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Hoermann et al., Sci. Adv. 8, eabo1733 (2022) 21 September 2022
SCIENCE ADVANCES | RESEARCH ARTICLE
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GENETICS
Gene drive mosquitoes can aid malaria elimination by
retarding Plasmodium sporogonic development
Astrid Hoermann1†, Tibebu Habtewold1†, Prashanth Selvaraj2, Giuseppe Del Corsano1,
Paolo Capriotti1, Maria Grazia Inghilterra1, Temesgen M. Kebede1,
George K. Christophides1*, Nikolai Windbichler1*
Gene drives hold promise for the genetic control of malaria vectors. The development of vector population
modification strategies hinges on the availability of effector mechanisms impeding parasite development in
transgenic mosquitoes. We augmented a midgut gene of the malaria mosquito Anopheles gambiae to secrete two
exogenous antimicrobial peptides, magainin 2 and melittin. This small genetic modification, capable of efficient
nonautonomous gene drive, hampers oocyst development in both Plasmodium falciparum and Plasmodium
berghei. It delays the release of infectious sporozoites, while it simultaneously reduces the life span of homo-
zygous female transgenic mosquitoes. Modeling the spread of this modification using a large-scale agent-based
model of malaria epidemiology reveals that it can break the cycle of disease transmission across a range of trans-
mission intensities.
INTRODUCTION
Malaria remains one of the most devastating human diseases. A surge
in insecticide-resistant mosquitoes and drug-resistant parasites has
brought a decades-long period of progress in reducing cases and
deaths to a standstill (1). Despite the availability of the first World
Health Organization–approved malaria vaccine (2) the necessity to
develop alternative intervention strategies remains pressing, partic-
ularly if malaria elimination is to remain the goal. Gene drive, based
on the super-Mendelian spread of endonuclease genes, is a promis-
ing new control strategy that has been under development for over
a decade (3). Suppressing mosquito populations by targeting female
fertility has remained a prime application of gene drives, and to date,
specific gene drives have been shown to eliminate caged mosquito
populations (4–6). Gene drives for population replacement (or mod-
ification), designed to propagate antimalarial effector traits, have also
seen notable development in the past years (7–9). To date, a range
of antimalarial effectors and tissue-specific drivers have been tested
in transgenic mosquitoes, and some of them have been shown to
reduce Plasmodium infection prevalence or infection intensity
(10–23). However, the pursuit of novel and effective mechanisms
is ongoing, especially in Anopheles gambiae where effectors so far
have shown only moderate reductions in parasite transmission
(12,15,20–22).
Antimicrobial peptides (AMPs) from reptiles, plants, or insects
have long been considered putative antimalarial effectors and have
been tested invitro and invivo for their efficacy against different
parasite life stages [for reviews, see (24–27); for other relevant stud-
ies, see (28,29)]. Although AMPs are very diverse in sequence and
structure, many are cationic and amphiphilic and thus tend to
adhere to negatively charged microbial membranes and, to a much
lesser extent, to membranes of animal cells (30). Permeabilization
mechanisms have been proposed, which rely on either pore forma-
tion or accumulation of peptides on the microbial surface, causing
disruption in a detergent-like manner (31). A subset of AMPs has
been suggested to act by mitochondrial uncoupling, interfering di-
rectly with mitochondria-dependent adenosine triphosphate (ATP)
synthesis (32–34). Two such peptides, magainin 2, found within
skin secretions of the African claw frog Xenopus laevis, and melittin,
a primary toxin component of the European honey bee Apis mellifera,
have been shown to both form pores on the microbial membrane
(35,36) and trigger uncoupling of mitochondrial respiration (37–40).
Intrathoracic injection of magainin 2 into Anopheles mosquitoes
has been demonstrated to cause Plasmodium oocyst degeneration
and shrinkage and a consequent reduction in the number of sporo-
zoites released (41), while a transmission blocking effect of magainin
2 has also been revealed when spiked into gametocytemic blood at
a 50 M concentration (29). Similarly, melittin has been shown
to reduce the number and prevalence of Plasmodium falciparum
oocysts in spike-in experiments at concentrations as low as 4 M
(28,29), while expression of melittin in transgenic Anopheles
stephensi mosquitoes as a part of a multieffector transgene, addi-
tionally including the AMPs TP10, EPIP, Shiva1, and Scorpine, has
led to a significant reduction in oocyst prevalence and infection
intensity (23).
RESULTS
Here, we augmented two host genes of A. gambiae to coexpress
magainin 2 and melittin following the previously described integral
gene drive (IGD) paradigm (42). This allowed for minimal genetic
modifications, capable of nonautonomous gene drive, to be intro-
duced into the host gene loci, making full use of the gene regulatory
regions for controlling tissue-specific expression of the AMPs. We used
the previously evaluated zinc carboxypeptidase A1 (CP; AGAP009593)
or the AMP gambicin 1 (Gam1; AGAP008645) as host genes for the
exogenous AMP integration, respectively (Fig.1A). The transcrip-
tional profile of these genes was expected to drive expression of the
1Department of Life Sciences, Imperial College London, London SW7 2AZ, UK.
2Institute for Disease Modeling, Bill and Melinda Gates Foundation, Seattle, WA
98109, USA.
