Publications by authors named "John M Marshall"

62 Publications

Population size estimation of seasonal forest-going populations in southern Lao PDR.

Sci Rep 2021 Jul 20;11(1):14816. Epub 2021 Jul 20.

Malaria Elimination Initiative, The Global Health Group, University of California, San Francisco, CA, USA.

Forest-going populations are key to malaria transmission in the Greater Mekong Sub-region (GMS) and are therefore targeted for elimination efforts. Estimating the size of this population is essential for programs to assess, track and achieve their elimination goals. Leveraging data from three cross-sectional household surveys and one survey among forest-goers, the size of this high-risk population in a southern province of Lao PDR between December 2017 and November 2018 was estimated by two methods: population-based household surveys and capture-recapture. During the first month of the dry season, the first month of the rainy season, and the last month of the rainy season, respectively, 16.2% [14.7; 17.7], 9.3% [7.2; 11.3], and 5.3% [4.4; 6.1] of the adult population were estimated to have engaged in forest-going activities. The capture-recapture method estimated a total population size of 18,426 [16,529; 20,669] forest-goers, meaning 61.0% [54.2; 67.9] of the adult population had engaged in forest-going activities over the 12-month study period. This study demonstrates two methods for population size estimation to inform malaria research and programming. The seasonality and turnover within this forest-going population provide unique opportunities and challenges for control programs across the GMS as they work towards malaria elimination.
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http://dx.doi.org/10.1038/s41598-021-94413-zDOI Listing
July 2021

Combating mosquito-borne diseases using genetic control technologies.

Nat Commun 2021 07 19;12(1):4388. Epub 2021 Jul 19.

Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, CA, USA.

Mosquito-borne diseases, such as dengue and malaria, pose significant global health burdens. Unfortunately, current control methods based on insecticides and environmental maintenance have fallen short of eliminating the disease burden. Scalable, deployable, genetic-based solutions are sought to reduce the transmission risk of these diseases. Pathogen-blocking Wolbachia bacteria, or genome engineering-based mosquito control strategies including gene drives have been developed to address these problems, both requiring the release of modified mosquitoes into the environment. Here, we review the latest developments, notable similarities, and critical distinctions between these promising technologies and discuss their future applications for mosquito-borne disease control.
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http://dx.doi.org/10.1038/s41467-021-24654-zDOI Listing
July 2021

Population modification strategies for malaria vector control are uniquely resilient to observed levels of gene drive resistance alleles.

Bioessays 2021 Aug 20;43(8):e2000282. Epub 2021 Jun 20.

Department of Microbiology & Molecular Genetics, University of California, Irvine, California, USA.

Cas9/guide RNA (gRNA)-based gene drive systems are expected to play a transformative role in malaria elimination efforts., whether through population modification, in which the drive system contains parasite-refractory genes, or population suppression, in which the drive system induces a severe fitness load resulting in population decline or extinction. DNA sequence polymorphisms representing alternate alleles at gRNA target sites may confer a drive-resistant phenotype in individuals carrying them. Modeling predicts that, for observed levels of SGV at potential target sites and observed rates of de novo DRA formation, population modification strategies are uniquely resilient to DRAs. We conclude that gene drives can succeed when fitness costs incurred by drive-carrying mosquitoes are low enough to prevent strong positive selection for DRAs produced de novo or as part of the SGV and that population modification strategies are less prone to failure due to drive resistance.
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http://dx.doi.org/10.1002/bies.202000282DOI Listing
August 2021

Engineered reproductively isolated species drive reversible population replacement.

Nat Commun 2021 06 2;12(1):3281. Epub 2021 Jun 2.

Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, San Diego, CA, USA.

Engineered reproductive species barriers are useful for impeding gene flow and driving desirable genes into wild populations in a reversible threshold-dependent manner. However, methods to generate synthetic barriers are lacking in advanced eukaryotes. Here, to overcome this challenge, we engineer SPECIES (Synthetic Postzygotic barriers Exploiting CRISPR-based Incompatibilities for Engineering Species), an engineered genetic incompatibility approach, to generate postzygotic reproductive barriers. Using this approach, we create multiple reproductively isolated SPECIES and demonstrate their reproductive isolation and threshold-dependent gene drive capabilities in D. melanogaster. Given the near-universal functionality of CRISPR tools, this approach should be portable to many species, including insect disease vectors in which confinable gene drives could be of great practical utility.
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http://dx.doi.org/10.1038/s41467-021-23531-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173020PMC
June 2021

MGDrivE 2: A simulation framework for gene drive systems incorporating seasonality and epidemiological dynamics.

PLoS Comput Biol 2021 May 21;17(5):e1009030. Epub 2021 May 21.

Divisions of Epidemiology and Biostatistics, School of Public Health, University of California, Berkeley, California, United States of America.

