Biology subjects

Capriotti, P.

Publications and source records attributed to Capriotti, P..

6 recordsLinked to original sources

bioRxiv2025

The nanosd integral gene drive enables population modification of the malaria vector Anopheles gambiae

The modification of mosquito populations at scale through CRISPR-Cas9-mediated homing gene drives is a promising route for malaria vector control. Integral gene drives (IGDs) are designed to utilize the regulatory sequences of endogenous genes to express only the minimal set of components required for gene drive. In this study, we describe the creation and characterization of the nanosd IGD targeting and inserted into the nanos gene of the malaria vector Anopheles gambiae and show that it achieves high rates of gene drive (97.7% in females, 99.0% in males). We find that homozygous nanosd females but not males show impaired fecundity, and a variable loss of ovary phenotype. Transcriptomic analysis of ovaries points to decreased transcript levels of the nanos gene when harbouring Cas9. As a minimal genetic modification, nanosd does not induce widespread transcriptomic perturbations, and its susceptibility to Plasmodium spp. and Onyong nyong virus infection remains similar to wild-type mosquitoes. Importantly, we find that nanosd propagates efficiently in caged mosquito populations and is maintained as a source of Cas9 after the emergence of drive resistant alleles whilst also mobilising a non-autonomous antiparasitic effector modification. The nanosd gene drive shows promise as a genetic tool for malaria vector control via population modification, and we outline steps towards its further optimization.

synthetic biology↗

bioRxiv2024

REL2 overexpression in the Anopheles gambiae midgut causes major transcriptional changes but fails to induce an immune response

The NF-{kappa}B-like transcription factor, REL2, is a key player in the mosquito Immunodeficiency (Imd) pathway and holds promise for controlling malaria parasite infections in genetically modified Anopheles gambiae mosquitoes. We engineered transgenic mosquitoes overexpressing REL2 from within the bloodmeal-inducible zinc carboxypeptidase A1 (CP) host gene in the adult posterior midgut. Our results confirmed elevated REL2 expression in the posterior midgut following a bloodmeal, with the corresponding protein localized within epithelial cell nuclei. While this induced overexpression triggered substantial transcriptional changes, accompanied by notable fitness costs, the resultant reduction in Plasmodium falciparum infection was modest. An in-depth analysis of regulatory regions of differentially regulated genes allowed us to identify direct REL2 target genes and revealed signatures indicative of potential transcriptional repressors. To account for potential impacts of host gene modification, we also created a CP knockout line that caused marginal effects on mosquito fitness. These findings shed light on the observed absence of transcriptional activation and, in some cases, induced repression of antimicrobial peptides (AMPs) presumed to be under Imd pathway control. In conclusion, our study suggests that elevated REL2 expression in the posterior midgut may induce the upregulation of negative immune regulators, facilitating control over an otherwise unrestrained immune response, and that concurrent transcriptional derepression may be needed to effectively induce the mosquito immune response. This work contributes valuable insights into the intricate regulation of midgut immunity in malaria vector mosquitoes.

immunology↗

bioRxiv2022

Gene drive mosquitoes can aid malaria elimination by retarding Plasmodium sporogonic development

Gene drives hold promise for the genetic control of malaria vectors. The development of vector population modification strategies hinges on the availability of effector mechanisms impeding parasite development in transgenic mosquitoes. We augmented a midgut gene of the malaria mosquito Anopheles gambiae to secrete two exogenous antimicrobial peptides, Magainin 2 and Melittin. This small genetic modification, capable of efficient non-autonomous gene drive, hampers oocyst development in both Plasmodium falciparum and Plasmodium berghei. It delays the release of infectious sporozoites while it simultaneously reduces the lifespan of homozygous female transgenic mosquitoes. Modeling the spread of this modification using a large-scale agent-based model of malaria epidemiology reveals that it can break the cycle of disease transmission across a range of endemic settings. One sentence summaryWe developed a gene drive effector that retards Plasmodium development in transgenic Anopheles gambiae mosquitoes via the expression of antimicrobial peptides in the midgut and which is predicted to eliminate malaria under a range of transmission scenarios.

