Gene-drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years [AU please provide a real estimate, this seems vague]. We describe CRISPR-Cas9 endonuclease constructs that function as gene-drive systems in Anopheles gambiae, the main vector for malaria [AU:OK?]. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene-drive constructs designed to target and edit each gene [AU:OK?]. For each locus targeted we observed strong gene drive at the molecular level, with transmission rates to progeny of 91 to 99.6%. Population modelling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to control suppress mosquito populations to levels that do not support malaria transmission.

Complete population collapse of malaria vector Anopheles gambiae in cages is achieved using a gene drive that targets doublesex.Supplementary informationThe online version of this article (doi:10.1038/nbt.4245) contains supplementary material, which is available to authorized users.

Gene drives have enormous potential for the control of insect populations of medical and agricultural relevance. By preferentially biasing their own inheritance, gene drives can rapidly introduce genetic traits even if these confer a negative fitness effect on the population. We have recently developed gene drives based on CRISPR nuclease constructs that are designed to disrupt key genes essential for female fertility in the malaria mosquito. The construct copies itself and the associated genetic disruption from one homologous chromosome to another during gamete formation, a process called homing that ensures the majority of offspring inherit the drive. Such drives have the potential to cause long-lasting, sustainable population suppression, though they are also expected to impose a large selection pressure for resistance in the mosquito. One of these population suppression gene drives showed rapid invasion of a caged population over 4 generations, establishing proof of principle for this technology. In order to assess the potential for the emergence of resistance to the gene drive in this population we allowed it to run for 25 generations and monitored the frequency of the gene drive over time. Following the initial increase of the gene drive we observed a gradual decrease in its frequency that was accompanied by the spread of small, nuclease-induced mutations at the target gene that are resistant to further cleavage and restore its functionality. Such mutations showed rates of increase consistent with positive selection in the face of the gene drive. Our findings represent the first documented example of selection for resistance to a synthetic gene drive and lead to important design recommendations and considerations in order to mitigate for resistance in future gene drive applications.

ex-chromosome drivers are genetic elements that interfere with chromosome segregation during meiosis and are over-represented in progeny 1. In heterogametic sex, they cause an unbalanced male-to-female ratio among offspring, which can potentially lead to population suppression or extinction. Relatively few sex-chromosome drives have been characterized, most likely because they produce an evolutionary conflict with the rest of the genome that selects for autosomal suppressors or resistant sex chromosomes 2,3. Mathematical modeling predicts that a driving sex distorter will spread in a population and, in the absence of resistance, cause eventual collapse 4,5. Population collapse using natural sex-chromosome drives has been reported in laboratory colonies of Drosophila 6,7. In the field, a population crash of the species Drosophila neotestacea was detected in Washington State due to a natural X-chromosome distorter that produced a female-only population 8. Therefore, sex-distorter drives could conceivably be harnessed for invasive pest or vector control 9,10. Although Y drives are less common than X drives, they have been described in Aedes aegypti and Culex pipiens mosquitoes 11,12. Y drives are particularly attractive for mosquito vector control because they can progressively reduce the number of females and hence disease transmission as they spread. In addition, Y drives are likely to be more effective than X drives because they can increase at a greater rate the fraction of heterogametic driving individuals 3-5. Synthetic sex distorters have been generated in A. gambiae mosquitoes by using site-specific nucleases such as I-PpoI or CRISPR-Cas9, which cleave conserved repeated sequences in the mosquito ribosomal DNA gene cluster located exclusively on the X chromosome 13,14. These nucleases, when expressed during spermatozoa development, selectively cleave the X chromosome, thereby favoring the production of Y-bearing gametes and causing a 95% male bias in the progeny 13,14. However, attempts to convert synthetic sex-ratio distorters into Y-chromosome drives have been unsuccessful so far. In most insect species, including A. gambiae, the sex chromosomes are transcriptionally shut down during gametogenesis, a process known as meiotic sex-chromosome inactivation 15,16 , which prevents the transcription of X-shredding nucleases if they are inserted into the Y chromosome (personal observation, A.C. and R.G.). Recently, a gene drive that targeted the dsx gene reached 100% frequency in 7-11 generations and crashed a caged population of 600 mosquitoes without inducing resistance 17. We hypothesized that it might be possible to circumvent meiotic sex-chromosome inactivation by developing an autosomal male-biased sex distorter and coupling sex-ratio distortion with drive. This could result in a quicker impact on disease transmission and a synergistic effect (robustness) between the sex distorter and gene-drive components. Here we report the design and validation of an SDGD to spread the X-chromosome-shredding I-PpoI endonu...

Genetic control aims to reduce the ability of insect pest populations to cause harm via the release of modified insects. One strategy is to bias the reproductive sex ratio towards males so that a population decreases in size or is eliminated altogether due to a lack of females. We have shown previously that sex ratio distortion can be generated synthetically in the main human malaria vector Anopheles gambiae, by selectively destroying the X-chromosome during spermatogenesis, through the activity of a naturally-occurring endonuclease that targets a repetitive rDNA sequence highly-conserved in a wide range of organisms. Here we describe a CRISPR-Cas9 sex distortion system that targets ribosomal sequences restricted to the member species of the Anopheles gambiae complex. Expression of Cas9 during spermatogenesis resulted in RNA-guided shredding of the X-chromosome during male meiosis and produced extreme male bias among progeny in the absence of any significant reduction in fertility. The flexibility of CRISPR-Cas9 combined with the availability of genomic data for a range of insects renders this strategy broadly applicable for the species-specific control of any pest or vector species with an XY sex-determination system by targeting sequences exclusive to the female sex chromosome.