*Corresponding author. Email: g.christophides@imperial.ac.uk (G.K.C.); n.windbichler@
imperial.ac.uk (N.W.)
†These authors contributed equally to this work.
Copyright © 2022
The Authors, some
rights reserved;
exclusive licensee
American Association
for the Advancement
of Science. No claim to
original U.S. Government
Works. Distributed
under a Creative
Commons Attribution
License 4.0 (CC BY).
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Hoermann et al., Sci. Adv. 8, eabo1733 (2022) 21 September 2022
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AMPs in the mosquito midgut upon ingestion of a blood meal
(CP) or in the anterior gut (Gam1). The use of 2A ribosome-skipping
peptides and secretion signals guaranteed separate secretion of the
exogenous AMPs and host gene products. For this purpose, we re-
placed the signal peptides and prepropeptides of magainin 2 and
melittin with the endogenous secretion signals of A. gambiae Cecropin 1
and 2 genes, respectively. An intron harboring the guide RNA
(gRNA) module that enables nonautonomous gene drive and the
fluorescent marker-module required for transgenesis was introduced
within the melittin coding sequence. Transgenesis of A. gambiae
G3 strain via CRISPR-Cas9–mediated homology-directed repair and
subsequent removal of the green fluorescent protein (GFP) transfor-
mation maker, resulting in the establishment of homozygous marker-
less strains, designated as Gam1-MM and MM-CP, were performed
as previously described (Fig.1A and fig. S1) (42). We validated and
tracked the correct transgene insertion by genomic polymerase
chain reaction (PCR; Fig.1B) and confirmed the expression of CP
or Gam1 host genes and the inserted AMP cassette by reverse tran-
scription PCR (RT-PCR), respectively (Fig.1C). Sequencing of the
cDNA amplicon over the splice site of the artificial intron revealed
the expected splicing pattern in 89.9% of all MM-CP reads but only
in 65.3% of all Gam1-MM reads (Fig.1D). A cryptic splice site re-
sulting in a loss of additional 25base pairs (bp) from the melittin
coding sequence accounted for most of the unexpected splicing
events (fig. S2).
Next, we performed infection experiments with the P. falciparum
NF54 strain to determine the effect of these modifications on para-
site transmission (Fig.2A). Both transgenic strains showed a signif-
icant reduction in the midgut oocyst loads on day 7 post infection
(pi; Fig.2B). While only a few oocysts of the MM-CP strain had the
expected size, a closer examination of infected midguts revealed the
presence of many smaller structures possibly representing stunted
or aborted oocysts (Fig.2C), prompting a more detailed investi-
gation of this strain. We performed infections and quantified the
number and diameter of all oocyst-like structures on days 7, 9, and
15 pi. Given that nutritional stress is a factor that recently emerged
as causing stunting of oocysts (43,44), a group of mosquitoes were
provided a supplemental blood meal on day 4 pi. We found that the
small oocyst-like structures were stunted oocysts that grew over time
and that the supplemental blood meal further boosted their growth
(Fig.2D). Overall, oocysts infecting the MM-CP strain were signifi-
cantly smaller than in the wild-type (WT) control by an average of
47.8, 41.1, and 59.8% on days 7, 9, and 15 pi, respectively (Fig.2D).
This was also the case for the cohort that received an additional blood
meal, but the difference with WT controls decreased over time to
50.4, 24.9, and 18.6% on days 7, 9, and 15 pi, respectively. We re-
peated this experiment with the rodent parasite Plasmodium berghei
to determine whether the observed infection phenotype would also
occur with a different parasite species and under different environmen-
tal conditions. Using a GFP-labeled P. berghei ANKA 2.34 strain,
Fig. 1. Generation of gene drive effector strains expressing AMPs. (A) Schematic showing the design and integration strategy of the effector cassette coding for
magainin 2 and melittin at the endogenous loci Gam1 and CP. AMP integration is targeted to the C terminus of Gam1 and the N terminus of CP, respectively. The gRNA
target sequences (red) or gRNA module (red circle) is indicated, including the protospacer adjacent motif (bold) and the stop and start codons (underlines). Coding
sequences (CDS) and signal peptides (CDS-SP) are indicated by light shading. Endogenous secretion signals of the A. gambiae Cecropin 1 and 2 genes (Cec1 SP and Cec2
SP) and ribosomal skipping signals (P2A and T2A) are indicated. Half arrows indicate primer binding sites for genomic PCR and RT-PCR. (B) PCR on genomic DNA (gDNA)
of 15 pooled homozygous Gam1-MM, MM-CP, or wild-type (WT) individuals. (C) RT-PCR of midguts from WT, Gam1-MM or MM-CP mosquitoes that were either non–
blood-fed (NBF) or dissected 3 hours post blood feeding (3 hours PBF), respectively. (D) Analysis of cDNA amplicons over the splice site subjected to next-generation
sequencing showing the predicted splicing outcomes for strains Gam1-MM and MM-CP.
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fluorescent imaging revealed clearly stunted oocysts that, on day 14 pi,
were about two times smaller than in the control (Fig.2,EandF).
