As gene drives are in an early stage of development, the discussion about their possible consequences and risks is still largely speculative. However, numerous critical points are already emerging that need to be considered before a possible release.
Uncontrollability
Once released into the wild, a gene drive organism actively spreads in free-living populations and can spread rapidly over large distances. The vast diversity of natural habitats and ecosystems affected will make the prediction and control of potential risks massively more difficult.
In 2016, the U.S. Academy of Sciences recommended that gene drive organisms first be tested on small and remote islands.¹ However, calculations using models show that this form of testing would not be sufficiently isolated: individual GDOs can reach other regions through water, wind or unintentional transport and spread the gene drive further.² Moreover, GDO could be spread deliberately. A group of researchers led by gene drive developer Kevin Esvelt at the Massachusetts Institute of Technology (MIT) in Boston, USA, is working on a gene drive variant that can be limited in its spatial spread. They call this gene drive Daisy Chain Drive.³ So far, however, this gene drive variant exists only in theory.
What is a Daisy Chain Drive?
The Daisy Chain Drive is a variant of gene drive based on CRISPR/Cas9 that hasnot yet been implemented. However, here the CRISPR/Cas-based gene drive would consist of individual elements located on different chromosomes.⁴ Element C consists of the gene ‘scissors’ and a guidepost for B. Element B is the gene ‘scissors’ Cas9 plus guidepost for C. C is the target site of the gene drive, an essential gene that is knocked out by the DNA double-strand break and replaced by a new gene if necessary. Component C is inherited according to Mendelian rules. Therefore, the process should stop on its own at a certain point, which could greatly limit its spatial and temporal distribution.
Irreversibility
A gene drive causes a permanent genetic modification of the genetic material, which is passed on to all subsequent generations. Even if a gene drive encounters resistance and no longer spreads under its own power, these changes could continue to be inherited according to Mendelian rules and persist for a long time in the genome of the population. Only if the deactivated gene drive severely impairs the survivability of the individuals do the mechanisms of natural selection take effect, which could eliminate the change in the natural populations again.
As early as 2014, a discussion started about the need for a so-called Reversal Drive, which is intended to reverse the changes of a Gene Drive in the manipulated populations. In principle, this is a modified version of the original gene drive that overwrites the genetic manipulations and prevents their further spread. However, even such a Reversal Drive could not restore the original genetic state of the population, but only introduce further genetic modifications into the genome of these populations.
In a study on fruit flies, genetic elements were presented that are designed to switch off or completely remove CRISPR/Cas-based gene drives from the genome. Specific signposts of the CRISPR/Cas9 gene scissors are used to terminate the chain reaction of a CRISPR/Cas-based gene drive. The result: the gene ‘scissors’ paralyze themselves. Results from cage experiments show that these elements have prevailed after 10 generations. However, synthetic genetic elements remain in the genome and are inherited according to Mendelian rules. In addition, unintended changes to the genome occur. It is difficult to estimate how these remaining genetic changes will behave in the wild populations in the long term and whether they will be influenced by external factors.⁵ According to current knowledge, any release of a gene drive carries the risk of irreversibly and uncontrollably altering the genetic material of a natural population.⁶
Outcrossing across species boundaries
Gene drives are tailored to the genome of a single species, but in many cases outcrossing across species boundaries could be inevitable. For example, the malaria-carrying mosquito Anopheles gambiae belongs to a complex of seven different subspecies that are genetically very similar and can produce fertile offspring with each other.⁷ A gene drive by Target Malaria targets disruption of the gene Doublesex, which has undergone little change during the evolution of the mosquito species. This approach could drive all seven related mosquito species to the brink of extinction, although at least one species does not transmit malaria.⁸
A similar risk exists in fruit flies of the genus Drosophila, which have played a central role in the development and application of gene drives. It has been known for over 90 years that different species of Drosophila can interbreed and produce fertile offspring.⁹ Thousands of other animal and plant species form natural hybrids, so the spread of gene drives would not be limited to one species but could also extend to its closer relatives.
Unexpected effects of CRISPR/Cas9
Many engineered gene drives use CRISPR/Cas9 to create a double-strand break at defined locations in the genome. However, this tool does not work flawlessly.¹⁰ CRISPR/Cas9 can change the activity of the target gene in unpredictable ways, increase the mutation rate in the genome, lead to unexpected mutations, or be disrupted in its function by emerging resistances. For example, there are increasing reports of so-called off-target effects, unintended changes to non-target sequences that can occur when the CRISPR/Cas system is applied.¹¹
Moreover, the genetic modifications not only affect the target area, but often also other areas in the genome.¹² One of the reasons for this is that in wild populations there are more sequences in the genome to which CRISPR/Cas9 can dock than the computer programs used for this purpose were able to determine in the laboratory. Gene drives can therefore lead to the development of organisms with unpredictable characteristics.¹³
Resistance
CRISPR-based gene drives search for a clearly defined DNA sequence at which they should cut the genome. Even single mutations in this sequence can therefore make the target invisible to them. The organism thus becomes resistant to the gene drive. Such resistance can arise when CRISPR/Cas9 itself generates mutations that destroy the target sequence. However, they could also occur naturally, especially in populations with high genetic diversity.
