production-down-by-27-7-percent/.
7. Shi, J. et al. (2016). ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant Biotechnology Journal. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/27442592.
8. FAQs: Gene drives. (n.d.). Wyss Institute for Biologically Inspired Engineering at Harvard. Retrieved from https://wyss.harvard.edu/staticfiles/newsroom/pressreleases/Gene%20drives%20FAQ%20FINAL.pdf.
9. Isaacs, A et al. (2011). Engineered Resistance to Plasmodium falciparum Development in Transgenic Anopheles stephensi. PLoS Pathogens. Retrieved from http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1002017.
10. Fact sheet about Malaria. (2016). World Health Organization. Retrieved from http://www.who.int/mediacentre/factsheets/fs094/en/.
11. Gantz, V. M., Jasinskiene, N., Tatarenkova, O., Fazekas, A., Macias, V. M., Bier, E., & James, A. A. (2015). Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. Proceedings of the National Academy of Sciences. Retrieved from http://www.pnas.org/content/112/49/E6736.abstract.
12. Borell, B. (2016). When Evolution Fights Back Against Genetic Engineering. The Atlantic. Retrieved from https://www.theatlantic.com/science/archive/2016/09/gene-drives/499574/.
13. Callaway, E. (2017). Gene drives thwarted by emergence of resistant organisms. Nature. Retrieved from http://www.nature.com/news/gene-drives-thwarted-by-emergence-of-resistant-organisms-1.21397.
14. Food Security: Overview. (2016, March 21). The World Bank. Retrieved from http://www.worldbank.org/en/topic/foodsecurity/overview.