Meeting Report

New Genomic Solutions for Conservation Problems Workshop

April 6-9, 2015 – Sausalito, California

Opportunities to solve previously intractable conservation problems are emerging from new genetic tools being developed for human biomedicine. Such tools have the potential to provide new solutions to some of the most difficult problems facing species conservation including exotic diseases of wildlife and alien invasive species.

For example, extremely precise genome-editing techniques such as CRISPR now may make it possible to adjust the genes of endangered wildlife populations toward better resistance to exotic diseases. Or CRISPR could be employed to reduce or extirpate a population of rapidly reproducing disease vectors or non-native organisms or aggressively invasive plants and animals. The potential exists to develop a sophisticated genomic toolkit for minimally disruptive “precision conservation” if the techniques are developed responsibly and can be made to work exactly as intended.

In order to explore and map potential roles for new genomic technologies in alleviating difficult conservation problems, Revive & Restore and Archipelago Consulting convened a workshop bringing together experts from wildlife biology, botany, island conservation, population ecology, veterinary medicine, and pioneers of genomic engineering. Held April 6-9, 2015 in Sausalito, California the workshop included 52 participants from 43 organizations and eight countries.

The goal of the workshop was to begin to create a future in which genomic technologies can be considered thoughtfully and eventually deployed safely and effectively. The workshop had three purposes:

Educate conservationists about the application of these new tools and the potential benefits and risks;
Educate genomic technologists about urgent conservation problems that might benefit from the application of new genomic tools;
Determine a small set of the cases presented at the meeting that offer the best opportunity for tool development and application, and begin further exploration;

Using a case study methodology the workshop focused on some of conservation’s major challenges where genetic diagnosis and treatment might prove useful – non-native wildlife diseases and invasive species:

Non-native wildlife diseases transmitted by vectors:
native wildlife disease is emerging as a significant threat to species conservation through both direct and indirect pathways. Its impact appears to be increasing, and there has been very limited success in addressing the threat. The workshop focused on Hawaiian birds and avian malaria (vector: mosquitoes) and black-footed ferrets, prairie dogs and sylvatic plague (vector: fleas);
Non-native diseases with the potential for genetic resistance:
The workshop focused on Chytrid fungus of amphibians, white-nose syndrome of bats and Herpes virus in Asian elephants;
Alien invasive species:
The third focus, invasive species, cost the US $1.2+ billion every year and threaten ecosystems around the globe. This work group focused on rodents and ants on oceanic islands.

The workshop was structured to:

Present case studies on potential and successful uses of precision genomic technologies and orientation presentations on the different technologies and how they might be deployed;
Work in three separate groups, each focused on the biological dimensions of the three focal areas, looking for feasible interventions where genomic interventions might solve these problems;
Present these possible interventions to the entire group of participants and receive feedback;
Look at the interventions being proposed from regulatory, social and political frameworks and which ones might actually be able to move forward;
Present these to the entire group of participants, including participants serving as sounding boards from business, philanthropy and government – and receive feedback;
Craft the interventions that seemed most feasible both biologically and in a regulatory/social context into a 10 minute ‘pitch’ to a panel of experts in conservation funding.

Examples of Success:

Innovative Models of Genomic Technology Applied to Conservation Problems

Presented by Luke Alphey (mosquitoes), Bill Powell (chestnut tree), Josh Donlan (island restoration), Oliver Ryder (California condors)

Luke Alphey (The Pirbright Institute, United Kingdom) reviewed genetics-based methods that have the potential to provide powerful new tools for the control of invasive species. Several methods are being developed using a modified form of the pest as the control agent, hence turning the pest against itself. Released modified pests will seek out their wild counterparts to mate, and thereby deliver their genetic payload to the target population.

A genetics-based method targeting the dengue mosquito Aedes aegypti has moved to the field, with a series of successful field trials. Pioneered by Oxitec Ltd, this is based on the use of “genetically sterile” male mosquitoes. When these males mate wild females the offspring inherit an engineered gene that prevents them developing into functional adults. This approach is a next-generation enhancement of the tried-and-tested Sterile Insect Technique (SIT). Successful field trials have been conducted in the Cayman Islands, Brazil and Panama. In each case >90% suppression of the target population was observed; this compares with 30-50% reported for intensive application of current methods.

