The nanos d integral gene drive enables population modification of the malaria vector Anopheles gambiae
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Abstract
The modification of mosquito populations at scale through CRISPR-Cas9-mediated homing gene drives is a promising route for malaria vector control. Integral gene drives (IGDs) are designed to utilize the regulatory sequences of endogenous genes to express only the minimal set of components required for gene drive. In this study, we describe the creation and characterization of the nanos d IGD targeting and inserted into the nanos gene of the malaria vector Anopheles gambiae and show that it achieves high rates of gene drive (97.7% in females, 99.0% in males). We find that homozygous nanos d females but not males show impaired fecundity, and a variable loss of ovary phenotype. Transcriptomic analysis of ovaries points to decreased transcript levels of the nanos gene when harbouring Cas9. As a minimal genetic modification, nanos d does not induce widespread transcriptomic perturbations, and its susceptibility to Plasmodium spp. and O’nyong nyong virus infection remains similar to wild-type mosquitoes. Importantly, we find that nanos d propagates efficiently in caged mosquito populations and is maintained as a source of Cas9 after the emergence of drive resistant alleles whilst also mobilising a non-autonomous antiparasitic effector modification. The nanos d gene drive shows promise as a genetic tool for malaria vector control via population modification, and we outline steps towards its further optimization.
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Referee #2
Evidence, reproducibility and clarity
Summary:
This study demonstrates an improved integral gene drive (IGD) for use in Anopheles gambiae. Inserting the coding sequence for Cas9 in-frame with a germline-specific gene (nanos) improved the performance of this IGD relative to previously reported systems while reducing fitness costs. Integration of the gRNA cassette within a synthetic intron is an elegant solution to constraining the minimal elements of the IGD within a single insertion. The results of this study found that while the IGD can be used to propagate anti-malarial effectors (MM-CP) within a population, fitness costs and resistance alleles were higher than …
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Referee #2
Evidence, reproducibility and clarity
Summary:
This study demonstrates an improved integral gene drive (IGD) for use in Anopheles gambiae. Inserting the coding sequence for Cas9 in-frame with a germline-specific gene (nanos) improved the performance of this IGD relative to previously reported systems while reducing fitness costs. Integration of the gRNA cassette within a synthetic intron is an elegant solution to constraining the minimal elements of the IGD within a single insertion. The results of this study found that while the IGD can be used to propagate anti-malarial effectors (MM-CP) within a population, fitness costs and resistance alleles were higher than anticipated, potentially limiting the application of this particular IGD design without further optimisation.
The results comprehensively demonstrate the effective transmission and stability of the IGD over several generations, while also characterising the limitations of the system. Although I don't think the authors make any claims which are not supported by their results. It might be good to provide more of an explanation for how the performance of this IGD compares to the zpg IGD reported in Ellis et al 2022 for readers less familiar with the IGD literature.
The manuscript is overall very well written with clear results and methods. However, I found the descriptions referring to the effects of the maternal, paternal, and even grandmaternal inheritance hard to follow. The statistical analysis and replications are adequate as well.
Referee cross-commenting
I agree with the other reviewer's comments regarding the need to clarify a few points made in the overall well written manuscript.
Significance
Gene drives are the most promising genetic biocontrol method for controlling the spread of malaria. However, there are many technical challenges that have made the development of gene drives quite difficult. This study works to address one such challenge - constraining the expression of Cas9 to the germline by integrating it within an endogenous loci rather than using semi-synthetic promoters. While IGD have been demonstrated before, this study further improves on their performance while reducing off-target effects.
The manuscript is written for a highly specialized audience that is very familiar with the genetic biocontrol, and especially the gene-drive field of research.
My fields of research include genetic biocontrol and insect synthetic biology.
