Expansions to the MGDrivE suite for simulating the efficacy of novel gene-drive constructs in the control of mosquito-borne diseases.

OBJECTIVE: The MGDrivE (MGDrivE 1 and MGDrivE 2) modeling framework provides a flexible and expansive environment for testing the efficacy of novel gene-drive constructs for the control of mosquito-borne diseases. However, the existing model framework did not previously support several features necessary to simulate some types of intervention strategies. Namely, current MGDrivE versions do not permit modeling of small molecule inducible systems for controlling gene expression in gene drive designs or the inheritance patterns of self-eliminating gene drive mechanisms. RESULTS: Here, we demonstrate a new MGDrivE 2 module that permits the simulation of gene drive strategies incorporating small molecule-inducible systems and self-eliminating gene drive mechanisms. Additionally, we also implemented novel sparsity-aware sampling algorithms for improved computational efficiency in MGDrivE 2 and supplied an analysis and plotting function applicable to the outputs of MGDrivE 1 and MGDrivE 2.

Genetic conversion of a split-drive into a full-drive element.

The core components of CRISPR-based gene drives, Cas9 and guide RNA (gRNA), either can be linked within a self-contained single cassette (full gene-drive, fGD) or be provided in two separate elements (split gene-drive, sGD), the latter offering greater control options. We previously engineered split systems that could be converted genetically into autonomous full drives. Here, we examine such dual systems inserted at the spo11 locus that are recoded to restore gene function and thus organismic fertility. Despite minimal differences in transmission efficiency of the sGD or fGD drive elements in single generation crosses, the reconstituted spo11 fGD cassette surprisingly exhibits slower initial drive kinetics than the unlinked sGD element in multigenerational cage studies, but then eventually catches up to achieve a similar level of final introduction. These unexpected kinetic behaviors most likely reflect differing transient fitness costs associated with individuals co-inheriting Cas9 and gRNA transgenes during the drive process.

Close-kin mark-recapture methods to estimate demographic parameters of mosquitoes.

Close-kin mark-recapture (CKMR) methods have recently been used to infer demographic parameters such as census population size and survival for fish of interest to fisheries and conservation. These methods have advantages over traditional mark-recapture methods as the mark is genetic, removing the need for physical marking and recapturing that may interfere with parameter estimation. For mosquitoes, the spatial distribution of close-kin pairs has been used to estimate mean dispersal distance, of relevance to vector-borne disease transmission and novel biocontrol strategies. Here, we extend CKMR methods to the life history of mosquitoes and comparable insects. We derive kinship probabilities for mother-offspring, father-offspring, full-sibling and half-sibling pairs, where an individual in each pair may be a larva, pupa or adult. A pseudo-likelihood approach is used to combine the marginal probabilities of all kinship pairs. To test the effectiveness of this approach at estimating mosquito demographic parameters, we develop an individual-based model of mosquito life history incorporating egg, larva, pupa and adult life stages. The simulation labels each individual with a unique identification number, enabling close-kin relationships to be inferred for sampled individuals. Using the dengue vector Aedes aegypti as a case study, we find the CKMR approach provides unbiased estimates of adult census population size, adult and larval mortality rates, and larval life stage duration for logistically feasible sampling schemes. Considering a simulated population of 3,000 adult mosquitoes, estimation of adult parameters is accurate when ca. 40 adult females are sampled biweekly over a three month period. Estimation of larval parameters is accurate when adult sampling is supplemented with ca. 120 larvae sampled biweekly over the same period. The methods are also effective at detecting intervention-induced increases in adult mortality and decreases in population size. As the cost of genome sequencing declines, CKMR holds great promise for characterizing the demography of mosquitoes and comparable insects of epidemiological and agricultural significance.

Reversing insecticide resistance with allelic-drive in Drosophila melanogaster.

A recurring target-site mutation identified in various pests and disease vectors alters the voltage gated sodium channel (vgsc) gene (often referred to as knockdown resistance or kdr) to confer resistance to commonly used insecticides, pyrethroids and DDT. The ubiquity of kdr mutations poses a major global threat to the continued use of insecticides as a means for vector control. In this study, we generate common kdr mutations in isogenic laboratory Drosophila strains using CRISPR/Cas9 editing. We identify differential sensitivities to permethrin and DDT versus deltamethrin among these mutants as well as contrasting physiological consequences of two different kdr mutations. Importantly, we apply a CRISPR-based allelic-drive to replace a resistant kdr mutation with a susceptible wild-type counterpart in population cages. This successful proof-of-principle opens-up numerous possibilities including targeted reversion of insecticide-resistant populations to a native susceptible state or replacement of malaria transmitting mosquitoes with those bearing naturally occurring parasite resistant alleles.

Exploiting a Y chromosome-linked Cas9 for sex selection and gene drive.

CRISPR-based genetic engineering tools aimed to bias sex ratios, or drive effector genes into animal populations, often integrate the transgenes into autosomal chromosomes. However, in species with heterogametic sex chromsomes (e.g. XY, ZW), sex linkage of endonucleases could be beneficial to drive the expression in a sex-specific manner to produce genetic sexing systems, sex ratio distorters, or even sex-specific gene drives, for example. To explore this possibility, here we develop a transgenic line of Drosophila melanogaster expressing Cas9 from the Y chromosome. We functionally characterize the utility of this strain for both sex selection and gene drive finding it to be quite effective. To explore its utility for population control, we built mathematical models illustrating its dynamics as compared to other state-of-the-art systems designed for both population modification and suppression. Taken together, our results contribute to the development of current CRISPR genetic control tools and demonstrate the utility of using sex-linked Cas9 strains for genetic control of animals.

Suppressing mosquito populations with precision guided sterile males.

The mosquito Aedes aegypti is the principal vector for arboviruses including dengue/yellow fever, chikungunya, and Zika virus, infecting hundreds of millions of people annually. Unfortunately, traditional control methodologies are insufficient, so innovative control methods are needed. To complement existing measures, here we develop a molecular genetic control system termed precision-guided sterile insect technique (pgSIT) in Aedes aegypti. PgSIT uses a simple CRISPR-based approach to generate flightless females and sterile males that are deployable at any life stage. Supported by mathematical models, we empirically demonstrate that released pgSIT males can compete, suppress, and even eliminate mosquito populations. This platform technology could be used in the field, and adapted to many vectors, for controlling wild populations to curtail disease in a safe, confinable, and reversible manner.

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

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.

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

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 Drosophila 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.

Inherently confinable split-drive systems in Drosophila.

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.

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

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.

Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives.

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.

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

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 UDMEL-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. UDMEL 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.

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

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.

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

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.