Biology subjects

Nolan, T.

Publications and source records attributed to Nolan, T..

16 recordsLinked to original sources

bioRxiv2026

Functional disruption of vgsc reveals haplosufficiency with implications for insecticide resistance and genetic control in Anopheles gambiae

Pyrethroid insecticides on bednets have been the mainstay of malaria control since the millennium by killing mosquitoes. The target of these pyrethroids is the voltage-gated sodium channel (VGSC). Resistance mutations at the pyrethroid binding site (L995F/S) have spread widely, but functional tools to dissect vgsc itself have lagged behind the population-genetic surveillance data. Manipulation of vgsc via knockout or functional mutation would be a vital step towards understanding its contribution to insecticide resistance, as well as exploring its utility as a genetic control target. Here we generate and characterise two CRISPR-edited An. gambiae lines: an exonic knockout (vgscKO) and an intronic CRISPR-mediated cassette exchange intermediate (vgscInt) sited near SNPs linked to L995F. Both insertions are viable and fertile in heterozygosity but homozygous lethal, demonstrating that vgsc is haplosufficient and therefore the locus represents a viable target for population suppression genetic control strategies. These insertions can also be used as balancers for studying vgsc mutations without the confounding effects of wild type alleles. We investigate the insecticide resistance phenotype of vgscKO and demonstrate that knocking out one copy of vgsc produces reduced susceptibility to deltamethrin and DDT at sub-discriminating doses, with implications for vgsc-mediated mechanisms of insecticide resistance.

genetics↗

bioRxiv2026

Repair outcomes after germline homing endonuclease cleavage in Anopheles gambiae inform the design of synthetic gene drives

Homing endonuclease genes spread by cleaving homologous chromosomes that lack the endonuclease cassette, after which repair from the endonuclease-containing chromosome converts the cut allele into a copy of the drive allele. This mechanism has provided a conceptual foundation for synthetic gene drive systems, including CRISPR-based drives, that represent promising strategies for the genetic control of insect pests. However gene drive performance depends critically on the repair pathways available in the germline of the target organism. Here, we report a set of transgenic assays originally developed as part of an attempt to establish gene targeting in the malaria mosquito Anopheles gambiae using an in vivo-generated linear targeting molecule. Although the intended FLP-mediated excision step was not achieved in the mosquito germline, analysis of the component strains revealed efficient germline activity of the rare-cutting homing endonuclease I-SceI and a striking bias towards homology-based repair of I-SceI-induced double-strand breaks. Across reporter and donor configurations, cleavage outcomes were dominated by single-strand annealing, microhomology-mediated repair, synthesis-dependent strand annealing and gene conversion-like events, with comparatively limited evidence for classical non-homologous end joining. In reciprocal crosses designed to distinguish gene conversion from gamete loss, I-SceI cleavage also produced inheritance distortion consistent with both conversion of the cleaved allele and reduced recovery of gametes carrying extensively damaged donor alleles. These findings indicate that the An. gambiae germline can strongly favour homology-dependent repair following homing endonuclease cleavage and that cleavage can also generate meiotic drive-like distortion through selective loss of damaged gametes. The results have direct relevance for the design and interpretation of homing endonuclease and CRISPR-based gene drives in malaria mosquitoes, where the balance between homology-directed repair, end joining and gamete viability will determine drive efficiency, resistance formation and transmission bias. Author summaryGene drives depend on a simple but demanding principle: a nuclease cuts one chromosome, and the cell repairs the break using the homologous chromosome as a template, copying the drive element in the process. Before CRISPR, this type of system was explored using naturally occurring homing endonucleases such as I-SceI. We attempted to develop a gene targeting system in Anopheles gambiae based on the Rong and Golic strategy, in which FLP recombinase would excise a donor molecule and I-SceI would linearise it to stimulate recombination. The full knockout technology did not work because FLP-mediated excision was not detected in the mosquito germline. However, the component tests revealed something more broadly important: I-SceI-induced breaks were repaired predominantly through homology-based pathways rather than simple end joining. We also observed inheritance distortion consistent with both gene conversion and loss of damaged gametes. These results help explain why homing-based systems can work in mosquitoes, while also highlighting why repair pathway choice and gamete viability need to be measured directly in any new drive configuration.

