Biology subjects

Champer, J.

Publications and source records attributed to Champer, J..

At least 19 recordsLinked to original sources

bioRxiv2026

The effects of DNA repair factor-Cas9 fusions on homing drive performance

CRISPR-based homing gene drives achieve super-Mendelian inheritance by converting wild-type alleles into drive alleles through homology-directed repair (HDR). However, competing end-joining repair can generate resistance alleles that limit drive spread. Although fusing Cas9 to DNA repair-associated proteins has improved HDR in conventional genome editing systems, its effectiveness in gene drives remains unclear. Here, we systematically evaluated in Drosophila melanogaster eight Cas9 fusion constructs previously reported to improve HDR in other systems. Although none of the fusion constructs significantly increased HDR-associated signals in S2 cells, several candidates retained relatively high activity comparable to standard Cas9. These candidates were then tested in vivo across multiple split drive systems, regulatory elements, target loci, and parental sexes. Cas9-POLD3 significantly increased female drive inheritance in contexts with low baseline efficiency or substantial nonfunctional resistance formation, likely by increasing effective cutting rate and potentially also by biasing repair outcomes toward drive conversion. Cas9-Geminin degron generally maintained germline drive performance while reducing embryo resistance. Together, these findings identify Cas9-POLD3 and Cas9-Geminin degron as complementary, context-specific tools for enhancing drive conversion and limiting embryo resistance, respectively. These results highlight the importance of matching fusion modules to the regulatory and genetic context of individual drive systems.

genetics↗

bioRxiv2026

Confinement considerations for CRISPR toxin-antidote gene drive systems

Gene drives are potentially powerful tools, able to spread throughout target populations. They could be used to modify or suppress disease vectors, invasive species, and agricultural pests. Yet, in many scenarios, confinement of the drive to only a target population is required. Several types of drives are capable of this, among the most promising of which are CRISPR toxin-antidote drives. Though showing good performance in simple models, such drives have not been thoroughly assessed in spatially structured populations. Here we evaluate three modification drive variants with varying levels of confinement. We find that Toxin-Antidote Recessive Embryo (TARE) drive and 2-locus TARE drives can usually spread in connected populations or from a single sufficiently large release. However, they can still be stopped by a migration corridor or by a sufficiently high population density gradient. 1-locus 2-drive TARE, on the other hand, will not be able to spread outside of release areas and indeed will often retreat in the face of wild-type alleles. When these drives arrive at a point source such as a port, only the standard TARE drive has a significant chance of establishing if releases occur at sufficiently high frequency and quantity. However, in models with parental care among a limited number of offspring, TARE drives become more invasive, with lower introduction thresholds. Overall, we find that spatial and other ecological factors can substantially affect the outcome of a confined CRISPR toxin-antidote drive release.

ecology↗

bioRxiv2026

Two-locus CRISPR toxin-antidote gene drive for confined population modification

Gene drive systems enable rapid spread of desired transgenes throughout populations. Advances in CRISPR technology have facilitated the construction of toxin-antidote gene drives, which utilize a CRISPR nuclease as the toxin to disrupt an essential wild-type gene alongside a recoded version of the same gene as the antidote. Because these systems propagate by eliminating wild-type alleles rather than directly copying themselves like homing drives, they typically exhibit introduction thresholds, allowing them to be confined to target populations. Previous work developed the efficient Toxin-Antidote Recessive Embryo (TARE) drive, but its threshold may be too low in challenging confinement scenarios. Here, we constructed a 2-locus TARE drive system. It has underdominance characteristics, yielding a higher introduction threshold, even when drive performance is ideal. It targets the essential but haplosufficient genes hairy and sim using two different drives at different genomic locations, each targeting the gene that the other rescues. Our system involved two linked elements together with rare homology-directed repair-mediated drive conversion, reducing the threshold to compensate for fitness costs. The system showed high efficiency in individual crosses. When released into multigenerational cage populations above the introduction threshold, the drive successfully and rapidly modified the entire population, and when below this threshold, it was eliminated. Our findings indicate that 2-locus TARE drives represent promising tools for effective and strongly confined population modification.

