Malaria-transmitting mosquitoes cause over 245 million malaria cases and about 620,000 deaths annually (WHO, 2021). Vector control, mainly in the form of insecticide indoor residual spraying and insecticide-treated bed nets, has permitted a steady decrease in malaria cases during the last decades. However, this decline is stalling as mosquitoes become increasingly resistant to insecticides and as malaria parasites continue to evolve resistance to drugs. These ongoing genetic changes in vectors and parasites, under the selection pressure of two major pillars of malaria control, jeopardize the hard-won successes in the fight against malaria. To address the need for complementary malaria control approaches, intervention strategies based on intentional genetic changes in mosquitoes to decrease malaria transmission are under development. Recent advances in the field of gene drive (GD) provide a mean to push desirable transgenes to invade a target population of insects (Carballar-Lejarazú and James, 2017; Gantz and Akbari, 2018; Quinn and Nolan, 2020; James and Santos, 2023).

GD interventions against malaria mosquitoes are still in the laboratory phase of their development, and can be subdivided in two classes: (a) population suppression GDs that spread either female sterility or female killing in mosquito populations with the aim to eliminate them (Hammond et al., 2016; Kyrou et al., 2018; Simoni et al., 2020); (b) population modification GDs that leave mosquito populations in place, but genetically alter them to reduce their disease transmission capacity (Gantz et al., 2015; Pham et al., 2019; Carballar-Lejarazú et al., 2020; Carballar-Lejarazú et al., 2023). Both approaches raise ethical, safety and ecological concerns, and trigger controversy (e.g., National Academies of Science, Engineering and Medicine, 2016; Courtier-Orgogozo et al., 2017; de Graeff et al., 2021). One concern raised by elimination approaches is their possible consequences on the food web and ecosystems, even where dozens of sympatric mosquito species are potentially available to compensate for the loss of the target species. In some regions, elimination of one species could favor the installation of competing species that could also act as vectors, as can be feared for the Aedes aegypti / Ae. albopictus mosquito dyad vectoring dengue, Zika, and chikungunya viruses. Modification approaches alleviate these concerns by leaving the target species in its ecological niche, but in turn, the indefinite persistence of transgenes utilized in modification drives could have long-term secondary effects, so that both approaches should be planned with utmost caution. However, the option of rejecting gene drive technologies as a complementary tool to fight mosquito-borne pathogens also raises ethical issues, by ignoring a chance to curb the immense mortality and morbidity caused by mosquito-borne infections.

Anopheles gambiae (sensu lato), the major mosquito vector of malaria in Sub-Saharan Africa, is widely distributed, locally abundant during rainy seasons and its biomass might significantly contribute to the food chain, in a temporal fashion. Besides their possible negative impact on food webs, elimination approaches for this mosquito are likely to be complicated by re-colonization from residual pockets of surviving isolated populations, resulting in chasing dynamics of gene drive (Champer et al., 2021; Champer et al., 2022). For this species in its native ecosystem, population modification to decrease vector competence and preserve ecological functions could be a meaningful gene drive approach. Here, we present the engineering of the Lipophorin (Lp) essential gene in Anopheles coluzzii, a prominent member of the A. gambiae species complex and a major malaria vector in sub-Saharan Africa.

Lp naturally encodes an abundant lipid transport protein secreted in mosquito hemolymph. We modified Lp to co-express a single-chain antibody (ScFv) derived from 2A10, a well-established mouse monoclonal antibody that binds the Plasmodium falciparum circumsporozoite protein (CSP) (Zavala et al., 1983). Sporozoites are the parasite stage that mosquito females inject in vertebrate hosts while aquiring a blood meal. Once in the host, sporozoites quickly infect the liver and multiply to produce merozoites with a tropism for red blood cells. Through its binding to CSP (the major sporozoite surface antigen), 2A10 was shown to decrease sporozoite infectivity to cultured human hepatocytes (Hollingdale et al., 1984). Transgenic expression of a 2A10 ScFv, fused to the antimicrobial peptide Cecropin A and expressed under the control of the Vitellogenin promoter, decreased the number of salivary gland sporozoites in Anopheles stephensi (Isaacs et al., 2011; Isaacs et al., 2012) and in A. gambiae/coluzzii (Carballar-Lejarazú et al., 2023). Expression of a 2A10 ScFv directly in the salivary glands of An. stephensi reduced sporozoite infectivity to cultured hepatocytes, and transmission to mice of chimeric Plasmodium berghei expressing CSP from P. falciparum (Sumitani et al., 2013). Likewise, expression of a different CSP-binding ScFv in transgenic A. coluzzii decreased the sporozoite’s ability to infect cultured hepatocytes and to cause malaria in mice (Triller et al., 2017).

