Biology subjects

Mameli, E.

Publications and source records attributed to Mameli, E..

9 recordsLinked to original sources

bioRxiv2026

A pro-apoptotic selection strategy enables CRISPR screening in mosquitoes and identifies Lachesin as a chikungunya virus entry factor

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that is transmitted primarily by Aedes aegypti and Aedes albopictus mosquitoes. CHIKV infection can result in debilitating arthritis-like symptoms in humans. While determinants of CHIKV host cell entry into mammalian cells are known, their equivalents in mosquito cells remain largely elusive. To identify CHIKV entry factors, we performed a membrane-focused genome-scale CRISPR loss-of-function screen in Aedes albopictus mosquito cells. To enable selection of refractory cells, we engineered CHIKV to express the pro-apoptotic Drosophila Reaper protein. CHIKV-Reaper induced robust cell death in mosquito cells with only modest viral fitness loss. Using this selection strategy together with an Aedes albopictus C6/36-based CRISPR screening platform, we identified glycosylphosphatidylinositol (GPI)-anchored cell surface proteins and multiple enzymes involved in the GPI-anchor biosynthesis pathway as proviral candidates. Ectopic expression of the GPI-anchored cell-adhesion protein Lachesin rendered refractory mammalian cells susceptible to CHIKV and the related arthritogenic alphaviruses Semliki Forest virus and Ross River virus. In contrast, Lachesin-expressing cells remained refractory to the encephalitic alphavirus Venezuelan equine encephalitis virus. We demonstrate that Lachesin is essential for CHIKV infection in both Aedes aegypti and Aedes albopictus cells, as confirmed by gene silencing. Here, we identify Lachesin as a critical candidate entry receptor for CHIKV and establish pro-apoptotic arboviruses as a powerful and versatile strategy for functional CRISPR screening in mosquito cells.

microbiology↗

bioRxiv2026

Lachesin is a mosquito receptor for multiple arthritogenic alphaviruses

Arthritogenic alphaviruses cause acute febrile illnesses associated with rash and arthritis when they are transmitted to humans through the bite of infected mosquitoes. Among these, chikungunya virus (CHIKV), transmitted primarily through the bite of infected Aedes aegypti and Aedes albopictus mosquitoes, causes explosive outbreaks involving hundreds of thousands to millions of cases, with recent re-emergence in several global regions. While several cellular receptors that mediate alphavirus entry into mammalian cells have been identified, their mosquito counterparts remained unknown, largely due to a lack of functional genomics tools for these invertebrate species. Here, we established a CRISPR-based genetic screening platform in Aedes albopictus cells and used it to identify Lachesin, a conserved invertebrate cell adhesion molecule, as a receptor for CHIKV and multiple related alphaviruses including Semliki Forest virus (SFV), onyong-nyong virus (ONNV), Mayaro virus (MAYV), and Ross River virus (RRV). Lachesin depletion using RNA interference, anti-Lachesin antibody treatment, and soluble forms of Lachesin blocked CHIKV and SFV E2-E1 glycoprotein-mediated infection of mosquito cells. We show that alphavirus E2-E1 glycoproteins bind the first immunoglobulin domain of Lachesin, facilitating attachment and internalization of virus-like particles. Orthologs from divergent mosquito genera, but not from arachnids or other arthropods, also serve as alphavirus receptors, suggesting that cellular receptor binding is not the main obstacle to arthritogenic alphavirus vector host expansion. Our findings enhance understanding of the mechanisms of alphavirus emergence and vector competence and could aid in the development of broadly active, entry-targeted therapeutics against multiple alphaviruses that threaten public health.

microbiology↗

bioRxiv2026

Genome-wide CRISPR knockout cell screening platform for the disease vector tick species Ixodes scapularis

The black legged tick, Ixodes scapularis, is a vector of the bacterium that causes Lyme disease and several other illnesses, including anaplasmosis, babesiosis, and tick-borne encephalitis. Although high-quality genome annotations are available for I. scapularis, functional understanding of I. scapularis genes is limited. To address this, we developed a platform for genome-wide CRISPR-Cas9 knockout screening in I. scapularis cells. To evaluate the platform, we performed a screen to identify genes associated with cellular fitness, and screens for resistance to treatment with copper chloride, Antimycin A, or Destruxin A (DA), a cyclic hexadepsipeptide produced by the pathogenic fungus Metarhizium anisopliae. In each case, the screens implicate specific sets of conserved and non-conserved I. scapularis genes in relevant cellular functions, providing the first experimental evidence of function for a large set of I. scapularis genes. Altogether, in this first-of-its-kind effort for the arthropod subclass Acari, we present an unbiased genome-wide CRISPR-Cas9 knockout cell screening platform, related resources, and datasets that will be broadly useful to efficiently uncover cellular functions of I. scapularis genes.

genetics↗

bioRxiv2025

A family of lethal exotoxins defined by cell entry via the Attractin receptor

Although bacterial genomes encode numerous potential toxins, it is unclear how evolution drives the specificity of these important virulence factors. Using an insect CRISPR screen, we identified the transmembrane protein Attractin (ATRN) as the receptor for Nigritoxin (Ntx), a Vibrio toxin that causes seasonal shrimp pandemics. We found that Ntxs effector "warhead" inhibits translation via a previously uncharacterized mechanism. Moreover, we show that two related toxins require ATRN for entry but possess unrelated effector domains. One has a Rho-GTPase AMPylation function and the other an actin-targeting/proteolysis function. Our findings reveal the mechanism of Ntx entry and toxicity and show that the ATRN-targeting domain can deliver disparate effector domains, strongly indicating that this class of exotoxins can evolve as modular proteins using a common entry domain.

