Biology subjects

Shackleford, L.

Publications and source records attributed to Shackleford, L..

4 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↗

bioRxiv2025

Integrating multiplexing into confineable gene drives effectively overrides resistance in Anopheles stephensi

Anopheles stephensi is a major malaria vector mainly present in southern Asia and the Arabian Peninsula. Since 2012 it has invaded several countries of eastern Africa, stimulating urgent efforts to develop more efficient strategies for vector control such as CRISPR/Cas9-based homing gene drives. Target site resistance is a significant challenge to the deployment of these systems. When a double-stranded break is repaired by NHEJ, it can lead to mutations which destroy the target site, making that allele unrecognizable to the sgRNA and resistant to further cleavage. The use of multiple sgRNAs has the potential to solve this issue. We performed experimental crosses to assess the homing and cutting efficiency of two different multiplexing strategies targeting the cardinal locus, in the presence and absence of a resistance allele. We found pre-existing mutations at one sgRNA target site did not significantly reduce the homing efficiency for either strategy. Modelling indicates that while both strategies can overcome resistance allele formation, the fitness of the drive-carrying alleles is a critical factor in determining the overall performance and persistence of a split drive.

genetics↗

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↗

bioRxiv2021

CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say

Culex quinquefasciatus Say is a brown, medium sized mosquito distributed widely in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and is responsible for vectoring many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical / chemical) are increasingly ineffective; use of insecticides also imposes some hazards to both human and ecosystem health. Recent advances in genome editing have allowed the development of genetic methods of insect control, which is species-specific and, theoretically, highly effective. CRISPR/Cas9 is a bacteria-derived programmable gene editing tool that has been shown to be functional in a range of species. We demonstrate here, the first successful germline gene knock-in by homology dependent repair in C. quinquefasciatus. Using CRISPR/Cas9, we integrated exogenous sequence comprising a sgRNA expression cassette and marker gene encoding a fluorescent protein fluorophore (Hr5/IE1-DsRed, Cq7SK-sgRNA) into the kynurenine 3-monooxygenase (kmo) gene. We achieved a minimum transformation rate of 2.8% similar to rates achieved in other mosquito species. Precise knock-in at the intended locus was confirmed by sequencing. Insertion homozygotes displayed a white eye phenotype in early-mid stage larvae and a recessive lethal phenotype by pupation. This work shows an alternative and efficient method for genetic engineering of C. quinquefasciatus, providing a new tool for researchers interested in developing genetic control tools for this vector.

bioengineering↗