Interest in gene drive technology has continued to grow as promising new drive systems have been developed in the lab and discussions are moving towards implementing field trials. The prospect of field trials requires models that incorporate a significant degree of ecological detail, including parameters that change over time in response to environmental data such as temperature and rainfall, leading to seasonal patterns in mosquito population density. Epidemiological outcomes are also of growing importance, as: i) the suitability of a gene drive construct for release will depend on its expected impact on disease transmission, and ii) initial field trials are expected to have a measured entomological outcome and a modeled epidemiological outcome. We present MGDrivE 2 (Mosquito Gene Drive Explorer 2): a significant development from the MGDrivE 1 simulation framework that investigates the population dynamics of a variety of gene drive architectures and their spread through spatially-explicit mosquito populations. Key strengths and fundamental improvements of the MGDrivE 2 framework are: i) the ability of parameters to vary with time and induce seasonal population dynamics, ii) an epidemiological module accommodating reciprocal pathogen transmission between humans and mosquitoes, and iii) an implementation framework based on stochastic Petri nets that enables efficient model formulation and flexible implementation. Example MGDrivE 2 simulations are presented to demonstrate the application of the framework to a CRISPR-based split gene drive system intended to drive a disease-refractory gene into a population in a confinable and reversible manner, incorporating time-varying temperature and rainfall data. The simulations also evaluate impact on human disease incidence and prevalence. Further documentation and use examples are provided in vignettes at the project's CRAN repository. MGDrivE 2 is freely available as an open-source R package on CRAN (https://CRAN.R-project.org/package=MGDrivE2). We intend the package to provide a flexible tool capable of modeling gene drive constructs as they move closer to field application and to infer their expected impact on disease transmission.
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http://dx.doi.org/10.1371/journal.pcbi.1009030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8186770PMC
May 2021

Estimating the potential impact of Attractive Targeted Sugar Baits (ATSBs) as a new vector control tool for Plasmodium falciparum malaria.

Malar J 2021 Mar 17;20(1):151. Epub 2021 Mar 17.

Malaria Research and Training Centre, Faculty of Medicine, Pharmacy and Odonto-Stomatology, University of Sciences, Techniques and Technology of Bamako, BP, Bamako, Mali.

Background: Attractive targeted sugar baits (ATSBs) are a promising new tool for malaria control as they can target outdoor-feeding mosquito populations, in contrast to current vector control tools which predominantly target indoor-feeding mosquitoes.

Methods: It was sought to estimate the potential impact of these new tools on Plasmodium falciparum malaria prevalence in African settings by combining data from a recent entomological field trial of ATSBs undertaken in Mali with mathematical models of malaria transmission. The key parameter determining impact on the mosquito population is the excess mortality due to ATSBs, which is estimated from the observed reduction in mosquito catch numbers. A mathematical model capturing the life cycle of P. falciparum malaria in mosquitoes and humans and incorporating the excess mortality was used to estimate the potential epidemiological effect of ATSBs.

Results: The entomological study showed a significant reduction of ~ 57% (95% CI 33-72%) in mosquito catch numbers, and a larger reduction of ~ 89% (95% CI 75-100%) in the entomological inoculation rate due to the fact that, in the presence of ATSBs, most mosquitoes do not live long enough to transmit malaria. The excess mortality due to ATSBs was estimated to be lower (mean 0.09 per mosquito per day, seasonal range 0.07-0.11 per day) than the bait feeding rate obtained from one-day staining tests (mean 0.34 per mosquito per day, seasonal range 0.28-0.38 per day).

Conclusions: From epidemiological modelling, it was predicted that ATSBs could result in large reductions (> 30% annually) in prevalence and clinical incidence of malaria, even in regions with an existing high malaria burden. These results suggest that this new tool could provide a promising addition to existing vector control tools and result in significant reductions in malaria burden across a range of malaria-endemic settings.
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http://dx.doi.org/10.1186/s12936-021-03684-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968277PMC
March 2021

Spatio-temporal associations between deforestation and malaria incidence in Lao PDR.

Elife 2021 Mar 9;10. Epub 2021 Mar 9.

Malaria Elimination Initiative, The Global Health Group, University of California, San Francisco, San Francisco, United States.

As countries in the Greater Mekong Sub-region (GMS) increasingly focus their malaria control and elimination efforts on reducing forest-related transmission, greater understanding of the relationship between deforestation and malaria incidence will be essential for programs to assess and meet their 2030 elimination goals. Leveraging village-level health facility surveillance data and forest cover data in a spatio-temporal modeling framework, we found evidence that deforestation is associated with short-term increases, but long-term decreases confirmed malaria case incidence in Lao People's Democratic Republic (Lao PDR). We identified strong associations with deforestation measured within 30 km of villages but not with deforestation in the near (10 km) and immediate (1 km) vicinity. Results appear driven by deforestation in densely forested areas and were more pronounced for infections with () than for (). These findings highlight the influence of forest activities on malaria transmission in the GMS.
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http://dx.doi.org/10.7554/eLife.56974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024023PMC
March 2021

Inherently confinable split-drive systems in Drosophila.

