Bioinformatic and literature assessment of toxicity and allergenicity of a CRISPR-Cas9 engineered gene drive to control Anopheles gambiae the mosquito vector of human malaria.

BACKGROUND: Population suppression gene drive is currently being evaluated, including via environmental risk assessment (ERA), for malaria vector control. One such gene drive involves the dsxFCRISPRh transgene encoding (i) hCas9 endonuclease, (ii) T1 guide RNA (gRNA) targeting the doublesex locus, and (iii) DsRed fluorescent marker protein, in genetically-modified mosquitoes (GMMs). Problem formulation, the first stage of ERA, for environmental releases of dsxFCRISPRh previously identified nine potential harms to the environment or health that could occur, should expressed products of the transgene cause allergenicity or toxicity. METHODS: Amino acid sequences of hCas9 and DsRed were interrogated against those of toxins or allergens from NCBI, UniProt, COMPARE and AllergenOnline bioinformatic databases and the gRNA was compared with microRNAs from the miRBase database for potential impacts on gene expression associated with toxicity or allergenicity. PubMed was also searched for any evidence of toxicity or allergenicity of Cas9 or DsRed, or of the donor organisms from which these products were originally derived. RESULTS: While Cas9 nuclease activity can be toxic to some cell types in vitro and hCas9 was found to share homology with the prokaryotic toxin VapC, there was no evidence from previous studies of a risk of toxicity to humans and other animals from hCas9. Although hCas9 did contain an 8-mer epitope found in the latex allergen Hev b 9, the full amino acid sequence of hCas9 was not homologous to any known allergens. Combined with a lack of evidence in the literature of Cas9 allergenicity, this indicated negligible risk to humans of allergenicity from hCas9. No matches were found between the gRNA and microRNAs from either Anopheles or humans. Moreover, potential exposure to dsxFCRISPRh transgenic proteins from environmental releases was assessed as negligible. CONCLUSIONS: Bioinformatic and literature assessments found no convincing evidence to suggest that transgenic products expressed from dsxFCRISPRh were allergens or toxins, indicating that environmental releases of this population suppression gene drive for malaria vector control should not result in any increased allergenicity or toxicity in humans or animals. These results should also inform evaluations of other GMMs being developed for vector control and in vivo clinical applications of CRISPR-Cas9.

Leveraging eco-evolutionary models for gene drive risk assessment.

Engineered gene drives create potential for both widespread benefits and irreversible harms to ecosystems. CRISPR-based systems of allelic conversion have rapidly accelerated gene drive research across diverse taxa, putting field trials and their necessary risk assessments on the horizon. Dynamic process-based models provide flexible quantitative platforms to predict gene drive outcomes in the context of system-specific ecological and evolutionary features. Here, we synthesize gene drive dynamic modeling studies to highlight research trends, knowledge gaps, and emergent principles, organized around their genetic, demographic, spatial, environmental, and implementation features. We identify the phenomena that most significantly influence model predictions, discuss limitations of biological complexity and uncertainty, and provide insights to promote responsible development and model-assisted risk assessment of gene drives.

Recommendations for environmental risk assessment of gene drive applications for malaria vector control.

Building on an exercise that identified potential harms from simulated investigational releases of a population suppression gene drive for malaria vector control, a series of online workshops identified nine recommendations to advance future environmental risk assessment of gene drive applications.

Bayesian network-based risk assessment of synthetic biology: Simulating CRISPR-Cas9 gene drive dynamics in invasive rodent management.

Gene drive technology has been proposed to control invasive rodent populations as an alternative to rodenticides. However, this approach has not undergone risk assessment that meets criteria established by Gene Drives on the Horizon, a 2016 report by the National Academies of Sciences, Engineering, and Medicine. To conduct a risk assessment of gene drives, we employed the Bayesian network-relative risk model to calculate the risk of mouse eradication on Southeast Farallon Island using a CRISPR-Cas9 homing gene drive construct. We modified and implemented the R-based model "MGDrivE" to simulate and compare 60 management strategies for gene drive rodent management. These scenarios spanned four gene drive mouse release schemes, three gene drive homing rates, three levels of supplemental rodenticide dose, and two timings of rodenticide application relative to gene drive release. Simulation results showed that applying a supplemental rodenticide simultaneously with gene drive mouse deployment resulted in faster eradication of the island mouse population. Gene drive homing rate had the highest influence on the overall probability of successful eradication, as increased gene drive accuracy reduces the likelihood of mice developing resistance to the CRISPR-Cas9 homing mechanism.

iGEM and Gene Drives: A Case Study for Governance.

