Cas9/gRNA-mediated gene-drive systems have advanced development of genetic technologies for controlling vector-borne pathogen transmission. These technologies include population suppression approaches, genetic analogs of insecticidal techniques that reduce the number of insect vectors, and population modification (replacement/alteration) approaches, which interfere with competence to transmit pathogens. Here, we develop a recoded gene-drive rescue system for population modification of the malaria vector, Anopheles stephensi, that relieves the load in females caused by integration of the drive into the kynurenine hydroxylase gene by rescuing its function. Non-functional resistant alleles are eliminated via a dominantly-acting maternal effect combined with slower-acting standard negative selection, and rare functional resistant alleles do not prevent drive invasion. Small cage trials show that single releases of gene-drive males robustly result in efficient population modification with ≥95% of mosquitoes carrying the drive within 5-11 generations over a range of initial release ratios.

/pdf/efficient-population-modification-gene-drive-rescue-system-29qp8toif2.pdf

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

Gene drives based on CRISPR-Cas9 technology are increasingly being considered as tools for reducing the capacity of mosquito populations to transmit malaria, and one of the most promising options is driving endonuclease genes that reduce the fertility of female mosquitoes. In particular, there is much interest in constructs that target the conserved mosquito doublesex (dsx) gene such that the emergence of functional drive-resistant alleles is unlikely. Proof of principle that these constructs can lead to substantial population suppression has been obtained in population cages, and they are being evaluated for use in sub-Saharan Africa. Here, we use simulation modelling to understand the factors affecting the spread of this type of gene drive over a one million-square kilometre area of West Africa containing substantial environmental and social heterogeneity. We found that a driving endonuclease gene targeting female fertility could lead to substantial reductions in malaria vector populations on a regional scale. The exact level of suppression is influenced by additional fitness costs of the transgene such as the somatic expression of Cas9, and its deposition in sperm or eggs leading to damage to the zygote. In the absence of these costs, or of emergent drive-resistant alleles that restore female fertility, population suppression across the study area is predicted to stabilise at ~ 95% 4 years after releases commence. Small additional fitness costs do not greatly affect levels of suppression, though if the fertility of females whose offspring transmit the construct drops by more than ~ 40%, then population suppression is much less efficient. We show the suppression potential of a drive allele with high fitness costs can be enhanced by engineering it also to express male bias in the progeny of transgenic males. Irrespective of the strength of the drive allele, the spatial model predicts somewhat less suppression than equivalent non-spatial models, in particular in highly seasonal regions where dry season stochasticity reduces drive efficiency. We explored the robustness of these results to uncertainties in mosquito ecology, in particular their method of surviving the dry season and their dispersal rates. The modelling presented here indicates that considerable suppression of vector populations can be achieved within a few years of using a female sterility gene drive, though the impact is likely to be heterogeneous in space and time.

/pdf/modelling-the-suppression-of-a-malaria-vector-using-a-crispr-1w0d1abstk.pdf

Modelling the suppression of a malaria vector using a CRISPR-Cas9 gene drive to reduce female fertility

Author(s): Long, Kanya C; Alphey, Luke; Annas, George J; Bloss, Cinnamon S; Campbell, Karl J; Champer, Jackson; Chen, Chun-Hong; Choudhary, Amit; Church, George M; Collins, James P; Cooper, Kimberly L; Delborne, Jason A; Edwards, Owain R; Emerson, Claudia I; Esvelt, Kevin; Evans, Sam Weiss; Friedman, Robert M; Gantz, Valentino M; Gould, Fred; Hartley, Sarah; Heitman, Elizabeth; Hemingway, Janet; Kanuka, Hirotaka; Kuzma, Jennifer; Lavery, James V; Lee, Yoosook; Lorenzen, Marce; Lunshof, Jeantine E; Marshall, John M; Messer, Philipp W; Montell, Craig; Oye, Kenneth A; Palmer, Megan J; Papathanos, Philippos Aris; Paradkar, Prasad N; Piaggio, Antoinette J; Rasgon, Jason L; Rasic, Gordana; Rudenko, Larisa; Saah, J Royden; Scott, Maxwell J; Sutton, Jolene T; Vorsino, Adam E; Akbari, Omar S

/pdf/core-commitments-for-field-trials-of-gene-drive-organisms-1pzgtynyn0.pdf

Core commitments for field trials of gene drive organisms.

Mosquito-borne diseases, such as dengue and malaria, pose significant global health burdens. Unfortunately, current control methods based on insecticides and environmental maintenance have fallen short of eliminating the disease burden. Scalable, deployable, genetic-based solutions are sought to reduce the transmission risk of these diseases. Pathogen-blocking Wolbachia bacteria, or genome engineering-based mosquito control strategies including gene drives have been developed to address these problems, both requiring the release of modified mosquitoes into the environment. Here, we review the latest developments, notable similarities, and critical distinctions between these promising technologies and discuss their future applications for mosquito-borne disease control.

