A small-molecule approach to restore female sterility phenotype targeted by a homing suppression gene drive in the fruit pest Drosophila suzukii.

CRISPR-based gene drives offer promising prospects for controlling disease-transmitting vectors and agricultural pests. A significant challenge for successful suppression-type drive is the rapid evolution of resistance alleles. One approach to mitigate the development of resistance involves targeting functionally constrained regions using multiple gRNAs. In this study, we constructed a 3-gRNA homing gene drive system targeting the recessive female fertility gene Tyrosine decarboxylase 2 (Tdc2) in Drosophila suzukii, a notorious fruit pest. Our investigation revealed only a low level of homing in the germline, but feeding octopamine restored the egg-laying defects in Tdc2 mutant females, allowing easier line maintenance than for other suppression drive targets. We tested the effectiveness of a similar system in Drosophila melanogaster and constructed additional split drive systems by introducing promoter-Cas9 transgenes to improve homing efficiency. Our findings show that genetic polymorphisms in wild populations may limit the spread of gene drive alleles, and the position effect profoundly influences Cas9 activity. Furthermore, this study highlights the potential of conditionally rescuing the female infertility caused by the gene drive, offering a valuable tool for the industrial-scale production of gene drive transgenic insects.

Harnessing Wolbachia cytoplasmic incompatibility alleles for confined gene drive: A modeling study.

Wolbachia are maternally-inherited bacteria, which can spread rapidly in populations by manipulating reproduction. cifA and cifB are genes found in Wolbachia phage that are responsible for cytoplasmic incompatibility, the most common type of Wolbachia reproductive interference. In this phenomenon, no viable offspring are produced when a male with both cifA and cifB (or just cifB in some systems) mates with a female lacking cifA. Utilizing this feature, we propose new types of toxin-antidote gene drives that can be constructed with only these two genes in an insect genome, instead of the whole Wolbachia bacteria. By using both mathematical and simulation models, we found that a drive containing cifA and cifB together creates a confined drive with a moderate to high introduction threshold. When introduced separately, they act as a self-limiting drive. We observed that the performance of these drives is substantially influenced by various ecological parameters and drive characteristics. Extending our models to continuous space, we found that the drive individual release distribution has a critical impact on drive persistence. Our results suggest that these new types of drives based on Wolbachia transgenes are safe and flexible candidates for genetic modification of populations.

Symbionts and gene drive: two strategies to combat vector-borne disease.

Mosquitoes bring global health problems by transmitting parasites and viruses such as malaria and dengue. Unfortunately, current insecticide-based control strategies are only moderately effective because of high cost and resistance. Thus, scalable, sustainable, and cost-effective strategies are needed for mosquito-borne disease control. Symbiont-based and genome engineering-based approaches provide new tools that show promise for meeting these criteria, enabling modification or suppression approaches. Symbiotic bacteria like Wolbachia are maternally inherited and manipulate mosquito host reproduction to enhance their vertical transmission. Genome engineering-based gene drive methods, in which mosquitoes are genetically altered to spread drive alleles throughout wild populations, are also proving to be a potentially powerful approach in the laboratory. Here, we review the latest developments in both symbionts and gene drive-based methods. We describe some notable similarities, as well as distinctions and obstacles, relating to these promising technologies.

Performance characteristics allow for confinement of a CRISPR toxin-antidote gene drive for population suppression in a reaction-diffusion model.

Gene drive alleles that can bias their own inheritance could engineer populations for control of disease vectors, invasive species and agricultural pests. There are successful examples of suppression drives and confined modification drives, but developing confined suppression drives has proven more difficult. However, CRISPR-based toxin-antidote dominant embryo (TADE) suppression drive may fill this niche. It works by targeting and disrupting a haplolethal target gene in the germline with its gRNAs while rescuing this target. It also disrupts a female fertility gene by driving insertion or additional gRNAs. Here, we used a reaction-diffusion model to assess drive performance in continuous space, where outcomes can be substantially different from those in panmictic populations. We measured drive wave speed and found that moderate fitness costs or target gene disruption in the early embryo from maternally deposited nuclease can eliminate the drive's ability to form a wave of advance. We assessed the required release size, and finally we investigated migration corridor scenarios. It is often possible for the drive to suppress one population and then persist in the corridor without invading the second population, a potentially desirable outcome. Thus, even imperfect variants of TADE suppression drive may be excellent candidates for confined population suppression.