We inserted the 2A10 ScFv coding sequence within the endogenous open-reading frame of the essential Lp gene, preserving Lp function and exploiting a natural proteolytic cleavage site to separate the fusion protein moieties during their secretion. Using a mouse model of malaria, in which a transgenic rodent Plasmodium berghei strain expresses CSP from P. falciparum, we show a strong decrease in the ability of the genetically engineered mosquitoes to transmit Plasmodium. In a second step, we tested different CRISPR/Cas9-based GD designs to force the spread of the Lp::Sc2A10 transgene in laboratory mosquito populations. A first, suppression GD disrupting the wild-type (WT) Lp locus revealed haploinsufficiency of the Lp gene, precluding this approach. Other GD versions were designed to home into and disrupt the Saglin pro-parasitic gene on the X chromosome. These GD also expressed guide RNAs targeting WT Lp, promoting homing of Lp::Sc2A10 on the second chromosome. This dual homing at both the Saglin and Lp loci resulted in the simultaneous spread of the two transgenes in caged mosquito populations. In modified mosquitoes, sporozoite transmission should be reduced by two distinct mechanisms, i.e., depriving Plasmodium from its Saglin agonist plus attacking sporozoites with the ScFv. Each of the Saglin and the Lp loci were targeted with multiplexes of three gRNAs, to increase the chance that failed homing events result in loss-of-function Lp mutations that would be eliminated by natural selection, and loss-of-function, GD-resistant Saglin alleles that should still contribute to decrease vector competence. One homing-refractory functional allele was however observed at the Lp locus.

We hijacked an essential gene, Lipophorin (Lp), to force Anopheles coluzzii mosquitoes to co-express an anti-malarial factor. The choice of the Lp gene was motivated by its exceptionally strong expression level (Lp being the most abundant constitutively expressed protein in mosquito hemolymph) and by its endogenous signal peptide, which we could exploit to obtain concomitant secretion of Sc2A10 with Lp protein. The existence of an endogenous proteolytic cleavage site within the Lp protein, that we duplicated to detach Sc2A10 from Lp, was also advantageous, as well as the natural intron immediately following the signal peptide-encoding exon 1 that could accept a visual selection marker for easy tracking of the genetic modification. This approach is related to the integral gene drive strategy (Hoermann et al., 2021) in that anti-malarial factors are expressed together with an endogenous gene, but here the modified endogenous gene does not comprise any component of a gene drive —in the case of integral gene drives, gRNAs are inserted in a synthetic intron. The genetic modification does not disrupt expression or function of the Lp essential gene. On the contrary, Lp protein expression is intended to remain as unaffected as possible, but obligatorily proceeds through translation of the Sc2A10 antimalarial factor inserted between the Lp signal peptide and the rest of the Lp protein. This design minimizes the risk of spontaneous transgene loss by mutation, since frameshift or nonsense mutations in the Sc2A10 coding sequence would prevent Lp expression, resulting in their elimination by natural selection. Furthermore, the absence of repeated motifs in the synthetic sequence decreases the risk of in-frame deletions. The possibility of a loss of the transgene’s anti-Plasmodium activity should therefore be limited to missense mutations or in-frame deletions arising in the Sc2A10 coding sequence, or to Plasmodium evolution, notably under selection pressure imposed by the transgene itself (Marshall et al., 2019). However, the 2A10 antibody targets a highly repeated epitope on the CSP protein (the NANP motif), that, in addition to being repetitive, is also conserved across P. falciparum strains. A combination of mutations leading to the complete loss of this epitope in Plasmodium therefore seems unlikely, and would probably cause a dramatic loss of parasite fitness as CSP is required in several processes in the sporozoite’s journey between two hosts (Coppi et al., 2011; Balaban et al., 2021).