genetics↗

bioRxiv2026

Assessment of homing gene drive efficiency using multiplexed sgRNAs targeting doublesex in the global crop pest Drosophila suzukii

Drosophila suzukii (Matsumura, 1931, Diptera: Drosophilidae) is a globally invasive pest of soft-skinned fruits that is currently controlled largely through the use of broad-spectrum insecticides. Increasing resistance to insecticides and regulatory pressures have motivated the development of genetic control strategies. We previously developed a CRISPR/Cas9-based homing gene drive targeting the coding sequence of the female-specific exon of the sex-determination gene doublesex, achieving highly efficient inheritance (94-99%) in both male and female germlines. A major limitation of homing gene drives is the formation of resistant alleles that evade cleavage yet retain gene function. Multiplexing guide RNAs (gRNAs) could reduce the formation of such functional resistance alleles. Here, we generated and tested homing constructs expressing one, two, or three gRNAs targeting different regions of the female-specific exon of doublesex, including the intron-exon splice junction. A single gRNA targeting the splice junction supported high inheritance in males but showed reduced efficiency in females. Combining this gRNA with a coding sequence-targeting guide further reduced drive efficiency, particularly in the female germline. Constructs expressing two gRNAs performed similarly whether guides were linked by transfer RNA (tRNA) sequences or expressed from independent promoters. Constructs expressing three gRNAs using tRNA processing showed consistently low drive inheritance in both sexes and low frequencies of target-site modification among non-drive progeny, consistent with reduced cleavage activity. Inheritance was significantly higher in male than female germlines for several constructs, indicating that germline context strongly influences drive performance. Our findings show that the strategy used for multi-gRNA expression, target site choice and sex-specific germline environments can influence gene drive efficiency, emphasizing the need to optimize construct design in the target species. Author SummarySpotted wing drosophila (Drosophila suzukii) is an invasive pest that damages soft skinned fruits such as berries and cherries. Control currently relies heavily on insecticides, but resistance and regulatory concerns are increasing the need for alternative approaches. We are developing genetic strategies for suppression of pest populations. We previously developed a gene editing system targeting a female-essential gene that showed very high biased inheritance (gene drive). However, a key challenge is that the system can sometimes create resistant individuals that are unaffected. One possible solution is to use using multiple gRNAs--molecules that direct gene editing to specific DNA sequences--to reduce the formation of these resistant individuals. In this study we found that using more than one gRNA reduced the efficiency of inheritance, especially in females. However, activity could be influenced by exactly where the DNA is targeted and how the guide RNAs are expressed. These results show that gene drive performance depends strongly on biological context and design choices, and that strategies must be carefully optimized in the target species.

genetics↗

bioRxiv2026

Comparative assessment of gene drive release patterns and spread with a hex-based model for large-scale simulations

Spatial population genetic and ecological modeling is often necessary to predict outcomes accurately. One example is gene drive, a rapid process involving spread of gene drive alleles through a population, usually to suppress pests or reduce transmission of vector-borne disease. Several existing models have been used to assess gene drive and other spatial processes. However, each of these has limitations, such as high computational cost and limited scalability, difficulty in incorporating environmental factors and complex lifecycles, or potentially simplified spatial structure. To overcome these challenges, we propose a hexagon-based computational framework that is designed to mimic continuous space for rapid genetic wave advances. This allows us to accurately simulate a larger spatial domain with lower computational investment. We implemented this model and compared the wave speeds of different gene drives with those obtained from other models. The results showed good agreement when hexagon width and dispersal were properly calibrated. We then determined optimal circular and linear (along roads) release patterns for a variety of gene drives and Wolbachia bacteria. To demonstrate the application of our framework to a hypothetical scenario, we constructed a model Culex quinquefasciatus mosquitoes on Hainan Island. We then evaluated the outcome of different gene drive release strategies, showing the transgenic insect release level necessary to achieve high gene drive coverage and how this could be further optimized based on mosquito and human distribution. Overall, our hex-based population genetic framework provides a flexible platform for realistic and large-scale models for gene drive and related applications.

ecology↗

bioRxiv2026

Dominant resistance alleles accelerate population suppression by CRISPR-based gene drive