We reasoned that the detected retardation of oocyst development
would, in turn, cause a delayed release of sporozoites into the mos-
quito hemocoel and subsequent infection of the salivary glands. To
determine the sporozoite load over time, we determined the abun-
dance of parasite DNA on days 10 to 16 pi by quantifying the
P. falciparum cytochrome B (Cyt-B) gene in the head and thorax
of single mosquitos via quantitative PCR (qPCR), considering only
mosquitoes that were positive for parasite DNA in the midgut. We
found that sporozoite infection prevalence, i.e., the rate of head and
thorax samples with amplification above the amplification cycle thresh-
old, was significantly reduced in MM-CP mosquitoes (Fig.2G) when
compared to WT (on average, by 77.9% across time points), with a
significant number of positives detectable only on day 16. Although
a supplemental blood meal accelerated sporozoite release in the
MM-CP strain by 4 to 5 days (now detected on days 11 to 12 pi),
overall sporozoite prevalence was still reduced significantly relative
to the control group that had received a single blood meal by 67.8%
on average. This suggested that sporozoite release in the MM-CP
strain was delayed under both nutritional conditions. We also ana-
lyzed the relative parasite DNA content in positive samples as a proxy
Fig. 2. Plasmodium infection experiments. (A) Schematic overview of Plasmodium infection experiments. (B) P. falciparum oocyst intensity 7 days pi (dpi) in midguts
from WT, MM-CP and Gam1-MM mosquitoes dissected. Data from three biological replicates was pooled, and statistical analysis was performed using the Mann-Whitney
test. (C) Bright-field images of midguts showing typical oocysts in WT and MM-CP mosquitoes at 7 dpi. (D) Quantification of P. falciparum oocyst diameter in WT and MM-
CP mosquitoes 7, 9, and 15 dpi from three pooled biological replicates. Note that many oocysts in WT mosquitoes had ruptured on day 15 pi. Quantification of oocyst
diameter (E) and fluorescent imaging (F) of oocysts in WT and MM-CP mosquitoes infected with P. berghei at 14 dpi. Sporozoite prevalence 10 to 16 dpi (G) and infection
intensity across all days (H) was measured by qPCR of the P. falciparum Cyt-B gene in dissected heads and thoraces of individual MM-CP and WT mosquitoes (10 to 16 dpi).
Only mosquitoes positive for oocyst DNA in the midgut were included in the analysis performed in two biological replicates. Statistical analysis in (D), (E), and (H) was
performed by a t test assuming unequal variance. Statistical analysis in (G) was performed using a generalized linear model with a quasibinomial error structure where
strain (P = 8.35 × 10−6) and dpi (P = 7.15 × 10−4) but not blood meal status (P = 0.0894) were found to be significant coefficients. In all panels, the provision of a supplemen-
tal blood meal 4 dpi is indicated by an additional blood drop. **P ≤ 0.01 and ***P ≤ 0.001; ns, not significant.
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for sporozoite numbers and found that these were significantly
higher in WT compared to MM-CP mosquitoes both after a single
(14.0-fold) or double (37.8-fold) blood meals (Fig.2H). To rule
out any founder effect in the homozygous MM-CP strain, we out-
crossed MM-CP mosquitoes to mosquitoes of the A. gambiae Ifakara
strain. After an F1 sibling cross, F2 mosquitoes were provided an
infected blood meal and dissected on day 9 to assess the midgut
oocyst size and be genotyped by PCR for the presence of the trans-
gene. The results showed that the effect on parasite development
was attributable to the presence of the transgene and that the devel-
opmental retardation of oocysts was reduced in hemizygous indi-
viduals (fig. S3).
Next, we measured fitness parameters of MM-CP mosquitoes.
The number of eggs laid by blood-fed females (a measure of fecun-
dity) and the number of eggs that hatched among those laid (a mea-
sure of fertility) were counted. The results showed a 14.1% difference
in the number of eggs laid (P=0.0299, two-sample t test assuming
unequal variances) but unaffected larval hatching rates between the
MM-CP and WT control mosquitoes (Fig.3,AandB). Overall, lar-
val output per female is shown in fig. S4. Pupal sex ratio (Fig.3C)
and pupation time did not significantly deviate between MM-CP and
control mosquitoes (fig. S5). However, a significant effect on the
life span of sugar-fed mosquitoes was detected for MM-CP females
(median life span, 15 days) and, to a lesser extent, in males (23 days)
compared to control females (26 days) and males (27 days; Fig.3D).
We repeated this experiment with females that were now also pro-
vided with regular blood meals. CP is only weakly expressed in the
sugar-fed midgut, but baseline expression of the AMPs is nevertheless
the most likely explanation for the observed fitness cost. In contrast,
CP is strongly induced following a blood feed, and thus, any effect
of the transgene was expected to be elevated by the blood meals.
As before, to blind the experiment and rule out that genetic back-
ground accounted for this effect, we first performed a backcross
to the Ifakara strain and genotyped individual F2 mosquitoes that
were raised as a mixed population at the end of the experiment. The
results confirmed a significant life span reduction in homozygous
MM-CP females under these conditions, but no significant effect
was detected in hemizygous individuals compared to nontransgenic
controls (Fig.3E).
