Complete population collapse of malaria vector Anopheles gambiae in cages is achieved using a gene drive that targets doublesex. In the human gambiae, doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), control differentiation sexes. The female transcript, unlike male, contains an exon (exon 5) whose sequence highly conserved all mosquitoes so far analyzed. We found CRISPR–Cas9-targeted disruption intron 4–exon 5 boundary aimed at...

Gene drives have enormous potential for the control of insect populations medical and agricultural relevance. By preferentially biasing their own inheritance, gene can rapidly introduce genetic traits even if these confer a negative fitness effect on population. We recently developed based CRISPR nuclease constructs that are designed to disrupt key genes essential female fertility in malaria mosquito. The construct copies itself associated disruption from one homologous chromosome another...

Homing-based gene drives use a germline source of nuclease to copy themselves at specific target sites in genome and bias their inheritance. Such can be designed spread deliberately suppress populations malaria mosquitoes by impairing female fertility. However, strong unintended fitness costs the drive propensity generate resistant mutations limit drive’s potential spread. Alternative regulatory sequences element confer improved fecundity carrier individuals reduced for site resistance. This...

CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading suppression of mosquito populations in the laboratory. This class relies on CRISPR-Cas9 cleavage a target sequence and copying ('homing') therein drive element from homologous chromosome. However, site mutations that are resistant yet maintain function expected strongly selected for. Targeting functionally constrained regions where not easily tolerated should lower...

CRISPR-Cas9 nuclease-based gene drives have been developed toward the aim of control human malaria vector Anopheles gambiae . Gene are based on an active source Cas9 nuclease in germline that promotes super-Mendelian inheritance transgene by homology-directed repair (“homing”). Understanding whether CRISPR-induced off-target mutations generated mosquitoes is important aspect risk assessment before any potential field release this technology. We compared frequencies and propensity events to...

Abstract Although of high priority for the development genetic tools to control malaria-transmitting mosquitoes, only a few germline-specific regulatory regions have been characterised date and presence global mechanisms, such as dosage compensation meiotic sex chromosome inactivation (MSCI), are mostly assumed from transcriptomic analyses reproductive tissues or whole gonads. In studies, samples include significant portion somatic inevitably complicating reconstruction defined...

Abstract CRISPR-Cas9 nuclease-based gene drives rely on inducing chromosomal breaks in the germline that are repaired ways lead to a biased inheritance of drive. Gene designed impair female fertility can suppress populations mosquito vector malaria. However, strong unintended fitness costs, due ectopic nuclease expression, and high levels resistant mutations, limited potential first generation spread. Here we show changes regulatory sequences drive element, contain expression germline,...

Abstract 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 major vectors malaria. While Anopheline had previously been implicated male mating behavior, recent data from gambiae complex suggests that, apart putative primary sex-determiner, no other genes are conserved on Y. Studying functional basis evolutionary divergence complicated by complete...

Genetic control strategies aimed to bias the sex of progenies towards males present a promising new paradigm eliminate malaria-transmitting mosquitoes. A synthetic sex-ratio distortion (SD) system was successfully engineered in Anopheles gambiae by exploiting meiotic activity I-PpoI endonuclease targeting ribosomal DNA (rDNA) repeats, exclusively located on X chromosome. Males carrying SD construct produce highly male-biased without evident reduction fertility. In this study, we investigated...

The increase in molecular tools for the genetic engineering of insect pests and disease vectors, such as Anopheles mosquitoes that transmit malaria, has led to an unprecedented investigation genomic landscape these organisms. understanding genome variability wild mosquito populations is primary importance vector control strategies. This particularly case gene drive systems, which look introduce traits into a population by targeting specific regions. Gene targets with functional or structural...

The availability of the genomic sequence malaria mosquito Anopheles gambiae has in recent years sparked development transgenic technologies with potential to be used as novel vector control tools. These rely on genome editing that confer traits able affect vectorial capacity. This can achieved by either reducing population or making mosquitoes refractory parasite infection. For any genetically modified organism is regarded for release, molecular characterization transgene and flanking sites...