CRISPR-based gene drive is a promising strategy for pest control. However, when targeting a recessive fertility gene, nonfunctional resistance alleles generated by end-joining slow the spread of drive and can even impair suppressive power. It was recently discovered that a doublesex target site can result in dominant female-sterile alleles when disrupted. It could serve as an ideal target for suppression drives, as disrupted alleles are efficiently removed when inherited by a female, increasing the suppressive power of the drive. Here, to better understand how dominant sterile target sites can improve suppression, we used an individual-based, continuous space model to investigate chasing dynamics of homing drives that produce dominant female-sterile resistance. Our results indicate that dominant-sterile resistance allows homing drives to achieve higher population elimination rates and a lower probability of chasing over a broader range of performance parameters. In addition, we propose a confined gene drive system, termed TADFI (Toxin-Antidote Dominant Female Intersex), that is also based on formation of dominant female sterile alleles. TADFI has two variants, designed for population suppression and modification, respectively. We estimate the introduction frequency threshold of TADFI in a panmictic model, and evaluate its suppression dynamics in a spatial model. Though spreading less rapidly than Toxin-Antidote Dominant Embryo (TADE) suppression drive, it has similar favorable dynamics related to confinement and suppressive power, while potentially being easier to construct. Together these results show that drives generating dominant-female sterile alleles may be promising candidates for a variety of population suppression applications.

genetics↗

bioRxiv2026

A confined gene drive for population modification in the malaria vector Anopheles stephensi

Gene drives are genetic elements that bias their own inheritance to spread desired traits in target populations, enabling population modification or suppression. Although homing-based drives can propagate efficiently, their potential for uncontrolled spread may present a challenge for field deployment. Thus, confined drive systems are needed. Here, we developed a confined modification drive, called Toxin-Antidote Recessive Embryo (TARE) drive, in the globally important malaria vector Anopheles stephensi. This drive works by cleaving and disrupting wild-type alleles in the germline or early embryo from maternally deposited Cas9. Disrupted alleles are recessive lethal, thus increasing the drive in a frequency-dependent manner. Inheritance bias was moderate in crosses between drive heterozygote mosquitoes, possibly due to low gRNA activity and thus moderate germline cleavage rates. Single-release cage trials confirmed the TARE drives ability to spread, although the drive ultimately declined due to fitness costs and resistance alleles associated with repetitive elements. Nonetheless our modelling analysis indicate that this TARE system could achieve population spread if the resistance issue is addressed. These findings demonstrate a functional prototype TARE drive in Anopheles stephensi and highlight key parameters governing its performance. Minor design optimizations could substantially improve efficiency and integrity, enabling rapid but confined population modification.

genetics↗

bioRxiv2026

Optimal spatial release strategies for confined gene drives and Wolbachia

Gene drives are genetic elements that can rapidly spread through populations, offering potential solutions for controlling disease vectors and pests. In some scenarios, it is necessary to utilize drives that can be confined to only target populations. The success of these threshold-dependent gene drives, which require a minimum local frequency to establish, depends critically on the spatial strategy used for introduction. Here, we use a reaction-diffusion model to systematically identify optimal release patterns that maximize the per-capita efficiency for four distinct gene drive designs as well as use of Wolbachia bacteria, which spread similarly to frequency-dependent gene drives. We find that the most efficient release strategy is highly dynamic, transitioning from a broad "everywhere" release for short timeframes to a "multiple-ring" pattern for intermediate times, and finally to a focused "center" release for longer timeframes. These timeframes depend on the specific type of drive, with more powerful variants transitioning more quickly to center releases. Our results demonstrate that these optimized, variable release strategies can be substantially more effective than simple uniform releases. This study provides a quantitative framework for designing effective gene drive implementations, highlighting that a carefully planned spatial strategy is essential for maximizing impact, making optimal use of available resources.

genetics↗

bioRxiv2025

Yuanyang allele pairs enable engineered obligate co-inheritance for population replacement and suppression

