RESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
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.
Asunto(s)
Anopheles/genética , Tecnología de Genética Dirigida/métodos , Malaria/transmisión , Mosquitos Vectores/genética , Procesos de Determinación del Sexo/genética , Animales , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Femenino , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Malaria/prevención & control , Masculino , Control de Mosquitos , Cromosoma X/genética , Cromosoma X/metabolismoRESUMEN
Gene drive is a natural process of biased inheritance that, in principle, could be used to control pest and vector populations. As with any form of pest control, attention should be paid to the possibility of resistance evolving. For nuclease-based gene drive aimed at suppressing a population, resistance could arise by changes in the target sequence that maintain function, and various strategies have been proposed to reduce the likelihood that such alleles arise. Even if these strategies are successful, it is almost inevitable that alleles will arise at the target site that are resistant to the drive but do not restore function, and the impact of such sequences on the dynamics of control has been little studied. We use population genetic modelling of a strategy targeting a female fertility gene to demonstrate that such alleles may be expected to accumulate, and thereby reduce the reproductive load on the population, if nuclease expression per se causes substantial heterozygote fitness effects or if parental (especially paternal) deposition of nuclease either reduces offspring fitness or affects the genotype of their germline. All these phenomena have been observed in synthetic drive constructs. It will, therefore, be important to allow for non-functional resistance alleles in predicting the dynamics of constructs in cage populations and the impacts of any field release.
Asunto(s)
Tecnología de Genética Dirigida , Genética de Población , Alelos , Animales , Femenino , Células Germinativas , ReproducciónRESUMEN
A first generation of CRISPR-based gene drives has now been tested in the laboratory in a number of organisms, including malaria vector mosquitoes. Challenges for their use in the area-wide genetic control of vector-borne disease have been identified, including the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. We have evaluated theoretically the concept of integral gene drive (IGD) as an alternative paradigm for population replacement. IGDs incorporate a minimal set of molecular components, including drive and anti-pathogen effector elements directly embedded within endogenous genes - an arrangement that in theory allows targeting functionally conserved coding sequences without disrupting their function. Autonomous and non-autonomous IGD strains could be generated, optimized, regulated and imported independently. We performed quantitative modeling comparing IGDs with classical replacement drives and show that selection for the function of the hijacked host gene can significantly reduce the establishment of resistant alleles in the population, while drive occurring at multiple genomic loci prolongs the duration of transmission blockage in the face of pre-existing target site variation. IGD thus has potential as a more durable and flexible population replacement strategy.
RESUMEN
In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. We found that CRISPR-Cas9-targeted disruption of the intron 4-exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR-Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7-11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive.