While these trials have used the method in isolation, for experimental clarity, such methods should not be seen as “magic bullets,” rather as powerful new tools to be combined with the best of current methods in an integrated pest management program. Though the field trials have been relatively small so far, the operationally similar Sterile Insect Technique has been successfully used to eliminate pests on various scales up to continent-wide. Public support has been good at the field sites so far, following careful public engagement.

Though not yet in field trials, several other genetic control methods are in development. These can be classified according to outcome – population suppression or population replacement. Population suppression methods – such as the use of sterile males – aim to reduce the numerical size of the pest population, possibly to zero. Population replacement methods aim to change individuals within it to a less harmful form without killing them, for example making mosquitoes less able to transmit a mosquito-borne pathogen.

Another classification relates to the degree of persistence or invasiveness of the genetic modification after release. Strongly self-limiting constructs such as the “sterility” gene above will disappear from the environment rapidly if releases cease; this is reassuring in terms of controllability and reversibility but typically requires frequent release of additional modified males during the program. “Self-sustaining” are designed to persist in the environment and perhaps spread within the target population or even through the entire species. Models suggest that far fewer modified pests would need to be released, on the other hand the ability to control such constructs post-release, or to assure their on-going function in the face of strong evolutionary pressures, is less clear; it may therefore be harder to gain regulatory and public approval for self-sustaining approaches.

Bill Powell (State University of New York College of Environmental Science and Forestry) presented his work on the American chestnut tree (Castanea dentata) and the exotic chestnut blight that drove it to virtual extension. They have enhanced blight resistance in chestnut trees by producing both transgenic and cisgenic American chestnut trees. The most promising of these transgenes encodes an oxalate oxidase (OxO) from bread wheat (Triticum aestivum). According to leaf and small stem assays that predict the level of blight resistance, this OxO has raised resistance levels at least as high as those found in the blight-resistant Chinese chestnut (C. mollissima). The next step will be to stack these two genes to determine if they can work synergistically and enhance blight resistance even higher. These results show that genetic engineering is a useful tool that can significantly enhance pathogen resistance while otherwise making very small changes in a tree. This is ideal for forest conservation because there would be less chance of unintended consequences and more benefits for the ecosystem than traditional hybrid breeding methods.

Josh Donlan (Advanced Conservation Strategies, Utah and France) laid out the potential to eradicate invasive species on islands using genomic technology. Invasive alien species continue to have substantial impacts globally, economically, culturally, and biologically. Invasive species are responsible for over 50 and 70 percent of recorded vertebrate and plant extinctions respectively, the overwhelming majority of which have occurred on islands. While our abilities to manage invasive species and their impacts have improved over the past decades, the current tools to do so are generally imprecise, blunt and not effective at scale. The development of species-specific toxicants or alternative approaches to specifically target invasive species would be transformative in the sense of drastically improving the conservation utility of invasive species eradication. Synthetic biology, through approaches such as of Ribonucleic acid (RNA) interference and gene drives, could potentially revolutionize the field of invasive species eradication. If developed safely and collaboratively, synthetic biology could potentially provide conservation practitioners with safer and more precise tools to manage invasive species.

Oliver Ryder (San Diego Zoo Institute for Conservation Research, California) provided a perspective on the use of genomic technology in the conservation of the California condor, an iconic example of bringing an endangered species back from the brink of extinction. In 1986 with only 21 birds descending from 13 individuals, the rapid decline of the wild population led to bringing all the birds into captivity for breeding, reintroduction, and recovery. The recovery program for the California condor faced many challenges, but fortunately the species has reproduced extraordinarily well in captivity, facilitating reintroduction efforts. At the end of 2014, there were 423 California condors, 230 of which were free-flying in reintroduced populations in California, Arizona/ Utah, and Baja California. Recently, investigators undertook whole genome sequencing efforts for 36 California condors that include the entire genetic diversity of the species and provides a dataset that will allow identification of markers linked to the recessive lethal disease. As a result of these studies, better estimates of kinship than were previously available have been utilized to guide the pairing of birds for captive breeding. The California condor is now the first endangered species recovery program for which breeding management is guided by whole genome sequence data. It is the first endangered species for which a test to identify carriers of a genetic disease has been established. The genome-wide genetic diversity of this endangered species can now be evaluated and monitored, making feasible a new era in endangered species population management and providing a genomic toolkit that can assist recovery efforts.

