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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: a) effectively spread into partially isolated populations in a confineable manner, and b) 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. 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. Our 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. https://youtu.be/lNcIHcJYI_0
Inheritance and landscape features of the modeling framework. (A) Reciprocal translocations (T1 and T2) result from the mutual exchange between terminal segments of two non-homologous chromosomes (N1 and N2). The cross here depicts possible parental gametes, with respect to the translocation, and offspring that result from matings between them. Matings between wild-type organisms or translocation homozygotes result in viable offspring; but translocation heterozygotes produce unbalanced gametes, and many of the resulting offspring are unviable (shaded). This results in a heterozygote disadvantage and threshold-dependent population dynamics. (B) UD MEL is composed of two unlinked constructs (here referred to as A and B), each consisting of a maternally-expressed toxin and a zygotically-expressed antidote for the toxin on the opposite construct. The cross here represents matings between two of the nine possible parental genotypes ("+" represents the wild-type allele, and "A" and "B" represent alleles corresponding to the two UD MEL constructs). The complete inheritance pattern is depicted in Figure S1. Offspring lacking the antidotes to the maternal toxins produced by their mother are unviable (shaded). At high population frequencies, the selective advantage on the constructs, by virtue of the antidotes, outweighs the fitness load due to the toxins, and hence results in frequency-dependent spread. (C) Distribution of households in Yorkeys Knob (blue) and Trinity Park (red) in Cairns, Queensland, Australia. Households serve as Aedes aegypti metapopulation nodes in our simulations, with movement of adult Ae. aegypti between them. Yorkeys Knob serves as a simulated release site, and Trinity Park as a simulated control site.
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Replacement, remediation and confinement outcomes for translocations and UDMEL. Outcomes are depicted for the proportion of 50 stochastic simulations of population replacement, remediation and confinement of translocations and UDMEL that result in fixation of each system. (A-D) For replacement and remediation, each cell corresponds to a given number of releases (horizontal axis) and coverage level (vertical axis), given 20 adult Ae. aegypti per household per release. For replacement, releases are of males homozygous for the system into a wild-type population. For remediation of translocations, releases are of wild-type males into a population homozygous for the translocation, and for mixed remediation of UDMEL, releases are of wild-type females and males into a population homozygous for UDMEL. Light blue cells represent cases where all simulations result in fixation of the system, and dark blue cells represent cases where the wild-type is fixed in all simulations. (E-F) For confinement, each cell corresponds to a daily number of batch migration events (horizontal axis) of a given size (vertical axis) from Yorkeys Knob, where the system is fixed, to Trinity Park, where the system is initially absent. White cells represent cases where all simulations result in fixation of the system in Trinity Park, and dark pink cells represent cases where the wild-type is fixed in all simulations. These results are encouraging for translocations as systems for introducing transgenes in a local and reversible way as: i) they can be remediated through an achievable number of male-only releases, and ii) they require more batch migration events to spread to neighboring communities.
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1
Confinement and reversibility of threshold-dependent gene drive systems in
spatially-explicit Aedes aegypti populations
Héctor M. Sánchez C. 1, Jared B. Bennett 2, Sean L. Wu 1, Gordana Rašić 3, Omar S. Akbari 4,
John M. Marshall 1,5,†
1 Division of Epidemiology and Biostatistics, School of Public Health, University of California,
Berkeley, CA 94720, USA
2 Biophysics Graduate Group, University of California, Berkeley, CA 94720, USA
3 Mosquito Control Laboratory, QIMR Berghofer Medical Research Institute, Brisbane,
Australia
4 Cell and Developmental Biology Section, Division of Biological Sciences, University of
California, San Diego, CA 92093, USA
5 Innovative Genomics Institute, Berkeley, CA 94720, USA
To whom correspondence should be addressed:
John M. Marshall
Division of Epidemiology & Biostatistics, School of Public Health
University of California, Berkeley, CA 94720, USA
Email: john.marshall@berkeley.edu
Key words:
Chromosomal translocations, underdominance, metapopulations, population dynamics,
population replacement, biosafety, field trials
Abstract:
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: a) effectively spread into partially isolated populations in a confineable manner,
and b) 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. 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.
Our 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.
.CC-BY-NC-ND 4.0 International licenseIt is made available under a
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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2
Introduction:
The discovery of CRISPR and its application as a gene editing tool has enabled gene drive
systems to be engineered with much greater ease (1, 2). Recent attention has focused on homing-
based drive systems and their potential to control mosquito-borne diseases on a wide scale, either
by spreading disease-refractory genes (3) or by spreading genes that confer a fitness load or sex
bias and thereby suppress mosquito populations (4, 5). The increased ease of gene editing has
also advanced the entire field of gene drive, including systems appropriate during the trial phase
of the technology (6). Such systems would ideally be capable of enacting local population
control by: a) effectively spreading into populations to achieve the desired epidemiological
effect, and b) being recallable from the environment in the event of unwanted consequences,
public disfavor, or the end of a trial period.
As gene drive technology has progressed, a number of systems have been proposed with
the potential to enact localized population control without spreading on a wide scale (6, 7).
Sterile male releases provide one option (8), a recent version of which is based on the same
molecular components as CRISPR gene drive systems (8, 9). At the interface between homing-
based and localized suppression systems, an autosomal X chromosome-shredding system has
been proposed that displays transient drive and suppression before being selected out of the
population (10). Population modification drive systems that display transient drive activity
before being eliminated by virtue of a fitness cost, could also spread disease-refractory genes
into populations in a localized way. Examples of this variety of drive systems include split-gene
drive (11), daisy drive (12) and killer-rescue systems (13). Each system has its own strengths and
weaknesses, and could be suited to a different situation. In this paper, we theoretically explore
the potential for two recently-engineered threshold-dependent gene drive systems to achieve
localized and reversible population modification in structured populations - reciprocal
chromosomal translocations (14) and a toxin-antidote-based system known as UDMEL (15).
Threshold-dependent gene drive systems must exceed a critical threshold frequency in a
population in order to spread. Based on this dynamic, simple population models, in which two
randomly mating populations exchange small numbers of migrants with each other, predict that
these systems can be released at high frequencies in one population and spread to near-fixation
there, but never take off in the neighboring population because they do not exceed the required
threshold there (16, 17). These systems can also be eliminated through dilution with wild-type
organisms at the release site, making them excellent candidates for the trial phase of a population
modification gene drive strategy, or when localized population modification is desired. However,
whether these dynamics hold in real ecosystems depends crucially on the dispersal patterns and
population structure of the species being considered. First steps towards modeling the spatial
dynamics of these systems have been taken by Champer et al. (18), who model spatially-
structured releases of various threshold-dependent systems without life history, and Huang et al.