Battles between ants (Hymenoptera: Formicidae): a review.

With their unique colony structure, competition between ants (Hymenoptera: Formicidae) can be particularly intense, with colonies potentially willing to sacrifice large number of individuals to obtain resources or territory under the right circumstances. In this review, we cover circumstances in which ant competition escalates into combat, battle strategies and tactics, and analysis methods for these battles. The trends for when colonies choose to fight can vary greatly dependent on the species and situation, which we review in detail. Because of their large group sizes, ant conflicts can follow different patterns than many other species, with a variety of specialist adaptations and battle strategies, such as specialized worker classes and the need to rapidly recruit large number of compatriots. These same large group sizes also can make ant fighting amenable to mathematical analysis, particularly in the context of Lanchester's laws that consider how total numbers influence the outcome of a confrontation. Yet, dynamic behavior can often disrupt idealized mathematical predictions in real-world scenarios, even though these can still shed light on the explanations for such behavior. We also systematically cover the literature on battles between groups of ants, presenting several other interesting studies on species with unique colony organization, such as army ants and leafcutter ants.

Adversarial interspecies relationships facilitate population suppression by gene drive in spatially explicit models.

Suppression gene drives bias their inheritance to spread through a population, potentially eliminating it when they reach high frequency. CRISPR homing suppression drives have already seen success in the laboratory, but several models predict that success may be elusive in population with realistic spatial structure due to extinction-recolonization cycles. Here, we extend our continuous space framework to include two competing species or predator-prey pairs. We find that in both general and mosquito-specific models, competing species or predators can facilitate drive-based suppression, albeit at the cost of an increased rate of drive loss outcomes. These results are robust in mosquito models with seasonal fluctuations. Our study illustrates the difficulty of predicting outcomes in complex ecosystems. However, our results are promising for the prospects of less powerful suppression gene drives to successfully eliminate target mosquito and other pest populations.

Making waves: Comparative analysis of gene drive spread characteristics in a continuous space model.

With their ability to rapidly increase in frequency, gene drives can be used to modify or suppress target populations after an initial release of drive individuals. Recent advances have revealed many possibilities for different types of drives, and several of these have been realized in experiments. These drives have advantages and disadvantages related to their ease of construction, confinement and capacity to be used for modification or suppression. Though characteristics of these drives have been explored in modelling studies, assessment in continuous space environments has been limited, often focusing on outcomes rather than fundamental properties. Here, we conduct a comparative analysis of many different gene drive types that have the capacity to form a wave of advance in continuous space using individual-based simulations in continuous space. We evaluate the drive wave speed as a function of drive performance and ecological parameters, which reveals substantial differences between drive performance in panmictic versus spatial environments. In particular, we find that suppression drive waves are uniquely vulnerable to fitness costs and undesired CRISPR cleavage activity in embryos by maternal deposition. Some drives, however, retain robust performance even with widely varying efficiency parameters. To gain a better understanding of drive waves, we compare their panmictic performance and find that the rate of wild-type allele removal is correlated with drive wave speed, though this is also affected by other factors. Overall, our results provide a useful resource for understanding the performance of drives in spatially continuous environments, which may be most representative of potential drive deployment in many relevant scenarios.

A CRISPR homing gene drive targeting a haplolethal gene removes resistance alleles and successfully spreads through a cage population.

Engineered gene drives are being explored as a new strategy in the fight against vector-borne diseases due to their potential for rapidly spreading genetic modifications through a population. However, CRISPR-based homing gene drives proposed for this purpose have faced a major obstacle in the formation of resistance alleles that prevent Cas9 cleavage. Here, we present a homing drive in Drosophila melanogaster that reduces the prevalence of resistance alleles below detectable levels by targeting a haplolethal gene with two guide RNAs (gRNAs) while also providing a rescue allele. Resistance alleles that form by end-joining repair typically disrupt the haplolethal target gene and are thus removed from the population because individuals that carry them are nonviable. We demonstrate that our drive is highly efficient, with 91% of the progeny of drive heterozygotes inheriting the drive allele and with no functional resistance alleles observed in the remainder. In a large cage experiment, the drive allele successfully spread to all individuals within a few generations. These results show that a haplolethal homing drive can provide an effective tool for targeted genetic modification of entire populations.

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.

Modelling homing suppression gene drive in haplodiploid organisms.

