Biology subjects

Akbari, O.

Publications and source records attributed to Akbari, O..

6 recordsLinked to original sources

bioRxiv2018

Site-specific transgenesis of the D. melanogaster Y-chromosome using CRISPR/Cas9

Despite the importance of Y-chromosomes in evolution and sex determination, their heterochromatic, repeat-rich nature makes them difficult to sequence and genetically manipulate, and therefore they generally remain poorly understood. For example, the D. melanogaster Y-chromosome, one of the best understood, is widely heterochromatic and composed mainly of highly repetitive sequences, with only a handful of expressed genes scattered throughout its length. Efforts to insert transgenes on this chromosome have thus far relied on either random insertion of transposons (sometimes harboring landing sites for subsequent integrations) with limited success or on chromosomal translocations, thereby limiting the types of Y-chromosome related questions that could be explored. Here we describe a versatile approach to site-specifically insert transgenes on the Y-chromosome in D. melanogaster via CRISPR/Cas9-mediated HDR. We demonstrate the ability to insert, and detect expression from, fluorescently marked transgenic transgenes at two specific locations on the Y-chromosome, and we utilize these marked Y-chromosomes to detect and quantify rare chromosomal nondisjunction effects. Finally, we discuss how this Y-docking technique could be adapted to other insects to aid in the development of genetic control technologies for the management of insect disease vectors and pests.

genetics↗

bioRxiv2017

Can CRISPR-based gene drive be confined in the wild? A question for molecular and population biology

The recent discovery of CRISPR and its application as a gene editing tool has enabled a range of gene drive systems to be engineered with much greater ease. In order for the benefits of this technology to be realized, drive systems must be developed that are capable of both spreading into populations to achieve their desired impact, and being recalled in the event of unwanted consequences or public disfavor. We review the performance of three broad categories of drive systems at achieving these goals - threshold-dependent drives, homing-based drive and remediation systems, and temporally self-limiting systems such as daisy-chain drives.

genetics↗

bioRxiv2017

Modulation of host learning in Aedes aegypti mosquitoes

How mosquitoes determine which individuals to bite has important epidemiological consequences. This choice is not random; most mosquitoes specialize in one or a few vertebrate host species, and some individuals in a host population are preferred over others. Here we show that aversive olfactory learning contributes to mosquito preference both between and within host species. Combined electrophysiological and behavioural recordings from tethered flying mosquitoes demonstrated that these odours evoke changes in both behaviour and antennal lobe (AL) neuronal responses. Using electrophysiological and behavioural approaches, and CRISPR gene editing, we demonstrate that dopamine plays a critical role in aversive olfactory learning and modulating odour-evoked responses in AL neurons. Collectively, these results provide the first experimental evidence that olfactory learning in mosquitoes can play an epidemiological role.

neuroscience↗

bioRxiv2017

Highly efficient site-specific mutagenesis in Malaria mosquitoes using CRISPR

Anopheles mosquitoes transmit at least 200 million annual malaria infections worldwide. Despite considerable genomic resources, mechanistic understanding of biological processes in Anopheles has been hampered by a lack of tools for reverse genetics. Here, we report successful application of the CRISPR/Cas9 system for highly efficient, site-specific mutagenesis in the diverse malaria vectors Anopheles albimanus, Anopheles coluzzii, and Anopheles funestus. When guide RNAs and Cas9 are injected at high concentration, germline mutations are common and usually bi-allelic allowing for the rapid creation of stable, mutant lines for reverse genetic analysis. Our protocol should enable researchers to dissect the molecular and cellular basis of anopheline traits critical to successful disease transmission, potentially exposing new targets for malaria control.

genetics↗

bioRxiv2017

Germline Cas9 Expression Yields Highly Efficient Genome Engineering in a Major Worldwide Disease Vector, Aedes aegypti

The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti. Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR. Using these strains, we disrupted numerous genes important for normal morphological development, and even generated triple mutants from a single injection. We have also managed to increase the rates of homology directed repair by more than an order of magnitude. Given the exceptional mutagenic efficiency and specificity of the Cas9 strains we built, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a first step towards the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives.\n\nSignificance StatementAedes aegypti is the principal vector of multiple arboviruses that significantly affect human health including dengue, chikungunya, and zika. Development of tools for efficient genome engineering in this mosquito will not only lay the foundation for the application of novel genetic control strategies that do not rely on insecticides, but will also accelerate basic research on key biological processes involved in disease transmission. Here, we report the development of a transgenic CRISPR approach for rapid gene disruption in this organism. Given their high editing efficiencies, the Cas9 strains we developed can be used to quickly generate novel genome modifications allowing for high-throughput gene targeting, and can possibly facilitate the development of gene drives, thereby accelerating comprehensive functional annotation and development of innovative population control strategies for Ae. aegypti.

bioengineering↗

bioRxiv2016

Engineered reciprocal chromosome translocations drive high threshold, reversible population replacement in Drosophila

Replacement of wild insect populations with transgene-bearing individuals unable to transmit disease or survive under specific environmental conditions provides self-perpetuating methods of disease prevention and population suppression, respectively. Gene drive mechanisms that require the gene drive element and linked cargo exceed a high threshold frequency to spread are attractive because they offer several points of control: they bring about local, but not global population replacement; and transgenes can be eliminated by reintroducing wildtypes into the population so as to drive the frequency of transgenes below the threshold required for drive. It has long been recognized that reciprocal chromosome translocations could, in principal, be used to bring about high threshold gene drive through a form of underdominance. However, translocations able to drive population replacement have not been reported, leaving it unclear if translocation-bearing strains fit enough to mediate gene drive can easily be generated. Here we use modeling to identify a range of conditions under which translocations should spread, and the equilibrium frequencies achieved, given specific introduction frequencies, fitness costs and migration rates. We also report the creation of engineered translocation-bearing strains of Drosophila melanogaster, generated through targeted chromosomal breakage and homologous recombination. By several measures translocation-bearing strains are fit, and drive high threshold, reversible population replacement in laboratory populations. These observations, together with the generality of the tools used to generate translocations, suggest that engineered translocations may be useful for controlled population replacement in many species.

evolutionary biology↗