(19), who model engineered underdominance (20) on a grid-based landscape incorporating life
history for Aedes aegypti, the mosquito vector of dengue, Zika and other arboviruses.
Here, we present a detailed ecological analysis of the expected population dynamics of
two recently-engineered threshold-dependent drive systems, translocations and UDMEL, in Ae.
aegypt in a well-characterized landscape - Yorkeys Knob, a suburb ~17 km northwest of Cairns,
Australia (Figure 1C) - where field trials could conceivably be conducted. Yorkeys Knob and the
nearby town of Gordonvale were field sites for releases of Wolbachia-infected mosquitoes in
2011 (21) and the prevalence of Wolbachia infection over time provided information on the
number of adult Ae. aegypti mosquitoes per household and other mosquito demographic
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parameters for that location (22). Wolbachia is an intracellular bacterium and biocontrol agent
that biases inheritance in its favor if infected females are present, and blocks transmission of
dengue and other arboviruses (23). Yorkeys Knob is a partially isolated suburb, separated by a 1-
2 km-wide uninhabited, vegetated area from the nearest suburb, Trinity Park, a control site for
the Wolbachia trial. This allowed us to simulate trials of transgenic mosquitoes in a well-
characterized population, while also theoretically exploring their potential of spread to a
neighboring community.
A number of other ecological details are relevant to the spread of threshold-dependent
gene drive systems that have not been considered in previous modeling studies. Perhaps of
greatest importance, the frequency of the introduced transgene is markedly different from the
frequency of introduced adults. It is typical to release only male mosquitoes as part of an
intervention, as only females are involved in human disease transmission. Life cycle and mating
structure therefore become relevant, as immature life stages are not available for mating, and
female adults are thought to mostly mate only once soon after emergence (24). This means that
many of the released adult males will not find a mating partner, and hence larger releases will be
required to exceed threshold frequencies than predicted in simple population frequency models.
The nature of mosquito dispersal behavior is also relevant to the spatial dispersal of
transgenes. Our species of interest, Ae. aegypti, is understood to display leptokurtic dispersal
behavior in a suburban setting, in which mosquitoes tend to remain in the same household for the
majority of their lifespan, while a few mosquitoes disperse over larger distances (25). With these
landscape, dispersal and life cycle considerations accounted for, we theoretically explore the
ability to drive two threshold-dependent systems, translocations and UDMEL, into populations of
Ae. aegypti in one community, Yorkeys Knob, without them spreading in significant numbers to
a neighboring community, Trinity Park, and to be remediated from Yorkeys Knob at the end of
the simulated trial period.
Results:
Model framework. We use the Mosquito Gene Drive Explorer (MGDrivE) modeling
framework (26) to model the spread of translocations and UDMEL through spatially-structured
mosquito populations (Figure 1). This is a genetic and spatial extension of the lumped age-class
model of mosquito ecology (27) modified and applied by Deredec et al. (28) and Marshall et al.
(29) to the spread of homing gene drive systems. The framework incorporates the egg, larval,
pupal and adult life stages, with egg genotypes being determined by maternal and paternal
genotypes and the allelic inheritance pattern of the gene drive system. Spatial dynamics are
accommodated through a metapopulation structure in which lumped age-class models run in
parallel and migrants are exchanged between metapopulations according to a specified dispersal
kernel. Further details of the framework are described in the Materials and Methods section.
Applying the MGDrivE modeling framework to our research questions, we incorporate
the inheritance patterns of reciprocal chromosomal translocations and UDMEL into the inheritance
module of the model (Figure 1A-B), the life cycle parameters of Aedes aegypti (Table S1) into
the life history module, and the distribution of households in Yorkeys Knob and Trinity Park
along with their expected mosquito population sizes and movement rates between them into the
landscape module (Figure 1C). The suburb of Trinity Park served as a control site for field
releases of Wolbachia-infected mosquitoes, to quantify the extent to which the Wolbachia
infection could spread from one community to another, and plays a similar role for our simulated
releases of threshold-dependent gene drive systems.
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(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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The inheritance patterns that result from chromosomal translocations are depicted in
Figure 1A. Chromosomal translocations result from the mutual exchange between terminal
segments of two nonhomologous chromosomes. When translocation heterozygotes mate, several
crosses result in unbalanced genotypes and hence unviable offspring, resulting in a heterozygote
reproductive disadvantage. This results in bistable, threshold-dependent population dynamics,
confirmed in laboratory drive experiments (14). The inheritance patterns produced by the UDMEL
system are depicted in Figure 1B. This system consists of two unlinked constructs, each
possessing a maternally-expressed toxin active during oogenesis, and a zygotically-active
antidote expressed by the opposite construct. The resulting dynamic, confirmed in laboratory
drive experiments (15), is gene drive for allele frequencies greater than ~24%, in the absence of a
fitness cost.
Population replacement and remediation for translocations. The use of translocations
for transforming pest populations was initially suggested by Serebrovskii (30) and later Curtis
(31) for the introduction of disease-refractory genes into mosquito populations. A number of
models have been proposed to describe their spread through randomly-mating populations (14,
16, 32, 33); however, with one recent exception addressing spatial structure (18), these have
largely ignored insect life history and mating structure. Such models suggest that the
translocation need only exceed a population frequency of 50%, in the absence of a fitness cost
associated with the translocation, to spread to fixation in a population, which could conceivably
be achieved through a single seeding release round. Here, we find that incorporating life history
and population structure into mosquito population dynamic models significantly increases
release requirements.
In Figures 2-3, based on the precedent set by the World Mosquito Program
(https://www.worldmosquitoprogram.org/), we consider weekly releases of 20 adult Ae. aegypti
males homozygous for the translocation for given durations and coverage levels, where coverage
level is the proportion of households that receive the releases. Releases are simulated in the
community of Yorkeys Knob, in which prior releases of Wolbachia-infected mosquitoes
suggested a local population of ~15 adult Ae. aegypti per household (22), and for mosquito
movement rates inferred from previous studies (25, 34, 35) (Table S1). For a coverage level of
100%, and in the absence of a fitness cost, five weekly releases of 20 Ae. aegypti males (~3:1
release to local males) are required for the translocation to spread to fixation in the community
(Figure 2), as opposed to the single release expected when ignoring life history and population
structure (32). As coverage is reduced to 50%, the required number of releases increases to 11,
and for a coverage level of 25%, as seen for the World Mosquito Program in Yorkeys Knob, the
required number of releases increases to 20 (Figures 2-3). Although large, these releases are
achievable, considering the much larger releases conducted for sterile insect programs (36).