Hijacking Lp and its signal peptide is an approach that could be widely applicable to other possible factors acting in the hemolymph to attack various parasite stages, such as ookinetes, which represent a particularly vulnerable stage of parasite development in the mosquito. To make mosquitoes that express and combine additional secreted factors against parasites, Vitellogenin (AGAP004203) could also be engineered in the same manner, taking advantage of its own signal peptide, extremely high expression level but also transient induction following a blood meal. Blood meal inducibility would be particularly suitable for antimalarial factors specifically targeting ookinetes, as they emerge on the hemolymph-bathed side of the midgut exactly within the short time window when Vitellogenin is expressed. Mosquitoes would not be exposed during their development to a factor expressed from Vg, limiting potential fitness costs. As proposed by Hoermann et al., 2021 Carboxipeptidase (Cp) represents an additional attractive host locus for antimalarial factors, sharing many of the advantages of Vg. A single GD expressing multiplexed gRNAs could stimulate the simultaneous spread of Lp, Vg and Cp modified, anti-parasitic alleles.

In spite of the high Lp protein secretion in mosquito hemolymph, we only detected less than 1/10 of this molecular amount for Sc2A10 in hemolymph by mass spectrometry. Apart from technical limitations in detection sensitity (e.g. biased trypsin digestion of some peptides) this could be explained by faster removal from the hemolymph and /or faster degradation of Sc2A10 compared to Lp. Different, more stable anti-Plasmodium peptides that could accumulate to higher levels may show even better efficacy as transmission-blocking agents compared to Sc2A10. This also highlights the benefit of choosing a strong promoter such as that of Lp to ensure maximal production of antimalarial effectors in the hemolymph. A single copy of the Lp::Sc2A10 transgene still showed a reduction in transmission. Of note, the rodent model of malaria used here (P. berghei expressing CSP from P. falciparum) is characterized by very high infection rates, typically producing dozens of oocysts per midgut, each of which can release hundreds of sporozoites in mosquito hemolymph. The ability of secreted Sc2A10 to decrease sporozoite transmission even in this high infection context is highly encouraging, given the much lower levels of P. falciparum (typically 1 to a few oocysts) in field infections. An even greater anti-Plasmodium activity of Sc2A10 may be reached by fusing it to antimicrobial peptides such as CecropinA, as achieved by Isaacs et al. in conventional transgenic An. stephensi mosquitoes (Isaacs et al., 2011; Isaacs et al., 2012) and subsequently in the first published GD for An. stephensi (Gantz et al., 2015). In these studies, the Sc2A10::CecropinA fusion was expressed intermittently under the control of the blood-meal inducible Vitellogenin promoter, and combined with a second scFv targeting Plasmodium falciparum ookinete Chitinase 1 also expressed after blood meals under the control of the Cp promoter. While the insertion of Sc2A10::CecropinA within Lp coding sequences may further improve the efficiency of our approach, there is a risk that CecropinA expressed at all developmental stages, and an increased complexity of the Lp fusion protein, worsen the fitness cost already observed in Lp::Sc2A10 mosquitoes.

To force the spread of the Lp::Sc2A10 transgene in mosquito populations, we tested several gene drive (GD) designs. A same-locus suppression drive was also installed in the Lp gene, creating a loss-of-function allele. The Lp::Sc2A10 allele was intended to act as an R1-type, gene drive refractory functional allele (Champer et al., 2018) that would supplant the suppression gene drive and invade the population. This was unsuccessful, as Lp proved largely haplo-insufficient. In contrast, distant-locus GDs disrupting Saglin, a non-essential but pro-parasitic gene, were efficient at promoting invasion of both the Saglin, and particularly the Lipophorin, modified alleles. Unlike the first approach, this design may allow Cas9 and gRNA-coding genes to persist indefinitely within the invaded mosquito population (unless nonfunctional resistance alleles outcompete the drive allele in the long run). Although for this reason we initially preferred the same-locus approach in which Lp function disruption by the insertion of CRISPR/Cas9 components caused their gradual disappearance by natural selection, the added benefit of a GD installed in Saglin is that it should further reduce the transgenic mosquitoes’ vectorial capacity, a combination of two distinct mechanisms to decrease sporozoite transmission being more robust to combat Plasmodium durably.