Nat Commun 2021 03 5;12(1):1480. Epub 2021 Mar 5.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, USA.

CRISPR-based gene-drive systems, which copy themselves via gene conversion mediated by the homology-directed repair (HDR) pathway, have the potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway, resistant to Cas9 cleavage, can interrupt the spread of gene-drive elements. We hypothesized that drives targeting genes essential for viability or reproduction also carrying recoded sequences that restore endogenous gene functionality should benefit from dominantly-acting maternal clearance of NHEJ alleles combined with recessive Mendelian culling processes. Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that are inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multigenerational cage trials, sGDs follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage and/or lethal/sterile mosaic Cas9-dependent phenotypes, leading to inherently confinable drive outcomes.
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http://dx.doi.org/10.1038/s41467-021-21771-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935863PMC
March 2021

A confinable home-and-rescue gene drive for population modification.

Elife 2021 Mar 5;10. Epub 2021 Mar 5.

Section of Cell and Developmental Biology, University of California, San Diego, San Diego, United States.

Homing-based gene drives, engineered using CRISPR/Cas9, have been proposed to spread desirable genes throughout populations. However, invasion of such drives can be hindered by the accumulation of resistant alleles. To limit this obstacle, we engineer a confinable population modification home-and-rescue (HomeR) drive in targeting an essential gene. In our experiments, resistant alleles that disrupt the target gene function were recessive lethal and therefore disadvantaged. We demonstrate that HomeR can achieve an increase in frequency in population cage experiments, but that fitness costs due to the Cas9 insertion limit drive efficacy. Finally, we conduct mathematical modeling comparing HomeR to contemporary gene drive architectures for population modification over wide ranges of fitness costs, transmission rates, and release regimens. HomeR could potentially be adapted to other species, as a means for safe, confinable, modification of wild populations.
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http://dx.doi.org/10.7554/eLife.65939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968924PMC
March 2021

Application of the Relationship-Based Model to Engagement for Field Trials of Genetically Engineered Malaria Vectors.

Am J Trop Med Hyg 2020 Dec 21. Epub 2020 Dec 21.

Department of Molecular Biology and Biochemistry, University of California, Irvine, California.

The transition of new technologies for public health from laboratory to field is accompanied by a broadening scope of engagement challenges. Recent developments of vector control strategies involving genetically engineered mosquitoes with gene drives to assist in the eradication of malaria have drawn significant attention. Notably, questions have arisen surrounding community and regulatory engagement activities and of the need for examples of models or frameworks that can be applied to guide engagement. A relationship-based model (RBM) provides a framework that places stakeholders and community members at the center of decision-making processes, rather than as recipients of predetermined strategies, methods, and definitions. Successful RBM application in the transformation of healthcare delivery has demonstrated the importance of open dialogue and relationship development in establishing an environment where individuals are actively engaged in decision-making processes regarding their health. Although guidelines and recommendations for engagement for gene drives have recently been described, we argue here that communities and stakeholders should lead the planning, development, and implementation phases of engagement. The RBM provides a new approach to the development of ethical, transparent, and effective engagement strategies for malaria control programs.
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http://dx.doi.org/10.4269/ajtmh.20-0868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941841PMC
December 2020

Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi.

Nat Commun 2020 11 3;11(1):5553. Epub 2020 Nov 3.

Department of Microbiology & Molecular Genetics, University of California, Irvine, CA, 92697-3900, USA.

Cas9/gRNA-mediated gene-drive systems have advanced development of genetic technologies for controlling vector-borne pathogen transmission. These technologies include population suppression approaches, genetic analogs of insecticidal techniques that reduce the number of insect vectors, and population modification (replacement/alteration) approaches, which interfere with competence to transmit pathogens. Here, we develop a recoded gene-drive rescue system for population modification of the malaria vector, Anopheles stephensi, that relieves the load in females caused by integration of the drive into the kynurenine hydroxylase gene by rescuing its function. Non-functional resistant alleles are eliminated via a dominantly-acting maternal effect combined with slower-acting standard negative selection, and rare functional resistant alleles do not prevent drive invasion. Small cage trials show that single releases of gene-drive males robustly result in efficient population modification with ≥95% of mosquitoes carrying the drive within 5-11 generations over a range of initial release ratios.
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http://dx.doi.org/10.1038/s41467-020-19426-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609566PMC
November 2020

Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives.