Mendels law of segregation, which dictates independent assortment of alleles, is a cornerstone of genetics. Here, we present the Yuanyang allele pair (YYAP), an engineered underdominance gene drive system that enforces obligate co-dependency between homologous alleles via synthetic toxin-antitoxin circuits. This design produces a striking deviation from classical Mendelian segregation: YYAP alleles cannot segregate independently, reshaping inheritance outcomes without altering the physical mechanics of meiosis. In Drosophila melanogaster, optimized YYAP strain K711 exhibits >99% hemizygous lethality when crossed with wild type, while maintaining stable transmission and high fitness in transheterozygotes. Cage experiments demonstrate efficient population replacement, positioning YYAP as a confined, resistance-proof alternative to CRISPR homing gene drives. Release of only males represents a self-limiting suppression strategy that was also successful in cage experiments. By imposing a near-complete postzygotic incompatibility, YYAP establishes a programmable framework that not only supports pest management, but also enables modeling of reproductive isolation and allelic co-dependency, thereby creating opportunities to explore speciation and broader synthetic inheritance systems in ecology and evolution.

molecular biology↗

bioRxiv2025

Variants in Cas9 and nanos regulatory elements modulate activity and reduce resistance allele formation in homing gene drive

Gene drive is a novel approach for controlling vector borne disease via either population modification or suppression. Even with high efficiency, though, overall drive performance can be reduced by somatic Cas9 expression and by maternal deposition of Cas9, leading to resistance allele formation. The nanos promoter for Cas9 shows very little leaky somatic expression, but it causes high rates of embryo resistance allele formation in Drosophila melanogaster. By truncating the promoter, we reduced rates of embryo resistance to undetectable levels, but germline cutting in females decreased by over half. Germline cutting and successful drive conversion was eventually lost when only the 5' UTR was present, though males still retained moderate germline drive efficiency. Several additional methods were tested to improve performance, including additional suppressor elements to the 3' UTR and introns to increase expression level. The most successful of these was the addition of a second nuclear localization signal, which substantially increased activity when coupled with a full-length or truncated nanos promoter. Overall, these experiments show the potential to modulate Cas9 regulatory elements to achieve desired expression for gene drive applications, while also showcasing the difficulty of obtaining an optimal activity profile.

genetics↗

bioRxiv2025

Finding the perfect promoter: Correlating single-cell transcriptome data with gene drive performance

Gene drive can control pathogen transmission or suppress vector populations by spreading drive alleles with super-Mendelian inheritance. CRISPR homing drive currently represents the most powerful type, and regulating Cas9 expression with specific promoters has been effective for improving drive performance. However, selecting these is often a major challenge. Here, we evaluated 35 Cas9 constructs driven by distinct promoters in different gene drive systems and identified associations between drive performance and single-cell RNA expression patterns of the promoter-associated genes. Our results indicate that higher drive conversion is significantly associated with elevated expression of the promoter-associated gene in the respective reproductive cells, but embryo resistance allele formation correlates with excessive female germline expression. For males, early germline expression produces superior performance. Thus, we find that optimal drive performance requires restricting Cas9 expression to a tight quantitative and spatiotemporal window. In addition, found that in situ integrated rhino-Cas9 constructs significantly reduce somatic expression, underscoring the importance of genomic locus. On the basis of these results, we propose criteria for selecting promoters, providing a theoretical rationale and practical guidance for optimization of promoter elements in homing gene drive systems.

genetics↗

bioRxiv2025

Experimental demonstration of daisy chain gene drive and modelling of daisy suppression systems

CRISPR-based gene drive can address ecological problems by biasing their inheritance coupled with an effector for either population modification of suppression. However, the risk of uncontrolled spread impedes some applications of gene drive. Daisy chain drives have received much attention as a potential approach to overcome this problem. They potentially allow efficient spread in a target population but are ultimately self-limiting. This is achieved by splitting a standard gene drive into multiple dependent elements, where each element can bias the inheritance of another, except one non-driving element. With the successive loss of each chain link, spread of transgenic elements will slow down and eventually stop. Here, we use modelling to assess the population dynamics of suppression daisy chain drives in both panmictic and continuous space models. We find that achieving population elimination through a single release of daisy chain gene drives is possible but difficult, with relatively high requirements for drive performance and release size. These effects are substantially amplified in spatial models. We also constructed two configurations of daisy chain gene drives in Drosophila melanogaster as a proof-of-principle. One is a rescue drive for population modification, and the other aims for population suppression by targeting a female fertility gene. Both functioned within expectations at moderate efficiency in individual crosses. However, the system failed to spread in cage populations because of higher than expected fitness costs. Overall, our study demonstrates that daisy chain systems may be promising candidates for both modification and suppression, but challenges remain in both construction and potential deployment in large regions.