Presentations of case studies of genomic technology applications to conservation

The three work groups each focused on two or three case studies and chose one intervention to propose as a compelling business case, identifying the problem, the proposed solution, the value proposition and strategic opportunities.

In order to hone the arguments for possible application of the discussed genomic tools, each work group narrowed its choices to one or two applications and prepared a 10-minute “pitch” to a panel that provided viewpoints from business, philanthropy and government. Panelists included:

Bob Cook, Program Director, Conservation Program and the Basic Medical Research Program, Helmsley Charitable Trust
Drew Endy, Associate Professor of Engineering, Stanford University
Alicia Jackson, Deputy Director, Biological Technologies Office, Defense Advanced Research Projects Agency
Ed Schulak, Private Investor

Read the Case Studies’ Final Pitches here.

What Genomics Can Do –

A Brief Description of a Variety of Tools That Might be Applied to Help Deliver Desired Conservation Outcomes

Presented by George Church (CRISPR), Kevin Esvelt (Gene Drive), Fred Gould (RNAi), Ronald Thresher (Engineered Sterility)

Kevin Esvelt (Harvard University, Massachusetts) spoke about gene drives — types of genetic elements that drive themselves through populations by distorting inheritance in their favor. Gene drive systems can spread even if they decrease the odds of the host organism surviving and reproducing. They consequently represent a potential way to edit the characteristics of wild populations. Until recently, technical limitations precluded the construction of synthetic gene drives systems capable of functioning in the wild.

The recent development of the CRISPR/Cas9 system for genome editing enables the construction of RNA-guided gene drives that may be capable of spreading nearly any alteration that can be made with Cas9. This equates to nearly any genomic change. The ability to target multiple sequences with Cas9 makes it possible to render these gene drives evolutionarily robust to mutations that would otherwise block the spread of the drive in the wild. While differences in the molecular biology of species make it difficult to predict whether gene drives will work in any given species, the >97% efficiency observed to date in both yeast and flies suggests that the technology may be broadly applicable.

RNA-guided gene drives can theoretically be used to delete any existing gene, to edit any gene important for fitness, or to add new transgenes. Edits and transgenes could mutate during or after the spread to fixation, though subsequent gene drive releases could periodically overwrite the mutated versions with a new copy. RNA-guided gene drives could also cause a population crash due to the buildup of recessive mutations causing infertility or inviability or by biasing the sex ratio. However, creating ‘population suppression’ gene drives is likely to be more difficult than standard ‘editing’ gene drives. Genomic changes can be reversed with subsequent gene drives, though the cassette encoding the guide RNAs and possibly cas9 will remain and any ecological changes resulting from the original genomic alteration will not necessarily be reversed. In principle, alterations can be limited to species carrying particular genetic sequences and will not spread into related species with different sequences, although this will require verification in the laboratory.

These capabilities could be used for conservation purposes. For example, a population suppression gene drive could control or remove an ecologically damaging invasive species. Alternatively, a basic gene drive could spread protective alleles through a population in advance of an impending ecological or disease-based challenge.

The primary limitation of RNA-guided gene drives is that they are likely to spread to fixation in all populations connected by gene flow. The possibility of humans deliberately transporting organisms carrying a population suppression drive from the invasive population to the native population deserves careful consideration; such an event would necessitate the release of an immunizing reversal drive into the native population to protect it from the misused suppression drive.