Gene drives have already challenged governance systems. In this case study, we explore the International Genetically Engineered Machine (iGEM) competition's experiences in gene drive-related research and lessons in developing, revising, and implementing a governance system. iGEM's experiences and lessons are distilled into 6 key insights for future gene drive policy development in the United States: (1) gene drives deserve special attention because of their potential for widescale impact and remaining uncertainty about how to evaluate intergenerational and transboundary risks; (2) an adaptive risk management approach is logical for gene drives because of the rapidly changing technical environment; (3) review by individual technical experts is limited and may fail to incorporate other forms of expertise and, therefore, must be complemented with a range of alternative governance methods; (4) current laboratory biosafety and biosecurity review processes may not capture gene drive research or its components in practice even if they are covered theoretically; (5) risk management for research and development must incorporate discussions of values and broader implications of the work; and (6) a regular technology horizon scanning capacity is needed for the early identification of advances that could pose governance system challenges.

Stakeholder engagement to inform the risk assessment and governance of gene drive technology to manage spotted-wing drosophila.

Emerging biotechnologies, such as gene drive technology, are increasingly being proposed to manage a variety of pests and invasive species. As one method of genetic biocontrol, gene drive technology is currently being developed to manage the invasive agricultural pest spotted-wing drosophila (Drosophila suzukii, SWD). While there have been calls for stakeholder engagement on gene drive technology, there has been a lack of empirical work, especially concerning stakeholder engagement to inform risk assessment. To help address this gap and inform future risk assessments and governance decisions for SWD gene drive technology, we conducted a survey of 184 SWD stakeholders to explore how they define and prioritize potential benefits and potential adverse effects from proposed SWD gene drive technology. We found that stakeholders considered the most important potential benefits of SWD gene drive technology to be: 1) Decrease in the quantity or toxicity of pesticides used, and 2) Decrease in SWD populations. Stakeholders were most concerned about the potential adverse effects of: 1) Decrease in beneficial insects, 2) Increase in non-SWD secondary pest infestations, and 3) Decrease in grower profits. Notably, we found that even stakeholders who expressed support for the use of SWD gene drive technology expressed concerns about potential adverse effects from the technology, emphasizing the need to move past simplistic, dichotomous views of what it means to support or oppose a technology. These findings suggest that instead of focusing on the binary question of whether stakeholders support or oppose SWD gene drive technology, it is more important to identify and assess the factors that are consequential to stakeholder decision making - including, for example, exploring whether and under what conditions key potential adverse effects and potential benefits would result from the use of SWD gene drive technology.

Risk management recommendations for environmental releases of gene drive modified insects.

The ability to engineer gene drives (genetic elements that bias their own inheritance) has sparked enthusiasm and concerns. Engineered gene drives could potentially be used to address long-standing challenges in the control of insect disease vectors, agricultural pests and invasive species, or help to rescue endangered species. However, risk concerns and uncertainty associated with potential environmental release of gene drive modified insects (GDMIs) have led some stakeholders to call for a global moratorium on such releases or the application of other strict precautionary measures to mitigate perceived risk assessment and risk management challenges. Instead, we provide recommendations that may help to improve the relevance of risk assessment and risk management frameworks for environmental releases of GDMIs. These recommendations include: (1) developing additional and more practical risk assessment guidance to ensure appropriate levels of safety; (2) making policy goals and regulatory decision-making criteria operational for use in risk assessment so that what constitutes harm is clearly defined; (3) ensuring a more dynamic interplay between risk assessment and risk management to manage uncertainty through closely interlinked pre-release modelling and post-release monitoring; (4) considering potential risks against potential benefits, and comparing them with those of alternative actions to account for a wider (management) context; and (5) implementing a modular, phased approach to authorisations for incremental acceptance and management of risks and uncertainty. Along with providing stakeholder engagement opportunities in the risk analysis process, the recommendations proposed may enable risk managers to make choices that are more proportionate and adaptive to potential risks, uncertainty and benefits of GDMI applications, and socially robust.