If a gene drive encounters resistance, it will stop at this point and only alter part of the population. However, whether it disappears completely depends on the number of individuals that have already been altered and the disadvantages that the gene drive has for their survival. It is therefore quite possible that the gene drive will continue to survive for a long time in an animal species despite resistance.
Unpredictable effects on ecosystems
Every living creature, even if it appears dangerous or harmful to humans, performs important tasks in its habitat. The extinction or even manipulation of one species will therefore have consequences for the entire ecosystem.
This can be well illustrated by the example of mosquitoes. In the course of their life cycle, they form important food sources for various animals. For example, mosquito larvae living in water are a food source for water bugs, beetles, flies, spiders, flatworms, tadpoles, fish and crustaceans. It is assumed that 95 percent of the larvae of the African malaria mosquito Anopheles gambiae are being eaten before becoming adults.¹⁴ Adult mosquitoes are also an important food source and are consumed by dragonflies, spiders, bats and birds, among others. In the Camargue, a nature reserve in southern France, decimation of mosquitoes with a biological control agent has also led to a reduction in the number and diversity of birds and dragonflies.¹⁵ A role in plant pollination also cannot be ruled out, as adult mosquitoes feed on nectar, among other things.¹⁶ The role of mosquitoes in their tightly interwoven ecosystem has hardly been studied so far, so the consequences of a possible extinction are not foreseeable.
These consequences can also affect humans: If one mosquito species is displaced, other species, which may transmit even more dangerous diseases, can spread more widely. Such risk scenarios are known with regard to the control of the dengue fever-transmitting yellow fever mosquito (Aedes aegypti) in North America and Brazil, which competes with the invasive Asian tiger mosquito (Aedes albopictus)¹⁷. If the yellow fever mosquito disappears, this could further promote the spread of the tiger mosquito, which is no less dangerous and also transmits dengue fever.¹⁸
But even if a species is not wiped out, gene drives harbor considerable risks: If the characteristics of the organisms change unintentionally, they can, for example, become more vital, change their behavior, transmit more diseases, or even disturb or destroy the habitat of other species. Because the respective species are closely linked to their ecosystems, the effects of uncontrolled spreads cannot be predicted reliably.¹⁹
Sources:
[1] National Academies of Science, Engineering and Medicine (2016). Gene Drives on the Horizon: Advancing Science, Navigating Uncertainty, and Aligning Research with Public Values. Washington, DC: The National Academies Press
[2] Noble C, Adlam B, Church GM, Esvelt KM, Nowak MA (2017). Current CRISPR gene drive systems are likely to be highly invasive in wild populations. Elife 7:e33423
[3] Noble C, Min J, Olejarz J, Buchthal J, Chavez A, Smidler AL, DeBenedictis EA, Church GM, Nowak MA, Esvelt KM (2019). Daisy-chain gene drives for the alteration of local populations. Proc Natl Acad Sci USA 116:8275
[4] Cf. ibid.
[5] Xu X-RS, Bulger EA, Gantz VM, Klanseck C, Heimler SR, Auradkar A, Bennett JB, Miller LA, Leahy S, Juste SS, Buchman A, Akbari OS, Marshall JM, Bier E (2020) Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives. Molecular Cell.
[6] Esvelt KM, Smidler AL, Catteruccia F, Church GM (2014). Concerning RNA-guided gene drives for the alteration of wild populations. Elife 17:e03401
[7] Coluzzi M, Sabatini A, Petrarca V, Di Deco MA (1979). Chromosomal differentiation and adaptation to human environments in the Anopheles gambiae complex. Trans R SocTrop Med Hyg 73:483
[8]Critical Scientists Switzerland (CSS), European Network of Scientists for Social and Environmental Responsibility (ENSSER), Vereinigung Deutscher Wissenschaftler (VDW) (2019). Gene Drives. A report on their science, applications, social aspects, ethics and regulations. p. 101. Online: genedrives.ch/report [letzter Zugriff:last accessed: 22.10.2020]
[9] Barbash DA (2010). Ninety years of Drosophila melanogaster hybrids. Genetics 186:1
[10] Kosicki M, Tomberg K, Bradley A (2018). Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol 36:765
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[16] Foster WA (1995). Mosquito sugar feeding and reproductive energetics. Annu Rev Entomol 40:443
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[18] Lwande OW, Obanda V, Lindström A, Ahlm C, Evander M, Näslund J, Bucht G (2020). Globe-trotting Aedes aegypti and Aedes albopictus: risk factors for arbovirus pandemics. Vector Borne Zoonotic Dis. 20(2):71–81
[19] Then C, Kawall K, Valenzuela N (2020). Spatiotemporal Controllability and Environmental Risk Assessment of Genetically Engineered Gene Drive Organisms from the Perspective of European Union Genetically Modified Organism Regulation. Integr Environ Assess Manag.