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Referee #1
Evidence, reproducibility and clarity
In this study, the authors develop a complete integral drive system in Anopheles gambiae malaria mosquitoes. This type of gene drive is interesting, with special advantages and disadvantages compared to more common designs. Here, the authors develop the Cas9 element and combine it with a previously developed antimalaria effector element. The new element performs very well in terms of drive efficiency, but it has unintended fitness costs, and a higher than desirable rate of functional resistance allele formation. Nevertheless, this study represents a very good step forward toward developing effective gene drives and is thus of high …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #1
Evidence, reproducibility and clarity
In this study, the authors develop a complete integral drive system in Anopheles gambiae malaria mosquitoes. This type of gene drive is interesting, with special advantages and disadvantages compared to more common designs. Here, the authors develop the Cas9 element and combine it with a previously developed antimalaria effector element. The new element performs very well in terms of drive efficiency, but it has unintended fitness costs, and a higher than desirable rate of functional resistance allele formation. Nevertheless, this study represents a very good step forward toward developing effective gene drives and is thus of high impact.
The format of the manuscript is a bit suboptimal for review. Please add line numbers next time for easy reference. It would also help to have spaces between paragraphs and to have figures (with legends) added to the text where they first appear.
It might be useful to add subsections to the results, just like in the methods section. It could even be expanded a bit with some specific parts from the discussion, through this is optional.
Abstract: The text says: "As a minimal genetic modification, nanosd does not induce widespread transcriptomic perturbations." However, it does seem to change things based on Figure 3c.
Page 2: "drive technologies for public health and pest control applications" needs a period afterward.
Page 2: "The fitness costs, homing efficiency, and resistance rate of the gene drive is" should be "The fitness costs, homing efficiency, and resistance rate of the gene drive are".
Page 2: "When they target important mosquito genes, gene drives are designed to ensure that the nuclease activity window (germline) does not overlap with that of the target gene (somatic)." is note quite correct. This is, of course, sensible for suppression drives, but it's not a necessary requirement for modification drives with rescue elements in many situations.
Page 2: "recessive somatic fitness cost phenotypes" is unclear. I think that you are trying to avoid the recessive fitness cost of null alleles becoming a dominant fitness cost from a gene drive allele (in drive-wild-type heterozygotes).
Page 2: "This optimization approach has had only limited success, and suboptimal performance is commonly attributed to not capturing all the regulatory elements specific to the germline gene's expression9,12". I don't think this is correct. There are several examples where a new promoter helped a lot. The zpg promoter in Anopheles gambiae allowed success at the dsx site in suppression cage studies (Kyrou et al 2018), and nanos gave big improvement to modification drives at the cardinal locus (Carballer et al 2020). In flies, several promoters were tested, and one allowed success in cage experiments (Du et al 2024). In Aedes, the shu promoter allowed for high drive performance (Anderson et al 2023), though this last one hasn't been tested in more difficult situations. I think you could certainly argue in the general case that not all promoters will work the way their transcriptome says, but there are many examples where they seem to be pretty good.
Page 2: "make it more likely that mutations that disrupt the drive components are selected against though loss of function of the host gene." I think that this needs a bit more explanation. You are referring to mutations in regulatory elements or frameshift mutations. This will make it more resistant to mutation. Also, these mutations would tend to have a minor effect expect perhaps in the cargo gene of a modification drive. By using a cargo gene in an integral drive, you could still keep it somewhat safer, but whether this is 1.2x or 10x safer is unclear.
Page 3: "they can incur severe unintended fitness costs". This is central to integral drives and this manuscript. It's worth elaborating on.
Page 3: "Regulatory elements from germline genes that have worked sub-optimally in traditional gene drive designs for the reasons outlined above may work well in an IDG design20." This is setting up the integral drive with nanos, but nanos DOES work well in traditional Anopheles gambiae gene drive designs. It is possible that you might end up with less somatic expression than Hammond et al 2020 (though the comparison is unclear due to batch effects in that study), but there is no direct comparison in this manuscript to that.