genetics↗

bioRxiv2026

An auditory advantage of Rdl-resistant mosquitoes may promote its persistence in urban environments

Despite dieldrin being discontinued, resistance mutations in the GABA-gated chloride channel RDL (Resistance to dieldrin) continue at high frequencies in natural malaria mosquitoes, suggesting a selective advantage. We showed that RDL modulates the mosquito auditory sensitivity. Because acoustic recognition is essential for mate acquisition, we hypothesized that resistance mutations confer a pleiotropic effect on mating success, with potential consequences for sexual selection. We provide laboratory evidence that resistance mutations enhance auditory and mating behaviours. We then conduct field collections in the city of Bangui (Central African Republic) and rural areas, revealing an association between Rdl resistance genotypes and urban settings, and within the city, with the noisiest locations. We also show decreased mating success of susceptible females with increasing noise levels, suggesting detrimental effects. Together, our findings support that Rdl resistance mutations enhance auditory function and mating success. We propose that this auditory advantage may contribute, together with other selective pressures such as cross-selection by other insecticides, to the persistence of these alleles in nature and may facilitate urban colonization by malaria vectors.

evolutionary biology↗

bioRxiv2025

Targeted genomic surveillance of insecticide resistance in African malaria vectors

The emergence of insecticide resistance is threatening the efforts of malaria control programmes, which rely heavily on a limited arsenal of insecticidal tools, such as insecticide-treated bed nets. Importantly, genomic surveillance of malaria vectors can provide critical, policy-relevant insights into the presence and evolution of insecticide resistance, allowing us to maintain and extend the shelf life of these interventions. Yet the complex genetic architecture of resistance, combined with resource constraints in malaria-endemic settings, have thus far precluded the widespread use of genomics in routine surveillance. Meanwhile, stakeholders in sub-Saharan Africa are moving towards locally driven, decentralised generation of genomic data, underscoring the need for standardised and robust genomics workflows. To address this need, we demonstrate an approach to targeted genomic surveillance in Anopheles gambiae s.l with Illumina sequencing. We target 90 genomic loci in the Anopheles gambiae s.l genome, including 55 resistance-associated mutations and 35 ancestry informative markers. This protocol is coupled with advanced, automated software for accurate and reproducible variant analysis. We are able to elucidate population structure and ancestry in our cohorts and accurately identify most species in the An. gambiae species complex. We report frequencies of variants at insecticide-resistance loci and explore the continued evolution of the pyrethroid target site, the Voltage-gated sodium channel. Applying the platform to a recently established colony of field-caught resistant mosquitoes (Siaya, Kenya), we identified seven independent resistance-associated variants contributing to reduced efficacy of insecticide-treated nets in East Africa. Additionally, we leverage a machine learning algorithm (XGBoost) to demonstrate the possibility of predicting bioassay mortality using genotypes alone. This achieved high accuracy (75%), demonstrating the potential of targeted genomics to predictively monitor insecticide resistance. Together these tools provide a practical, scalable solution for resistance monitoring while advancing the goal of building local genomic surveillance capacity in sub-Saharan Africa.

genomics↗

bioRxiv2024

Engineering Resilient Gene Drives Towards Sustainable Malaria Control: Predicting, Testing and Overcoming Target Site Resistance

CRISPR-based gene drives are selfish genetic elements with the potential to spread through entire insect populations for sustainable vector control. Gene drives designed to disrupt the reproductive capacity of females can suppress laboratory populations of the malaria mosquito. However, any suppressive intervention will inevitably exert an evolutionary pressure for resistance. Here, we present a pipeline for the accelerated discovery, engineering, and testing of both natural and drive-induced variants that could reverse gene drive spread. We applied our method to stress-test a highly effective gene drive that has evaded resistance in all laboratory-contained releases to date, known as Ag(QFS)1. We showed that previously undetected resistant alleles can arise at low frequency, and discovered novel, partially resistant alleles that can perturb drive-invasion dynamics. We then engineered next-generation gene drives that can actively remove resistant alleles by targeting several highly conserved and non-overlapping sites in the female-specific exon of the doublesex gene. Our models predict that such gene drive designs could suppress large, natural populations of the malaria mosquito in the field.