To simulate remediation of a translocation, we consider weekly releases of 20 adult Ae.
aegypti wild-type males in the community of Yorkeys Knob, whereby the translocation has
already reached fixation in that community. In the absence of a fitness cost associated with the
translocation, translocations are symmetrical in their threshold dynamics and so, for a coverage
level of 100%, five weekly releases are required for the translocation to be completely
remediated from the community, and for a coverage of 25%, 20 weekly releases are required for
the translocation to be completely remediated (Figures 2-3). Encouraging features of these
results are that: i) remediation can be achieved through releases of non-biting, non-disease-
transmitting males, ii) release sizes are achievable, and iii) despite the spatial household
structure, both replacement and remediation are complete within the community. The time to
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replacement is highly dependent on the coverage level and number of releases; but is reasonably
quick given sufficient releases. At a coverage of 50%, 20 weekly releases led to the translocation
spreading to a frequency >99% within a year of the final release (or within 500 days of the first
release). For equivalent wild-type releases, this is the same as the time to >99% elimination.
Population replacement and remediation for UDMEL. UDMEL was the first synthetic
gene drive system to be engineered that displays threshold-dependent dynamics (15). The system
consists of two unlinked constructs, each possessing a maternally expressed toxin active during
oogenesis, and a zygotically active antidote expressed by the opposite construct (Figure 1B). At
low population frequencies, the maternal toxin confers a significant fitness cost, leading to
elimination, while at high population frequencies, the zygotic antidote confers a selective benefit
in the context of a prevalent toxin, leading to fixation. The dynamics of this system in randomly-
mating populations have been characterized by Akbari et al. (15), suggesting that the system
need only exceed a population frequency of ~24%, in the absence of a fitness cost, to spread to
fixation, while the wild-type must exceed a population frequency of ~76% to eliminate the
construct. Both replacement and remediation should therefore be achievable with 1-2 releases of
transgenic and wild-type organisms, respectively; however, as for translocations, we find that
incorporating life history and population structure into our models increases release requirements
in both cases.
In Figures 2-3, we consider weekly releases of 20 adult Ae. aegypti males homozygous at
both loci for the UDMEL system in the community of Yorkeys Knob. The lower threshold for
UDMEL as compared to translocations means that replacement is much easier to achieve for
UDMEL. For a coverage level of 75% or higher, and in the absence of a fitness cost, a single
release of 20 Ae. aegypti males leads to the UDMEL system spreading to fixation throughout the
community (Figure 2). As coverage is reduced to 25%, the required number of releases to
achieve fixation increases to three (Figures 2-3). As for translocations, the time to replacement is
highly dependent on the coverage level and number of releases. From Figure 2, it is apparent that
UDMEL reaches total allele fixation slowly, although the number of individuals having at least
one copy of the transgene increases quickly. At a coverage of 50%, 10 weekly releases lead to
wild-type individuals falling to a frequency <2% within a year of the final release (or within 500
days of the first release).
Remediation, however, is more difficult to achieve for UDMEL compared to translocations
due to the higher threshold that wild-type organisms must exceed to eliminate UDMEL. To
simulate remediation, we first consider weekly releases of 20 adult Ae. aegypti wild-type males
in the community of Yorkeys Knob. This reveals that remediation of UDMEL is not possible with
male-only releases (Figure 2), and so we next consider weekly releases of 10 adult Ae. aegypti
females and 10 adult males. In the absence of a fitness cost associated with the UDMEL construct,
and for a coverage level of 75%, 10 weekly releases are required for the UDMEL construct to be
completely removed from the community. As coverage is reduced to 50%, the required number
of releases increases to 17, and for a coverage level of 25%, remediation is not possible with 20
releases (Figures 2-3). These results make a strong case for translocations as preferred systems to
introduce transgenes in a local and reversible way as: i) remediation of UDMEL requires releases
of biting, vector-competent females, and ii) release requirements for these biting, vector-
competent females are large due to the higher threshold that must be surpassed.
Confinement of translocations & UDMEL to release site. Confinement of translocations
and UDMEL to partially-isolated populations has previously been modeled by Marshall & Hay
(16) and Akbari et al. (15). In both cases, two randomly-mating populations were modeled that
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exchange migrants at given rates. Population structure was otherwise ignored, as was mosquito
life history. Results from these analyses suggest that translocations would spread and remain
confined to populations for which the migration rate is less than ~5.8% per mosquito per
generation (16), and that UDMEL would remain confined to populations for which the migration
rate is less than ~1.6% per mosquito per generation (15). These migration rates are relatively
low, however this may be beneficial for the types of landscapes we are considering here,
whereby the system may spread between neighboring households, but not from one suburb to
another. Recently, Champer et al. (18) showed that translocations would remain confined to and
persist in a population connected to another by a “migration corridor” under a range of parameter
values.
For our landscape of interest - the suburbs of Yorkeys Knob and Trinity Park - it is very
unlikely that Ae. aegypti mosquitoes will travel from one suburb to another by their own flight.
Extrapolating the exponential dispersal kernel used in our simulations, fitted to data from mark-
release-recapture experiments collated by Guerra et al. (34), suggests these events to be
negligible, before accounting for the fact that the intervening vegetated area may serve as a
barrier to Ae. aegypti flight (37). Furthermore, rare migrant mosquitoes are unlikely to cause the
threshold frequency for either drive system to be exceeded, thus making spatial spread due to
such movements unlikely. In considering confineability to the release suburb, we therefore
model “batch migration,” in which several mosquitoes are carried, perhaps by a vehicle, from
one community to another at once. Batch migration events could be thought of as several adult
mosquitoes being carried at once, or perhaps more likely, as a larval breeding site, such as a tyre,
being carried from one household to another, with several adults emerging from the tyre
following transport. We model batch migration events as occurring between randomly chosen
households, and vary the number of daily migration events and the effective number of adults
carried per event. For computational simplicity, we focus on migration events from Yorkeys
Knob, in which either system has already reached fixation, to households in Trinity Park, which
is initially fixed for wild-type mosquitoes.