Page 3: "This suggests an impact of maternal deposition on drive efficiency only in female drive carriers." This is quite strange. Usually, I would expect to see an equal reduction in efficiency between male and female progeny. Could this be due to limited sample size? Random idea: It's also possible that almost all maternal deposition was mosaic and wouldn't be enough to direct affect drive conversion. However, it could cause enough of a fitness cost TOGETHER with new drive expression in females that perhaps only tissues with randomly low expression rates properly developed and led to progeny, reducing drive inheritance? Another possibility: Could the drive/resistance males have impaired fertility, so these ones are underrepresented in the batch cross? If nanos is needed in males and a single drive copy is not quite enough for good fertility or mating competitiveness, they may be underrepresented in your crosses. They might have worse fertility than drive homozygous males, which at least have two partially working copies of nanos rather than just one (in many cells, at least). Maybe check the testis for abnormal phenotypes?
Overall, it would be favorable if the drive allele was somewhere more fit than a nonfunctional resistance allele. This could already be achieved in this drive, but it doesn't get much mention.
Page 3: There should be a comma after, "Interestingly, while many of the observed mutations were predicted to abolish nanos expression" and "This could indicate that in these experiments".
Page 3 last sentence: Please improve the clarity.
Removing the EGFP is supposed to restore the fitness, and this was helpful in some previous integral drive constructs. This could get a bit more mention (it is possible that I missed this somewhere in the manuscript).
Page 4: The MM-CP line and it's association with the integral drive strategy could get a little more introduction. Maybe even a supplemental figure showing the general idea.
Page 5: "cassette is predicted to disrupt the CP function entirely (Fig. 5d)" also lacks a period.
Page 5: "The subsequent stabilization of the nanosd frequency and the lack of rapid loss suggests that any associated fitness cost is primarily recessive." This is not quite correct because by this point, drive/wild-type heterozygotes are rare, and this is where you'd find a potential dominant fitness cost. It should be correct in the end stages where it is a mix of drive and functional/nonfunctional resistance alleles (though the nonfunctional resistance alleles may cause greater fitness costs when together with a drive - see above).
Page 6: "Maternal deposition of Cas9, or Cas9;gRNA, into the zygote can lead to cutting at stages when homing is not favoured, and has been commonly observed for canonical Anopheles nanos drives9,10,35." Reference 35 (which is more suitable for referencing an example of nanos in other Anopheles) found some resistance alleles by deep sequencing, but the timing that they formed was unclear (it's not certain if it was maternal deposition). This study may be a more suitable reference: Carballar-Lejarazú R, Tushar T, Pham TB, James AA. Cas9-mediated maternal-effect and derived resistance alleles in a gene-drive strain of the African malaria vector mosquito, Anopheles gambiae. Genetics, 2022.
Page 8: "could further reduce the likelihood of resistance allele formation by increasing the frequency of HDR events." Multiple gRNAs would mostly help by reducing functional resistance allele formation, especially since drive conversion is already very high in Anopheles.
Page 8, last paragraph: This conclusion is perhaps a little optimistic considering the functional resistance alleles, which should get a little more attention in the summary or elsewhere in the discussion section.
Figure 1d: The vertical text saying "Non-WT" is confusing. The circles themselves show + and -. Also, "-" isn't necessarily a knockout allele, so I'm not sure if - is the best symbol for resistance.
Figure 2e: The vertical scale is not the most intuitive. Consider rearranging it to "Transition from larvae to pupae" starting at zero and going to 1 when all the larvae become pupae.
Figure 2e-f: For both of these, there are clear differences between males and females. Thus, when comparing drive homozygotes to wild-type, it would probably be better to have separate statistical comparisons for males and females.
Figure 3: Can any of these transcription results in individual genes potentially explain the observed fitness cost?