genetics↗

bioRxiv2021

Assessment of Plasmodium falciparum infection and fitness of genetically modified Anopheles gambiae aimed at mosquito population replacement

Genetically modified (GM) mosquitoes expressing anti-plasmodial effectors propagating through wild mosquito populations by means of gene drive is a promising tool to support current malaria control strategies. The process of generating GM mosquitoes involves genetic transformation of mosquitoes from a laboratory colony and, often, interbreeding with other GM lines to cross in auxiliary traits. These mosquito colonies and GM lines thus often have different genetic backgrounds and GM lines are invariably highly inbred, which in conjunction with their independent rearing in the laboratory may translate to differences in their susceptibility to malaria parasite infection and life history traits. Here, we show that laboratory Anopheles gambiae colonies and GM lines expressing Cas9 and Cre recombinase vary greatly in their susceptibility to Plasmodium falciparum NF54 infection. Therefore, the choice of mosquitoes to be used as a reference when conducting infection or life history trait assays requires careful consideration. To address these issues, we established an experimental pipeline involving genetic crosses and genotyping of mosquitoes reared in shared containers throughout their lifecycle. We used this protocol to examine whether GM lines expressing the antimicrobial peptide (AMP) Scorpine in the mosquito midgut interfere with parasite infection and mosquito survival. We demonstrate that Scorpine expression in the Peritrophin 1 (Aper1) genomic locus reduces both P. falciparum sporozoite prevalence and mosquito lifespan; both these phenotypes are likely to be associated with the disturbance of the midgut microbiota homeostasis. These data lead us to conclude that the Aper1-Sco GM line could be used in proof-of-concept experiments aimed at mosquito population replacement, although the impact of its reduced fitness on the spread of the transgene through wild populations requires further investigation.

microbiology↗

bioRxiv2020

Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for population replacement

Gene drives for mosquito population replacement are promising tools for malaria control. However, there is currently no clear pathway for safely testing such tools in endemic countries. The lack of well-characterized promoters for infection-relevant tissues and regulatory hurdles are further obstacles for their design and use. Here we explore how minimal genetic modifications of endogenous mosquito genes can convert them directly into non-autonomous gene drives without disrupting their expression. We co-opted the native regulatory sequences of three midgut-specific loci of the malaria vector Anopheles gambiae to host a prototypical antimalarial molecule and guide-RNAs encoded within artificial introns, that support efficient gene drive. We assess the propensity of these modifications to interfere with the development of Plasmodium falciparum and their effect on fitness. Because of their inherent simplicity and passive mode of drive such traits could form part of an accepted testing pathway of gene drives for malaria eradication.

synthetic biology↗

bioRxiv2018

Rationally-engineered reproductive barriers using CRISPR & CRISPRa: an evaluation of the synthetic species concept in Drosophila melanogaster

The ability to erect rationally-engineered reproductive barriers in animal or plant species promises to enable a number of biotechnological applications such as the creation of genetic firewalls, the containment of gene drives or novel population replacement and suppression strategies for genetic control. However, to date no experimental data exist that explores this concept in a multicellular organism. Here we examine the requirements for building artificial reproductive barriers in the metazoan model Drosophila melanogaster by combining CRISPR-based genome editing and transcriptional transactivation (CRISPRa) of the same loci. We directed 13 single guide RNAs (sgRNAs) to the promoters of 7 evolutionary conserved genes and used 11 drivers to conduct a miss-activation screen. We identify dominant-lethal activators of the eve locus and find that they disrupt development by strongly activating eve outside its native spatio-temporal context. We employ the same set of sgRNAs to isolate, by genome editing, protective INDELs that render these loci resistant to transactivation without interfering with target gene function. When these sets of genetic components are combined we find that complete synthetic lethality, a prerequisite for most applications, is achievable using this approach. However, our results suggest a steep trade-off between the level and scope of dCas9 expression, the degree of genetic isolation achievable and the resulting impact on fly fitness. The genetic engineering strategy we present here allows the creation of single or multiple reproductive barriers and could be applied to other multicellular organisms such as disease vectors or transgenic organisms of economic importance.

synthetic biology↗