In the first post on CRISPR-Cas9, I’ve explained how bacteria and archaea create a “database” of infections and use it as a form of prokaryotic immunization. This time, I’m going to concentrate on how biotechnology turns this natural phenomenon into a powerful tool.
CRISPR-Cas9 is probably the most popular of CRISPR systems because of its simplicity. The natural effector complex consists of only three components – crRNA, tracrRNA and Cas9 endonuclease, but the synthetic form is even simpler, engineered as a two-component system by fusing the crRNA and tracrRNA into a single guide RNA (sgRNA or gRNA).
Engineering genomic regions of choice just by supplying two ingredients is a recipe any lab can handle. Directions according to Sander & Joung (2014):
“Twenty nucleotides at the 5′ end of the gRNA (corresponding to the protospacer portion of the crRNA) direct Cas9 to a specific target DNA site using standard RNA-DNA complementarity basepairing rules. These target sites must lie immediately 5′ of a PAM sequence that matches the canonical form 5′-NGG (although recognition at sites with alternate PAM sequences (e.g., 5′-NAG) has also been reported, albeit at less efficient rates). Thus, with this system, Cas9 nuclease activity can be directed to any DNA sequence of the form N20-NGG simply by altering the first 20 nt of the gRNA to correspond to the target DNA sequence.”
CRISPR-based gene drive
Despite all the previously described magical properties of CRISPR-Cas9, its main strength comes with the connection to a gene drive. Gene drive is a genetic element that basically defies the Mendelian laws and via biased inheritance increases in frequency each generation (Champer et al. 2016).
“Although there is no single molecular mechanism underlying all gene drives, they typically induce biased inheritance patterns via one of two methods. The first strategy involves copying themselves onto the opposite chromosome (that is, homing), resulting in most or all offspring inheriting the gene drive allele. The second method involves reducing the viability of gametes that inherit the wild-type allele, thus giving the wildtype allele a fitness disadvantage compared to the gene drive allele.” (Champer et al. 2016)
The combination of gene drive and CRISPR-Cas9 technologies provides “the ability to disperse engineered genes throughout target populations much more quickly than would be possible via simple genetic inheritance” (Webber et al. 2015).
The potential of CRISPR-based gene drives for application in conservation is wide, but the most common examples are:
- Disease resistance
- Control of invasive species
- Pest management
- Supplementing genetic diversity
All of these examples fit into two categories of gene drives: suppression and modification. While the purpose of suppression drives is reducing the populations of disease vectors, pests, and invasive species, modification drives aim to spread beneficial alleles or increase adaptive potential (Champer et al. 2016).
Among the suppression drives, the genetically modified malaria mosquitoes represent one of the CRISPR-Cas9 applications in making. Studies working with the malaria mosquitoes Anopheles gambiae (Hammond et al. 2016) and Anopheles stephensi (Gantz et al. 2015) made a substantial progress, reporting introgression rates to progeny of up to 99.6%.
These malaria mosquito gene drives use different approaches of population suppression. The work of Hammond et al. (2016) is based on inducing female sterility and possibly eradicating the mosquito population. On the other hand, Gantz et al. (2015) created anitpathogen effector genes, making the mosquitoes resistant to the pathogen.
Genetically engineered dengue mosquitoes have already reached the stage of field tests and so far have been pretty successful, reducing the target population by ~90% (Carvalho et al. 2015). However, these transgenic mosquitoes didn’t include gene drives, which means that a long-term release of a tremendous amount of these would need to be secured to suppress the population. Nonetheless, gene-drive equipped malaria mosquitoes are not there yet (Alphey 2016).
This example was rather human-health oriented, but it’s analogous to what can be done in terms of eliminating animal diseases, invasive species and pests. To learn more on this topic, have a look at the in-depth reviews of Esvelt et al. (2014) and Gurr & You (2016).
“Most attention so far has focused on doing bad things to unwanted species, but it may also be possible to use gene drives to do good things to wanted species, such as helping the survival of threatened species by increasing resistance to novel biological or abiotic threats.” (Corlett 2016).
The idea of resupplying genetic diversity and resurrecting species currently seems much more far-fetched than releasing mosquitoes engineered by gene drives, but considering the speed with which this field is advancing, I have to be careful with my statements.
While the CRISPR-based gene drive could be used for “fixing” bottlenecked populations and genomic regions with low diversity (Johnson et al. 2016), such research is lacking at the moment. However, the topic of de-extinction has gained quite a momentum. It’s become a subject of newspaper headlines, popular-science articles, books, and last but not least, science articles, e.g. Shapiro (2015).