One potential method of controlling the spread of an RNA-guided suppression drive is to combine it with another form of gene drive that is threshold-dependent. For example, chromosomal translocations often result in an “underdominance” effect in which the offspring of translocated and wild organisms are extremely unfit or even nonviable. If the two are otherwise of equal fitness, whichever genomic version constitutes >50% of the population is predicted to drive the other to extinction. Releasing >50% of the local population, even over successive generations, can therefore replace the local population without the risk of spreading into other populations connected by low levels of gene flow. However, underdominance gene drives cannot be used for population suppression. This could be remedied by using a threshold-dependent underdominance gene drive to ‘recode’ specific sequences of a local population, which then could be selectively targeted for removal using a subsequent RNA-guided suppression drive. This may represent a method of removing invasive mosquitoes from islands as large as Hawaii at comparatively low cost.

Fred Gould (North Carolina State University, North Carolina) explained the use of RNA insecticides to target invasive species and pests. Messenger RNA (mRNA) is critical for translating the information coded by an organism’s DNA into the diverse proteins that enable cells and organisms to function. In its normal form, mRNA is a single strand molecule. Any molecule that prematurely degrades mRNA in the cell could cause malfunction or death. Cells of plants and animals have evolved over millennia to produce molecules that specifically degrade RNA of viruses that invade cells while not attacking the cell’s own mRNA. Recently, researchers have developed approaches for using this natural cellular machinery to target specific mRNA molecules of the cell, thereby interrupting production of specific functional proteins. This interference in metabolism is referred to as RNAi, with the “I” standing for interference.

Companies are also developing double stranded RNAs by fermentation processes and formulating the RNAs into pest-specific toxins. These RNAs can be sprayed on plants to target crop pests but can also be put into baits that will be attractive to and be eaten by pests. An RNA that targets a pest of a beneficial insect can be fed to the beneficial insect to protect it against the pest. The advantage of these RNA insecticides is that they are almost species specific, and so are expected to have few off-target effects. The RNA pesticides are not expected to work with all insects, but there is evidence that they can work with bees, ants, and beetles. The implications for conservation biology are important. Such RNA pesticides could be used to eradicate Argentine ants from islands and to protect endangered species from pathogens.

Ronald Thresher (Commonwealth Scientific and Industrial Research Organisation [CSIRO], Australia) presented on autocidal – or “self killing” methods to control species. Autocidal refers to modifying a species’ genome such that as the modification spreads through the population the species’ impacts or abundance are reduced. To date, eight autocidal approaches have been identified as having the potential to control invasive species. All appear to be genetically feasible. The furthest developed at this stage is a construct that is lethal to the offspring of the released carriers, essentially a genetic equivalent of a traditional Sterile Insect Technique (SIT) for managing insect pests. These technologies are currently being applied for certain insect species such as mosquitos and research is underway to assess their efficacy in invasive species, particularly on islands.

Ethical, Legal, Regulatory Considerations

Presented by Gregg Howald, Jennifer Kuzma, and Eleonore Pauwels

Gregg Howald (Island Conservation, Canada)

Historically ~80% of extinctions since 1500 have occurred on island ecosystems that today support ~40% of all threatened species. One of the greatest threats includes the presence of invasive alien species, particularly rats (Rattus) and mice (Mus pp.). Commensal rats and mice have been introduced onto >82% of the world’s islands or island archipelagos with new introductions documented every year. Fortunately, introduced rats and mice can be removed from islands and ecosystems can recover.

The removal of introduced rats from island ecosystems is a powerful conservation tool to protect threatened species, and has been adopted worldwide with ~500 successful eradications reported. With the exception of very small islands, each project follows the same fundamental process of eradication – the delivery of a bait containing an anticoagulant rodenticide into every potential territory, timed when rodents are more likely to consume the bait and/or to minimize risks to non‐target species. However, the anticoagulant rodenticides are not species specific, and can represent a toxicological risk for non‐target species, species that may inadvertently be exposed primarily or secondarily to the rodenticide. Thus, significant investments are usually made into assessing the environmental risks associated with the rodenticide use and designing appropriate mitigation, avoidance and risk minimization strategies.