microbiology↗

bioRxiv2025

Engineering gene drive docking sites in a haplolethal locus in Anopheles gambiae

Gene drives are selfish genetic elements which promise to be powerful tools in the fight against vector-borne diseases such as malaria. We previously proposed population replacement gene drives designed to better withstand the evolution of resistance by homing through haplolethal loci. Because most mutations in the wild-type allele that would otherwise confer resistance are lethal, only successful drive homing permits the cell to survive. Here we outline the development and characterization of two {Phi}C31-Recombination mediated cassette exchange (RMCE) gene drive docking lines with these features in Anopheles gambiae, a first step towards construction of robust gene drives in this important malaria vector. We outline adaption of the technique HACK (Homology Assisted CRISPR knockin) to knock-in two docking site sequences into a paired haplolethal-haplosufficient (Ribosome-Proteasome) locus, and confirm that these docking lines permit insertion of drive-relevant transgenes. We report the first anopheline proteasome knockouts, and identify ribosome mutants that reveal a major hurdle that such designs must overcome to develop robust drives in the future. Although we do not achieve drive, this work provides a new tool for constructing future evolution-robust drive systems and reveals critical challenges that must be overcome for future development of gene drives designed to target haplolethal loci in anophelines and, potentially, other metazoans.

synthetic biology↗

bioRxiv2024

A genome-wide CRISPR screen in Anopheles mosquito cells identifies essential genes and required components of clodronate liposome function

Anopheles mosquitoes are the sole vector of human malaria, the most burdensome vector-borne disease worldwide. Strategies aimed at reducing mosquito populations and limiting their ability to transmit disease show the most promise for disease control. Therefore, gaining an improved understanding of mosquito biology, and specifically that of the immune response, can aid efforts to develop new approaches that limit malaria transmission. Here, we use a genome-wide CRISPR screening approach for the first time in mosquito cells to identify essential genes in Anopheles and identify genes for which knockout confers resistance to clodronate liposomes, which have been widely used in mammals and arthropods to ablate immune cells. In the essential gene screen, we identified a set of 1280 Anopheles genes that are highly enriched for genes involved in fundamental cell processes. For the clodronate liposome screen, we identified several candidate resistance factors and confirm their roles in the uptake and processing of clodronate liposomes through in vivo validation in Anopheles gambiae, providing new mechanistic detail of phagolysosome formation and clodronate liposome function. In summary, we demonstrate the application of a genome-wide CRISPR knockout platform in a major malaria vector and the identification of genes that are important for fitness and immune-related processes.

genetics↗

bioRxiv2023

Tick hemocytes have pleiotropic roles in microbial infection and arthropod fitness

Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here, we describe immune cells or hemocytes from the clinically relevant tick Ixodes scapularis using bulk and single cell RNA sequencing combined with depletion via clodronate liposomes, RNA interference, Clustered Regularly Interspaced Short Palindromic Repeats activation (CRISPRa) and RNA-fluorescence in situ hybridization (FISH). We observe molecular alterations in hemocytes upon tick infestation of mammals and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We predict distinct hemocyte lineages and reveal clusters exhibiting defined signatures for immunity, metabolism, and proliferation during hematophagy. Furthermore, we perform a mechanistic characterization of two I. scapularis hemocyte markers: hemocytin and astakine. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, which impacts blood feeding and molting behavior of ticks. Hemocytin specifically affects the c-Jun N-terminal kinase (JNK) signaling pathway, whereas astakine alters hemocyte proliferation in I. scapularis. Altogether, we uncover the heterogeneity and pleiotropic roles of hemocytes in ticks and provide a valuable resource for comparative biology in arthropods.

immunology↗

bioRxiv2021

Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos

Mosquito-borne diseases present a worldwide public health burden. Genome-scale screening tools that could inform our understanding of mosquitos and their control are lacking. Here, we adapt a recombination-mediated cassette exchange system for delivery of CRISPR sgRNA libraries into cell lines from several mosquito species and perform pooled CRISPR screens in an Anopheles cell line. To implement this method, we engineered modified mosquito cell lines, validated promoters and developed bioinformatics tools for multiple mosquito species.

genetics↗

bioRxiv2021

Optimized CRISPR tools and site-directed transgenesis in Culex quinquefasciatus mosquitoes for gene drive development.

Culex mosquitoes are a global vector for multiple human and animal diseases, including West Nile virus, lymphatic filariasis, and avian malaria, posing a constant threat to public health, livestock, companion animals, and endangered birds. While rising insecticide resistance has threatened the control of Culex mosquitoes, advances in CRISPR genome-editing tools have fostered the development of alternative genetic strategies such as gene drive systems to fight disease vectors. However, though gene-drive technology has quickly progressed in other mosquitoes, advances have been lacking in Culex. Here, we developed a Culex-specific Cas9/gRNA expression toolkit and used site-directed homology-based transgenesis to generate and validate a Culex quinquefasciatus Cas9-expressing line. We showed that gRNA scaffold variants improve transgenesis efficiency in both Culex and Drosophila and boost gene-drive performance in the fruit fly. These findings support future technology development to control Culex mosquitoes and provide valuable insight for improving these tools in other species.

synthetic biology↗