Gene drives have shown great promise for suppression of pest populations. These engineered alleles can function by a variety of mechanisms, but the most common is the CRISPR homing drive, which converts wild-type alleles to drive alleles in the germline of heterozygotes. Some potential target species are haplodiploid, in which males develop from unfertilized eggs and thus have only one copy of each chromosome. This prevents drive conversion, a substantial disadvantage compared to diploids where drive conversion can take place in both sexes. Here, we study homing suppression gene drives in haplodiploids and find that a drive targeting a female fertility gene could still be successful. However, such drives are less powerful than in diploids and suffer more from functional resistance alleles. They are substantially more vulnerable to high resistance allele formation in the embryo owing to maternally deposited Cas9 and guide RNA and also to somatic cleavage activity. Examining spatial models where organisms move over a continuous landscape, we find that haplodiploid suppression drives surprisingly perform nearly as well as in diploids, possibly owing to their ability to spread further before inducing strong suppression. Together, these results indicate that gene drive can potentially be used to effectively suppress haplodiploid populations.

Modeling CRISPR gene drives for suppression of invasive rodents using a supervised machine learning framework.

Invasive rodent populations pose a threat to biodiversity across the globe. When confronted with these invaders, native species that evolved independently are often defenseless. CRISPR gene drive systems could provide a solution to this problem by spreading transgenes among invaders that induce population collapse, and could be deployed even where traditional control methods are impractical or prohibitively expensive. Here, we develop a high-fidelity model of an island population of invasive rodents that includes three types of suppression gene drive systems. The individual-based model is spatially explicit, allows for overlapping generations and a fluctuating population size, and includes variables for drive fitness, efficiency, resistance allele formation rate, as well as a variety of ecological parameters. The computational burden of evaluating a model with such a high number of parameters presents a substantial barrier to a comprehensive understanding of its outcome space. We therefore accompany our population model with a meta-model that utilizes supervised machine learning to approximate the outcome space of the underlying model with a high degree of accuracy. This enables us to conduct an exhaustive inquiry of the population model, including variance-based sensitivity analyses using tens of millions of evaluations. Our results suggest that sufficiently capable gene drive systems have the potential to eliminate island populations of rodents under a wide range of demographic assumptions, though only if resistance can be kept to a minimal level. This study highlights the power of supervised machine learning to identify the key parameters and processes that determine the population dynamics of a complex evolutionary system.

Cheating evolution: engineering gene drives to manipulate the fate of wild populations.

Engineered gene drives - the process of stimulating the biased inheritance of specific genes - have the potential to enable the spread of desirable genes throughout wild populations or to suppress harmful species, and may be particularly useful for the control of vector-borne diseases such as malaria. Although several types of selfish genetic elements exist in nature, few have been successfully engineered in the laboratory thus far. With the discovery of RNA-guided CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) nucleases, which can be utilized to create, streamline and improve synthetic gene drives, this is rapidly changing. Here, we discuss the different types of engineered gene drives and their potential applications, as well as current policies regarding the safety and regulation of gene drives for the manipulation of wild populations.

Suppression gene drive in continuous space can result in unstable persistence of both drive and wild-type alleles.

Rapid evolutionary processes can produce drastically different outcomes when studied in panmictic population models vs. spatial models. One such process is gene drive, which describes the spread of "selfish" genetic elements through a population. Engineered gene drives are being considered for the suppression of disease vectors or invasive species. While laboratory experiments and modelling in panmictic populations have shown that such drives can rapidly eliminate a population, it remains unclear if these results translate to natural environments where individuals inhabit a continuous landscape. Using spatially explicit simulations, we show that the release of a suppression drive can result in what we term "chasing" dynamics, in which wild-type individuals recolonize areas where the drive has locally eliminated the population. Despite the drive subsequently reconquering these areas, complete population suppression often fails to occur or is substantially delayed. This increases the likelihood that the drive is lost or that resistance evolves. We analyse how chasing dynamics are influenced by the type of drive, its efficiency, fitness costs, and ecological factors such as the maximal growth rate of the population and levels of dispersal and inbreeding. We find that chasing is more common for lower efficiency drives when dispersal is low and that some drive mechanisms are substantially more prone to chasing behaviour than others. Our results demonstrate that the population dynamics of suppression gene drives are determined by a complex interplay of genetic and ecological factors, highlighting the need for realistic spatial modelling to predict the outcome of drive releases in natural populations.

Reducing resistance allele formation in CRISPR gene drive.