Mol Cell 2020 10 18;80(2):246-262.e4. Epub 2020 Sep 18.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, USA; Tata Institute for Genetics and Society, University of California, San Diego, La Jolla, CA, USA. Electronic address:

CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. Here we describe two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. We compare the strengths of these two systems.
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http://dx.doi.org/10.1016/j.molcel.2020.09.003DOI Listing
October 2020

Translating gene drive science to promote linguistic diversity in community and stakeholder engagement.

Glob Public Health 2020 10 26;15(10):1551-1565. Epub 2020 Jun 26.

The Qualcomm Institute, Calit2, UC San Diego, La Jolla, CA, USA.

Information about genetic engineering (GE) for vector control in the United States is disseminated primarily in English, though non-English speakers are equally, and in some geographic regions even more affected by such technologies. Non-English-speaking publics should have equal access to such information, which is especially critical when the technology in question may impact whole communities. We convened an interdisciplinary workgroup to translate previously developed narrated slideshows on gene drive mosquitoes from English into Spanish, reviewing each iteration for scientific accuracy and accessibility to laypeople. Using the finalised stimuli, we conducted five online, chat-based focus groups with Spanish-speaking adults from California. Overall, participants expressed interest in the topic and were able to summarise the information presented in their own words. Importantly, participants asked for clarification and expressed scepticism about the information presented, indicating critical engagement with the material. Through collaboration with Spanish-speaking scientists engaged in the development of GE methods of vector control, we translated highly technical scientific information into Spanish that successfully engaged Spanish-speaking participants in conversations about this topic. In this manuscript, we document the feasibility of consulting Spanish-speaking publics about a complex emerging technology by drawing on the linguistic diversity of the scientific teams developing the technology.
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http://dx.doi.org/10.1080/17441692.2020.1779328DOI Listing
October 2020

Modeling confinement and reversibility of threshold-dependent gene drive systems in spatially-explicit Aedes aegypti populations.

BMC Biol 2020 05 12;18(1):50. Epub 2020 May 12.

Division of Epidemiology and Biostatistics, School of Public Health, University of California, Berkeley, CA, 94720, USA.

Background: The discovery of CRISPR-based gene editing and its application to homing-based gene drive systems has been greeted with excitement, for its potential to control mosquito-borne diseases on a wide scale, and concern, for the invasiveness and potential irreversibility of a release. Gene drive systems that display threshold-dependent behavior could potentially be used during the trial phase of this technology, or when localized control is otherwise desired, as simple models predict them to spread into partially isolated populations in a confineable manner, and to be reversible through releases of wild-type organisms. Here, we model hypothetical releases of two recently engineered threshold-dependent gene drive systems-reciprocal chromosomal translocations and a form of toxin-antidote-based underdominance known as UD-to explore their ability to be confined and remediated.

Results: We simulate releases of Aedes aegypti, the mosquito vector of dengue, Zika, and other arboviruses, in Yorkeys Knob, a suburb of Cairns, Australia, where previous biological control interventions have been undertaken on this species. We monitor spread to the neighboring suburb of Trinity Park to assess confinement. Results suggest that translocations could be introduced on a suburban scale, and remediated through releases of non-disease-transmitting male mosquitoes with release sizes on the scale of what has been previously implemented. UD requires fewer releases to introduce, but more releases to remediate, including of females capable of disease transmission. Both systems are expected to be confineable to the release site; however, spillover of translocations into neighboring populations is less likely.

Conclusions: Our analysis supports the use of translocations as a threshold-dependent drive system capable of spreading disease-refractory genes into Ae. aegypti populations in a confineable and reversible manner. It also highlights increased release requirements when incorporating life history and population structure into models. As the technology nears implementation, further ecological work will be essential to enhance model predictions in preparation for field trials.
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http://dx.doi.org/10.1186/s12915-020-0759-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218562PMC
May 2020

Vector bionomics and vectorial capacity as emergent properties of mosquito behaviors and ecology.

PLoS Comput Biol 2020 04 22;16(4):e1007446. Epub 2020 Apr 22.

Institute for Health Metrics and Evaluation, University of Washington, Seattle, Washington, United States of America.

Mosquitoes are important vectors for pathogens that infect humans and other vertebrate animals. Some aspects of adult mosquito behavior and mosquito ecology play an important role in determining the capacity of vector populations to transmit pathogens. Here, we re-examine factors affecting the transmission of pathogens by mosquitoes using a new approach. Unlike most previous models, this framework considers the behavioral states and state transitions of adult mosquitoes through a sequence of activity bouts. We developed a new framework for individual-based simulation models called MBITES (Mosquito Bout-based and Individual-based Transmission Ecology Simulator). In MBITES, it is possible to build models that simulate the behavior and ecology of adult mosquitoes in exquisite detail on complex resource landscapes generated by spatial point processes. We also developed an ordinary differential equation model which is the Kolmogorov forward equations for models developed in MBITES under a specific set of simplifying assumptions. While mosquito infection and pathogen development are one possible part of a mosquito's state, that is not our main focus. Using extensive simulation using some models developed in MBITES, we show that vectorial capacity can be understood as an emergent property of simple behavioral algorithms interacting with complex resource landscapes, and that relative density or sparsity of resources and the need to search can have profound consequences for mosquito populations' capacity to transmit pathogens.
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http://dx.doi.org/10.1371/journal.pcbi.1007446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197866PMC
April 2020

Toward the Definition of Efficacy and Safety Criteria for Advancing Gene Drive-Modified Mosquitoes to Field Testing.