Importantly, we observed that same-locus or distant-locus constructs carrying guide RNAs that target wild-type Lp are inviable, and rapidly go extinct in the absence of a recoded Lp allele such as Lp::Sc2A10 (or spontaneous Lp mutants immune to GD that may arise in GD populations). The genetic design established here can be viewed as a two-component GD in which one component (Lp::Sc2A10) has no drive capacity by itself but (i) rescues the lethality, and (ii) hitchhikes on the drive capacity of the other (SagGD^vasa) ensuring its own hyper-Mendelian spread. The two transgenic loci are, therefore, reciprocally dependent on each other: Lp::Sc2A10 depends on SagGD for its long-term persistence and spread in a population, and SagGD depends on Lp::Sc2A10 as a rescue allele of the essential Lp target for its survival. This design can be seen as a two-locus variation of rescue-type GDs (Adolfi et al., 2020; Champer et al., 2020a).

Because Saglin knockout mutants bear no fitness cost at least in laboratory conditions, GD-refractory Saglin alleles generated by failed homing events are certain to accumulate over time (all the more so as their lack of toxicity toward the WT Lp allele confers them a high selective advantage compared to SagGD). If the demise of the Saglin GD occurs sufficiently late, Lp-Sc2A10 could still reach high levels or even fixation in a mosquito population. If, however, GD refractory mutations in Saglin accumulate rapidly and no longer support Lp::Sc2A10 maintenance, the elimination of both SagGD^vasa and Lp::Sc2A10 will be inexorable. Thus wild-types alleles are likely to re-establish at both loci once the frequency of active Saglin GDs drops, especially under conditions of strong mosquito immigration from regions adjacent to the initial GD spread range. Still, even as genetic resistance to the GD develops, the resulting high frequency of Saglin loss-of-function mutant alleles arising from failed homing events, with their transient selective advantage while SagGD^vasa and wild Lp alleles co-exist, should decrease global vector competence.

Once a GD installed in the Saglin locus is defeated by the accumulation of GD-refractory mutations, new generations of GD installed at different loci could be launched to re-ignite the spread of the Lp::Sc2A10 allele. For instance, GDs built for the same mosquito species in the frame of other projects could also incorporate gRNAs re-igniting the spread of Lp::Sc2A10, promoting antimalarial approaches similar to combination therapies. In this respect, transgenic systems permitting the expression of multiplexes of many gRNAs would be instrumental. Some of the constructs presented here incorporate multiplex gRNAs combining individual U6-gRNA cassettes with a U6-gRNA::tRNA quadruple gRNA expression cassette, with the drawback of displaying many repeated motifs (U6 promoters, multiple units of tRNAglycine, gRNA invariable region). Genetic instability of these constructs, with loss of gRNA expression segments, was prominent in our experiments. This is illustrated by the high prevalence, in our tracked mosquito populations, of a GD derivative allele carrying only two residual U6-gRNA expression units, from the original tandem of six. Luckily, this deletion allele still retains driving potential for both the Saglin and Lp loci, but it would take only one additional recombination event to reduce the array to a single gRNA. This would result either in a Saglin-only, non-lethal, GD allele, which may continue to reduce vectorial capacity of the mosquito population by mutating Saglin, or in a non-driving Saglin mutant still able to promote some Lp::Sc2A10 spread by a split GD effect (but particularly able to generate Lp R1-type mutants given the intronic target of the single residual gRNA). We are currently working to improve gRNA multiplex design by increasing the number and variety of tRNA spacers, so that the 76 bp constant region of each gRNA would be the only remaining repeated element. To further optimize future GD design, modeling studies can now aid in determining the optimal number of gRNAs in a multiplex, depending on the specific GD design and purpose (Champer et al., 2020b).

