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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.

Continuous‐space population models can yield significantly different results from their panmictic counterparts when assessing evolutionary, ecological or population genetic processes. However, the computational burden of spatial models is typically much greater than that of panmictic models due to the overhead of determining which individuals interact with one another and how strongly they interact. While these calculations are necessary to model local competition that regulates the population density, they can lead to prohibitively long runtimes.Here, we present a novel modelling method in which the resources available to a population are abstractly represented as an additional layer of the simulation. Instead of interacting directly with one another, individuals interact indirectly via this resource layer.We find that this method closely matches other spatial models, yet can dramatically increase the speed of the model, allowing the simulation of much larger populations.In addition to improved runtimes, models structured in this manner exhibit other desirable characteristics, including more explicit control over population density near the edge of the simulated area, and an efficient route for modelling complex heterogeneous landscapes. [ABSTRACT FROM AUTHOR]

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Gene drives are alleles that can bias the inheritance of specific traits in target populations for the purpose of modification or suppression. Here, we construct a homing suppression drive in the major urban malaria vector Anopheles stephensi targeting the female-specific exon of doublesex, incorporating two gRNAs and a nanos-Cas9 to reduce functional resistance and improve female heterozygote fitness. Our results show that the drive was recessive sterile in both females and males, with various intersex phenotypes in drive homozygotes. Both male and female drive heterozygotes show only moderate drive conversion, indicating that the nanos promoter has lower activity in A. stephensi than in Anopheles gambiae. By amplicon sequencing, we detect a very low level of resistance allele formation. Combination of the homing suppression drive and a vasa-Cas9 line boosts the drive conversion rate of the homing drive to 100%, suggesting the use of similar systems for population suppression in a continuous release strategy with a lower release rate than SIT or fsRIDL techniques. This study contributes valuable insights to the development of more efficient and environmentally friendly pest control tools aimed at disrupting disease transmission. Gene drive alleles bias their inheritance to spread through populations. Here, we constructed a moderate efficiency 2-gRNA nanos-Cas9 homing suppression drive in the malaria mosquito Anopheles stephensi targeting doublesex. With vasa-Cas9, high efficiency and self-limiting suppression was achieved. [ABSTRACT FROM AUTHOR]

Mosquito-borne diseases, such as dengue and malaria, pose a significant burden to global health. Current control strategies with insecticides are only moderately effective. Scalable solutions are needed to reduce the transmission risk of these diseases. Symbionts and genome engineering–based mosquito control strategies have been proposed to address these problems. Bacterial, fungal, and viral symbionts affect mosquito reproduction, reduce mosquito lifespan, and block pathogen transmission. Field tests of endosymbiont Wolbachia-based methods have yielded promising results, but there are hurdles to overcome due to the large-scale rearing and accurate sex sorting required for Wolbachia-based suppression approaches and the ecological impediments to Wolbachia invasion in replacement approaches. Genome engineering–based methods, in which mosquitoes are genetically altered for the modification or suppression of wild populations, offer an additional approach for control of mosquito-borne diseases. In particular, the use of gene drive alleles that bias inheritance in their favor is a potentially powerful approach. Several drives are frequency dependent, potentially giving them broadly similar population dynamics to Wolbachia. However, public acceptance and the behavior of released drives in natural mosquito populations remain challenges. We summarize the latest developments and discuss the knowledge gaps in both symbiont- and gene drive–based methods. [ABSTRACT FROM AUTHOR]

