A genetically encoded anti-CRISPR protein constrains gene drive spread and prevents population suppression.

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.

Regulating the expression of gene drives is key to increasing their invasive potential and the mitigation of resistance.

Homing-based gene drives use a germline source of nuclease to copy themselves at specific target sites in a genome and bias their inheritance. Such gene drives can be designed to spread and deliberately suppress populations of malaria mosquitoes by impairing female fertility. However, strong unintended fitness costs of the drive and a propensity to generate resistant mutations can limit a gene drive's potential to spread. Alternative germline regulatory sequences in the drive element confer improved fecundity of carrier individuals and reduced propensity for target site resistance. This is explained by reduced rates of end-joining repair of DNA breaks from parentally deposited nuclease in the embryo, which can produce heritable mutations that reduce gene drive penetrance. We tracked the generation and selection of resistant mutations over the course of a gene drive invasion of a population. Improved gene drives show faster invasion dynamics, increased suppressive effect and later onset of target site resistance. Our results show that regulation of nuclease expression is as important as the choice of target site when developing a robust homing-based gene drive for population suppression.

Author Correction: 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.

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

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.

Gene drive for population genetic control: non-functional resistance and parental effects.

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.

Integral gene drives for population replacement.

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.

A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes.

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.

Vector control with driving Y chromosomes: modelling the evolution of resistance.

BACKGROUND: The introduction of new malaria control interventions has often led to the evolution of resistance, both of the parasite to new drugs and of the mosquito vector to new insecticides, compromising the efficacy of the interventions. Recent progress in molecular and population biology raises the possibility of new genetic-based interventions, and the potential for resistance to evolve against these should be considered. Here, population modelling is used to determine the main factors affecting the likelihood that resistance will evolve against a synthetic, nuclease-based driving Y chromosome that produces a male-biased sex ratio. METHODS: A combination of deterministic differential equation models and stochastic analyses involving branching processes and Gillespie simulations is utilized to assess the probability that resistance evolves against a driving Y that otherwise is strong enough to eliminate the target population. The model considers resistance due to changes at the target site such that they are no longer cleaved by the nuclease, and due to trans-acting autosomal suppressor alleles. RESULTS: The probability that resistance evolves increases with the mutation rate and the intrinsic rate of increase of the population, and decreases with the strength of drive and any pleiotropic fitness costs of the resistant allele. In seasonally varying environments, the time of release can also affect the probability of resistance evolving. Trans-acting suppressor alleles are more likely to suffer stochastic loss at low frequencies than target site resistant alleles. CONCLUSIONS: As with any other intervention, there is a risk that resistance will evolve to new genetic approaches to vector control, and steps should be taken to minimize this probability. Two design features that should help in this regard are to reduce the rate at which resistant mutations arise, and to target sequences such that if they do arise, they impose a significant fitness cost on the mosquito.

Requirements for Driving Antipathogen Effector Genes into Populations of Disease Vectors by Homing.

There is a need for new interventions against the ongoing burden of vector-borne diseases such as malaria and dengue. One suggestion has been to develop genes encoding effector molecules that block parasite development within the vector, and then use the nuclease-based homing reaction as a form of gene drive to spread those genes through target populations. If the effector gene reduces the fitness of the mosquito and does not contribute to the drive, then loss-of-function mutations in the effector will eventually replace functional copies, but protection may nonetheless persist sufficiently long to provide a public health benefit. Here, we present a quantitative model allowing one to predict the duration of protection as a function of the probabilities of different molecular processes during the homing reaction, various fitness effects, and the efficacy of the effector in blocking transmission. Factors that increase the duration of protection include reducing the frequency of pre-existing resistant alleles, the probability of nonrecombinational DNA repair, the probability of homing-associated loss of the effector, the fitness costs of the nuclease and effector, and the completeness of parasite blocking. For target species that extend over an area much larger than the typical dispersal distance, the duration of protection is expected to be highest at the release site, and decrease away from there, eventually falling to zero, as effector-less drive constructs replace effector-containing ones. We also model an alternative strategy of using the nuclease to target an essential gene, and then linking the effector to a sequence that restores the essential function and is resistant to the nuclease. Depending upon parameter values, this approach can prolong the duration of protection. Our models highlight the key design criteria needed to achieve a desired level of public health benefit.

Gene drive through a landscape: Reaction-diffusion models of population suppression and elimination by a sex ratio distorter.

Some genes or gene complexes are transmitted from parents to offspring at a greater-than-Mendelian rate, and can spread and persist in populations even if they cause some harm to the individuals carrying them. Such genes may be useful for controlling populations or species that are harmful. Driving-Y chromosomes may be particularly potent in this regard, as they produce a male-biased sex ratio that, if sufficiently extreme, can lead to population elimination. To better understand the potential of such genes to spread over a landscape, we have developed a series of reaction-diffusion models of a driving-Y chromosome in 1-D and radially-symmetric 2-D unbounded domains. The wild-type system at carrying capacity is found to be unstable to the introduction of driving-Y males for all models investigated. Numerical solutions exhibit travelling wave pulses and fronts, and analytical and semi-analytical solutions for the asymptotic wave speed under bounded initial conditions are derived. The driving-Y male invades the wild-type equilibrium state at the front of the wave and completely replaces the wild-type males, leaving behind, at the tail of the wave, a reduced- or zero-population state of females and driving-Y males only. In our simplest model of a population with one life stage and density-dependent mortality, wave speed depends on the strength of drive and the diffusion rate of Y-drive males, and is independent of the population dynamic consequences (suppression or elimination). Incorporating an immobile juvenile stage of fixed duration into the model reduces wave speed approximately in proportion to the relative time spent as a juvenile. If females mate just once in their life, storing sperm for subsequent reproduction, then wave speed depends on the movement of mated females as well as Y-drive males, and may be faster or slower than in the multiple-mating model, depending on the relative duration of juvenile and adult life stages. Numerical solutions are shown for parameter values that may in part be representative for Anopheles gambiae, the primary vector of malaria in sub-Saharan Africa.