Vector Borne Zoonotic Dis 2020 04 9;20(4):237-251. Epub 2020 Mar 9.

Liverpool School of Tropical Medicine, Liverpool, United Kingdom.

Mosquitoes containing gene drive systems are being developed as complementary tools to prevent transmission of malaria and other mosquito-borne diseases. As with any new tool, decision makers and other stakeholders will need to balance risks (safety) and benefits (efficacy) when considering the rationale for testing and deploying gene drive-modified mosquito products. Developers will benefit from standards for judging whether an investigational gene drive product meets acceptability criteria for advancing to field trials. Such standards may be formalized as preferred product characteristics and target product profiles, which describe the desired attributes of the product category and of a particular product, respectively. This report summarizes discussions from two scientific workshops aimed at identifying efficacy and safety characteristics that must be minimally met for an investigational gene drive-modified mosquito product to be deemed viable to move from contained testing to field release and the data that will be needed to support an application for first field release.
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http://dx.doi.org/10.1089/vbz.2019.2606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153640PMC
April 2020

Progress towards engineering gene drives for population control.

J Exp Biol 2020 02 7;223(Pt Suppl 1). Epub 2020 Feb 7.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92093, USA

Vector-borne diseases, such as dengue, Zika and malaria, are a major cause of morbidity and mortality worldwide. These diseases have proven difficult to control and currently available management tools are insufficient to eliminate them in many regions. Gene drives have the potential to revolutionize vector-borne disease control. This suite of technologies has advanced rapidly in recent years as a result of the availability of new, more efficient gene editing technologies. Gene drives can favorably bias the inheritance of a linked disease-refractory gene, which could possibly be exploited (i) to generate a vector population incapable of transmitting disease or (ii) to disrupt an essential gene for viability or fertility, which could eventually eliminate a population. Importantly, gene drives vary in characteristics such as their transmission efficiency, confinability and reversibility, and their potential to develop resistance to the drive mechanism. Here, we discuss recent advancements in the gene drive field, and contrast the benefits and limitations of a variety of technologies, as well as approaches to overcome these limitations. We also discuss the current state of each gene drive technology and the technical considerations that need to be addressed on the pathway to field implementation. While there are still many obstacles to overcome, recent progress has brought us closer than ever before to genetic-based vector modification as a tool to support vector-borne disease elimination efforts worldwide.
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http://dx.doi.org/10.1242/jeb.208181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790193PMC
February 2020

Development of a confinable gene drive system in the human disease vector .

Elife 2020 01 21;9. Epub 2020 Jan 21.

Section of Cell and Developmental Biology, University of California, San Diego, San Diego, United States.

is the principal mosquito vector for many arboviruses that increasingly infect millions of people every year. With an escalating burden of infections and the relative failure of traditional control methods, the development of innovative control measures has become of paramount importance. The use of gene drives has sparked significant enthusiasm for genetic control of mosquitoes; however, no such system has been developed in . To fill this void, here we develop several CRISPR-based split gene drives for use in this vector. With cleavage rates up to 100% and transmission rates as high as 94%, mathematical models predict that these systems could spread anti-pathogen effector genes into wild populations in a safe, confinable and reversible manner appropriate for field trials and effective for controlling disease. These findings could expedite the development of effector-linked gene drives that could safely control wild populations of to combat local pathogen transmission.
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http://dx.doi.org/10.7554/eLife.51701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6974361PMC
January 2020

A transcomplementing gene drive provides a flexible platform for laboratory investigation and potential field deployment.

Nat Commun 2020 01 17;11(1):352. Epub 2020 Jan 17.

Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, 92093, USA.

CRISPR-based gene drives can spread through wild populations by biasing their own transmission above the 50% value predicted by Mendelian inheritance. These technologies offer population-engineering solutions for combating vector-borne diseases, managing crop pests, and supporting ecosystem conservation efforts. Current technologies raise safety concerns for unintended gene propagation. Herein, we address such concerns by splitting the drive components, Cas9 and gRNAs, into separate alleles to form a trans-complementing split-gene-drive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This dual-component configuration allows for combinatorial transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a small-molecule-controlled version to investigate the biology of component inheritance and resistant allele formation, and to study the effects of maternal inheritance and impaired homology on efficiency. Lastly, mathematical modeling of tGD spread within populations reveals potential advantages for improving current gene-drive technologies for field population modification.
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http://dx.doi.org/10.1038/s41467-019-13977-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6969112PMC
January 2020

Experimental population modification of the malaria vector mosquito, Anopheles stephensi.