Recent experiments have produced several Anopheles gambiae homing gene drives that disrupt female fertility genes, thereby eventually inducing population collapse. Such drives may be highly effective tools to combat malaria. One such homing drive, based on the zpg promoter driving CRISPR/Cas9, was able to eliminate a cage population of mosquitoes. A second version, purportedly improved upon the first by incorporating an X-shredder element (which biases inheritance towards male offspring), was similarly successful. Here, we analyze experimental data from each of these gene drives to extract their characteristics and performance parameters and compare these to previous interpretations of their experimental performance. We assess each suppression drive within an individual-based simulation framework that models mosquito population dynamics in continuous space. We find that the combined homing/X-shredder drive is actually less effective at population suppression within the context of our mosquito population model. In particular, the combined drive often fails to completely suppress the population, instead resulting in an unstable equilibrium between drive and wild-type alleles. By contrast, otherwise similar drives based on the nos promoter may prove to be more promising candidates for future development than originally thought.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 provides a highly efficient and flexible genome editing technology with numerous potential applications ranging from gene therapy to population control. Some proposed applications involve the integration of CRISPR/Cas9 endonucleases into an organism’s genome, which raises questions about potentially harmful effects to the transgenic individuals. One example for which this is particularly relevant are CRISPR-based gene drives conceived for the genetic alteration of entire populations. The performance of such drives can strongly depend on fitness costs experienced by drive carriers, yet relatively little is known about the magnitude and causes of these costs. Here, we assess the fitness effects of genomic CRISPR/Cas9 expression in Drosophila melanogaster cage populations by tracking allele frequencies of four different transgenic constructs that allow us to disentangle ‘direct’ fitness costs due to the integration, expression, and target-site activity of Cas9, from fitness costs due to potential off-target cleavage. Using a maximum likelihood framework, we find that a model with no direct fitness costs but moderate costs due to off-target effects fits our cage data best. Consistent with this, we do not observe fitness costs for a construct with Cas9HF1, a high-fidelity version of Cas9. We further demonstrate that using Cas9HF1 instead of standard Cas9 in a homing drive achieves similar drive conversion efficiency. These results suggest that gene drives should be designed with high-fidelity endonucleases and may have implications for other applications that involve genomic integration of CRISPR endonucleases.

Abstract 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 is a genetic engineering technology that can enable super-mendelian inheritance of specific alleles, allowing them to spread through a population. New gene drive types have increased flexibility, offering options for confined modification or suppression of target populations. Among the most promising are CRISPR toxin-antidote gene drives, which disrupt essential wild-type genes by targeting them with Cas9/gRNA. This results in their removal, increasing the frequency of the drive. All these drives rely on having an effective rescue element, which consists of a recoded version of the target gene. This rescue element can be at the same site as the target gene, maximizing the chance of efficient rescue, or at a distant site, which allows useful options such as easily disrupting another essential gene or increasing confinement. Previously, we developed a homing rescue drive targeting a haplolethal gene and a toxin-antidote drive targeting a haplosufficient gene. These successful drives had functional rescue elements but suboptimal drive efficiency. Here, we attempted to construct toxin-antidote drives targeting these genes with a distant-site configuration from three loci in Drosophila melanogaster. We found that additional gRNAs increased cut rates to nearly 100%. However, all distant-site rescue elements failed for both target genes. Furthermore, one rescue element with a minimally recoded sequence was used as a template for homology-directed repair for the target gene on a different chromosomal arm, resulting in the formation of functional resistance alleles. Together, these results can inform the design of future CRISPR-based toxin-antidote gene drives.

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.

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.

Gene drives have great potential for suppression of pest populations and removal of exotic invasive species. CRISPR homing suppression drive is a powerful but unconfined drive, posing risks of uncontrolled spread. Thus, developing methods for confining a gene drive is of great significance. Tethered drive combines a confined system such as Toxin‐Antidote Recessive Embryo drive with a strong drive such as a homing suppression drive. It can prevent the homing drive from spreading beyond the confined drive and can be constructed readily, giving it good prospects for future development. However, we have found that care must be taken when deploying tethered drive systems in some scenarios. Simulations of tethered drive in a panmictic population model reveal that successful deployment requires a proper release ratio between the two components, tailored to prevent the suppression drive from eliminating the confined system before it has the chance to spread. Spatial models where the population moves over a one‐dimensional landscape display a more serious phenomenon of drive wave interference between the two tethered drive components. If the faster suppression drive wave catches up to the confined drive wave, success is still possible, but it is dependent on drive performance and ecological parameters. Two‐dimensional simulations further restrict the parameter range for drive success. Thus, careful consideration must be given to drive performance and ecological conditions, as well as specific release proposals for potential application of tethered drive systems. [ABSTRACT FROM AUTHOR]

Propionibacterium acnes is a skin commensal bacterium that contributes to the development of acne vulgaris and other infections. Recent work revealed that P. acnes clinical isolates can be classified into distinct phylotypes, several of which have associations with healthy skin or acne. We sought to determine if these phylotypes induce different immunological responses and express protein factors that may contribute to their disease associations. We found that acne-associated P. acnes phylotypes induced 2- to 3-fold higher levels of IFN-γ and IL-17 in peripheral blood mononuclear cells compared with healthy phylotypes. On the other hand, P. acnes phylotypes associated with healthy skin induced 2- to 4-fold higher levels of IL-10. Comparative proteomic analysis of P. acnes phylotypes revealed a differential expression of several proteins, including an adhesion protein that was expressed at least 10-fold higher in acne-associated phylotypes and a cell surface hydrolase expressed in all phylotypes except those associated with healthy skin. Taken together, our data provide insight into how specific P. acnes phylotypes influence immune responses and the pathogenesis of acne.