Figure 3b: The scale here also doesn't quite make sense. It's the fraction of underdeveloped ovaries, but the graph is also perhaps trying to show whether just 1-2 ovaries are present, or maybe how many ovaries are undeveloped, but then it would say "zero"? This should be clarified. Number of ovaries and how well-developed they are is separate (it can be put on the same graph, but needs to be more clear).
Figure 4f: The vertical axis should say "ONNV."
Figure 5c-d: These should be labeled as the most common resistance allele. Also, I'm not sure how relevant it is, but we also found an alternate start codon here: Hou S, Chen J, Feng R, Xu X, Liang N, Champer J. A homing rescue gene drive with multiplexed gRNAs reaches high frequency in cage populations but generates functional resistance. J Genet Genomics, 2024. Maybe this is a more common problem than one would expect?
Figure 5cd,S4,S5: They have a bit of a weird plot. Why not make four line graphs for each? Also, some alleles use the symbol. + is wild-type, which is well understood, but - as resistance is not always clear, and seeing them together may confuse readers. Additionally, the fact that you have the most common resistance allele in Figure 5cd might mean that you know more about the genotype? If so, it would be best to separate wild-type and resistance alleles in whatever the final figure looks like.
Some supplemental raw data files would be useful if they were available, but the figures are through enough that this isn't essential.
Review by:
Jackson Champer, with major assistance from Ruobing Feng (essentially section B) and Jie Du
Referee cross-commenting
We don't have any cross-comments, other than supporting the idea of slightly more comparisons to the authors' previous construct.
Significance
- Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.
A key innovation of the nanosd gene drive is its integral gene drive (IGD) design, which inserts the drive cassette directly into the A. gambiae nanos gene, incorporating only the minimal components necessary for drive function. The drive achieves high transmission rates, without causing widespread disruption of gene expression or increasing susceptibility to malaria parasites, and imposes an acceptable fitness cost-primarily a reduction in female fecundity when homozygous. The strong performance of nanosd can be attributed to its design: Cas9 is expressed in the correct cells and timing to induce efficient homing, effectively hijacking the nanos gene's natural expression profile. However, despite the careful design aimed at preserving nanos function, the rescue was incomplete: homozygous female drive carriers exhibited a clear reduction in ovarian function.
In caged population trials, both the drive and a co-introduced anti-malaria effector gene reached high frequencies, even in the presence of emerging resistance alleles. Because the drive is inserted into an essential gene, nonfunctional resistance alleles are selected against and tend to be purged over time. Nonetheless, functional resistance remains a concern. The use of a single, though precisely positioned gRNA targeting the native nanos gene ATG site increases the likelihood of generating functional resistance alleles. Over the long term, if the drive imposes fitness costs, it may be outcompeted by such functional resistance alleles, potentially undermining the goal of sustained population modification.
Overall, this study represent a notable advance in Anopheles mosquito gene drive development and can be considered as high impact.
- Place the work in the context of the existing literature (provide references, where appropriate).
Previous IGD efforts in Drosophila, mice and mosquitoes have demonstrated nearly super‐Mendelian inheritance but often at the expense of host fitness. For example, Nash et al. built an intronic‐gRNA Cas9 drive at the D. melanogaster rcd-1r locus that propagated efficiently through cage populations (Nash et al., 2022), and Gonzalez et al. reported that a Cas9 drive inserted at the germline zpg locus in Anopheles stephensi biased inheritance by ~99.8% (Gonzalez et al., 2025). However, these strong drives disrupted essential genes: in A. gambiae, inserting Cas9 into zpg produced efficient homing but rendered females largely sterile (Ellis et al., 2022). A similar germline Cas9 knock-in in Mus musculus enabled gene conversion in both sexes, albeit with only modest efficiency and potential fitness trade-offs (Weitzel et al., 2021). The current nanosd IGD is explicitly designed to overcome this limitation by selecting a more permissive gene target and using a minimal drive cassette, so as to preserve mosquito viability while maintaining robust drive efficiency, although still with reduced female drive homozygotes fertility.