synthetic biology↗

bioRxiv2024

A Y chromosome-linked genome editor for efficient population suppression in the malaria vector Anopheles gambiae

Genetic control - the deliberate introduction of genetic traits to control a pest or vector population - offers a powerful tool to augment conventional mosquito control tools that have been successful in reducing malaria burden but that are compromised by a range of operational challenges. Self-sustaining genetic control strategies have shown great potential in laboratory settings but hesitancy due to their invasive and persistent nature may delay their implementation. Here instead we describe a self-limiting strategy, designed to have geographically and/or temporally restricted effect, based on a Y chromosome-linked genome editor (YLE). The YLE comprises a CRISPR-Cas9 construct that is always inherited by males yet generates an autosomal dominant mutation that is transmitted to over 90% of the offspring and results in female-specific sterility. Males are unaffected. To our knowledge, our system represents the first engineering of the Y chromosome to generate a genetic control strain for mosquitoes. Mathematical modelling shows that this YLE technology is up to 8 times more efficient for population suppression than optimal versions of other self-limiting strategies.

synthetic biology↗

bioRxiv2023

Measuring the impact of genetic heterogeneity and chromosomal inversions on the efficacy of CRISPR-Cas9 gene drives in different strains of Anopheles gambiae

The human malaria vector Anopheles gambiae is becoming increasingly resistant to insecticides, spurring the development of genetic control strategies. CRISPR-Cas9 gene drives can modify a population by creating double-stranded breaks at highly specific targets, triggering copying of the gene drive into the cut site ( homing), ensuring its inheritance. The DNA repair mechanism responsible requires homology between the donor and recipient chromosomes, presenting challenges for the invasion of lab-developed gene drives into wild populations of target species An. gambiae species complex, which show high levels of genome variation. Two gene drives (vas2-5958 and zpg-7280) were introduced into three An. gambiae strains collected across Africa with 5.3-6.6% variation around the target sites, and the effect of this variation on homing was measured. Gene drive homing across different karyotypes of the 2La chromosomal inversion was also assessed. No decrease in gene drive homing was seen despite target site heterology, demonstrating the applicability of gene drives to wild populations.

genetics↗

bioRxiv2021

Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation

CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying ( homing) therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel gene drive in the malaria vector Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations. Author summaryGene drives have the potential to be applied as novel control strategy of disease-transmitting mosquitoes, by spreading genetic traits that suppress or modify the target population. Many gene drive elements work by recognising and cutting a specific target sequence in the mosquito genome and copying themselves into that target sequence allowing the gene drive to increase in frequency in the population. Like other mosquito control interventions, efficacy will greatly depend on minimising the development of resistance to the gene drive mechanism - most likely via a change in the target sequence that prevents further cutting. One strategy to reduce resistance is to target sequences that are highly conserved, which implies that changes cannot easily be tolerated. We developed a strategy that simulates high selection pressure, under which resistance is most likely to emerge, and therefore provides a stringent test of its propensity to arise. Unlike previous results with another gene drive, we recovered a resistant allele within a few generations of gene drive exposure and at high frequency. Our results show that conserved sequences can vary hugely in ability to tolerate mutations and highlights the need to functionally validate future candidate gene drive target sites for their robustness to resistance.

genetics↗

bioRxiv2021

CRISPR/Cas9 modified An. gambiae carrying kdr mutation L1014F functionally validate its contribution in insecticide resistance and interaction with metabolic enzymes.

