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.
CRISPR-based gene drives offer promising means to reduce the burden of pests and vector-borne diseases. These techniques consist of releasing genetically modified organisms carrying CRISPR-Cas nucleases designed to bias their inheritance and rapidly propagate desired modifications. Gene drives can be intended to reduce reproductive capacity of harmful insects or spread anti-pathogen effectors through wild populations, even when these confer fitness disadvantages. Technologies capable of halting the spread of gene drives may prove highly valuable in controlling, counteracting, and even reverting their effect on individual organisms as well as entire populations. Here we show engineering and testing of a genetic approach, based on the germline expression of a phage-derived anti-CRISPR protein (AcrIIA4), able to inactivate CRISPR-based gene drives and restore their inheritance to Mendelian rates in the malaria vector Anopheles gambiae. Modeling predictions and cage testing show that a single release of male mosquitoes carrying the AcrIIA4 protein can block the spread of a highly effective suppressive gene drive preventing population collapse of caged malaria mosquitoes.
(Supplementary Fig. S8) confirming that most of the genes required for the late stages are transcribed before meiosis, as previously observed also in other model organisms and human spermatogenesis 24,55,56 .
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