We performed transcriptomic analysis of dissected midguts
before and 6 and 20 hours after blood feeding. We quantified the
number of differentially expressed genes between MM-CP and con-
trol females and performed a gene ontology (GO) analysis to deter-
mine significantly enriched gene groups (table S1). The results
indicated that genes involved in mitochondrial function and located
at the inner mitochondrial membrane were disproportionally affected
in MM-CP females, particularly after the blood meal (Fig.3,FtoH).
Among most significant hits were genes encoding a member of the
ubiquinone complex (AGAP003900), a mitochondrial H+ adenosine
triphosphatase (AGAP012818), an ATP synthase subunit (AGAP004788),
and a protein belonging to a family of calcium channels (AGAP002578),
which control the rate of mitochondrial ATP production. These
findings offered a hypothesis that could explain the dual phenotype
regarding parasite development and adult female life span. Plasmo-
dium development in the mosquito is critically dependent on mito-
chondrial function including active respiration (45–48). Magainin
2 and melittin are known to trigger mitochondrial uncoupling and
could, upon secretion into the midgut lumen, interfere with ATP
Fig. 3. Life history traits and midgut transcriptome of MM-CP mosquitoes. (A) Fecundity of individual homozygous MM-CP females compared to the WT and corre-
sponding (B) larval hatching rates obtained during the first gonotrophic cycle. Data from three pooled biological replicates are shown. Statistical significance was deter-
mined by a t test assuming unequal variance. (C) Pupal sex ratios of MM-CP and WT strains analyzed using the chi-square test for equality. (D) Survival analysis of MM-CP
and WT male and female mosquitoes maintained on sugar and (E) of F2 genotyped MM-CP female mosquitoes following backcrossing to the Ifakara strain, intercrossing
of F1 mosquitoes, and provision of blood meals. Statistical significance was determined with a Mantel-Cox log rank test. Data from three biological replicates are pooled,
and the mean and 95% confidence intervals are plotted. *P ≤ 0.05 and ***P ≤ 0.001. (F) Volcano plots of an RNA sequencing (RNA-seq) experiment performed on midguts
dissected before or 6 and 20 hours after blood meal. Differentially expressed genes between MM-CP and WT mosquitoes (P ≤ 0.01) are indicated, and genes belonging to
enriched GO groups are highlighted in red.
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synthesis targeting the parasite mitochondrion. This effect would be-
come apparent as the parasite transforms into the energy-demanding
oocyst stage that undergoes several rounds of endomitosis and veg-
etative growth. As AMPs are unlikely to be able to access the oocyst,
the effect would wear off with time and partly offset by supplemental
blood meals. The AMPs, however, are likewise expected to affect the
mosquito midgut mitochondria, affecting energy homeostasis and
modulating life span. While further experiments are needed to un-
tangle these effects, the most significant knowledge gap, when it comes
to transmission blocking, is to what degree any effects observed with
laboratory strains of P. falciparum would be reproducible in infec-
tions with genetically diverse parasites isolated from patient blood.
The MM-CP strain is an excellent candidate to attempt to answer
this question, as the transmission-blocking mechanism that we
describe here appears to act across Plasmodium species. MM-CP is
incapable of autonomous gene drive, unless it mates with a mosquito
source of Cas9, and can thus be evaluated in an endemic setting under
standard mosquito confinement protocols.
Last, we predicted how deployment of the MM-CP effector trait
would modify malaria epidemiology using a mechanistic, agent-based
model of P. falciparum transmission that includes vector life cycle
and within-host parasite and immune dynamics. The model is based
on the EMOD framework that has recently been updated to enable
the simulation of gene drives (49). In our model, seasonality of rain-
fall and temperature ranges are characteristic of the Sahel for one
representative year and were kept the same across transmission set-
tings from year to year. Vector density was varied to match desired
transmission intensity. There remain knowledge gaps that preclude
a direct translation of experimental entomological or molecular data
into epidemiological parameters, for example, linking the observed
reduction in sporozoite DNA and its quantitative effect on onward
transmission. For the phenotypic effects, we thus estimated likely
parameter value ranges (a 30 to 70% increase in time until sporo-
zoites are released and a 40 to 100% reduction in infectious spo-
rozoites) that we considered to be within physiologically plausible
limits supported by our invivo experiments. As a final parameter
for the model, we experimentally determined the rate of nonauton-
omous gene drive of the MM-CP allele by pairing it with a source of
Cas9, which resulted in high levels of homing of the transgene in both
males and females: 96.01 and 98.91%, respectively (Fig.4A). MM-CP,
as a nonautonomous effector, could be flexibly deployed in con-
junction with nondriving, self-limiting, or, as we assumed in our
model, a fully autonomous Cas9 gene drive that is able to mobilize
MM-CP (fig. S6). A single release of 1000 mosquitoes carrying the
driver and MM-CP effector was conducted 6 months into the sim-
ulation, and simulations were run for a total of 6 years. We assumed
that functional resistance (R1 alleles) at both loci would arise at 1%
rate. We observed rapid propagation of MM-CP with all WT targets
replaced by the end of year 2 of the simulations. Subsequently, MM-CP
is gradually replaced by functional resistant alleles falling below
50% allele frequency by the end of year 4. We determined the prob-
ability of elimination by the last year of simulation, defined as the
number of simulations per parameter set that have zero prevalence
in the last year divided by the total number of simulations. Incidence
reduction was evaluated for the duration of the simulation following
gene drive releases compared to control scenarios with no releases.