Insecticide resistance in Anopheles mosquitoes is a major obstacle in maintaining the momentum in reducing the malaria burden; mitigating strategies require improved understanding of the underlying mechanisms. Mutations in the target site of insecticides (the voltage gated sodium channel for the most widely used pyrethroid class) and over-expression of detoxification enzymes are commonly reported, but their relative contribution to phenotypic resistance remain poorly understood. Here we present a genome editing pipeline to introduce single nucleotide polymorphisms in An. gambiae which we have used to study the effect of the classical kdr mutation L1014F (L995F based on An. gambiae numbering), one of the most widely distributed resistance alleles. Introduction of 1014F in an otherwise fully susceptible genetic background increased levels of resistance to all tested pyrethroids and DDT ranging from 9.9-fold for permethrin to >24-fold for DDT. The introduction of the 1014F allele was sufficient to reduce mortality of mosquitoes after exposure to deltamethrin treated bednets, even as the only resistance mechanism present. When 1014F was combined with over-expression of glutathione transferase Gste2, resistance to permethrin increased further demonstrating the critical combined effect between target site resistance and detoxification enzymes in vivo. We also show that mosquitoes carrying the 1014F allele in homozygosity showed fitness disadvantages including increased mortality at the larval stage and a reduction in fecundity and adult longevity, which can have consequences for the strength of selection that will apply to this allele in the field. Author SummaryEscalation of pyrethroid resistance in Anopheles mosquitoes threatens to reduce the effectiveness of our most important tools in malaria control. Studying the mechanisms underlying insecticide resistance is critical to design mitigation strategies. Here, using genome modified mosquitoes, we functionally characterize the most prevalent mutation in resistant mosquitoes, showing that it confers substantial levels of resistance to all tested pyrethroids and undermines the performance of pyrethroid-treated nets. Furthermore, we show that combining this mutation with elevated levels of a detoxification enzyme further increases resistance. The pipeline we have developed provides a robust approach to quantifying the contribution of different combinations of resistance mechanisms to the overall phenotype, providing the missing link between resistance monitoring and predictions of resistance impact.

genetics↗

bioRxiv2021

CRISPR-mediated knock-in of transgenes into the malaria vector Anopheles funestus

The ability to introduce mutations, or transgenes, of choice to precise genomic locations has revolutionised our ability to understand how genes and organisms work. In many mosquito species that are vectors of various human disease, the advent of CRISPR genome editing tools has shed light on basic aspects of their biology that are relevant to their efficiency as disease vectors. This allows a better understanding of how current control tools work and opens up the possibility of novel genetic control approaches, such as gene drives, that deliberately introduce genetic traits into populations. Yet for the Anopheles funestus mosquito, a significant vector of malaria in sub-Saharan Africa and indeed the dominant vector species in many countries, transgenesis has yet to be achieved. We describe herein an optimised transformation system based on the germline delivery of CRISPR components that allows efficient cleavage of a previously validated genomic site and preferential repair of these cut sites via homology-directed repair (HDR), which allows introduction of exogenous template sequence, rather than end-joining repair. The rates of transformation achieved are sufficiently high that it should be able to introduce alleles of choice to a target locus, and recover these, without the need to include additional dominant marker genes. Moreover, the high rates of HDR observed suggest that gene drives, which employ an HDR-type mechanism to ensure their proliferation in the genome, may be well suited to work in An. funestus.

genetics↗

bioRxiv2021

Ultra-conserved sequences in the genomes of highly diverse Anopheles mosquitoes, with implications for malaria vector control.

DNA sequences that are exactly conserved over long evolutionary time scales have been observed in a variety of taxa. Such sequences are likely under strong functional constraint and they have been useful in the field of comparative genomics for identifying genome regions with regulatory function. A potential new application for these ultra-conserved elements has emerged in the development of gene drives to control mosquito populations. Many gene drives work by recognising and inserting at a specific target sequence in the genome, often imposing a reproductive load as a consequence. They can therefore select for target sequence variants that provide resistance to the drive. Focusing on highly conserved, highly constrained sequences lowers the probability that variant, gene drive-resistant alleles can be tolerated. Here we search for conserved sequences of 18bp and over in an alignment of 21 Anopheles genomes, spanning an evolutionary timescale of 100 million years, and characterise the resulting sequences according to their location and function. Over 8000 ultra-conserved elements were found across the alignment, with a maximum length of 164 bp. Length-corrected gene ontology analysis revealed that genes containing Anopheles ultra-conserved elements were over-represented in categories with structural or nucleotide binding functions. Known insect transcription factor binding sites were found in 48% of intergenic Anopheles ultra-conserved elements. When we looked at the genome sequences of 1142 wild-caught mosquitoes we found that 15% of the Anopheles ultra-conserved elements contained no polymorphisms. Our list of Anopheles ultra-conserved elements should provide a valuable starting point for the selection and testing of new targets for gene-drive modification in the mosquitoes that transmit malaria.