Potential use of gene drive modified insects against disease vectors, agricultural pests and invasive species poses new challenges for risk assessment.

Potential future application of engineered gene drives (GDs), which bias their own inheritance and can spread genetic modifications in wild target populations, has sparked both enthusiasm and concern. Engineered GDs in insects could potentially be used to address long-standing challenges in control of disease vectors, agricultural pests and invasive species, or help to rescue endangered species, and thus provide important public benefits. However, there are concerns that the deliberate environmental release of GD modified insects may pose different or new harms to animal and human health and the wider environment, and raise novel challenges for risk assessment. Risk assessors, risk managers, developers, potential applicants and other stakeholders at many levels are currently discussing whether there is a need to develop new or additional risk assessment guidance for the environmental release of GD modified organisms, including insects. Developing new or additional guidance that is useful and practical is a challenge, especially at an international level, as risk assessors, risk managers and many other stakeholders have different, often contrasting, opinions and perspectives toward the environmental release of GD modified organisms, and on the adequacy of current risk assessment frameworks for such organisms. Here, we offer recommendations to overcome some of the challenges associated with the potential future development of new or additional risk assessment guidance for GD modified insects and provide considerations on areas where further risk assessment guidance may be required.

Small-scale release of non-gene drive mosquitoes in Burkina Faso: from engagement implementation to assessment, a learning journey.

BACKGROUND: Innovative tools are needed to complement the existing approach for malaria elimination. Gene drive mosquitoes are one potential new technology in the control of malaria vectors. Target Malaria is one of the research projects developing this technology, and in July 2019, the project proceeded to an important step for this evaluation pathway: the small-scale release of non-gene drive sterile male mosquitoes in a village in Burkina Faso. In addition to the entomological and laboratory work to prepare for this important milestone, significant community and stakeholder engagement work was done. The existing guidelines on gene drive mosquito provide an overall framework for such engagement work. However, they do not provide a road map on how to proceed or what benchmarks should be used to assess this work. METHODS: This study provides a review of engagement activities relevant to field trials on non-gene drive genetically-modified mosquitoes as well as an assessment framework-using both qualitative and quantitative studies as well as an audit procedure. The latter was implemented to evaluate whether the release activities could proceed with the appropriate level of agreement from the community. RESULTS: This paper shows the importance of this first phase of work to innovate and learn about engagement processes for responsible research in the field of genetic approaches for malaria vector control. The function of these assessments is crucial for the learning agenda. The assessments demonstrated ways to increase understanding and ensure effective progress with field studies and, therefore, the pathway for responsible research. CONCLUSION: Gene drive technology is increasingly considered as a promising approach to control vector borne diseases, in particular malaria. Stakeholders' involvement in this research process is one of the recurring requirements in international guidance documents. With this paper Target Malaria offers an opportunity to explore the practical achievements and challenges of stakeholder engagement during early phases of a technology evaluation, and in particular how it implemented an assessment framework to learn from its experience.

Experimenting with co-development: A qualitative study of gene drive research for malaria control in Mali.