CRISPR homing gene drives can convert heterozygous cells with one copy of the drive allele into homozygotes, thereby enabling super-Mendelian inheritance. Such a mechanism could be used, for example, to rapidly disseminate a genetic payload in a population, promising effective strategies for the control of vector-borne diseases. However, all CRISPR homing gene drives studied in insects thus far have produced significant quantities of resistance alleles that would limit their spread. In this study, we provide an experimental demonstration that multiplexing of guide RNAs can both significantly increase the drive conversion efficiency and reduce germline resistance rates of a CRISPR homing gene drive in Drosophila melanogaster We further show that an autosomal drive can achieve drive conversion in the male germline, with no subsequent formation of resistance alleles in embryos through paternal carryover of Cas9. Finally, we find that the nanos promoter significantly lowers somatic Cas9 expression compared with the vasa promoter, suggesting that nanos provides a superior choice in drive strategies where gene disruption in somatic cells could have fitness costs. Comparison of drive parameters among the different constructs developed in this study and a previous study suggests that, while drive conversion and germline resistance rates are similar between different genomic targets, embryo resistance rates can vary significantly. Taken together, our results mark an important step toward developing effective gene drives capable of functioning in natural populations and provide several possible avenues for further control of resistance rates.

Performance analysis of novel toxin-antidote CRISPR gene drive systems.

BACKGROUND: CRISPR gene drive systems allow the rapid spread of a genetic construct throughout a population. Such systems promise novel strategies for the management of vector-borne diseases and invasive species by suppressing a target population or modifying it with a desired trait. However, current homing-type drives have two potential shortcomings. First, they can be thwarted by the rapid evolution of resistance. Second, they lack any mechanism for confinement to a specific target population. In this study, we conduct a comprehensive performance assessment of several new types of CRISPR-based gene drive systems employing toxin-antidote (TA) principles, which should be less prone to resistance and allow for the confinement of drives to a target population due to invasion frequency thresholds. RESULTS: The underlying principle of the proposed CRISPR toxin-antidote gene drives is to disrupt an essential target gene while also providing rescue by a recoded version of the target as part of the drive allele. Thus, drive alleles tend to remain viable, while wild-type targets are disrupted and often rendered nonviable, thereby increasing the relative frequency of the drive allele. Using individual-based simulations, we show that Toxin-Antidote Recessive Embryo (TARE) drives targeting an haplosufficient but essential gene (lethal when both copies are disrupted) can enable the design of robust, regionally confined population modification strategies with high flexibility in choosing promoters and targets. Toxin-Antidote Dominant Embryo (TADE) drives require a haplolethal target gene and a germline-restricted promoter, but they could permit faster regional population modification and even regionally confined population suppression. Toxin-Antidote Dominant Sperm (TADS) drives can be used for population modification or suppression. These drives are expected to spread rapidly and could employ a variety of promoters, but unlike TARE and TADE, they would not be regionally confined and also require highly specific target genes. CONCLUSIONS: Overall, our results suggest that CRISPR-based TA gene drives provide promising candidates for flexible ecological engineering strategies in a variety of organisms.

Novel CRISPR/Cas9 gene drive constructs reveal insights into mechanisms of resistance allele formation and drive efficiency in genetically diverse populations.

A functioning gene drive system could fundamentally change our strategies for the control of vector-borne diseases by facilitating rapid dissemination of transgenes that prevent pathogen transmission or reduce vector capacity. CRISPR/Cas9 gene drive promises such a mechanism, which works by converting cells that are heterozygous for the drive construct into homozygotes, thereby enabling super-Mendelian inheritance. Although CRISPR gene drive activity has already been demonstrated, a key obstacle for current systems is their propensity to generate resistance alleles, which cannot be converted to drive alleles. In this study, we developed two CRISPR gene drive constructs based on the nanos and vasa promoters that allowed us to illuminate the different mechanisms by which resistance alleles are formed in the model organism Drosophila melanogaster. We observed resistance allele formation at high rates both prior to fertilization in the germline and post-fertilization in the embryo due to maternally deposited Cas9. Assessment of drive activity in genetically diverse backgrounds further revealed substantial differences in conversion efficiency and resistance rates. Our results demonstrate that the evolution of resistance will likely impose a severe limitation to the effectiveness of current CRISPR gene drive approaches, especially when applied to diverse natural populations.

The role of the cutaneous microbiome in skin cancer: lessons learned from the gut.