genomics↗

bioRxiv2020

An integrated mosquito small RNA genomics resource reveals dynamic evolution and host responses to viruses and transposons.

Although mosquitoes are major transmission vectors for pathogenic arboviruses, viral infection has little impact on mosquito health. This immunity is due in part to mosquito RNA interference (RNAi) pathways that generate antiviral small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). RNAi also maintains genome integrity by potently repressing mosquito transposon activity in the germline and soma. However, viral and transposon small RNA regulatory pathways have not been systematically examined together in mosquitoes. Therefore, we developed an integrated Mosquito Small RNA Genomics (MSRG) resource that analyzes the transposon and virus small RNA profiles in mosquito cell cultures and somatic and gonadal tissues across four medically important mosquito species. Our resource captures both somatic and gonadal small RNA expression profiles within mosquito cell cultures, and we report the evolutionary dynamics of a novel Mosquito-Conserved piRNA Cluster Locus (MCpiRCL) composed of satellite DNA repeats. In the larger culicine mosquito genomes we detected highly regular periodicity in piRNA biogenesis patterns coinciding with the expansion of Piwi pathway genes. Finally, our resource enables detection of crosstalk between piRNA and siRNA populations in mosquito cells during a response to virus infection. The MSRG resource will aid efforts to dissect and combat the capacity of mosquitoes to tolerate and spread arboviruses.

genomics↗

bioRxiv2018

Improved CRISPR-based suppression gene drives mitigate resistance and impose a large reproductive load on laboratory-contained mosquito populations

CRISPR-Cas9 nuclease-based gene drives rely on inducing chromosomal breaks in the germline that are repaired in ways that lead to a biased inheritance of the drive. Gene drives designed to impair female fertility can suppress populations of the mosquito vector of malaria. However, strong unintended fitness costs, due to ectopic nuclease expression, and high levels of resistant mutations, limited the potential of the first generation of gene drives to spread. Here we show that changes to regulatory sequences in the drive element, designed to contain nuclease expression to the germline, confer improved fecundity over previous versions and generate drastically lower rates of target site resistance. We employed a genetic screen to show that this effect is explained by reduced rates of end-joining repair of DNA breaks at the target site caused by deposited nuclease in the embryo. Highlighting the impact of deposited Cas9, many of the mutations arising from this source of nuclease activity in the embryo are heritable, thereby having the potential to generate resistant target sites that reduce the penetrance of the gene drive. Finally, in cage invasion experiments these gene drives show improved invasion dynamics compared to first generation drives, resulting in greater than 90% suppression of the reproductive output and a delay in the emergence of target site resistance, even at a resistance-prone target sequence. We shed light on the dynamics of generation and selection of resistant alleles in a population by tracking, longitudinally, the frequency of resistant alleles in the face of an invading gene drive. Our results illustrate important considerations for future gene drive design and should expedite the development of gene drives robust to resistance.

genetics↗

bioRxiv2017

Cross-species Y chromosome function between malaria vectors of the Anopheles gambiae species complex