AbstractGene drives are engineered alleles that can bias inheritance in their favor, allowing them to spread throughout a population. They could potentially be used to modify or suppress pest populations, such as mosquitoes that spread diseases. CRISPR/Cas9 homing drives, which copy themselves by homology-directed repair in drive/wild-type heterozygotes, are a powerful form of gene drive, but they are vulnerable to resistance alleles that preserve the function of their target gene. Such resistance alleles can prevent successful population suppression. Here, we constructed a homing suppression drive in Drosophila melanogaster

Propionibacterium acnes , plays an important role in acne vulgaris and other diseases. However, understanding of the exact mechanisms of P. acnes pathogenesis is limited. Few studies have investigated its proteome, which is essential for vaccine development. Here, we comprehensively investigate the proteome of P. acnes strain ATCC 6919, including secreted, cell wall, membrane, and cytosolic fractions in three types of growth media. A total of 531 proteins were quantified using an Orbitrap mass spectrometer and bioinformatically categorized for localization and function. Several, including PPA1939, a highly expressed surface and secreted protein, were identified as potential vaccine candidates.

Although antibiotics are a common treatment for acne, the difficulties inherent to effective antimicrobial penetration in sebum and selective antimicrobial action in the skin are compounded by increasing resistance of Propionibacterium acnes clinical isolates. To address these problems, we engineered Pentobra, a peptide-aminoglycoside molecule that has multiple mechanisms of antibacterial action and investigated whether it can be a potential candidate for the treatment of acne. Pentobra combines the potent ribosomal activity of aminoglycosides with the bacteria-selective membrane-permeabilizing abilities of antimicrobial peptides. Pentobra demonstrated potent and selective killing of P. acnes but not against human skin cells in vitro. In direct comparison, Pentobra demonstrated bactericidal activity and drastically outperformed free tobramycin (by 5-7 logs) against multiple P. acnes clinical strains. Moreover, electron microscopic studies showed that Pentobra had robust membrane activity, as treatment with Pentobra killed P. acnes cells and caused leakage of intracellular contents. Pentobra may also have potential anti-inflammatory effects as demonstrated by suppression of some P. acnes-induced chemokines. Importantly, the killing activity was maintained in sebaceous environments as Pentobra was bactericidal against clinical isolates in comedones extracts isolated from human donors. Our work demonstrates that equipping aminoglycosides with selective membrane activity is a viable approach for developing antibiotics against P. acnes that are effective in cutaneous environments.

CRISPR homing gene drives are highly invasive and can fail due to the rapid evolution of resistance. Here the authors present TARE drive, inspired by naturally occurring selfish genetic elements, which is less vulnerable to resistance and can potentially be confined to a target population.

IntroductionResveratrol (3,5,4'-trihydroxystilbene) is an antioxidant that has multiple biologic effects including antimicrobial properties. Acne vulgaris is a disease of the pilosebaceous unit, characterized by an inflammatory host immune response to the bacteria Propionibacterium acnes (P. acnes). This study sought to determine whether resveratrol may be a potential treatment for acne vulgaris.MethodsColony-forming unit (CFU) assays together with transmission electron microscopy using P. acnes treated with resveratrol or benzoyl peroxide were used to assess antibacterial effects. Blood was drawn from healthy human volunteers, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays were used to assess cytotoxicity in monocytes and keratinocytes.ResultsResveratrol demonstrated sustained antibacterial activity against P. acnes, whereas benzoyl peroxide, a commonly used antibacterial treatment for acne, demonstrated a short-term bactericidal response. A combination of resveratrol and benzoyl peroxide showed high initial antibacterial activity and sustained bacterial growth inhibition. Electron microscopy of P. acnes treated with resveratrol revealed altered bacterial morphology, with loss of membrane definition and loss of well-defined extracellular fimbrial structures. Resveratrol was less cytotoxic than benzoyl peroxide.ConclusionThe sustained antibacterial activity and reduced cytotoxicity versus benzoyl peroxide demonstrated by resveratrol in this study highlight its potential as a novel therapeutic option or adjuvant therapy in the treatment of acne vulgaris.