In Figure 3E-F, we see that both the number and size of daily batch migration events
affect the chance of either system establishing itself in the neighboring suburb, Trinity Park. For
translocations, ~20 daily migration events of batches of 5 adults are required for spread in Trinity
Park. For batches of 10 adults, ~10 daily migration events are required, and for batches of 20
adults, ~8 daily migration events are required. For UDMEL, ~4 daily migration events of batches
of 5 adults are required for spread in Trinity Park, and for batches of 10 adults, ~2 daily
migration events are required.
These results continue to make a strong case for translocations as preferred systems to
introduce transgenes in a local and reversible way as: i) many more batch migration events are
required to lead to spread for translocations as opposed to UDMEL, and ii) the rate of migration
events required for translocations to spread is higher than what would be expected between these
communities. Specifically, Wolbachia releases in Yorkeys Knob in 2011 provide evidence for
occasional batch migrations to the nearby suburb of Holloways Beach; however the spatio-
temporal pattern of Wolbachia spread, as inferred from monitored trap data, suggests only ~1-2
batch migration events over the course of a month, consisting of less than 5 adult females per
event (21).
Sensitivity analysis. A theoretical study by Khamis et al. (38) on toxin-antidote-based
underdominance gene drive systems, similar to UDMEL but for which the toxins are zygotic rather
than maternal (20), found that the gene drive threshold frequency is highly sensitive to: i) the
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increase in adult mortality rate in organisms having the transgene, ii) the duration of the larval
life stage, and iii) parameters determining the character or strength of larval density dependence.
In Figure 4 and Figure S2, we explore the sensitivity of our model outcomes of replacement,
remediation and confinement for translocations and UDMEL as we vary: i) the duration of the
larval life stage, ii) the baseline adult mortality rate, and iii) the fitness cost associated with the
gene drive system. For translocations, we model a 10% fitness cost as a 10% reduction in mean
adult lifespan for organisms homozygous for the translocation, and a 5% reduction for organisms
heterozygous for the translocation. For UDMEL, since its inheritance bias is induced through the
action of maternal toxins, we model a 10% fitness cost as a 10% reduction in female fecundity
for organisms homozygous for UDMEL at both loci, with 2.5% additive fitness costs contributed
by each transgenic allele.
For translocations, the associated fitness cost had the greatest impact on the release
scheme required for the system to be fixed or remediated from the population, given the life
parameters considered (Figure 4). A 10% fitness cost led to 14 weekly releases at a coverage of
50% being required for the translocation to reach fixation (an increase of 3 releases), while a
20% fitness cost led to 18 weekly releases being required (an increase of 7 releases). Small
changes in the duration of the larval life stage and baseline adult mortality had minor impacts on
the release requirements, with an increase in larval lifespan of 2 days or a 2% decrease in the
adult mortality rate leading to one more weekly release being required for the translocation to
reach fixation, and vice versa. Remediation, on the other hand, requires fewer wild-type releases
when there is a fitness cost associated with the translocation. A 10% fitness cost led to 8 weekly
releases at a coverage of 50% being sufficient to eliminate the translocation (a decrease of 3
releases), and a 20% fitness cost led to 6 weekly releases at a coverage of 50% being sufficient
for elimination (a decrease of 5 releases). Small changes in the duration of the larval life stage
and baseline adult mortality had minor impacts on the wild-type release requirements for
elimination, with an increase in larval lifespan of 2 days or a 2% decrease in the adult mortality
rate leading to one fewer release being required.
The sensitivity of our predictions regarding confinement to the release site are of
particular interest, as invasion of a neighboring community may be more likely under some
parameter values than others. Fortunately, a fitness cost associated with the translocation leads to
a higher threshold, and hence more batch migration events required for invasion of a neighboring
community. A 10% fitness cost led to ~2-3 additional daily migration events of 10 adults
required for spread to Trinity Park, and a 20% fitness cost led to ~6-7 additional daily migration
events required (Figure 4). Also noteworthy, a 2% increase in the adult mortality rate led to ~2
fewer daily migration events required for spread to Trinity Park - i.e. ~8 migration events for
batches of 10 adults, and ~18 migration events for batches of 5 adults. While still above inferred
batch migration rates, this highlights that there could exist parameter sets beyond those explored
for which invasion is feasible.
UDMEL displays similar parameter sensitivities regarding fixation, remediation and batch
migration outcomes as for translocations, with the exception that these outcomes are less
sensitive to fitness costs (Figure S2), likely due to the fact that fitness is accommodated through
a reduction in female fecundity rather than an increase in adult mortality. A 20% fitness cost led
to ~1 additional weekly release being required for the system to spread to fixation, whether at a
coverage of 25% or 50%. Similarly, for an invasion of Trinity Park, a 10% fitness cost required
~1 additional daily migration event of 5 adults, and a 20% fitness cost required ~2 additional
daily migration events. Of note, a 2% increase in the adult mortality rate led to ~1 fewer daily
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migration event required for spread to Trinity Park, making this now very achievable - i.e. ~3
migration events for batches of 5 adults, and ~2 migration events for batches of 10 adults.
Discussion:
The idea of using threshold-dependent gene drive systems to replace local populations of disease
vectors with varieties that are unable to transmit diseases has been discussed for over half a
century now, since Curtis (31) famously proposed the use of translocations to control diseases
transmitted by mosquitoes. While Curtis had been primarily concerned with introducing and
spreading genes into a population, as the technology nears implementation, confining them is
becoming of equal concern. As CRISPR-based homing gene drive technology edges closer to
field application, concerns are increasingly being raised regarding the invasiveness of these
systems (39, 40), and systems such as split drive (11), daisy drive (12) and threshold-dependent
underdominant systems (7) are gaining interest, at least during the trial phase of population
replacement technology (6). In this paper, we model the introduction of two drive systems,
chromosomal translocations and UDMEL, that have been engineered in the laboratory and shown
to display threshold-dependent spread (14, 15). While previous papers have described the
population dynamics of these two systems in randomly-mating populations ignoring life history
(14–16, 31), with one recent paper including spatial structure (18), we present the first analysis
of these systems in a spatially-structured population including mosquito life history and
reflecting a well-characterized landscape where field trials could conceivably be conducted (21).
