Experimental demonstration of tethered gene drive systems for confined population modification or suppression.

BACKGROUND: Homing gene drives hold great promise for the genetic control of natural populations. However, current homing systems are capable of spreading uncontrollably between populations connected by even marginal levels of migration. This could represent a substantial sociopolitical barrier to the testing or deployment of such drives and may generally be undesirable when the objective is only local population control, such as suppression of an invasive species outside of its native range. Tethered drive systems, in which a locally confined gene drive provides the CRISPR nuclease needed for a homing drive, could provide a solution to this problem, offering the power of a homing drive and confinement of the supporting drive. RESULTS: Here, we demonstrate the engineering of a tethered drive system in Drosophila, using a regionally confined CRISPR Toxin-Antidote Recessive Embryo (TARE) drive to support modification and suppression homing drives. Each drive was able to bias inheritance in its favor, and the TARE drive was shown to spread only when released above a threshold frequency in experimental cage populations. After the TARE drive had established in the population, it facilitated the spread of a subsequently released split homing modification drive (to all individuals in the cage) and of a homing suppression drive (to its equilibrium frequency). CONCLUSIONS: Our results show that the tethered drive strategy is a viable and easily engineered option for providing confinement of homing drives to target populations.

Gene Drive Dynamics in Natural Populations: The Importance of Density Dependence, Space, and Sex.

The spread of synthetic gene drives is often discussed in the context of panmictic populations connected by gene flow and described with simple deterministic models. Under such assumptions, an entire species could be altered by releasing a single individual carrying an invasive gene drive, such as a standard homing drive. While this remains a theoretical possibility, gene drive spread in natural populations is more complex and merits a more realistic assessment. The fate of any gene drive released in a population would be inextricably linked to the population's ecology. Given the uncertainty often involved in ecological assessment of natural populations, understanding the sensitivity of gene drive spread to important ecological factors is critical. Here we review how different forms of density dependence, spatial heterogeneity, and mating behaviors can impact the spread of self-sustaining gene drives. We highlight specific aspects of gene drive dynamics and the target populations that need further research.

Locally Fixed Alleles: A method to localize gene drive to island populations.

Invasive species pose a major threat to biodiversity on islands. While successes have been achieved using traditional removal methods, such as toxicants aimed at rodents, these approaches have limitations and various off-target effects on island ecosystems. Gene drive technologies designed to eliminate a population provide an alternative approach, but the potential for drive-bearing individuals to escape from the target release area and impact populations elsewhere is a major concern. Here we propose the "Locally Fixed Alleles" approach as a novel means for localizing elimination by a drive to an island population that exhibits significant genetic isolation from neighboring populations. Our approach is based on the assumption that in small island populations of rodents, genetic drift will lead to alleles at multiple genomic loci becoming fixed. In contrast, multiple alleles are likely to be maintained in larger populations on mainlands. Utilizing the high degree of genetic specificity achievable using homing drives, for example based on the CRISPR/Cas9 system, our approach aims at employing one or more locally fixed alleles as the target for a gene drive on a particular island. Using mathematical modeling, we explore the feasibility of this approach and the degree of localization that can be achieved. We show that across a wide range of parameter values, escape of the drive to a neighboring population in which the target allele is not fixed will at most lead to modest transient suppression of the non-target population. While the main focus of this paper is on elimination of a rodent pest from an island, we also discuss the utility of the locally fixed allele approach for the goals of population suppression or population replacement. Our analysis also provides a threshold condition for the ability of a gene drive to invade a partially resistant population.

Tethered homing gene drives: A new design for spatially restricted population replacement and suppression.

Optimism regarding potential epidemiological and conservation applications of modern gene drives is tempered by concern about the possibility of unintended spread of engineered organisms beyond the target population. In response, several novel gene drive approaches have been proposed that can, under certain conditions, locally alter characteristics of a population. One challenge for these gene drives is the difficulty of achieving high levels of localized population suppression without very large releases in the face of gene flow. We present a new gene drive system, tethered homing (TH), with improved capacity for both localization and population suppression. The TH drive is based on driving a payload gene using a homing construct that is anchored to a spatially restricted gene drive. We use a proof-of-concept mathematical model to show the dynamics of a TH drive that uses engineered underdominance as an anchor. This system is composed of a split homing drive and a two-locus engineered underdominance drive linked to one part of the split drive (the Cas endonuclease). We use simple population genetic simulations to show that the tethered homing technique can offer improved localized spread of costly transgenic payload genes. Additionally, the TH system offers the ability to gradually adjust the genetic load in a population after the initial alteration, with minimal additional release effort. We discuss potential solutions for improving localization and the feasibility of creating TH drive systems. Further research with models that include additional biological details will be needed to better understand how TH drives would behave in natural populations, but the preliminary results shown here suggest that tethered homing drives can be a useful addition to the repertoire of localized gene drives.

Invasion and migration of spatially self-limiting gene drives: A comparative analysis.