/pdf/combating-mosquito-borne-diseases-using-genetic-control-gueh3emai8.pdf

Combating mosquito-borne diseases using genetic control technologies.

A dominant male-determining locus (M-locus) establishes the male sex (M/m) in the yellow fever mosquito, Aedes aegypti. Nix, a gene in the M-locus, was shown to be a male-determining factor (M factor) as somatic knockout of Nix led to feminized males (M/m) while transient expression of Nix resulted in partially masculinized females (m/m), with male reproductive organs but retained female antennae. It was not clear whether any of the other 29 genes in the 1.3-Mb M-locus are also needed for complete sex-conversion. Here, we report the generation of multiple transgenic lines that express Nix under the control of its own promoter. Genetic and molecular analyses of these lines provided insights unattainable from previous transient experiments. We show that the Nix transgene alone, in the absence of the M-locus, was sufficient to convert females into males with all male-specific sexually dimorphic features and male-like gene expression. The converted m/m males are flightless, unable to perform the nuptial flight required for mating. However, they were able to father sex-converted progeny when presented with cold-anesthetized wild-type females. We show that myo-sex, a myosin heavy-chain gene also in the M-locus, was required for male flight as knockout of myo-sex rendered wild-type males flightless. We also show that Nix-mediated female-to-male conversion was 100% penetrant and stable over many generations. Therefore, Nix has great potential for developing mosquito control strategies to reduce vector populations by female-to-male sex conversion, or to aid in a sterile insect technique that requires releasing only non-biting males.

https://www.pnas.org/content/pnas/117/30/17702.full.pdf

Nix alone is sufficient to convert female Aedes aegypti into fertile males and myo-sex is needed for male flight.

Only female insects transmit diseases such as malaria, dengue and Zika; therefore, control methods that bias the sex ratio of insect offspring have long been sought. Genetic elements such as sex-chromosome drives can distort sex ratios to produce unisex populations that eventually collapse, but the underlying molecular mechanisms are unknown. We report a male-biased sex-distorter gene drive (SDGD) in the human malaria vector Anopheles gambiae. We induced super-Mendelian inheritance of the X-chromosome-shredding I-PpoI nuclease by coupling this to a CRISPR-based gene drive inserted into a conserved sequence of the doublesex (dsx) gene. In modeling of invasion dynamics, SDGD was predicted to have a quicker impact on female mosquito populations than previously developed gene drives targeting female fertility. The SDGD at the dsx locus led to a male-only population from a 2.5% starting allelic frequency in 10–14 generations, with population collapse and no selection for resistance. Our results support the use of SDGD for malaria vector control.

/pdf/a-male-biased-sex-distorter-gene-drive-for-the-human-malaria-2cx84bddfi.pdf

A male-biased sex-distorter gene drive for the human malaria vector Anopheles gambiae

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

/pdf/author-correction-a-male-biased-sex-distorter-gene-drive-for-1mizvkhcu1.pdf

Author Correction: A male-biased sex-distorter gene drive for the human malaria vector Anopheles gambiae

In vertebrates, enzymes responsible for DNA methylation, one of the epigenetic mechanisms, are encoded by genes falling into the cytosine methyltransferases genes family (Dnmt1, Dnmt3a,b and Dnmt3L). However, in Diptera, only the methyltransferase Dnmt2 was found, suggesting that DNA methylation might act differently for species in this order. Moreover, genes involved in epigenetic dynamics, such as Ten-eleven Translocation dioxygenases (TET) and Methyl-CpG-binding domain (MBDs), present in vertebrates, might play a role in insects. This work aimed at investigating nucleic acids methylation in the malaria vector Anopheles gambiae (Diptera: Culicidae) by analysing the expression of Dnmt2, TET2 and MBDs genes using quantitative real-time polymerase chain reaction (qRT-PCR) at pre-immature stages and in reproductive tissues of adult mosquitoes. In addition, the effect of two DNA methylation inhibitors on larval survival was evaluated. The qPCR results showed an overall low expression of Dnmt2 at all developmental stages and in adult reproductive tissues. In contrast, MBD and TET2 showed an overall higher expression. In adult mosquito reproductive tissues, the expression level of the three genes in males' testes was significantly higher than that in females' ovaries. The chemical treatments did not affect larval survival. The findings suggest that mechanisms other than DNA methylation underlie epigenetic regulation in An. gambiae.

Dario Meacci

Papers

A male-biased sex-distorter gene drive for the human malaria vector Anopheles gambiae

Author Correction: A male-biased sex-distorter gene drive for the human malaria vector Anopheles gambiae

Relative expression of key genes involved in nucleic acids methylation in Anopheles gambiae sensu stricto.