Biology subjects

Purusothaman, D.-K.

Publications and source records attributed to Purusothaman, D.-K..

4 recordsLinked to original sources

bioRxiv2026

Telomere-to-telomere genome assembly of Microsporidia sp. MB, a microsporidian symbiont of Anopheles coluzzii isolated from Burkina Faso

BackgroundMicrosporidia sp. MB is an intracellular parasite of anopheline mosquitoes identified across the African continent. Microsporidia sp. MB infections appear to have no significant effect on host fitness and vertically transmit, whilst infected individuals have exhibited significantly reduced levels of Plasmodium falciparum transmission. These combined characteristics make Microsporidia sp. MB a promising candidate for use in malaria control. A comprehensive genome would greatly facilitate investigation into the evolution and biological pathways underlying important phenotypes, such as the mechanism of malarial inhibition. ResultsIn this study, we present the de novo assembly of the first complete genome of Microsporidia sp. MB SOUVK7. Multi-platform sequencing was performed on ovary samples of laboratory established Anopheles coluzzii collected from Burkina Faso. The SOUVK7 genome has a total size of 9.16Mb, encodes 2,435 genes and is organised into 13 chromosomes with telomeres identified at all flanks. Telomeric repeats exhibit a 4-mer motif due to a glutamine deletion previously unobserved in Microsporidia. Ploidy analysis of Illumina reads predicts MB as tetraploid, whilst analysis of CpG methylation and retroelements highlights loci in all chromosomes with characteristics consistent with regional centromeres. Orthology analysis identifies several key genes in pathways associated to telomeric and centromeric maintenance, along with methylation and host invasion machinery. A loss of several components of the infection machinery is observed in Microsporidia sp. MB and the wider Enterocytozoonida, consistent with a general trend towards genome size reduction in the clade. ConclusionThis study provides the first complete, telomere to telomere assembly of Microsporidia sp. MB, offering new insight into the genomic architecture of Microsporidia sp. MB and the broader Mrazekiidae family.

genomics↗

bioRxiv2023

CRISPR-based gene drives generate super-Mendelian inheritance in the disease vector Culex quinquefasciatus.

Culex mosquitoes pose a significant public health threat as vectors for a variety of diseases including West Nile virus and lymphatic filariasis, and transmit pathogens threatening livestock, companion animals, and endangered birds. Rampant insecticide resistance makes controlling these mosquitoes challenging and necessitates the development of new control strategies. Gene drive technologies have made significant progress in other mosquito species, although similar advances have been lagging in Culex. Here we test the first CRISPR-based homing gene drive for Culex quinquefasciatus, demonstrating the possibility of using this technology to control Culex mosquitoes. Our results show that the inheritance of two split-gene-drive transgenes, targeting different loci, are biased in the presence of a Cas9-expressing transgene although with modest efficiencies. Our findings extend the list of disease vectors where engineered homing gene drives have been demonstrated to include Culex alongside Anopheles and Aedes, and pave the way for future development of these technologies to control Culex mosquitoes.

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↗

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↗