Growing evidence links tumor progression with chronic inflammatory processes and dysregulated activity of various immune cells. In this study, we demonstrate that various types of macrophages internalize microvesicles, called exosomes, secreted by breast cancer and non-cancerous cell lines. Although both types of exosomes targeted macrophages, only cancer-derived exosomes stimulated NF-κB activation in macrophages resulting in secretion of pro-inflammatory cytokines such as IL-6, TNFα, GCSF, and CCL2. In vivo mouse experiments confirmed that intravenously injected exosomes are efficiently internalized by macrophages in the lung and brain, which correlated with upregulation of inflammatory cytokines. In mice bearing xenografted human breast cancers, tumor-derived exosomes were internalized by macrophages in axillary lymph nodes thereby triggering expression of IL-6. Genetic ablation of Toll-like receptor 2 (TLR2) or MyD88, a critical signaling adaptor in the NF-κB pathway, completely abolished the effect of tumor-derived exosomes. In contrast, inhibition of TLR4 or endosomal TLRs (TLR3/7/8/9) failed to abrogate NF-κB activation by exosomes. We further found that palmitoylated proteins present on the surface of tumor-secreted exosomes contributed to NF-κB activation. Thus, our results highlight a novel mechanism used by breast cancer cells to induce pro-inflammatory activity of distant macrophages through circulating exosomal vesicles secreted during cancer progression.

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. [ABSTRACT FROM AUTHOR]

Fertility‐targeted gene drives have been proposed as an ethical genetic approach for managing wild populations of vertebrate pests for public health and conservation benefit. This manuscript introduces a framework to identify and evaluate target gene suitability based on biological gene function, gene expression and results from mouse knockout models. This framework identified 16 genes essential for male fertility and 12 genes important for female fertility that may be feasible targets for mammalian gene drives and other non‐drive genetic pest control technology. Further, a comparative genomics analysis demonstrates the conservation of the identified genes across several globally significant invasive mammals. In addition to providing important considerations for identifying candidate genes, our framework and the genes identified in this study may have utility in developing additional pest control tools such as wildlife contraceptives. [ABSTRACT FROM AUTHOR]

Propionibacterium acnes and Staphylococcus aureus are cutaneous pathogens that have become increasingly resistant to antibiotics. We sought to determine if chitosan, a polymer of deacetylated chitin, could be used as a potential treatment against these bacteria. We found that higher molecular weight chitosan had superior antimicrobial properties compared to lower molecular weights, and that this activity occurred in a pH dependent manner. Electron and fluorescence microscopy revealed that chitosan forms aggregates and binds to the surface of bacteria, causing shrinkage of the bacterial membrane from the cell wall. Of special relevance, clinical isolates of P. acnes were vulnerable to chitosan, which could be combined with benzoyl peroxide for additive antibacterial effect. Chitosan also demonstrated significantly less cytotoxicity to monocytes than benzoyl peroxide. Overall, chitosan demonstrates many promising qualities for treatment of cutaneous pathogens.

Advances in nanotechnology have demonstrated potential application of nanoparticles (NPs) for effective and targeted drug delivery. Here we investigated the antimicrobial and immunological properties and the feasibility of using NPs to deliver antimicrobial agents to treat a cutaneous pathogen. NPs synthesized with chitosan and alginate demonstrated a direct antimicrobial activity in vitro against Propionibacterium acnes, the bacterium linked to the pathogenesis of acne. By electron microscopy (EM) imaging, chitosan-alginate NPs were found to induce the disruption of the P. acnes cell membrane, providing a mechanism for the bactericidal effect. The chitosan-alginate NPs also exhibited anti-inflammatory properties as they inhibited P. acnes-induced inflammatory cytokine production in human monocytes and keratinocytes. Furthermore, benzoyl peroxide (BP), a commonly used antiacne drug, was effectively encapsulated in the chitosan-alginate NPs and demonstrated superior antimicrobial activity against P. acnes compared with BP alone while demonstrating less toxicity to eukaryotic cells. Together, these data suggest the potential utility of topical delivery of chitosan-alginate NP-encapsulated drug therapy for the treatment of dermatologic conditions with infectious and inflammatory components.