genetics↗

bioRxiv2025

Self-limiting population suppression gene drive in the West Nile vector mosquito, Culex quinquefasciatus

Culex mosquitoes transmit major pathogens including West Nile virus, encephalitis, filariasis, and avian malaria, threatening public health, poultry, and ecosystems. We engineered a CRISPR-based population suppression gene drive targeting a conserved exon of the doublesex (dsx) gene. The drive incorporates a recoded dsxM segment to preserve male function while converting genetic females into sterile intersexes, enabling male-biased propagation and removal of fertile females. It achieves super-Mendelian inheritance ([~]71%) and generates partially dominant sterile resistance alleles via end-joining, resulting in intersex phenotypes with reduced fertility and hatchability. Modeling predicts that this RIDD (Release of Insects carrying a Dominant-sterile Drive) system can suppress populations at low intrinsic growth rates and release ratios, outperforming SIT and fs-RIDL strategies in persistence and efficiency, with further gains achievable through improved cleavage rates. This study establishes a self-limiting gene drive framework for Culex suppression, highlightling the potential of targeting conserved sex-determination pathways for sustainable vector control.

genetics↗

bioRxiv2025

Strategies to improve the efficiency of homing gene drives with multiplexed gRNAs

CRISPR homing gene drive holds great potential for pest control, but its success is challenged by the generation of resistance alleles. To mitigate the impact of resistance, multiplexed gRNA strategies have been demonstrated. However, unless both outmost sites are cleaved simultaneously, poor homology during DNA repair may compromise efficiency, leading to decline in drive conversion efficiency when the number of gRNAs is higher than two. Here, to better estimate the rate of drive efficiency decline, we designed and assessed the efficiency of single gRNA drives with imperfect homologous arms, refining a detailed gRNA multiplexing model. To mitigate the greater than expected efficiency loss, we further evaluated two new strategies: (1) extended homology arms to span all target sites with mutations in the PAMs and (2) a population-level multiplexing gRNAs system involving two or more drives, each carrying two gRNAs. Specifically, the population-level multiplexing system has four adjacent gRNA target sites, and the drives have small mutations in their homologous arms to prevent cleavage by the other drive. Mutations in both strategies did not impair efficiency, but they were not consistently inherited, and undesired cutting in the homologous arms decreased drive efficiency. We simulated homing suppression drive using a dual 2-gRNA population-level gRNA multiple xing strategy based on our experimental evaluation. Despite being somewhat more vulnerable to functional resistance than a standard 4-gRNA drive, the higher individual drive efficiency of the population-level multiplexing system increased successful population elimination outcomes. Thus, population-level multiplexing can be a useful for improving suppression drive power.

genetics↗

bioRxiv2025

Investigating multiple types of resistance against a homing gene drive in European populations of Drosophila melanogaster

Gene drive technology may be a valuable tool for addressing several contemporary challenges, including combating disease vectors, conserving biodiversity, and controlling agricultural pests. Homing gene drives spread through a population by copying themselves onto the homologous chromosome in the germline of heterozygous individuals. However, it is possible that resistance will evolve against homing gene drives, especially if the goal is to suppress or eliminate a pest population so that resistance alleles have a large selective advantage over the gene drive. Resistance can result from a simple mutation at the drives target site, which is found in many studies but can potentially be avoided by improving drive design. However, a more complex polygenic type of resistance could also evolve through selection on standing genetic variation that affects the efficiency of the spread of the gene drive. In this study, we test an efficient homing gene drive in genetically diverse lines of Drosophila melanogaster, collected from across Europe by the DrosEU Consortium. We find that the gene drive shows considerable variability in homing efficiency, but that none of this variability can be ascribed to heritable genetic effects. Selection for complex resistance is thus unlikely and will be inefficient, probably still giving a gene drive enough time to fixate in the population. However, although our tested gene drive targets a highly conserved haploinsufficient gene with two gRNAs, we find simple resistance alleles in viable offspring. Half of these are the product of end-joining repair instead of homing and may still carry heavy fitness costs. However, the other half are the result of partial homing events. These alleles indicate that resistance could likely evolve against this gene drive in a simple, non-polygenic way. Therefore, more effective strategies may be required to address simple resistance mutations, whereas complex resistance may be unlikely to pose a substantial barrier to the employment of at least certain types of gene drive.