PLoS Genet 2019 12 19;15(12):e1008440. Epub 2019 Dec 19.

Department of Microbiology & Molecular Genetics, University of California, Irvine, California, United States of America.

Small laboratory cage trials of non-drive and gene-drive strains of the Asian malaria vector mosquito, Anopheles stephensi, were used to investigate release ratios and other strain properties for their impact on transgene spread during simulated population modification. We evaluated the effects of transgenes on survival, male contributions to next-generation populations, female reproductive success and the impact of accumulation of gene drive-resistant genomic target sites resulting from nonhomologous end-joining (NHEJ) mutagenesis during Cas9, guide RNA-mediated cleavage. Experiments with a non-drive, autosomally-linked malaria-resistance gene cassette showed 'full introduction' (100% of the insects have at least one copy of the transgene) within 8 weeks (≤ 3 generations) following weekly releases of 10:1 transgenic:wild-type males in an overlapping generation trial design. Male release ratios of 1:1 resulted in cages where mosquitoes with at least one copy of the transgene fluctuated around 50%. In comparison, two of three cages in which the malaria-resistance genes were linked to a gene-drive system in an overlapping generation, single 1:1 release reached full introduction in 6-8 generations with a third cage at ~80% within the same time. Release ratios of 0.1:1 failed to establish the transgenes. A non-overlapping generation, single-release trial of the same gene-drive strain resulted in two of three cages reaching 100% introduction within 6-12 generations following a 1:1 transgenic:wild-type male release. Two of three cages with 0.33:1 transgenic:wild-type male single releases achieved full introduction in 13-16 generations. All populations exhibiting full introduction went extinct within three generations due to a significant load on females having disruptions of both copies of the target gene, kynurenine hydroxylase. While repeated releases of high-ratio (10:1) non-drive constructs could achieve full introduction, results from the 1:1 release ratios across all experimental designs favor the use of gene drive, both for efficiency and anticipated cost of the control programs.
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http://dx.doi.org/10.1371/journal.pgen.1008440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6922335PMC
December 2019

Winning the Tug-of-War Between Effector Gene Design and Pathogen Evolution in Vector Population Replacement Strategies.

Front Genet 2019 30;10:1072. Epub 2019 Oct 30.

Section of Cell and Developmental Biology, University of California, San Diego, CA, United States.

While efforts to control malaria with available tools have stagnated, and arbovirus outbreaks persist around the globe, the advent of clustered regularly interspaced short palindromic repeat (CRISPR)-based gene editing has provided exciting new opportunities for genetics-based strategies to control these diseases. In one such strategy, called "population replacement", mosquitoes, and other disease vectors are engineered with effector genes that render them unable to transmit pathogens. These effector genes can be linked to "gene drive" systems that can bias inheritance in their favor, providing novel opportunities to replace disease-susceptible vector populations with disease-refractory ones over the course of several generations. While promising for the control of vector-borne diseases on a wide scale, this sets up an evolutionary tug-of-war between the introduced effector genes and the pathogen. Here, we review the disease-refractory genes designed to date to target malaria transmitted by , and arboviruses transmitted by , including dengue serotypes 2 and 3, chikungunya, and Zika viruses. We discuss resistance concerns for these effector genes, and genetic approaches to prevent parasite and viral escape variants. One general approach is to increase the evolutionary hurdle required for the pathogen to evolve resistance by attacking it at multiple sites in its genome and/or multiple stages of development. Another is to reduce the size of the pathogen population by other means, such as with vector control and antimalarial drugs. We discuss lessons learned from the evolution of resistance to antimalarial and antiviral drugs and implications for the management of resistance after its emergence. Finally, we discuss the target product profile for population replacement strategies for vector-borne disease control. This differs between early phase field trials and wide-scale disease control. In the latter case, the demands on effector gene efficacy are great; however, with new possibilities ushered in by CRISPR-based gene editing, and when combined with surveillance, monitoring, and rapid management of pathogen resistance, the odds are increasingly favoring effector genes in the upcoming evolutionary tug-of-war.
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http://dx.doi.org/10.3389/fgene.2019.01072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831721PMC
October 2019

Reply to 'Concerns about the feasibility of using "precision guided sterile males" to control insects'.

Nat Commun 2019 09 2;10(1):3955. Epub 2019 Sep 2.

Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, United States of America.

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http://dx.doi.org/10.1038/s41467-019-11617-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6718384PMC
September 2019

Genome-wide divergence among invasive populations of Aedes aegypti in California.

BMC Genomics 2019 Mar 12;20(1):204. Epub 2019 Mar 12.