The major limitations to rodent eradication projects are typically technological limits of the tools available, sociopolitical and regulatory. Over the last 15 years in the United States, a series of rat eradications from islands has been implemented using aerial broadcast approaches. The key lessons learned and strategies to consider are:

Engage the regulatory agencies early and frequently during the development of a product or project, and seek guidance and concurrence on the process.
Start with small projects; conduct demonstration and experimental projects to collect data to inform regulatory requirements.
Replicate projects with increasing complexity to demonstrate efficacy and the continued absence (or presence) of known or new risks in increasing complexity.
Don’t underestimate the opportunity to form unique partnerships. Highly effective and needed conservation requires the cooperation and collaboration of both the public and private sectors.
Think programmatically but implement project to project.
Start with the end goal in mind, how the technology is to be used, and lay out the path to get there. Typically for large complex projects, a “stepping stone” approach is necessary.

Jennifer Kuzma (North Carolina State University, North Carolina) discussed her team’s research on governance systems for genetically engineered organisms, ranging from work on policy processes to public perception of existing GE products and systems. She described how, today, changes in the engineering process, including the use of gene editing, gene drives, and synthetic biology are and will continue to challenge US oversight of GEOs. Work in this area has been marked by legal, media, and public policy challenges from non-governmental, advocacy groups concerned about US oversight being too weak. Furthermore, her research on evaluating oversight through a multi-criteria analytical approach indicates that the US system has strengths in its use of natural science data, but is weak in transparency in operation, inclusivity of a broad range of stakeholders and perspectives, and capacity of regulatory agencies to dealing with changing environments and technologies. This context has contributed to growing movements against GM food in the US, sparked also by public desires for local, organic foods.

Genomic approaches to conservation are emerging against this background. Her observations were that the community at the workshop was very different than those associated with the development of the first generation of GEOs. Given its roots in conservation and ecology, she noted her impression that the participants were explicit about the values behind their statements or views and had greater tendencies to acknowledge the uncertainties associated with planned releases of GEOs. She recommended that this community work with these strengths in moving forward by including stakeholder and public dialogues as part of pre-release efforts and early field research and by continuing to address uncertainties through risk-related research. She showed some of her public perception data indicating that regular citizens can make nuanced decisions and formulate sophisticated opinions, although they draw upon a variety of factors to do so including history, trust, controllability, familiarity, being informed, and cultural world views. In summary, she noted that the conservation community has an opportunity to do things differently with GEO releases. If they openly communicate and earn the trust of the public, and the public sees a great benefit to releasing GEOs in their environments, the positive and cooperative atmosphere for deployment may be achieved.

Eleonore Pauwels (Woodrow Wilson Center, Washington DC) spoke on how experts in genomics navigate the complex societal contexts and issues of trust as they engage with publics about their work. Over the past few years, the debate about genomic technologies has been characterized by battles between those who have sought to reassure us about the technology and those who have raised concerns about possible impacts, environmental and otherwise. Though fears about the impacts may be exaggerated, the pro-technology forces will not be able to erase fears from the public because genomics is becoming integrated into the collective narrative we share about technology and its place in our lives.

The scientific community can provide little counterweight to these storylines because the narratives imply failures inherent in our larger society – ethical failures, failures to anticipate, and failures to develop adequate controls for complex technological systems. These powerful narratives of failure to control are reinforced by the fact that there is no clear consensus among scientists about the uncertainties raised by the use of genomic technologies in our ecosystems. Questions remain about how, and by whom, genomics is represented. And how this determines what we learn about the field and what synthetic biologists do actually know. We need strategies for managing public expectations and understandings of unknowns and unpredictable technological developments, benefits and risks.

Conclusion

The workshop was the first to bring together conservation experts focused on specific intractable problems with key developers of new genomic tools. There was strong support for the potential to develop and apply novel genomic approaches to addressing the identified conservation issues. At the same time there was a marked concurrence in the need to consult broadly with publics and regulators to seek the ‘social license to operate’ that is required before actually carrying out any of the work that was outlined.