“While extinction is forever, there is little doubt that genome engineering can and will be used to resurrect extinct traits.” (Shapiro 2015)
More on de-extinction in my post: How to Clone a Mammoth: When science fiction becomes reality.
The moral dilemma
”Conservationists have been wary of using genetic modification for wild species because of both restrictive government regulations and continued public suspicion, as well as lingering concerns in the conservation community itself.” (Corlett 2016)
So far, classical biological control of pests and invasive species has been the only cost-effective management tool (Webber et al. 2015); however, conservation biology has a long history of “biocontrol gone wrong” cases, e.g. the introduction of cane toads to Australia (Phillips et al. 2006).
There are many weak aspects of the CRISPR-based gene drive, which need to be addressed before any of this becomes reality.
“The biggest concern is that, once a gene drive is released, it may be impossible to stop, although ‘reverse gene drives’ that cancel the original mutation are being developed. There is also the possibility that the deleterious trait could spread into the natural range of the species, leading to its global extinction.” (Corlett 2016)
This time conservation biologists cannot afford to fail. And knowing that, there is a wide range of attitudes towards how the gene-engineering should be applied to practice. From cautious highlighting the need for ethical and legal considerations…
“Practical conservation requires that practitioners balance the risks of active intervention with those of doing nothing. With endangered species, the risks of doing nothing are, by definition, high. However, before using any new technology, practitioners need clear guidance.” (Corlett 2016)
… to calling CRISPR-based gene drive a potential global threat.
“Irrespective of how these biosecurity risks are perceived, we caution that without a regulatory framework that provides a mechanism to work through these issues with clarity and transparency for CRISPRCas9 gene drive, this putative silver bullet technology could become a global conservation threat.” (Webber et al. 2015)
Alphey, L. (2016). Nature Biotechnology 34, 149–150. DOI:10.1038/nbt.3473
Carvalho, D.O., McKemey, A.R., Garziera, L., Lacroix, R., Donnelly, C.A., et al. (2015) Suppression of a field population of Aedes aegypti in Brazil by sustained release of transgenic male mosquitoes. PLoS Negl. Trop. Dis. 9(7): e0003864. DOI: 10.1371/journal.pntd.0003864
Champer, J., Buchman, A. & Akbari, O.S. (2016). Cheating evolution: engineering gene drives to manipulate the fate of wild populations. Nat. Rev. Genet. 17, 146–159. DOI: 10.1038/nrg.2015.34
Corlett, R.T. (2016). A Bigger Toolbox: Biotechnology in Biodiversity Conservation. Trends Biotechnol. In press. DOI: http://dx.doi.org/10.1016/j.tibtech.2016.06.009
Esvelt, K.M., Smidler, A.L., Catteruccia, F., & Church, G.M. (2014). Concerning RNA-guided gene drives for the alteration of wild populations. eLife, 3, e03401. DOI: http://doi.org/10.7554/eLife.03401
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. Proc. Natl. Acad. Sci. 112, E6736–E6743. DOI: 10.1073/pnas.1521077112
Gurr, G.M. & You, M. (2016). Conservation biological control of pests in the molecular era: New opportunities to address old constraints. Front. Plant. Sci. 6, 1255. DOI: http://dx.doi.org/10.3389/fpls.2015.01255
Hammond, A., Galizi, R., Kyrou, K., Simoni, A., Siniscalchi, C., Katsanos, D., Gribble, M., Baker, D., Marois, E., Russell, S. et al. (2016). A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat Biotechnol 34, 78–83. DOI: 10.1038/nbt.3439
Johnson, J.A., Altwegg, R., Evans, D.M., Ewen, J.G., Gordon, I.J., Pettorelli, N. & Young, J.K. (2016). Is there a future for genome-editing technologies in conservation? Anim. Conserv. 19, 97–101. DOI: 10.1111/acv.12273
Phillips, B.L., Brown, G.P., Webb, J.K. and Shine, R. 2006. Invasion and the evolution of speed in toads. Nature 439, 803. DOI: 10.1038/439803a
Sander, J.D. & Joung, J.K. (2014). CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32(4):347–355. DOI: 10.1038/nbt.2842
Shapiro, B. (2015). Mammoth 2.0: will genome engineering resurrect extinct species. Genome Biol. 16, 228. DOI: 10.1186/s13059-015-0800-4
Webber, B.L., Raghu, S. & Edwards, O.R. (2015). Opinion: is CRISPR-based gene drive a biocontrol silver bullet or global conservation threat? Proc. Natl Acad. Sci. USA 112, 10565–10567. DOI: 10.1073/pnas.1514258112