This nanosd gene drive like previous homing drives in Anopheles, is capable of achieving a high level of inheritance bias. Although it uses the endogenous nanos regulatory elements, which have less leaky somatic expression compared to using vasa (Gantz et al., 2015; Hammond et al., 2016; Hammond et al., 2017) or zpg promoters(Hammond et al., 2021; Kyrou et al., 2018), to drive Cas9 expression and thereby reduces somatic expression-induced female sterility, the incomplete rescue of nanos function still leads to reduced female fertility in drive homozygotes.
- State what audience might be interested in and influenced by the reported findings.
It's worth noting the broad audience that will find this work relevant. Gene drive developers and molecular geneticists will be impressed by the good drive performance and directly influenced by the design principles showcased here. The study's integral gene drive architecture that leverages the endogenous nanos regulatory elements, in-frame E2A peptide linkage for co-expression, and intronic insertion of gRNA and selectable markers addresses long-standing challenges in promoter leakage, somatic fitness costs, and resistance allele evolution. What's more, vector biologists and malaria researchers will be interested in the successful deployment of a gene drive in A. gambiae that actually carries a disease-blocking trait.
- Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.
We have worked on CRISPR gene drive development in both fruit flies and Anopheles mosquitoes and have experience with modeling their spread.
References
Ellis, D.A., Avraam, G., Hoermann, A., Wyer, C.A.S., Ong, Y.X., Christophides, G.K., and Windbichler, N. (2022). Testing non-autonomous antimalarial gene drive effectors using self-eliminating drivers in the African mosquito vector Anopheles gambiae. PLOS Genetics 18, e1010244-e1010244.
Gantz, V.M., Jasinskiene, N., Tatarenkova, O., Fazekas, A., Macias, V.M., Bier, E., and James, A.A. (2015). Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. Proc Natl Acad Sci U S A 112, E6736-E6743.
Gonzalez, E., Anderson, M.A.E., Ang, J.X.D., Nevard, K., Shackleford, L., Larrosa-Godall, M., Leftwich, P.T., and Alphey, L. (2025). Optimization of SgRNA expression with RNA pol III regulatory elements in Anopheles stephensi. Scientific Reports 15, 13408.
Hammond, A., Galizi, R., Kyrou, K., Simoni, A., Siniscalchi, C., Katsanos, D., Gribble, M., Baker, D., Marois, E., Russell, S., et al. (2016). A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat Biotechnol 34, 78-83.
Hammond, A., Karlsson, X., Morianou, I., Kyrou, K., Beaghton, A., Gribble, M., Kranjc, N., Galizi, R., Burt, A., Crisanti, A., et al. (2021). Regulating the expression of gene drives is key to increasing their invasive potential and the mitigation of resistance. PLOS Genetics 17, e1009321-e1009321.
Hammond, A.M., Kyrou, K., Bruttini, M., North, A., Galizi, R., Karlsson, X., Kranjc, N., Carpi, F.M., D'Aurizio, R., Crisanti, A., et al. (2017). The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito. PLOS Genetics 13, e1007039-e1007039.
Kyrou, K., Hammond, A.M., Galizi, R., Kranjc, N., Burt, A., Beaghton, A.K., Nolan, T., and Crisanti, A. (2018). A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes. Nature Biotechnology 36, 1062-1066.
Nash, A., Capriotti, P., Hoermann, A., Papathanos, P.A., and Windbichler, N. (2022). Intronic gRNAs for the construction of minimal gene drive systems. Frontiers in Bioengineering and Biotechnology 0, 570-570. Weitzel, A.J., Grunwald, H.A., Ceri, W., Levina, R., Gantz, V.M., Hedrick, S.M., Bier, E., and Cooper, K.L. (2021). Meiotic Cas9 expression mediates gene conversion in the male and female mouse germline. Plos Biol 19, e3001478-e3001478.
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