Our results provide strong support for the use of translocations to implement confineable
and reversible population replacement in structured Ae. aegypti populations. Regarding
reversibility, translocations are preferable to UDMEL as: i) they can be remediated through
releases of non-disease-transmitting male Ae. aegypti, and ii) required releases sizes are
achievable (~10 weekly releases at a coverage level of 50%). UDMEL requires less effort to
introduce into a population; but is much more difficult to remove once it has been introduced,
requiring a large number of both males and disease-transmitting females to be released. This
highlights the benefit of a ~50% threshold for reversible population replacement: the symmetry
allows both replacement and remediation to be achieved with similar effort. Extreme
underdominance is another example of system with a 50% threshold (20, 41). Regarding
confineability, translocations again outperform UDMEL as ~20 daily migration events of batches
of 5 Ae. aegypti adults are required for translocations to spread to the neighboring suburb of
Trinity Park, while UDMEL can spread to Trinity Park given only ~4 daily migration events (or
~3 daily migration events for alternative model parameterizations). The true batch migration rate
between suburbs is expected to be smaller than either of these (21), however the rate required for
translocations to spread is highly unlikely to be reached, while the rate for UDMEL is conceivable.
As with any modeling study, there are limitations inherent in our analysis. Several of the
parameters we assumed to be constant here would indeed be dynamic in a real intervention
scenario. At the genetic level, lab experiments suggest non-outbred individuals homozygous for
the translocation had a fitness cost that largely disappeared once offspring were produced that
were the product of at least one wild-type individual (14). Models fitted to data from UDMEL
drive experiments also suggested dynamic fitness costs that depended on the frequency of
transgenic organisms in the population (15). At the ecological level, our model of Ae. aegypti life
history (26), based on the lumped age-class model of Hancock & Godfray (27), assumes a
constant population size, and other constant ecological parameters, such as adult death rate and
larval development times. These parameters have indeed been shown to vary in space and time,
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9
and in response to local mosquito density (42, 43), which our sensitivity analyses suggest could
have significant impacts on release thresholds and gene drive outcomes (38) (Figure 4 & S2). At
the landscape level, we have assumed a relatively homogenous distribution of mosquitoes per
household, and movement rates between households that depend only on distance and household
distribution. Extensive landscape heterogeneities have been shown to slow and alter the spread of
Wolbachia (44, 45), and would likely impact the spread of translocations and UDMEL as well.
Future work that helps to characterize the environmental drivers of mosquito population
dynamics will inform iterative model development to address this.
In conclusion, our analysis supports the use of translocations as a threshold-dependent
drive system capable of spreading disease-refractory genes into structured Ae. aegypti
populations in a confineable and reversible manner. If such a system were engineered in Ae.
aegypti, it would be an excellent candidate for the introduction of disease-refractory genes during
the trial phase of population replacement technology, or whenever a localized release were
otherwise desired. As the technology nears implementation, further ecological work
characterizing the density-dependencies, seasonalities and spatial heterogeneities of Ae. aegypti
populations will be essential to enhance model predictions in preparation for field trials.
Materials and methods:
To model the expected performance of threshold-dependent gene drive systems - reciprocal
chromosomal translocations and UDMEL - at functioning in a confineable and reversible way, we
simulated releases of adult Ae. aegypti males homozygous for each system in the community of
Yorkeys Knob in Queensland, Australia using the MGDrivE simulation framework (26)
(https://marshalllab.github.io/MGDrivE/). To simulate remediation, we modeled releases of
wild-type adult Ae. aegypti into populations in Yorkeys Knob already fixed for the gene drive
system. To determine confineability, we simulated batch migration events from Yorkeys Knob
(fixed for the gene drive system) to the neighboring community of Trinity Park (initially wild-
type). The MGDrivE framework models the egg, larval, pupal and adult (male and female)
mosquito life stages implementing a daily time step, overlapping generations and a mating
structure in which adult males mate throughout their lifetime, while adult females mate once
upon emergence, retaining the genetic material of the adult male with whom they mate for the
duration of their adult lifespan. Density-independent mortality rates for the juvenile life stages
are assumed to be identical and are chosen for consistency with the population growth rate in the
absence of density-dependent mortality. Additional density-dependent mortality occurs at the
larval stage, the form of which is taken from Deredec et al. (28). Full details of the modeling
framework are available in the S1 Text of Sánchez et al. (26), and in the software documentation
available at https://marshalllab.github.io/MGDrivE/docs/reference/. Parameters describing Ae.
aegypti life history and the gene drive systems and landscape of interest are listed in Table S1.
The inheritance patterns for reciprocal chromosomal translocations (depicted in Figure
1A) and UDMEL (depicted in Figure S1) are modeled within the inheritance module of the
MGDrivE framework (26), along with their impacts on female fecundity and adult lifespan. The
distribution of households in Yorkeys Knob and Trinity Park were taken from OpenStreetMap
(https://www.openstreetmap.org/) (Figure 1C). We implement the stochastic version of the
MGDrivE framework to capture the randomness associated with events that occur in small
populations, such as households, which serve as nodes in the landscape modeled here. In the
stochastic implementation of the model, the number of eggs produced per day by females
follows a Poisson distribution, the number of eggs having each genotype follows a multinomial
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distribution, all survival/death events follow a Bernoulli distribution, and female mate choice
follows a multinomial distribution with probabilities given by the relative frequency of each
adult male genotype in the population.
Acknowledgements:
The authors would like to thank Dr. Gregory Lanzaro, Dr. Yoosook Lee and Ms. Partow Imani
for discussions on Aedes aegypti life history and dispersal behavior. This work was supported by
a DARPA Safe Genes Program Grant (HR0011-17-2-0047), awarded to OSA and JMM, and
funds from the Innovative Genomics Institute, awarded to JMM.
Author contributions:
JMM and HMSC conceptualized the study. HMSC, JBB and SLW performed the experiments
and visualized the results. GR and OSA contributed to the interpretation of the results. JMM
wrote the first draft of the manuscript. All authors contributed to the writing of and approved the
final manuscript.
Competing interests:
All authors declare no competing financial, professional or personal interests that might have
influenced the performance or presentation of the work described in this manuscript.