Targeting genes involved in sexual determinism, for vector or pest control purposes, requires a better understanding of their polymorphism natural populations order to ensure rapid spread the construct. By using genomic data from An. gambiae s.l., we analyzed genetic variation and conservation score fru gene 18 across Africa. A total 34,339 SNPs were identified, including 3.11% non-synonymous segregating sites. Overall, nucleotide diversity was low, Tajima’s D neutrality test negative,...

Abstract Despite efforts to explore the genome of malaria vector Anopheles gambiae , Y chromosome this species remains enigmatic. The large number repetitive and heterochromatic DNA sequences makes exceptionally difficult fully assemble, hampering progress gene editing techniques functional studies for chromosome. In study, we made use a bioinformatic platform identify Y-specific that served as target site CRISPR/Cas9 system. activity Cas9 in reproductive organs males caused damage Y-bearing...

Despite efforts to explore the genome of malaria vector Anopheles gambiae , Y chromosome this species remains enigmatic. The large number repetitive and heterochromatic DNA sequences makes exceptionally difficult fully assemble, hampering progress gene editing techniques functional studies for chromosome. In study, we made use a bioinformatic platform identify Y-specific that served as target site CRISPR/Cas9 system. activity Cas9 in reproductive organs males caused damage Y-bearing sperm...

The crucial molecular factors that shape the interfaces of lipid-binding proteins with their target ligands and surfaces remain unknown due to complex makeup biological membranes. Cholesterol, major modulator bilayer structure in mammalian cell membranes, is recognized by various proteins, including well-studied cholesterol-dependent cytolysins. Here, we use vitro evolution investigate adaptations preserve cholesterol specificity perfringolysin O, prototypical cytolysin from Clostridium...

<title>Abstract</title> Background The recent success of a population control gene drive targeting the <italic>doublesex</italic> in <italic>Anopheles gambiae</italic> paved way for development self-sustaining and self-limiting genetic strategies sex determination pathway to reduce and/or distort reproductive capacity insect vectors. However, these genes purposes requires better understanding their variation natural populations ensure effective spread. Using whole genome sequencing data from...

Abstract Background The recent success of a population control gene drive targeting the doublesex in Anopheles gambiae paved way for developing self-sustaining and self-limiting genetic strategies sex determination pathway to reduce and/or distort reproductive capacity insect vectors. However, these genes requires better understanding their variation natural populations ensure effective spread. Using whole genome sequencing (WGS) data from Ag1000G project (Ag3.0, 3.4 3.8), Illumina pooled...

By using developed ribosomal display, we discovered variants of perfringolysin O, a pore forming toxin from bacteria<italic>Clostridium perfringens</italic>, with non-conserved amino acid substitutions at regions crucial for cholesterol recognition.

Abstract 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 major vectors malaria. While Anopheline had previously been implicated male mating behavior, recent data from gambiae complex suggests that, apart putative primary sex-determiner, no other genes are conserved on Y. Studying functional basis evolutionary divergence complicated by complete...

Targeting genes involved in sexual determinism for vector or pest control purpose, requires a better understanding of their polymorphism natural populations order to ensure rapid spread the construct. By using genomic data from An. gambiae s.l., we analyzed genetic variation and conservation score fru gene 18 across Africa. A total 34339 SNPs were identified including 3.11% non-synonymous segregating sites. Overall, nucleotide diversity was low Tajima's D neutrality test negative indicating...

Abstract CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading suppression of mosquito populations in the laboratory. This class relies on CRISPR-Cas9 cleavage a target sequence and copying (‘homing’) therein drive element from homologous chromosome. However, site mutations that are resistant yet maintain function expected strongly selected for. Targeting functionally constrained regions where not easily tolerated should...