We investigate how technology 'co-development' (between researchers, stakeholders and local communities) is framed in practice by those developing gene drive mosquitos for malaria eradication. Our case study focuses on UK and Mali-based researchers planning to undertake the first field trials in Mali of gene drive mosquitos for malaria control. While they and the wider gene drive research community are explicitly committed to the principle of co-development, how this is framed and practiced is not clear. Through qualitative analysis of 34 interviews complemented by observation and documentary research conducted in 2018, we identify and compare ten framings of co-development mobilised by UK and Malian researchers and stakeholders. For Malians, co-development reflected Mali's broader socio-political context and a desire for African scientific independence and leadership. It was mobilised to secure community and stakeholder support for gene drive mosquito field trials, through outreach, building local scientific capacity and developing those institutions (e.g. regulatory) necessary for field trials to go ahead. For UK participants, co-development was also concerned with scientific capacity-building, knowledge exchange between researchers, and stakeholder and community outreach to secure consent for field trials. Overall, our findings suggest co-development is opening up previously expert-dominated spaces as researchers attempt to take responsibility for the societal implications of their work. However, its main function is as a project management tool to enable and instrumentally support technological development, field trials and eventual deployment. This function extends into areas which are traditionally the responsibility of the state, such as regulatory development, facilitated by Mali's fragile political and economic situation. Paradoxically, co-development simultaneously depoliticises gene drive, masking power relations and closing down substantive debate and agency. Characterised by extreme poverty, conflict and weak institutions, Mali may become a site for technological experimentation where there is little interrogation of gene drive or its governance.

Embracing Dynamic Models for Gene Drive Management.

Robust methods of predicting how gene drive systems will interact with ecosystems is essential for safe deployment of gene drive technology. We describe how quantitative tools can reduce risk uncertainty, streamline empirical research, guide risk management, and promote cross-sector collaboration throughout the process of gene drive technology development and implementation.

Gene Drive-Modified Organisms: Developing Practical Risk Assessment Guidance.

Risk assessors, risk managers, developers, potential applicants, and other stakeholders at many levels discuss the need for new or further risk assessment guidance for deliberate environmental releases of gene drive-modified organisms. However, preparing useful and practical guidance entails challenges, to which we offer recommendations based on our experience drafting guidance.

Spatiotemporal Controllability and Environmental Risk Assessment of Genetically Engineered Gene Drive Organisms from the Perspective of European Union Genetically Modified Organism Regulation.

Gene drive organisms are a recent development created by using methods of genetic engineering; they inherit genetic constructs that are passed on to future generations with a higher probability than with Mendelian inheritance. There are some specific challenges inherent to the environmental risk assessment (ERA) of genetically engineered (GE) gene drive organisms because subsequent generations of these GE organisms might show effects that were not observed or intended in the former generations. Unintended effects can emerge from interaction of the gene drive construct with the heterogeneous genetic background of natural populations and/or be triggered by changing environmental conditions. This is especially relevant in the case of gene drives with invasive characteristics and typically takes dozens of generations to render the desired effect. Under these circumstances, "next generation effects" can substantially increase the spatial and temporal complexity associated with a high level of uncertainty in ERA. To deal with these problems, we suggest the introduction of a new additional step in the ERA of GE gene drive organisms that takes 3 criteria into account: the biology of the target organisms, their naturally occurring interactions with the environment (biotic and abiotic), and their intended biological characteristics introduced by genetic engineering. These 3 criteria are merged to form an additional step in ERA, combining specific "knowns" and integrating areas of "known unknowns" and uncertainties, with the aim of assessing the spatiotemporal controllability of GE gene drive organisms. The establishment of assessing spatiotemporal controllability can be used to define so-called "cut-off criteria" in the risk analysis of GE gene drive organisms: If it is likely that GE gene drive organisms escape spatiotemporal controllability, the risk assessment cannot be sufficiently reliable because it is not conclusive. Under such circumstances, the environmental release of the GE gene drive organisms would not be compatible with the precautionary principle (PP). Integr Environ Assess Manag 2020;16:555-568. © 2020 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

The ethics of genetic engineering and gene drives in conservation.

The ethical issues associated with using genetic engineering and gene drives in conservation are typically described as consisting of risk assessment and management, public engagement and acceptance, opportunity costs, risk and benefit distributions, and oversight. These are important, but the ethical concerns extend beyond them because the use of genetic engineering has the potential to significantly alter the practices, concepts, and value commitments of conservation. I sought to elucidate the broader set of ethical issues connected with a potential genetic engineering turn in conservation and provide an approach to ethical analysis of novel conservation technologies. The primary rationales offered in support of using genetic engineering and gene drives in conservation are efficiency and necessity for achieving conservation goals. The instrumentalist ethical perspective associated with these rationales involves assessing novel technologies as a means to accomplish desired ends. For powerful emerging technologies the instrumentalist perspective needs to be complemented by a form-of-life perspective frequently applied in the philosophy of technology. The form-of-life perspective involves considering how novel technologies restructure the activities into which they are introduced. When the form-of-life perspective is applied to creative genetic engineering in conservation, it brings into focus a set of ethical issues, such as those associated with power, meaning, relationships, and values, that are not captured by the instrumentalist perspective. It also illuminates why the use of gene drives in conservation is so ethically and philosophically interesting.