In addition to this and to the stabilization of multiplex gRNA arrays, other paths to improvement of the system presented here include avoiding gRNAs targeting intronic sequences, and the use of promoters better restricted to the germline to control Cas9 expression and limit GD refractory mutation emergence. Interestingly, the zpg promoter that in theory possesses this advantage showed low activity once inserted in the Lp and Saglin loci, in contrast to the DSX or Nudel loci (Hammond et al., 2016; Kyrou et al., 2018). This suggests that the zpg promoter is more sensitive to the local chromatin context than the vasa2 promoter, similarly to the sds3 promoter in Aedes aegypti (Anderson et al., 2023), and that its usability in GD constructs may be restricted to a subset of genomic loci. The nanos promoter (Gantz et al., 2015; Terradas et al., 2022; but see Hammond et al., 2021) may represent an interesting alternative. Finally, tighter clustering of gRNA target sites at target homing loci, especially Saglin, should improve gene drive performance by reducing the length of DNA sequences flanking the cut site that bear no homology to the repair template on the sister chromosome and need to be resected by the repair machinery to allow homing (López Del Amo et al., 2020).

A simpler design to promote the spread of antimalarial factors such as Sc2A10 in mosquito populations would be a single-locus GD (for example installed in Saglin or other pro-parasitic genes if the idea of consolidating the anti-Plasmodium activity via two mechanisms is retained). Few loci, however, can provide the benefits offered here by Lp that include a suitable expression pattern, high expression, an endogenous secretion signal, endogenous proteolytic cleavage, a natural intron ideally positioned to host a fluorescent marker, and the status of essential gene that a translational fusion exploits to limit spontaneous transgene loss by mutation. Besides, adding the Cas9 and gRNA-coding cassettes to an intron of an essential gene chosen as GD host would add >5 kb of foreign DNA sequence, thereby significantly increasing the complexity of the modification and the risk of affecting functionality of the essential gene. For example, inadvertent intron splice sites in synthetic sequences can preclude proper mRNA splicing (Hoermann et al., 2022); we also encountered this problem when inserting a different anti-Plasmodium ScFv in Lp (Green, 2019).

In conclusion, the Sag-GD^vasa transgene presented here constitutes a viable GD only in the presence of an engineered Lp allele, which by itself has no drive capacity but will hitchhike with Sag-GD^vasa to spread at hyper-Mendelian rates. In caged mosquito populations, both genetic loci initially spread together and decrease mosquito capacity to transmit Plasmodium parasites expressing CSP from P. falciparum. Mutations at both loci, especially in Saglin that is under low selection pressure, arise due to failed homing events, have a selective advantage over the toxic SagGD^vasa allele, and over time should replace the GD, so that the system carries the seeds of its own removal. The anti-Plasmodium Lp::Sc2A10 allele will likely slowly disappear due to its fitness cost, unless its spread is re-ignited by updated distant-locus GDs. Although such a reversibility could be seen as a safety advantage for modification GDs (and may be enhanced by intentionally releasing laboratory-selected resistance alleles), future work should focus on establishing GD constructs in Saglin that limit the emergence of GD refractory alleles to prolong their potential field lives.

All data generated or analysed during this study are included in the manuscript and supplementary files.

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Author details

1. Emily I Green

   Inserm U1257, CNRS UPR9022, University of Strasbourg, Strasbourg, France

   Contribution

   Investigation, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

2. Etienne Jaouen

   Inserm U1257, CNRS UPR9022, University of Strasbourg, Strasbourg, France

   Contribution

   Investigation

   Competing interests

   No competing interests declared

3. Dennis Klug

   Inserm U1257, CNRS UPR9022, University of Strasbourg, Strasbourg, France

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9108-454X

4. Roenick Proveti Olmo

   Inserm U1257, CNRS UPR9022, University of Strasbourg, Strasbourg, France

   Contribution

   Data curation, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3849-8591

5. Amandine Gautier

   Inserm U1257, CNRS UPR9022, University of Strasbourg, Strasbourg, France

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Stéphanie Blandin

   Inserm U1257, CNRS UPR9022, University of Strasbourg, Strasbourg, France

   Contribution

   Formal analysis, Funding acquisition, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4566-1200

7. Eric Marois

   Inserm U1257, CNRS UPR9022, University of Strasbourg, Strasbourg, France

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   e.marois@unistra.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4147-3747

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