Biology subjects

Nevard, K.

Publications and source records attributed to Nevard, K..

3 recordsLinked to original sources

bioRxiv2026

Knock-in = knock-out: differential fitness effects of cardinal mutations in Anopheles stephensi

The kynurenine pathway metabolizes tryptophan into 3-hydroxykynurenine (3-HK), a precursor for ommochrome eye pigments synthesized via the cardinal (cd) gene in mosquitoes. While cd disruption was presumed neutral, we observed fitness costs in Anopheles stephensi knock-in but not knock-out cd mutants. Here we investigated this anomaly further by assessing survival, fecundity, and midgut integrity across multiple cd mutant lines. Heterozygous knock-in lines, expressing a fluorescent marker and guide RNA for CRISPR/Cas9, exhibited reduced survival post-blood feeding, larva-to-adult survival deficits, and midgut barrier dysfunction, whereas knock-outs showed no such costs. Oral supplementation with xanthurenic acid partially rescued knock-in mortality, implicating oxidative stress linked to 3-HK metabolism. Expression analyses suggest transgene insertion effects, rather than cd disruption, underlie these fitness costs. These findings highlight the importance of evaluating insertional effects in gene drive target selection and support cd as a viable target for genetic control strategies in An. stephensi.

genetics↗

bioRxiv2023

Germline transformation of the West Nile Virus and avian malaria vector Culex quinquefasciatus Say using the piggyBac transposon system

Culex quinquefasciatus Say is a mosquito which acts as a vector for numerous diseases including West Nile Virus, lymphatic filariasis and avian malaria, over a broad geographical range. As the effectiveness of insecticidal mosquito control methods declines, the need has grown to develop genetic control methods to curb the spread of disease. The piggyBac transposon system - the most widely used genetic transformation tool in insects, including mosquitoes - generates quasi-random insertions of donor DNA into the host genome. However, despite the broad reported species range of piggyBac, previous attempts to use this tool to transform Culex quinquefasciatus mosquitoes have failed. Here we report the first successful transformation of Culex quinquefasciatus with the piggyBac transposon system. Using commercially synthesised piggyBac mRNA as a transposase source, we were able to generate three independent insertions of a ZsGreen fluorescent marker gene, with transformation efficiencies of up to 5%. Through this work, we have expanded the genetic toolkit available for the genetic manipulation of Culex mosquitoes and thus removed a barrier to developing novel genetic control methods in this important disease vector.

synthetic biology↗

bioRxiv2022

A multiplexed, confinable CRISPR/Cas9 gene drive propagates in caged Aedes aegypti populations

Aedes aegypti, the yellow fever mosquito, is the main vector of several major pathogens including yellow fever, dengue, Zika and chikungunya viruses. Classical mosquito control strategies, mainly utilizing insecticides, have had success in controlling other mosquito vectors in recent years, but are much less useful against Ae. aegypti, and even these methods are threatened by rising insecticide resistance. This has stimulated interest in new mosquito control mechanisms, notably genetic systems such as gene drives. However, the development of CRISPR/Cas9 gene drive systems has faced challenges such as low inheritance biasing rate, the emergence of resistance alleles, and the possibility of spreading beyond the intended population. Here, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm (bgcn) homolog to express Cas9 in the germline to find an expression timing more conducive to homing. We also created a separate multiplexing (targeting multiple different sites within the target gene) sgRNA-expressing homing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene to limit the consequences of resistance alleles. This creates a split gene drive such that one part does not drive, allowing control over geographic spread and temporal persistence. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element was driven through a cage trial population such that carrier frequency of the element increased from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system. Significance statementMosquito-borne diseases affect millions of people worldwide, with the yellow fever mosquito (Aedes aegypti) being the principal vector of many viral diseases. Effective measures for controlling this mosquito are sorely needed. Gene drive systems have arisen as a potential tool for mosquito control due to their ability of biasing inheritance of a trait into a target population. Here, we assess a split gene drive, based on CRISPR/Cas9 endonuclease technology driving a target element into the mosquito population. Evaluated over successive generations in a replicated cage trial, the drive successfully biased its inheritance, increasing in frequency from 50% to up to 89%. Our results are encouraging for the potential use of this type of contained gene drive system for mosquito control in endemic areas.

synthetic biology↗