Biology subjects

Arien, Y.

Publications and source records attributed to Arien, Y..

4 recordsLinked to original sources

bioRxiv2026

Spermatogenic context controls outcomes of engineered sex distortion in malaria mosquitoes

Sex-ratio distortion systems are promising genetic tools for mosquito population control. Two strategies have been proposed: prezygotic elimination of X-bearing sperm by X-shredding, which can drive invasive Y-chromosome transmission when sex distorters are Y-linked, and postzygotic daughter killing through disruption of X-linked haploinsufficient genes, a self-limiting approach known as X-poisoning. Previous attempts to implement X-poisoning in the malaria mosquito Anopheles gambiae unexpectedly produced prezygotic distortion, with sex bias arising from loss of X-bearing sperm rather than daughter lethality. Here we use a split CRISPR-Cas9 system to systematically compare sex-ratio distortion outcomes across germline Cas9 drivers and X-linked target genes. Meiotic X-chromosome targeting induced preferential Y-chromosome transmission regardless of target identity, function, or number of sgRNA target sites. In contrast, shifting Cas9 expression to earlier spermatogenic stages altered outcomes dramatically: targeting X-linked ribosomal protein genes caused severe developmental or reproductive toxicity, whereas targeting the haplolethal muscle gene wupA produced daughter-specific post-embryonic lethality, with the majority of surviving females emerging flightless. Tracking offspring using a Y-linked fluorescent marker confirmed that sex chromosome segregation remained unbiased, with female mortality accumulating progressively from the first larval instar, reaching near-complete lethality by adulthood. These results demonstrate that the timing of Cas9 expression during spermatogenesis, rather than target gene identity alone, determines the outcome of X-chromosome targeting in malaria mosquitoes, and establish the conditions required for genuine X-poisoning. Identification of wupA as an effective X-poisoning target provides a solid foundation for the future development of self-limiting Y-linked sex-ratio distortion systems for malaria vector control.

genetics↗

bioRxiv2025

Developing Y chromosome sex ratio distorters in the model insect Drosophila melanogaster

CRISPR-Cas9 sex ratio distortion (SRD) systems can suppress insect populations by biasing progeny toward males, but realizing such systems requires reliable Cas9 expression from insect Y chromosomes. Here, we tested whether the spermatocyte-specific {beta}Tub85D promoter can drive functional Cas9 expression when inserted on the Drosophila melanogaster Y chromosome. Using CRISPR-mediated homology-directed repair, we generated a Y-linked {beta}Tub85D-Cas9-T2A-eGFP construct and compared its activity with an autosomal counterpart. Whereas autosomal {beta}Tub85D-Cas9 induced strong male-biased sex ratios when paired with an X-poisoning gRNA, the Y-linked construct failed to distort sex ratios and exhibited approximately 2,000-fold reduction in Cas9 transcript abundance. Nonetheless, weak but detectable GFP fluorescence and Cas9 transcripts confirmed partial Y-linked promoter activity. These findings provide the first direct experimental evidence of meiotic sex chromosome inactivation (MSCI) acting on the Drosophila Y chromosome, revealing that meiotic promoters can remain weakly active despite strong repression. This work defines transcriptional limits of the Drosophila Y chromosome and informs the design of next-generation Y-linked gene drives for sustainable insect control.

genetics↗

bioRxiv2025

Mosquito Sex Separation using Complementation of Selectable Traits and Engineered Neo-Sex Chromosomes

Effective and scalable sex separation remains a critical challenge for mosquito genetic control strategies. Genetic sexing strains (GSS) address this by genetically linking maleness with selectable traits, enabling efficient removal of females before release. Here, we describe a robust platform for the development of GSSs in the invasive Aedes albopictus mosquito by integrating a CRISPR-engineered selectable phenotype with sex conversion via nix, the male-determining factor. As a proof-of-concept, we disrupt the yellow gene to generate a vivid pigmentation marker, then rescue its function in males using nix-containing transgenes, creating a stable strain where all females are yellow and all engineered males are dark. The resulting GSS males are fertile, robust, and despite lacking the ancestral M locus, exhibit gene expression profiles closely resembling wild-type males. We benchmark sex separation based on pigmentation and discover that yellow mutant females exhibit slower larval development, enhancing protandry-based sorting. The GSS strain is compatible with existing size-based sex sorting systems, allowing for improved separation accuracy through the integration of natural and engineered sexually dimorphic traits. Additionally, we find that GSS females lay desiccation-sensitive eggs, reducing the risk of accidental female releases. Our approach is the first to engineer a sex-linked selectable trait by precisely targeting an endogenous gene and restoring its function in males, establishing a versatile platform for GSS development in Aedes mosquitoes.

synthetic biology↗

bioRxiv2023

Targeting mosquito X-chromosomes reveals complex transmission dynamics of sex ratio distorting gene drives

Engineered sex ratio distorters (SRDs) have been proposed as a powerful component of genetic control strategies designed to suppress harmful insect pests. Two types of CRISPR-based SRD mechanisms have been proposed: X-shredding eliminates X-bearing sperm, while X-poisoning eliminates daughters inheriting disrupted X-chromosomes. These differences can have a profound impact on the population dynamics of SRDs when linked to the Y-chromosome: an X-shredder is invasive constituting a classical meiotic Y-drive, whereas X-poisoning is self-limiting, unable to invade but also insulated from selection. Here, we established the first X-poisoning strains in the malaria vector Anopheles gambiae targeting three X-linked genes during spermatogenesis resulting in male bias. Surprisingly, we found that sex distortion was primarily driven by a loss of X-bearing sperm with limited evidence for postzygotic lethality of daughters. By leveraging a Drosophila melanogaster model, we show unambiguously that engineered SRD traits can operate differently in these two insects. Unlike X-shredding, X-poisoning could theoretically operate at early stages of spermatogenesis. We therefore explored premeiotic Cas9 expression to target the mosquito X-chromosome. We find that, by pre-empting the onset of meiotic sex chromosome inactivation, this approach may enable the development of Y-linked SRDs if mutagenesis of spermatogenesis-essential genes is functionally balanced.

synthetic biology↗