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13
Figure 1. Inheritance and landscape features of the modeling framework. (A) Reciprocal
translocations (T1 and T2) result from the mutual exchange between terminal segments of two
non-homologous chromosomes (N1 and N2). The cross here depicts possible parental gametes,
with respect to the translocation, and offspring that result from matings between them. Matings
between wild-type organisms or translocation homozygotes result in viable offspring; but
translocation heterozygotes produce unbalanced gametes, and many of the resulting offspring are
unviable (shaded). This results in a heterozygote disadvantage and threshold-dependent
population dynamics. (B) UDMEL is composed of two unlinked constructs (here referred to as A
and B), each consisting of a maternally-expressed toxin and a zygotically-expressed antidote for
the toxin on the opposite construct. The cross here represents matings between two of the nine
possible parental genotypes (“+” represents the wild-type allele, and “A” and “B” represent
alleles corresponding to the two UDMEL constructs). The complete inheritance pattern is depicted
in Figure S1. Offspring lacking the antidotes to the maternal toxins produced by their mother are
unviable (shaded). At high population frequencies, the selective advantage on the constructs, by
virtue of the antidotes, outweighs the fitness load due to the toxins, and hence results in
frequency-dependent spread. (C) Distribution of households in Yorkeys Knob (blue) and Trinity
Park (red) in Cairns, Queensland, Australia. Households serve as Aedes aegypti metapopulation
nodes in our simulations, with movement of adult Ae. aegypti between them. Yorkeys Knob
serves as a simulated release site, and Trinity Park as a simulated control site.
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Figure 2. Replacement and remediation results for translocations and UDMEL. Time-series
results are shown for a given number of weekly releases of 20 adult Ae. aegypti per household
with the intent of population replacement or remediation in the community of Yorkeys Knob
(Figure 1C), and at given coverage levels, where coverage is the proportion of households that
receive the releases. For population replacement, releases are of males homozygous for the
translocation or UDMEL into a wild-type population. For remediation, releases are of wild-type
males into a population homozygous for the translocation or UDMEL. (Top) Replacement and
remediation are symmetric for translocations. At a coverage of 50%, 11 or more releases result in
the translocation being driven to fixation or remediated from the population. (Bottom) For
UDMEL, remediation is not possible through releases of males only, and so “mixed remediation”
is considered, in which releases consist of 10 females and 10 males. Release requirements for
UDMEL are smaller for population replacement, but larger for mixed remediation. At a coverage
of 50%, two or more releases result in UDMEL being driven to fixation; however, 17 releases of
both females and males are required to remediate UDMEL from the population.
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Figure 3. Replacement, remediation and confinement outcomes for translocations and
UDMEL. Outcomes are depicted for the proportion of 50 stochastic simulations of population
replacement, remediation and confinement of translocations and UDMEL that result in fixation of
each system. (A-D) For replacement and remediation, each cell corresponds to a given number of
releases (horizontal axis) and coverage level (vertical axis), given 20 adult Ae. aegypti per
household per release. For replacement, releases are of males homozygous for the system into a
wild-type population. For remediation of translocations, releases are of wild-type males into a
population homozygous for the translocation, and for mixed remediation of UDMEL, releases are
of wild-type females and males into a population homozygous for UDMEL. Light blue cells
represent cases where all simulations result in fixation of the system, and dark blue cells
represent cases where the wild-type is fixed in all simulations. (E-F) For confinement, each cell
corresponds to a daily number of batch migration events (horizontal axis) of a given size
(vertical axis) from Yorkeys Knob, where the system is fixed, to Trinity Park, where the system
is initially absent. White cells represent cases where all simulations result in fixation of the
system in Trinity Park, and dark pink cells represent cases where the wild-type is fixed in all
simulations. These results are encouraging for translocations as systems for introducing
transgenes in a local and reversible way as: i) they can be remediated through an achievable
number of male-only releases, and ii) they require more batch migration events to spread to
neighboring communities.
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16
Figure 4. Sensitivity of model outcomes for replacement, remediation and confinement of
translocations. Changes are depicted in the proportion of 50 stochastic simulations that result in
fixation for replacement, remediation and confinement of translocations. Proportions are
compared to those in the first row of Figure 3 as we vary: i) the duration of the larval life stage
(+/- 2 days), ii) the adult daily mortality rate (+/- 2%), and iii) the fitness cost associated with
being homozygous for the translocation (+10% or +20%). Fitness costs have the greatest impact
on the release scheme required for the system to be fixed or remediated from the population,
given the life parameters considered. Fitness costs also lead to more batch migration events
being required for invasion of Trinity Park. A small increase in the baseline adult mortality rate
leads to slightly fewer batch migration events being required for invasion of Trinity Park;
however, comparison to migration rates inferred from field data suggests that confinement is still
expected.
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17
Figure S1. Complete inheritance pattern of UDMEL. UDMEL is composed of two unlinked
constructs (here referred to as A and B), each consisting of a maternally-expressed toxin and a
zygotically-expressed antidote for the toxin on the opposite construct (see Figure 1B). The cross
here represents matings between all nine possible parental genotypes (“+” represents the wild-
type allele, and “A” and “B” represent alleles corresponding to the two UDMEL constructs).
Offspring lacking the antidotes to the maternal toxins produced by their mother are unviable
(shaded). At high population frequencies, the selective advantage on the constructs, by virtue of
the antidotes, outweighs the fitness load due to the toxins, and hence results in frequency-
dependent spread.
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(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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18
Figure S2. Sensitivity of model outcomes for replacement, remediation and confinement of
UDMEL. Changes are depicted in the proportion of 50 stochastic simulations that result in fixation
for replacement, remediation and confinement of UDMEL. Proportions are compared to those in
the second row of Figure 3 as we vary: i) the duration of the larval life stage (+/- 2 days), ii) the
adult daily mortality rate (+/- 2%), and iii) the fitness cost associated with being homozygous for
the UDMEL (+10% or +20%). UDMEL displays similar parameter sensitivities regarding fixation,
remediation and batch migration outcomes as for translocations (Figure 4), with the exception
that these outcomes are less sensitive to fitness costs, likely due to the fact that fitness is
accommodated through a reduction in female fecundity rather than an increase in adult mortality.
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19
Table S1. Life history, population size and movement parameters for Aedes aegypti in
Cairns, Australia.
Parameter
Symbol
Value
Reference
Egg production per female (day-1)
β
20
(1)
Duration of egg stage (days)
TE
5
(2)
Duration of larval stage (days)
TL
6
(2)
Duration of pupa stage (days)
TP
4
(2)
Daily population growth rate (day-1)
r
1.175
(3)
Daily mortality rate of adult stage (day-1)
µM
0.090
(4–6)
Adult population size per household
NH
15
(7)
Daily probability adult leaves household
p
0.28
(8, 9)
Mean dispersal distance of adult
conditional upon movement (meters)
d
54.1
(10)
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recapture data to inform the control of mosquito-borne pathogens. Parasites & Vectors
7(1):276.