Department of Entomology and Nematology, College of Agricultural and Environmental Sciences, University of California - Davis, Davis, CA, 95616, USA.

Background: In the summer of 2013, Aedes aegypti Linnaeus was first detected in three cities in central California (Clovis, Madera and Menlo Park). It has now been detected in multiple locations in central and southern CA as far south as San Diego and Imperial Counties. A number of published reports suggest that CA populations have been established from multiple independent introductions.

Results: Here we report the first population genomics analyses of Ae. aegypti based on individual, field collected whole genome sequences. We analyzed 46 Ae. aegypti genomes to establish genetic relationships among populations from sites in California, Florida and South Africa. Based on 4.65 million high quality biallelic SNPs, we identified 3 major genetic clusters within California; one that includes all sample sites in the southern part of the state (South of Tehachapi mountain range) plus the town of Exeter in central California and two additional clusters in central California.

Conclusions: A lack of concordance between mitochondrial and nuclear genealogies suggests that the three founding populations were polymorphic for two main mitochondrial haplotypes prior to being introduced to California. One of these has been lost in the Clovis populations, possibly by a founder effect. Genome-wide comparisons indicate extensive differentiation between genetic clusters. Our observations support recent introductions of Ae. aegypti into California from multiple, genetically diverged source populations. Our data reveal signs of hybridization among diverged populations within CA. Genetic markers identified in this study will be of great value in pursuing classical population genetic studies which require larger sample sizes.
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http://dx.doi.org/10.1186/s12864-019-5586-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6417271PMC
March 2019

Transforming insect population control with precision guided sterile males with demonstration in flies.

Nat Commun 2019 01 8;10(1):84. Epub 2019 Jan 8.

Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92093, California, USA.

The sterile insect technique (SIT) is an environmentally safe and proven technology to suppress wild populations. To further advance its utility, a novel CRISPR-based technology termed precision guided SIT (pgSIT) is described. PgSIT mechanistically relies on a dominant genetic technology that enables simultaneous sexing and sterilization, facilitating the release of eggs into the environment ensuring only sterile adult males emerge. Importantly, for field applications, the release of eggs will eliminate burdens of manually sexing and sterilizing males, thereby reducing overall effort and increasing scalability. Here, to demonstrate efficacy, we systematically engineer multiple pgSIT systems in Drosophila which consistently give rise to 100% sterile males. Importantly, we demonstrate that pgSIT-generated sterile males are fit and competitive. Using mathematical models, we predict pgSIT will induce substantially greater population suppression than can be achieved by currently-available self-limiting suppression technologies. Taken together, pgSIT offers to potentially transform our ability to control insect agricultural pests and disease vectors.
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http://dx.doi.org/10.1038/s41467-018-07964-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325135PMC
January 2019

Estimating the elimination feasibility in the 'end game' of control efforts for parasites subjected to regular mass drug administration: Methods and their application to schistosomiasis.

PLoS Negl Trop Dis 2018 11 12;12(11):e0006794. Epub 2018 Nov 12.

Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Australia.

Progress towards controlling and eliminating parasitic worms, including schistosomiasis, onchocerciasis, and lymphatic filariasis, is advancing rapidly as national governments, multinational NGOs, and pharmaceutical companies launch collaborative chemotherapeutic control campaigns. Critical questions remain regarding the potential for achieving elimination of these infections, and analytical methods can help to quickly estimate progress towards-and the probability of achieving-elimination over specific timeframes. Here, we propose the effective reproduction number, Reff, as a proxy of elimination potential for sexually reproducing worms that are subject to poor mating success at very low abundance (positive density dependence, or Allee effects). Reff is the number of parasites produced by a single reproductive parasite at a given stage in the transmission cycle, over the parasite's lifetime-it is the generalized form of the more familiar basic reproduction number, R0, which only applies at the beginning of an epidemic-and it can be estimated in a 'model-free' manner by an estimator ('ε'). We introduce ε, demonstrate its estimation using simulated data, and discuss how it may be used in planning and evaluation of ongoing elimination efforts for a range of parasitic diseases.
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http://dx.doi.org/10.1371/journal.pntd.0006794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258430PMC
November 2018

Consequences of resistance evolution in a Cas9-based sex conversion-suppression gene drive for insect pest management.

Proc Natl Acad Sci U S A 2018 06 29;115(24):6189-6194. Epub 2018 May 29.