The human microbiome has recently gained prominence as a major factor in health and disease. Here we review the literature regarding the microbiome and cancer and suggest how the microbiome may be manipulated for improved health outcomes. The gut microbiome has been relatively well studied, and the mechanisms of how it may increase or decrease the risk of certain cancers may apply to the skin microbiome. Additionally, the gut microbiome may directly impact the risk of cancer in the skin and other organs by promoting systemic inflammation. The skin microbiome itself is as diverse as the gut microbiome, but research has just begun to unravel its influence on the host. Like the gut microbiome, it affects the risk for several diseases, including cancer. By using healthpromoting strains from the microbiome in oral or topical probiotics, it may be possible to reduce the risk of skin cancer and perhaps even increase the likelihood of successful treatment.

Overriding Mendelian inheritance in Arabidopsis with a CRISPR toxin-antidote gene drive that impairs pollen germination.

Synthetic gene drives, inspired by natural selfish genetic elements and transmitted to progeny at super-Mendelian (>50%) frequencies, present transformative potential for disseminating traits that benefit humans throughout wild populations, even facing potential fitness costs. Here we constructed a gene drive system in plants called CRISPR-Assisted Inheritance utilizing NPG1 (CAIN), which uses a toxin-antidote mechanism in the male germline to override Mendelian inheritance. Specifically, a guide RNA-Cas9 cassette targets the essential No Pollen Germination 1 (NPG1) gene, serving as the toxin to block pollen germination. A recoded, CRISPR-resistant copy of NPG1 serves as the antidote, providing rescue only in pollen cells that carry the drive. To limit potential consequences of inadvertent release, we used self-pollinating Arabidopsis thaliana as a model. The drive demonstrated a robust 88-99% transmission rate over two successive generations, producing minimal resistance alleles that are unlikely to inhibit drive spread. Our study provides a strong basis for rapid genetic modification or suppression of outcrossing plant populations.

Involvement of miR-8510a-3p in response to Cry1Ac protoxin by regulating PxABCG3 in Plutella xylostella.

Overuse of insecticides has accelerated the evolution of insecticide resistance and created serious environmental concerns worldwide, thus incentivizing development of alternative methods. Bacillus thuringiensis (Bt) is an insecticidal bacterium that has been developed as a biopesticide to successfully control multiple species of pests. It operates by secreting several insect toxins such as Cry1Ac. However, metabolic resistance based on ATP-binding cassette (ABC) transporters may play a crucial role in the development of metabolic resistance to Bt. Here, we characterized an ABCG gene from the agricultural pest Plutella xylostella (PxABCG3) and found that it was highly expressed in a Cry1Ac-resistant strain, up-regulated after Cry1Ac protoxin treatment. Binding miR-8510a-3p to the coding sequence (CDS) of PxABCG3 was then confirmed by a luciferase reporter assay and RNA immunoprecipitation. miR-8510a-3p agomir delivery markedly reduced PxABCG3 expression in vivo and consequently decreased the tolerance of P. xylostella to Cry1Ac, while reduction of miR-8510a-3p significantly increased PxABCG3 expression, accompanied by an increased tolerance to Cry1Ac. Our results suggest that miR-8510a-3p could potentially be used as a novel molecular target against P. xylostella or other lepidopterans, providing novel insights into developing effective and environmentally friendly pesticides.

A homing rescue gene drive with multiplexed gRNAs reaches high frequency in cage populations but generates functional resistance.

CRISPR homing gene drives have considerable potential for managing populations of medically and agriculturally significant insects. They operate by Cas9 cleavage followed by homology-directed repair, copying the drive allele to the wild-type chromosome and thus increasing in frequency and spreading throughout a population. However, resistance alleles formed by end-joining repair pose a significant obstacle. To address this, we create a homing drive targeting the essential hairy gene in Drosophila melanogaster. Nonfunctional resistance alleles are recessive lethal, while drive carriers have a recoded "rescue" version of hairy. The drive inheritance rate is moderate, and multigenerational cage studies show drive spread to 96%-97% of the population. However, the drive does not reach 100% due to the formation of functional resistance alleles, despite using four gRNAs. These alleles have a large deletion but likely utilize an alternate start codon. Thus, revised designs targeting more essential regions of a gene may be necessary to avoid such functional resistance. Replacement of the rescue element's native 3' UTR with a homolog from another species increases drive inheritance by 13%-24%. This was possibly because of reduced homology between the rescue element and surrounding genomic DNA, which could also be an important design consideration for rescue gene drives.