.CC-BY-NC-ND 4.0 International licenseIt is made available under a
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint. http://dx.doi.org/10.1101/607267doi: bioRxiv preprint first posted online Apr. 12, 2019;
... Similarly, stable introduction of a virus-blocking Wolbachia may fail if the release area of Wolbachia-infected Ae. aegypti is too small and too vulnerable to immigration by wild-type mosquitoes [8]. For the emerging genetic-based control approaches such as gene drive systems [9], well characterized mosquito dispersal is crucial for addressing the biosafety concerns around the systems' confineability and reversibility in the field [10,11]. ...
... Moreover, globally collated MRR experimental data for Ae. aegypti [14] produced an exponential kernel with σ = 54.1 m [10]. Such high congruence with our genetic-based inferences indicates the robustness of spatial-genetic patterns in reflecting the dispersal characteristics of this mosquito, and it demonstrates the utility of our genetic-based method as a viable alternative to conventional, operationally-demanding MRR experiments. ...
... The above mentioned theoretical approximation of the conditions for Wolbachia initiation and spread [61] assumes isotropic dispersal in a 2-dimensional habitat. Optimal release strategy for the Sterile Insect Technique programs [62], different suppression and replacement strategies [63,64], the effect of larval habitat fragmentation on population crash [65], and confinement and reversibility conditions for threshold-dependent gene drive systems [10] have all been simulated in the spatially explicit models of mosquito populations that applied the exponential dispersal kernel in a 2-dimensional landscape. The approximation of the parametrized dispersal kernel for high-rise landscapes could be achieved by considering the releases of mosquitoes from multiple floors rather than from the ground level only. ...
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Background: Hundreds of millions of people get a mosquito-borne disease every year, of which nearly one million die. Mosquito-borne diseases are primarily controlled and mitigated through the control of mosquito vectors. Accurately quantified mosquito dispersal in a given landscape is critical for the design and optimization of the control programs, yet the field experiments that measure dispersal of mosquitoes recaptured at certain distances from the release point (mark-release-recapture MRR studies) are challenging for such small insects and often unrepresentative of the insect's true field behavior. Using Singapore as a study site, we show how mosquito dispersal patterns can be characterized from the spatial analyses of genetic relatedness among individuals sampled over a short time span without interruption of their natural behaviors. Methods and Findings: We captured ovipositing females of Aedes aegypti, a major arboviral disease vector, across floors of high-rise apartment blocks and genotyped them using thousands of genome-wide SNP markers. We developed a methodology that produces a dispersal kernel for distance that results from one generation of successful breeding (effective dispersal), using the distances separating full siblings, 2 nd and 3 rd degree relatives (close kin). In Singapore, the estimated dispersal distance kernel was exponential (Laplacian), giving the mean effective dispersal distance (and dispersal kernel spread σ) of 45.2 m (95%CI: 39.7-51.3 m), and 10% probability of dispersal >100 m (95%CI: 92-117 m). Our genetic-based estimates matched the parametrized dispersal kernels from the previously reported MRR experiments. If few close-kin are captured, a conventional genetic isolation-by-distance analysis can be used, and we show that it can produce σ estimates congruent with the close-kin method, conditioned on the accurate estimation of effective population density. We also show that genetic patch size, estimated with the spatial autocorrelation analysis, reflects the spatial extent of the dispersal kernel 'tail' that influences e.g. predictions of critical radii of release zones and Wolbachia wave speed in mosquito replacement programs. Conclusions: We demonstrate that spatial genetics (the newly developed close-kin analysis, and conventional IBD and spatial autocorrelation analyses) can provide a detailed and robust characterization of mosquito dispersal that can guide operational vector control decisions. With the decreasing cost of next generation sequencing, acquisition of spatial genetic data will become increasingly accessible, and given the complexities and criticisms of conventional MRR methods, but the central role of dispersal measures in vector control programs, we recommend genetic-based dispersal characterization as the more desirable means of parameterization.
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If they are able to spread in wild populations, CRISPR-based gene-drive elements would provide new ways to address ecological problems by altering the traits of wild organisms, but the potential for uncontrolled spread tremendously complicates ethical development and use. Here, we detail a self-exhausting form of CRISPR-based drive system comprising genetic elements arranged in a daisy chain such that each drives the next. “Daisy-drive” systems can locally duplicate any effect achievable by using an equivalent self-propagating drive system, but their capacity to spread is limited by the successive loss of nondriving elements from one end of the chain. Releasing daisy-drive organisms constituting a small fraction of the local wild population can drive a useful genetic element nearly to local fixation for a wide range of fitness parameters without self-propagating spread. We additionally report numerous highly active guide RNA sequences sharing minimal homology that may enable evolutionarily stable daisy drive as well as self-propagating CRISPR-based gene drive. Especially when combined with threshold dependence, daisy drives could simplify decision-making and promote ethical use by enabling local communities to decide whether, when, and how to alter local ecosystems.
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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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A promising strategy for reducing the transmission of dengue and other arboviral human diseases by Aedes aegypti mosquito vector populations involves field introductions of the endosymbiotic bacteria Wolbachia . Wolbachia infections inhibit viral transmission by the mosquito, and can spread between mosquito hosts to reach high frequencies in the vector population. Wolbachia spreads by maternal transmission, and spread dynamics can be variable and highly dependent on natural mosquito population dynamics, population structure and fitness components. We develop a mathematical model of an Ae. aegypti metapopulation that incorporates empirically validated relationships describing density-dependent mosquito fitness components. We assume that density dependence relationships differ across subpopulations, and construct heterogeneous landscapes for which model-predicted patterns of variation in mosquito abundance and demography approximate those observed in field populations. We then simulate Wolbachia release strategies similar to that used in field trials. We show that our model can produce rates of spatial spread of Wolbachia similar to those observed following field releases. We then investigate how different types of spatio-temporal variation in mosquito habitat, as well as different fitness costs incurred by Wolbachia on the mosquito host, influence predicted spread rates. We find that fitness costs reduce spread rates more strongly when the habitat landscape varies temporally due to stochastic and seasonal processes. Our empirically based modelling approach represents effects of environmental heterogeneity on the spatial spread of Wolbachia. The models can assist in interpreting observed spread patterns following field releases and in designing suitable release strategies for targeting spatially heterogeneous vector populations.