Department of Developmental Biology, Johann Friedrich Blumenbach Institute of Zoology and Anthropology, Göttingen Center of Molecular Biosciences, University of Göttingen, 37077 Göttingen, Germany;

The use of a site-specific homing-based gene drive for insect pest control has long been discussed, but the easy design of such systems has become possible only with the recent establishment of CRISPR/Cas9 technology. In this respect, novel targets for insect pest management are provided by new discoveries regarding sex determination. Here, we present a model for a suppression gene drive designed to cause an all-male population collapse in an agricultural pest insect. To evaluate the molecular details of such a sex conversion-based suppression gene drive experimentally, we implemented this strategy in to serve as a safe model organism. We generated a Cas9-based homing gene-drive element targeting the gene and showed its high efficiency for sex conversion from females to males. However, nonhomologous end joining increased the rate of mutagenesis at the target site, which resulted in the emergence of drive-resistant alleles and therefore curbed the gene drive. This confirms previous studies that simple homing CRISPR/Cas9 gene-drive designs will be ineffective. Nevertheless, by performing population dynamics simulations using the parameters we obtained in and by adjusting the model for the agricultural pest , we were able to identify adequate modifications that could be successfully applied for the management of wild Mediterranean fruit fly populations using our proposed sex conversion-based suppression gene-drive strategy.
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http://dx.doi.org/10.1073/pnas.1713825115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004448PMC
June 2018

Mathematical models of human mobility of relevance to malaria transmission in Africa.

Sci Rep 2018 05 16;8(1):7713. Epub 2018 May 16.

MRC Centre for Outbreak Analysis and Modelling, Department of Infectious Disease Epidemiology, Imperial College London, London, UK.

As Africa-wide malaria prevalence declines, an understanding of human movement patterns is essential to inform how best to target interventions. We fitted movement models to trip data from surveys conducted at 3-5 sites throughout each of Mali, Burkina Faso, Zambia and Tanzania. Two models were compared in terms of their ability to predict the observed movement patterns - a gravity model, in which movement rates between pairs of locations increase with population size and decrease with distance, and a radiation model, in which travelers are cumulatively "absorbed" as they move outwards from their origin of travel. The gravity model provided a better fit to the data overall and for travel to large populations, while the radiation model provided a better fit for nearby populations. One strength of the data set was that trips could be categorized according to traveler group - namely, women traveling with children in all survey countries and youth workers in Mali. For gravity models fitted to data specific to these groups, youth workers were found to have a higher travel frequency to large population centers, and women traveling with children a lower frequency. These models may help predict the spatial transmission of malaria parasites and inform strategies to control their spread.
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http://dx.doi.org/10.1038/s41598-018-26023-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955928PMC
May 2018

Synthetically engineered gene drive system in the worldwide crop pest .

Proc Natl Acad Sci U S A 2018 05 17;115(18):4725-4730. Epub 2018 Apr 17.

Department of Entomology, University of California, Riverside, CA 92521;

Synthetic gene drive systems possess enormous potential to replace, alter, or suppress wild populations of significant disease vectors and crop pests; however, their utility in diverse populations remains to be demonstrated. Here, we report the creation of a synthetic gene drive system in a major worldwide crop pest, We demonstrate that this drive system, based on an engineered maternal "toxin" coupled with a linked embryonic "antidote," is capable of biasing Mendelian inheritance rates with up to 100% efficiency. However, we find that drive resistance, resulting from naturally occurring genetic variation and associated fitness costs, can be selected for and hinder the spread of such a drive. Despite this, our results suggest that this gene drive could maintain itself at high frequencies in a wild population and spread to fixation if either its fitness costs or toxin resistance were reduced, providing a clear path forward for developing future such systems in this pest.
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http://dx.doi.org/10.1073/pnas.1713139115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5939061PMC
May 2018

Engineered Reciprocal Chromosome Translocations Drive High Threshold, Reversible Population Replacement in Drosophila.

ACS Synth Biol 2018 05 13;7(5):1359-1370. Epub 2018 Apr 13.

Division of Biology and Biological Engineering , California Institute of Technology , Pasadena , California 91125 , United States.

Replacement of wild insect populations with transgene-bearing individuals unable to transmit disease or survive under specific environmental conditions using gene drive provides a self-perpetuating method of disease prevention. Mechanisms that require the gene drive element and linked cargo to exceed a high threshold frequency in order for spread to occur are attractive because they offer several points of control: they bring about local, but not global population replacement; and transgenes can be eliminated by reintroducing wildtypes into the population so as to drive the frequency of transgenes below the threshold frequency required for drive. Reciprocal chromosome translocations were proposed as a tool for bringing about high threshold population replacement in 1940 and 1968. However, translocations able to achieve this goal have only been reported once, in the spider mite Tetranychus urticae, a haplo-diploid species in which there is strong selection in haploid males for fit homozygotes. We report the creation of engineered translocation-bearing strains of Drosophila melanogaster, generated through targeted chromosomal breakage and homologous recombination. These strains drive high threshold population replacement in laboratory populations. While it remains to be shown that engineered translocations can bring about population replacement in wild populations, these observations suggest that further exploration of engineered translocations as a tool for controlled population replacement is warranted.
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http://dx.doi.org/10.1021/acssynbio.7b00451DOI Listing
May 2018