In a low transmission setting [annual entomological inoculation
rate (EIR), ~15 infectious bites per person], most simulations within
the space of parameter estimates for strain MM-CP (gray bars)
resulted in the elimination of malaria transmission (Fig.4B and fig.
S7A). As transmission intensity increased (annual EIR, ~30), we de-
tected a reduction in probability of elimination in the lower end of
Fig. 4. Gene drive and predicted epidemiological impact of strain MM-CP deployment. (A) Assessment of nonautonomous gene drive in the progeny of male and
female hemizygous MM-CP mosquitoes in the presence or absence of a vasa-Cas9 driver crossed to the WT. Larval offspring were subjected to multiplex PCR genotyping,
and the mean and standard error (SEM) from three biological replicates is plotted, and the total number n is indicated. Statistical significance was determined using a
one-way analysis of variance (ANOVA) with Tukey’s correction. ***P ≤ 0.001. (B) Heatmaps depicting elimination probabilities (top) and number of clinical cases reduced
(bottom) at the end of 6 years following a single release of 1000 homozygous MM-CP mosquitoes that also carry a Cas9 IGD. Three transmission scenarios with varying
EIRs for P. falciparum as a measure of exposure to infectious mosquitoes were explored. Homozygous transgenic mosquitoes are released 6 months after the start of the
simulation in highly seasonal transmission settings of varying intensities. The parameter range that we explored for the reduction of the number of infectious sporozoites
is represented on the major y axis, while the range for the average increase in time until sporozoites are released is represented on the major x axis. Parameter range es-
timates based on the experimental data for strain MM-CP are indicated next to the axes (gray bars).
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the parameter estimate range (Fig.4B and fig. S7B). In a high trans-
mission scenario, a high reduction in sporozoite production in com-
bination with a large delay was necessary for elimination to be triggered
in the model (Fig.4B and fig. S7C). However, note that significant
reductions in clinical cases occurred even when elimination was
not achieved. Therefore, in high transmission settings, even when
not achieving elimination alone, MM-CP could open a window
for elimination by strategically deploying other interventions that
could act synergistically to drive transmission to zero.
DISCUSSION
CRISPR-Cas9 gene drives aimed at suppressing mosquito popula-
tions have seen much progress and publicity. Gene drives aimed at
modifying mosquito populations, by directly interfering with their
vectorial capacity, have not been in the spotlight to the same degree
as they face an additional hurdle: the lack of a robust mechanism
to interfere with Plasmodium development in genetically modified
mosquitoes. This is despite research on such mechanisms predating
the gene drive field by more than a decade. Any such mechanism
must eventually hold up against the high genetic diversity of malaria
parasites. The effector mechanism that we present here can bridge
this gap. It is based on a minimal modification of an endogenous
A. gambiae genomic locus to express two exogenous AMPs acting
against malaria parasites in the mosquito midgut. We show that this
modification impedes transmission of two different Plasmodium
species, the deadliest human parasite P. falciparum and the rodent
parasite P. berghei. It achieves this by hampering parasite sporogonic
development that occurs in the oocyst, markedly delaying the emer-
gence of infectious sporozoites, and we attribute this effect to the
known propensity of these AMPs for interfering with mitochondrial
function. As the modification additionally reduces female mosquito
life span, the possibility of infectious sporozoites to be transmitted
to a new host is reduced markedly.
Modeling suggests that propagation of this modification via
gene drive promises to break the malaria transmission cycle across
a range of epidemiological scenarios in sub-Saharan Africa even if the
effector itself is eventually replaced by resistant alleles because of the
fitness cost that it imposes. This modification is already designed for
gene drive and requires no further adjustment before deployment,
while, at the same time, it is inert on its own and thus can be safely
tested in an endemic setting under standard containment protocols.
It thus enables the next step for testing antimalarial effectors, i.e.,
to evaluate their transmission blocking modifications against par-
asites directly sampled from patients in malaria endemics countries.
MATERIALS AND METHODS
Design and generation of constructs
Annotated DNA sequence files for the final transformation con-
structs pD-Gam1-MM and pD-MM-CP are provided in file S1.