Y chromosome function, structure and evolution is poorly understood in many species including the Anopheles genus of mosquitoes, an emerging model system for studying speciation that also represents the major vectors of malaria. While the Anopheline Y had previously been implicated in male mating behavior, recent data from the Anopheles gambiae complex suggests that, apart from the putative primary sex-determiner, no other genes are conserved on the Y. Studying the functional basis of the evolutionary divergence of the Y chromosome in the gambiae complex is complicated by complete F1 male hybrid sterility. Here we used an F1xF0 crossing scheme to overcome a severe bottleneck of male hybrid incompatibilities and enabled us to experimentally purify a genetically labelled A. gambiae Y chromosome in an A. arabiensis background. Whole genome sequencing confirmed that the A. gambiae Y retained its original sequence content in the A. arabiensis genomic background. In contrast to comparable experiments in Drosophila, we find that the presence of a heterospecific Y chromosome has no significant effect on the expression of A. arabiensis genes and transcriptional differences can be explained almost exclusively as a direct consequence of transcripts arising from sequence elements present on the A. gambiae Y chromosome itself. We find that Y hybrids show no obvious fertility defects and no substantial reduction in male competitiveness. Our results demonstrate that, despite their radically different structure, Y chromosomes of these two species of the gambiae complex that diverged an estimated 1.85Myr ago function interchangeably, thus indicating that the Y chromosome does not harbor loci contributing to hybrid incompatibility. Therefore, Y chromosome gene flow between members of the gambiae complex is possible even at their current level of divergence. Importantly, this also suggests that malaria control interventions based on sex-distorting Y drive would be transferable, whether intentionally or contingent, between the major malaria vector species.

genetics↗

bioRxiv2017

The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito

Gene drives have enormous potential for the control of insect populations of medical and agricultural relevance. By preferentially biasing their own inheritance gene drives can rapidly introduce genetic traits even if these confer a negative fitness on the population.\n\nWe have recently developed gene drives based on a CRISPR nuclease construct that is designed to disrupt key genes essential for female fertility in the malaria mosquito. The construct copies itself and the associated genetic disruption from one homologous chromosome to another during gamete formation, in a process called homing that ensures the majority of offspring inherit the drive. Such drives have the potential to cause long-lasting, sustainable population suppression though they are also expected to impose a large selection pressure for resistance in the mosquito. One of these population suppression gene drives showed rapid invasion of a caged population over 4 generations, establishing proof of principle for this technology. In order to assess the potential for the emergence of resistance to the gene drive in this population we allowed it to run for 25 generations and monitored the frequency of the gene drive over time. Following the initial increase of the gene drive we noticed a gradual decrease in its frequency that was accompanied emergence of small, nuclease-induced mutations at the target gene that are resistant to further cleavage and restore its functionality. Such mutations show rates of increase consistent with positive selection in the face of the gene drive. Our findings represent the first documented example of selection for resistance to a synthetic gene drive and lead to important design recommendations and considerations in order to mitigate for resistance for future gene drive applications.

evolutionary biology↗

bioRxiv2016

Rapid evolution of female-biased genes among four species of Anopheles malaria mosquitoes

Understanding how phenotypic differences between males and females arise from the sex-biased expression of nearly identical genomes can often reveal important insights into the biology and evolution of a species. Among Anopheles mosquito species, these phenotypic differences include vectorial capacity, as it is only females that blood feed and thus transmit human malaria. Here, we use RNA-seq data from multiple tissues of four vectors spanning the Anopheles phylogeny to explore the genomic and evolutionary properties of sex-biased genes. We find that in these mosquitoes, in contrast to what has been found in many other organisms, female-biased genes are more rapidly evolving in sequence, expression, and genic turnover, than male-biased genes. Our results suggests that this atypical pattern may be due to the combination of sex-specific life history challenges encountered by females, such as blood feeding. Furthermore, female propensity to only mate once in nature in male swarms likely diminishes sexual selection of post-reproductive traits related to sperm competition among males. We also develop a comparative framework to systematically explore tissue- and sex-specific splicing, to document its conservation throughout the genus and identify a set of candidate genes for future functional analyses of sex-specific isoform usage. Finally, our data reveals that the deficit of male-biased genes on the X chromosomes in Anopheles is a conserved feature in this genus and can be directly attributed to chromosome-wide transcriptional regulation that demasculinizes the X in male reproductive tissues.

genomics↗