Ética de la Ingeniería Genética y la Transmisión de Genes en la Conservación Resumen Los temas éticos asociados con el uso de la ingeniería genética y la transmisión de genes en la conservación están típicamente descritos como evaluación y manejo de riesgos, participación pública y aceptación, costos de oportunidad, distribuciones de riesgos y beneficios, y omisiones. Todo lo anterior es importante, pero las preocupaciones éticas se extienden más allá de los temas previos porque el uso de la ingeniería genética tiene el potencial de alterar significativamente las prácticas, conceptos y compromisos de valor de la conservación. Busqué elucidar los temas éticos conectados con un giro potencial hacia la ingeniería genética en la conservación y también proporcionar una estrategia para el análisis ético de las novedosas tecnologías para la conservación. Las razones principales ofrecidas como apoyo para el uso de la ingeniería genética y la transmisión de genes en la conservación son la eficacia y la necesidad de lograr los objetivos de conservación. La perspectiva ética instrumentalista asociada con estas razones involucra la evaluación de las tecnologías novedosas como medio para obtener los fines deseados. Para las emergentes tecnologías poderosas, la perspectiva instrumentalista necesita estar complementada con una perspectiva de forma de vida, la cual se aplica frecuentemente en la filosofía de la tecnología. La perspectiva de forma de vida involucra la consideración de cómo las tecnologías novedosas reestructuran las actividades a las cuales son introducidas. Cuando en la conservación se aplica la perspectiva de forma de vida a la ingeniería genética creativa, trae hacia el foco un conjunto de temas éticos, como aquellos asociados con el poder, el significado, las relaciones y los valores, que no captura la perspectiva instrumentalista. Esta perspectiva también ilustra sobre el por qué el uso de la transmisión de genes en la conservación es tan interesante ética y filosóficamente.

Assessment of a Split Homing Based Gene Drive for Efficient Knockout of Multiple Genes.

Homing based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that host encoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent disease transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate genes has not been thoroughly explored. To test this approach, we generated a split-HGD in Drosophila melanogaster that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host-encoded gene, which we term a gRNA-mediated effector (GME). This design enabled us to assess homing and knockout efficiencies of two target genes simultaneously, and also explore the timing and tissue specificity of Cas9 expression on cleavage/homing rates. We demonstrate that inclusion of a GME can result in high efficiency of disruption of both genes during super-Mendelian propagation of split-HGD. Furthermore, both genes were knocked out one generation earlier than expected indicating the robust somatic expression of Cas9 driven by Drosophila germline-limited promoters. We also assess the efficiency of 'shadow drive' generated by maternally deposited Cas9 protein and accumulation of drive-induced resistance alleles along multiple generations, and discuss design principles of HGD that could mitigate the accumulation of resistance alleles while incorporating a GME.

Considerations for the governance of gene drive organisms.

Governance is a broader and more flexible concept than statute-driven regulations as it incorporates components outside the latter's remit. Considerations of governance are critical in the development of emerging biotechnologies such as gene drive organisms. These have been proposed or are being developed to address public and environmental health issues not addressed easily by conventional means. Here, we consider how the concept of governance differs from statute-driven regulation with reference to the role each may play in the development of gene drive organisms. First, we discuss existing statute-based regulatory systems. Second, we consider whether novel risks or different concerns derive from gene drive organisms, concentrating on characteristics that contribute to public health or environmental risk and uncertainties that may affect risk perceptions. Third, we consider public engagement, outlining how existing statute-driven regulatory systems and other governance mechanisms may provide opportunities for constructive interactions. Finally, we provide some observations that may help address science- and values-based concerns in a governance space larger than that of statute-driven regulatory systems.