Briefly, the 23 N-terminal amino acids of Cecropin 1 (Cec1, CecA,
and AGAP000693) and Cecropin 2 (Cec2, CecB, and AGAP000692)
served as secretion signals. Magainin 2, melittin, T2A, and P2A
were codon usage optimized for A. gambiae, and the intron located
within the melittin coding sequence was previously described (42)
except for the SV40 terminator within the 3xP3-EGFP (enhanced GFP)
marker module for which we swapped in the trypsin terminator
(50). We first neutralized a Bsa I site in the ampicillin resistance
cassette and gene-synthesized (Genewiz) a fragment ranging from
the Cecropin 1 secretion signal to the trypsin terminator, including
18-bp overlaps with the vector backbone and EGFP for subsequent
Gibson Assembly. The marker-module and the U6 promoter were
PCR-amplified from pI-Scorpine (42) with primers 78-GFP-R and
167-U6-R. The fragment from the Bsa I spacer to the P2A was syn-
thesized (Genewiz), including 18-bp overlaps to the U6 promoter
and the vector backbone. The vector backbone was PCR-amplified
from pAmpR_SDM with primers 168-BBmut-F and 169-BBmut-R,
and last, the four fragments were joined via Gibson Assembly to
yield the intermediate plasmid pI-MM. The CP gRNA spacer (42)
was inserted via the Bsa I sites, and the cassette was amplified with
primers 172- CP-HA3-F-degen and 173-CP-HA5-R-Cec1 and fused
with the CP homology arms and backbone amplified from pD-Sco-
CP (42) with primers 170-Cec1-SS-F and 171-P2A-R to assemble the
donor plasmid pD-MM-CP. A 5′ P2A was added to the cassette via
Golden Gate cloning of the annealed oligos 182-P2Aanneal-F and
183-P2Aanneal-R into Bgl II digested plasmid pI-MM, and subsequently,
the Gam1 gRNA spacer (5′-TACAGAATGTTTCTTCTGAG-3′)
was inserted via Bsa I. The gRNA sequence was chosen using Deskgen
(Desktop Genetics, LTD) with an activity score of 54 and an off-target
score of 99. The effector cassette was amplified with primers 184-
P2Adegen-F and 185-Mel-R and fused via Gibson Assembly with the
gambicin homology arms amplified from G3 genomic DNA (gDNA)
and the backbone to generate the donor plasmid pD-Gam1-MM. For
primers, see table S2.
Transgenesis and establishment of markerless strains
A. gambiae G3 eggs were injected with the corresponding donor
plasmids pD-MM-CP or pD-Gam1-MM and the Cas9 helper plas-
mid p155 (4). Twenty-five F1 transgenics were obtained for MMGFP-
CP and one female F1 transgenic for Gam1-MMGFP. MMGFP-CP
was established from a founder cage with nine females, and F1 indi-
viduals were confirmed by Sanger sequencing with primers EGFP-
C-For, 117-CP-ctrl-R, and 163-P3-probe-F and Gam1-MMGFP with
primers EGFP-C-For and EGFP-N. Transgenics were outcrossed to
G3 WT over three generations for Gam1-MMGFP and two genera-
tions for MMGFP-CP before crossing to the vasa-Cre (51) strain in
the KIL background. Larval offspring were screened for GFP and
DsRed, and siblings were mated. The progeny was screened against
GFP and DsRed; pupae were singled out, and the exuviate was col-
lected for genotyping with primers 99-CP-locus-F and 100-CP-
locus-R or 241-Gam-locus-F and 242-Gam-locus-R, respectively, to
identify homozygotes. The markerless line MM-CP was established
from 9 males and 11 females. For Gam1-MM, three cups with one
female and one male and six cups with two males and two females
were set up and pooled after confirmation via Sanger sequencing. A
G3-KIL mixed colony was used as WT control for all experiments,
unless otherwise stated. All experiments were performed with cow
blood [First Link (UK) Ltd.], unless otherwise stated.
RT-PCR and splicing analysis
MM-CP and the WT control were fed with human blood, and mid-
guts were dissected after 3hours. For Gam1-MM, this experiment
was performed on unfed females. Thirty guts were lysed in TRIzol
and homogenized with 2.8-mm ceramic beads (CK28R, Precellys)
for 30 s at 6800rpm in a Precellys 24 homogenizer (Bertin). RNA was
extracted with the Direct-zol RNA Mini-prep kit (Zymo Research)
including on-column deoxyribonuclease (DNase) treatment and
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Hoermann et al., Sci. Adv. 8, eabo1733 (2022) 21 September 2022
SCIENCE ADVANCES | RESEARCH ARTICLE
7 of 9
transcribed into cDNA with the qScript cDNA Synthesis Kit (Quantabio).
RT-PCR was performed with a Phire Tissue Direct PCR Master Mix
kit (Thermo Scientific) using primers 429-Gambicin-F and 430-
Gambicin-R, 270-qCP-F1 and 271-qCP-R3, 484-qMag-both-F and
485-qMel-both-R, and 447-S7-F and 448-S7-R for the S7 reference
gene. To quantify splicing efficiency, PCRs were performed on above
cDNAs with Q5 High-Fidelity DNA Polymerase (NEB) using 484-
qMag-both-F as forward primer and 242-Gam-locus-R (365-bp
amplicon) or 246-qCP-R2 (309 bp) as reverse primer, respectively.
Annealing temperature, extension time, and cycle number were set
to 67°C, 5s, and 27 cycles, respectively. Amplicons were purified
with the QIAquick PCR Purification Kit (QIAGEN) and submitted to
Amplicon-EZ NGS (Genewiz), and the data (GenBank accession
PRJNA778891) were analyzed using Geneious Prime (Biomatters).