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Underdominance gene drive systems promise a mechanism for rapidly spreading payload alleles through a local population while otherwise remaining confined, unable to spread into neighboring populations due to their frequency-dependent dynamics. Such systems could provide a new tool in the fight against vector-borne diseases by disseminating transgenic payloads through vector populations. If local confinement can indeed be achieved, the decision-making process for the release of such constructs would likely be considerably simpler compared to other gene drive mechanisms such as CRISPR homing drives. So far, the confinement ability of underdominance systems has only been demonstrated in models of panmictic populations linked by migration. How such systems would behave in realistic populations where individuals move over continuous space remains largely unknown. Here, we study several underdominance systems in continuous-space population models and show that their dynamics are drastically altered from those in panmictic populations. Specifically, we find that all underdominance systems we studied can fail to persist in such environments, even after successful local establishment. At the same time, we find that a two-locus two-toxin-antitoxin system can still successfully invade neighboring populations in many scenarios even under weak migration. This suggests that the parameter space for underdominance systems to both establish in a given region and remain confined to that region would likely be highly limited. Overall, these results indicate that spatial context must be considered when assessing strategies for the deployment of underdominance systems.
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In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. We found that CRISPR–Cas9-targeted disruption of the intron 4–exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR–Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7–11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive.
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Mosquito-borne diseases, such as malaria, dengue and chikungunya, cause morbidity and mortality around the world. Recent advances in gene drives have produced control methods that could theoretically modify all populations of a disease vector, from a single release, making whole species less able to transmit pathogens. This ability has caused both excitement, at the prospect of global eradication of mosquito-borne diseases, and concern around safeguards. Drive mechanisms that require individuals to be released at high frequency before genes will spread can therefore be desirable as they are potentially localised and reversible. These include underdominance-based strategies and use of the reproductive parasite Wolbachia Here, we review recent advances in practical applications and mathematical analyses of these threshold-dependent gene drives with a focus on implementation in Aedes aegypti, highlighting their mechanisms and the role of fitness costs on introduction frequencies. Drawing on the parallels between these systems offers useful insights into practical, controlled application of localised drives, and allows us to assess the requirements needed for gene drive reversal.
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Recent reports have suggested that self-propagating CRISPR-based gene drive systems are unlikely to efficiently invade wild populations due to drive-resistant alleles that prevent cutting. Here we develop mathematical models based on existing empirical data to explicitly test this assumption for population alteration drives. Our models show that although resistance prevents spread to fixation in large populations, even the least effective drive systems reported to date are likely to be highly invasive. Releasing a small number of organisms will often cause invasion of the local population, followed by invasion of additional populations connected by very low rates of gene flow. Hence, initiating contained field trials as tentatively endorsed by the National Academies report on gene drive could potentially result in unintended spread to additional populations. Our mathematical results suggest that self-propagating gene drive is best suited to applications such as malaria prevention that seek to affect all wild populations of the target species.
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Malaria, dengue, Zika, and other mosquito-borne diseases continue to pose a major global health burden through much of the world, despite the widespread distribution of insecticide-based tools and antimalarial drugs. The advent of CRISPR/Cas9-based gene editing and its demonstrated ability to streamline the development of gene drive systems has reignited interest in the application of this technology to the control of mosquitoes and the diseases they transmit. The versatility of this technology has also enabled a wide range of gene drive architectures to be realized, creating a need for their population-level and spatial dynamics to be explored. To this end, we present MGDrivE (Mosquito Gene Drive Explorer): a simulation framework designed to investigate the population dynamics of a variety of gene drive architectures and their spread through spatially-explicit mosquito populations. A key strength of the MGDrivE framework is its modularity: a) a genetic inheritance module accommodates the dynamics of gene drive systems displaying user-defined inheritance patterns, b) a population dynamic module accommodates the life history of a variety of mosquito disease vectors and insect agricultural pest species, and c) a landscape module accommodates the distribution of insect metapopulations connected by migration in space. Example MGDrivE simulations are presented to demonstrate the application of the framework to CRISPR/Cas9-based homing gene drive for: a) driving a disease-refractory gene into a population (i.e. population replacement), and b) disrupting a gene required for female fertility (i.e. population suppression), incorporating homing-resistant alleles in both cases. We compare MGDrivE with other genetic simulation packages, and conclude with a discussion of future directions in gene drive modeling.
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Gene drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years. We describe CRISPR-Cas9 endonuclease constructs that function as gene drive systems in Anopheles gambiae, the main vector for malaria. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female-sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene drive constructs designed to target and edit each gene. For each targeted locus we observed a strong gene drive at the molecular level, with transmission rates to progeny of 91.4 to 99.6%. Population modeling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to suppress mosquito populations to levels that do not support malaria transmission.
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Underdominance gene drives are frequency-dependent drives that aim to spread a desired homozygote genotype within a population. When the desired homozygote is released above a threshold frequency, heterozygote fitness disadvantage acts to drive the desired trait to fixation. Underdominance drives have been proposed as a way to control vector-borne disease through population suppression and replacement in a spatially contained and reversible way-benefits that directly address potential safety concerns with gene drives. Here, ecological and epidemiological dynamics are coupled to a model of mosquito genetics to investigate theoretically the impact of different types of underdominance gene drive on disease prevalence. We model systems with two engineered alleles carried either on the same pair of chromosomes at the same locus or homozygously on different pairs at different loci, genetic lethality that affects both sexes or only females, and bi-sex or male-only releases. Further, the different genetic and ecological fitness costs that can arise from genetic modification and artificial rearing are investigated through their effect on the population threshold frequency that is required to trigger the drive mechanism. We show that male-only releases must be significantly larger than bi-sex releases to trigger the underdominance drive. In addition, we find that female-specific lethality averts a higher percentage of disease cases over a control period than does bi-sex lethality. Decreases in the genetic fitness of the engineered homozygotes can increase the underdominance threshold substantially, but we find that the mating success of transgenic mosquitoes with wild-type females (influenced by a lack of competitiveness or the evolution of behavioural resistance in the form of active female mate preference) and the longevity of artificially-reared mosquitoes are vitally important to the success chances of underdominance based gene drive control efforts.