genetics↗

bioRxiv2025

A systematic delineation of 3'UTR regulatory elements and their contextual associations

The 3'UTR encodes regulatory information that shapes transcript abundance and subcellular distribution, yet the underlying sequence rules remain incompletely defined. Using SEERS, we quantified the effects of [~]2 million synthetic 3'UTR inserts in A549 and HCT116 cells on RNA output and nuclear-cytoplasmic partitioning. We identify a broad repertoire of short (2[~]8 nt) elements whose effects largely align along a major coupled axis that links high expression with cytoplasmic enrichment and low expression with nuclear enrichment, and contribute predominantly in an additive manner. A context-aware deep learning model (TALE) captures most of this behavior while revealing that strong context dependence is uncommon and emerges mainly in rare, extreme contexts consistent with higher-order constraints. Applying TALE to ClinVar 3'UTR variants prioritizes a subset of pathogenic SNVs with aberrant predicted effects, frequently explained by creation or disruption of splice-site-like U1 telescripting signals, highlighting a potent route to 3'UTR-driven disease mechanisms.

genomics↗

bioRxiv2025

Gene drive dynamics in plants: the role of seedbanks

Gene drives offer revolutionary potential for the management of problematic plant populations, such as invasive weeds and herbicide-resistant species, by rapidly spreading desired genetic alterations. Two recent studies have provided the first experimental demonstrations of engineered CRISPR gene drive systems in plants (CAIN and ClvR). However, the successful application of such systems in the field will critically depend on an accurate understanding of plant-specific life-history traits, especially seed dormancy, a ubiquitous yet frequently overlooked eco-evolutionary force. In this study, we develop the first comprehensive modeling framework for gene drives in plant populations that incorporates a persistent soil seedbank. We show how the presence of a seedbank can significantly slow gene drive spread but also reduce the genetic load required to achieve population elimination. Furthermore, we show that seedbanks substantially increase the required introduction frequency of threshold-dependent gene drives, which could prevent establishment in some cases, yet also provide an intrinsic biosafety mechanism for confining a highly efficient drive to a target population. Our study highlights the need to incorporate seedbank dynamics into gene drive strategies to ensure realistic predictions and successful field applications.

evolutionary biology↗

bioRxiv2025

Stronger population suppression by gene drive targeting doublesex from dominant female-sterile resistance alleles

CRISPR homing drives can be used to suppress a population by targeting female fertility genes. They convert wild-type alleles to drive alleles in the germline of drive heterozygotes by homology-directed repair after DNA cleavage. However, resistance alleles produced by end-joining pose a great threat to homing drive. They prevent further recognition by Cas9, and therefore weaken suppressive power, or even stop suppression if they preserve the function of the target gene. We used multiplexed gRNAs targeting doublesex in Drosophila to avoid functional resistance and create resistance alleles that were dominant female-sterile. This occurred because the male dsx transcript was generated in females by disruption of the female-specific splicing acceptor site. We rescued dominant sterility of the drive by providing an alternate splicing site. As desired, the drive was recessive female sterile and yielded high drive inheritance among the progeny of both male and female drive heterozygotes. The dominant-sterile resistance alleles enabled stronger suppression in computational models, even in the face of modest drive efficiency and fitness costs. However, we found that male drive homozygotes were also sterile because they used the rescue splice site. Attempts to rescue males with alternate expression arrangements were not successful, though some male homozygotes had less severe intersex phenotypes. Though this negatively impacted the drive, models showed that it still had significantly improved suppressive power. Therefore, this design may have wide applicability to dsx-based suppression gene drives in a variety of organisms with intermediate homing drive performance.

genetics↗