Mosquito infection assays
Transgenic or control mosquitos were infected with mature
P. falciparum NF54 gametocyte cultures (2 to 6% gametocytemia)
as described previously using the streamlined standard membrane
feeding assay (29) or with P. berghei ANKA 2.34 that constitutive-
ly expresses GFP by direct feeding on infected mice. Engorged
mosquitoes were provided 10% sucrose and maintained at 27°C for
P. falciparum infections and 21°C/75% relative humidity for P. berghei
infections until dissections were performed. Supplemental blood
meals on human blood were provided via membrane feeding. For
infections of mosquitoes with mature P. falciparum, mosquitoes were
starved without sugar for 48 hours after the infective or supplemen-
tal blood meal to eliminate unfed individuals.
Analysis of parasite infection intensity and prevalence
We dissected midguts at the indicated days and microscopically ex-
amined them for the presence of oocysts after staining with 0.1%
mercurochrome. We measured the diameter of oocyst using ImageJ
(52). For measuring the prevalence and intensity of sporozoites, the
head, thorax, and the midgut were dissected for each female.
The gDNA was extracted separately from head/thorax samples and
the corresponding midgut samples with the DNeasy 96 Blood and
Tissue Kit (QIAGEN) and was used for qPCR 20-l reactions
using a QIAGEN QuantiNova SYBR Green PCR kit to quantify the
P. falciparum Cyt-B gene fragment using primers and methods de-
scribed previously (43,53). Standard curves for the target gene and the
A. gambiae S7 reference gene were calculated after serial dilution of
nucleic acid templates. Ct values were converted using their respec-
tive standard curves, and the target gene Ct value was normalized to
the reference gene (A. gambiae S7 ribosomal gene).
Generation of backcross populations and genotyping
Fifty homozygous MM-CP males and females were crossed to 50
A. gambiae s.s. Ifakara strain females and males, respectively. F1 siblings
were mass-mated in a single cage to obtain F2 progeny. F2 females
were used for infection experiments or survival assays as described.
For genotyping of individual mosquitoes, we used multiplex genomic
PCR with primers CP-multi-F, CP-multi-R, and Mag-R, which re-
sults in a 356-bp amplicon for the MM-CP transgene and 670 bp for
the unmodified CP locus.
Fitness and survival assays
For each replicate, 20 females were individually transferred to cups
1 day after being offered an uninfected blood meal. Spermathecae
were dissected from females that failed to produce eggs or larvae
and thus determine whether they were fertilized by sperm. Unfertil-
ized females were excluded from the analysis. Eggs and larvae were
counted on day 7 after blood feed. To determine the pupal sex ratio
and pupation time, 100 L1 larvae per tray were reared to the pupal
stage where pupae were being collected and sexed once a day. Three
biological replicates were performed, and the data were analyzed
via the chi-square test for deviations from the expected sex ratio of
50%. The average pupation time in days was calculated and tested
for statistical significance with the Mann-Whitney test. For the
survival analysis including both sexes, a total of 274 WT and
276 MM-CP male and 275 WT and 304 MM-CP female pupae were
placed in six separate cages with bottles containing 10% filtered
fructose solution, and accumulated dead mosquitoes were counted
daily. Survival was monitored daily for 44 days on three inde-
pendent replicates. For the survival analysis of backcrossed fe-
males, F2 mosquitoes were placed in W24.5cm by D24.5cm by
H24.5cm cages as pupae. Mosquitoes were offered a 10% sugar
solution, and they were also offered a blood meal and allowed
to deposit eggs 72 hours after every blood meal. Dead mosquitoes
were collected every 24 hours from the cage and preserved to be
genotyped. Survival was monitored daily for 25 days on two inde-
pendent replicates.
RNA-seq analysis
Females were fed with human blood, and 15 guts were dissected
into TRIzol after 6 hours, after 20 hours, and from unfed females.
After homogenization with 2.8-mm ceramic beads (CK28R, Precellys),
RNA was extracted with the Direct-zol RNA Mini-prep Kit (Zymo
Research) including on-column DNase treatment. Four biological
replicates per condition were subjected to RNA sequencing (RNA-seq).
Libraries were prepared with the NEB Next Ultra RNA Library Prep
Kit and sequenced on a NovaSeq 6000 Illumina platform (instrument
HWI-ST1276) generating 150-bp paired-end reads (GenBank acces-
sion PRJNA822650). Replicate 1 for MM-CP without blood meal
(MMCP_N_1) was identified as outlier with squared Pearson cor-
relation coefficients with the other three biological replicates below
0.84 and hence removed from further analysis. Sequencing reads were
mapped to the A. gambiae PEST genome (AgamP4.13, GCA_000005575.2
supplemented with the MM-CP construct reference) using HISAT2
software v2.0.5 (with parameters --dta --phred33) (54). Differential
expression was assessed with DESeq2 v1.20.0. GO enrichment anal-
ysis was performed using TopGO (55) with a pruning factor of 50
using a P value cutoff of P=0.01.