Recent advances in research on gene drives have produced genetic constructs that could theoretically spread a desired gene (payload) into all populations of a species, with a single release in one place. This attribute has advantages, but also comes with risks and ethical concerns. There has been a call for research on gene drive systems that are spatially and/or temporally self-limiting. Here, we use a population genetics model to compare the expected characteristics of three spatially self-limiting gene drive systems: one-locus underdominance, two-locus underdominance and daisy-chain drives. We find large differences between these gene drives in the minimum release size required for successfully driving a payload into a population. The daisy-chain system is the most efficient, requiring the smallest release, followed by the two-locus underdominance system, and then the one-locus underdominance system. However, when the target population exchanges migrants with a nontarget population, the gene drives requiring smaller releases suffer from higher risks of unintended spread. For payloads that incur relatively low fitness costs (up to 30%), a simple daisy-chain drive is practically incapable of remaining localized, even with migration rates as low as 0.5% per generation. The two-locus underdominance system can achieve localized spread under a broader range of migration rates and of payload fitness costs, while the one-locus underdominance system largely remains localized. We also find differences in the extent of population alteration and in the permanence of the alteration achieved by the three gene drives. The two-locus underdominance system does not always spread the payload to fixation, even after successful drive, while the daisy-chain system can, for a small set of parameter values, achieve a temporally limited spread of the payload. These differences could affect the suitability of each gene drive for specific applications.

Direct detection of male quality can facilitate the evolution of female choosiness and indicators of good genes: Evolution across a continuum of indicator mechanisms.

The evolution of mating displays as indicators of male quality has been the subject of extensive theoretical and empirical research for over four decades. Research has also addressed the evolution of female mate choice favoring such indicators. Yet, much debate still exists about whether displays can evolve through the indirect benefits of female mate choice. Here, we use a population genetic model to investigate how the extent to which females can directly detect male quality influences the evolution of female choosiness and male displays. We use a continuum framework that incorporates indicator mechanisms that are traditionally modeled separately. Counter to intuition, we find that intermediate levels of direct detection of male quality can facilitate, rather than impede, the evolution of female choosiness and male displays in broad regions of this continuum. We examine how this evolution is driven by selective forces on genetic quality and on the display, and find that direct detection of male quality results in stronger indirect selection favoring female choosiness. Our results imply that displays maybe more likely to evolve when female choosiness has already evolved to discriminate perceptible forms of male quality. They also highlight the importance of considering general female choosiness, as well as preference, in studies of "good genes."

Not just a theory--the utility of mathematical models in evolutionary biology.

Progress in science often begins with verbal hypotheses meant to explain why certain biological phenomena exist. An important purpose of mathematical models in evolutionary research, as in many other fields, is to act as "proof-of-concept" tests of the logic in verbal explanations, paralleling the way in which empirical data are used to test hypotheses. Because not all subfields of biology use mathematics for this purpose, misunderstandings of the function of proof-of-concept modeling are common. In the hope of facilitating communication, we discuss the role of proof-of-concept modeling in evolutionary biology.

Sperm competition and the evolution of seminal fluid composition.

Male ejaculates include large amounts of seminal fluid proteins (Sfps) that influence male sperm competitive success. In spite of their diverse proximate functions, Sfps involved in sperm competition increase male fitness in one of three ways: (1) "avoidance" proteins help males avoid sperm competition, (2) "defense" proteins help males defend their sperm from displacement by the female's subsequent mate, and (3) "offense" proteins aid males in displacing sperm of preceding males. Here, we present a population genetic model of the evolution of allocation of finite resources by males to the three kinds of Sfps. We analyze the influence of relative efficiencies of different Sfps, of plasticity in resource allocation, and of differences in viability costs of Sfps. We find that in absence of plasticity or different viability costs, equal investment in defense and offense Sfps evolves, irrespective of their relative efficiency. In all cases, males evolve to invest more in avoidance when avoidance proteins are increasingly efficient, and when offense is more efficient than defense. Differences in viability costs result in lower investment in costly proteins, whereas plasticity has complex effects, influencing both the optimal seminal fluid composition and maintenance of variation in investment in these proteins across populations.

Age-dependent male mating investment in Drosophila pseudoobscura.

Male mating investment can strongly influence fitness gained from a mating. Yet, male mating investment often changes with age. Life history theory predicts that mating investment should increase with age, and males should become less discriminatory about their mate as they age. Understanding age-dependent changes in male behavior and their effects on fitness is important for understanding how selection acts in age-structured populations. Although the independent effects of male or female age have been studied in many species, how these interact to influence male mating investment and fitness is less well understood. We mated Drosophila pseudoobscura males of five different age classes (4-, 8-, 11-, 15-, 19-day old) to either young (4-day) or old (11-day) females, and measured copulation duration and early post-mating fecundity. Along with their independent effects, we found a strong interaction between the effects of male and female ages on male mating investment and fitness from individual matings. Male mating investment increased with male age, but this increase was more prominent in matings with young females. Male D. pseudoobscura made smaller investments when mating with old females. The level of such discrimination based on female age, however, also changed with male age. Intermediate aged males were most discriminatory, while the youngest and the oldest males did not discriminate between females of different ages. We also found that larger male mating investments resulted in higher fitness payoffs. Our results show that male and female ages interact to form a complex pattern of age-specific male mating investment and fitness.