Assessment of nonautonomous gene drive
At least 60 homozygous MM-CP or WT females were crossed to males
of the vasa-Cas9 strain. F1 progeny were screened for the presence
of the 3xP3-YFP marker, and transhemizygotes were then sexed and
crossed to WTs. gDNA was isolated from the progeny at the L2-L3
larval stage according to the protocol of the Phire Tissue Direct
PCR Kit (Thermo Scientific). Multiplex PCR was performed with
primers 260-q-Mag-Mel-R, 531-CP-multi-R, and 532-CP-multi-F,
yielding a 356-bp band if the construct is present and a WT band of
670 bp as control. Two 96-well-plates per parent (paternal or mater-
nal transhemizygotes) and replicate were analyzed, and four nega-
tive controls were included on each plate. From the control crosses,
46 offspring per parent were analyzed for each replicate. The hom-
ing rate was calculated as (n × 0.5 – Eneg)/(n × 0.5) ×100, with Eneg
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Hoermann et al., Sci. Adv. 8, eabo1733 (2022) 21 September 2022
SCIENCE ADVANCES | RESEARCH ARTICLE
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being the individuals negative for the effector and n being the total
number of samples successfully analyzed by PCR.
Transmission modeling using EMOD
Simulations were performed using EMOD v2.20 (56), a mechanistic,
agent-based model of P. falciparum malaria transmission that include
vector life cycle dynamics and within host-parasite and immune
dynamics. Seasonality of rainfall and temperature as well as vec-
tor species were kept the same across transmission settings, but
vector density was varied to match desired transmission intensity.
A. gambiae, the only vector considered, was assumed as being 95%
endophilic and 65% anthropophilic. Each simulation contained
1000 representative people with birth and death rates appropriate
to the demography without considering importation of malaria. We
include baseline health seeking for symptomatic cases as an interven-
tion where human agents can seek treatment with 80% artemether-
lumefantrine of the time within 2 days of severe symptom onset and
50% of the time within 3 days of the onset of a clinical but nonsevere
case. Mosquitoes within EMOD contain simulated genomes that
can model up to 10 genes with eight alleles per gene with phenotyp-
ic traits that map onto different genotypes (49). Here, we modeled
an IGD system (57) aimed at population replacement with an effector
that results in delayed sporozoite production in infected mosqui-
toes and overall reduction in the number of sporozoites produced
by infected mosquitoes. The model was further parameterized us-
ing the experimentally determined measures of fitness, life span,
and homing. One thousand male IGD mosquitoes homozygous for
the autonomous drive (Cas9) and nonautonomous effector (MM-CP)
were released just before the wet season begins to pick up in trans-
mission intensity (table S2). Apart from the sex chromosomes, two
loci representing the effector and driver were modeled (57), with each
locus having four possible alleles (WT, resistant, effector, or nuclease
and loss of gene function for the effector or driver locus). To evalu-
ate the performance of these drives in a range of transmission set-
tings, we vary transmission intensity via annual entomological rates
(EIR) ranging from 15 infectious bites per person to 60 infectious
bites per person. We also vary the final phenotypic effect of express-
ing the effector gene that leads to delayed and reduced sporozoite
formation. We evaluate average increases in time until sporozoite
formation ranging from no increase in time compared to a WT
mosquito up to 70% increase in sporozoite formation time. As for
the reduced sporozoite effect, we evaluated to full range of possible
effects compared to a WT mosquito. Mosquitoes carrying the drive
are released 6 months into the simulation, and simulations are run
for a total of 6 years. The outputs represent the mean of 25 stochas-
tic realizations per parameter set.
SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at https://science.org/doi/10.1126/
sciadv.abo1733
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Acknowledgments: We thank E. Marois for sharing the vasa-Cre and vasa-Cas9 lines. We
thank D. Bridenbecker for software support and A. Burt for suggestions on the manuscript.
Funding: The work was funded by the Bill and Melinda Gates Foundation grant OPP1158151
to N.W. and G.K.C. and Wellcome Trust Investigator Award 107983/Z/15/Z to G.K.C. Author
contributions: Conceptualization, data curation, formal analysis, investigation, methodology,
visualization, and writing (preparation of original draft): A.H. and T.H. Investigation,
methodology, software, visualization, and writing (preparation of original draft):
P.S. Investigation and resources: G.D.C., P.C., M.G.I., and T.M.K. Conceptualization, funding
acquisition, project management, supervision, and writing (review and editing):
G.K.C. Conceptualization, funding acquisition, project management, supervision, writing
(review and editing), formal analysis, and visualization: N.W. Competing interests: The
authors declare that they have no competing interests. Data and materials availability: All
data needed to evaluate the conclusions in the paper are present in the paper and/or the
Supplementary Materials. Input data and scripts to generate the figure panels presented in the
paper are available at Dryad (doi:10.5061/dryad.rfj6q57dq) or https://github.com/
genome-traffic/anopheles_MM-CP_paper. As an additional resource, the EMOD gene drive
model has been made available at https://github.com/InstituteforDiseaseModeling/
sporogonic_retardation_2022. The RNA-seq raw data have been deposited to the National
Center for Biotechnology Information NRA (PRJNA822650).
Submitted 18 January 2022
Accepted 4 August 2022
Published 21 September 2022
10.1126/sciadv.abo1733
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to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).
Gene drive mosquitoes can aid malaria elimination by retarding Plasmodium
sporogonic development
Astrid Hoermann, Tibebu Habtewold, Prashanth Selvaraj, Giuseppe Del Corsano, Paolo Capriotti, Maria Grazia Inghilterra,
Temesgen M. Kebede, George K. Christophides, and Nikolai Windbichler
Sci. Adv., 8 (38), eabo1733.
DOI: 10.1126/sciadv.abo1733
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