Biology subjects

Guerra, F.

Publications and source records attributed to Guerra, F..

4 recordsLinked to original sources

bioRxiv2025

SYNCAS-Mediated CRISPR-Cas9 Genome Editing in the Jewel Wasp, Nasonia vitripennis

Genetic engineering is a formidable approach to study biology. The development of CRISPR-Cas9 has allowed the genetic engineering of insect species from several orders, and in some species, this tool is used routinely for genetic research. However, insect gene editing often relies on the delivery of CRISPR-Cas9 components via embryo injection. This technique has a limitation: some species lay their eggs inside hard substrates or living hosts, making embryo collection impossible or labour-intensive. Recently, a variety of techniques that exploit maternal injection of nucleases have been developed to circumvent embryo injection. Yet, despite this variety of maternal delivery techniques, some insects remain refractory to gene editing. One of these is the parasitoid wasp, Nasonia vitripennis, an important hymenopteran model species. In this study, a recently developed method termed SYNCAS was used to perform knock-out (KO) of the cinnabar gene in this wasp, obtaining KO efficiencies up to ten times higher than reported for other maternal injection approaches. We found up to 2.73% of all offspring to display a KO phenotype, and we obtained up to 68 KO offspring per 100 injected mothers. The optimal timing of injection and provision of hosts for egg laying was determined. With this protocol, routine applications of CRISPR-Cas9 become feasible in this species, allowing reverse genetics studies of genes with unknown associated phenotypes and paving the way for more advanced editing techniques. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/643426v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@170bdd6org.highwire.dtl.DTLVardef@bf3cbaorg.highwire.dtl.DTLVardef@12be928org.highwire.dtl.DTLVardef@1d85e6a_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIMaternal injection of Cas9 RNP together with BAPC and saponins (SYNCAS) results in efficient gene editing of Nasonia vitripennis offspring C_LIO_LITiming of injection and egg collection is important for maximising efficiency and reducing screening effort C_LIO_LIThe efficient genetic engineering of Nasonia vitripennis suggests the applicability of SYNCAS to other parasitoid wasps, a category of insects particularly difficult to genetically engineer with embryonic Cas9 injections. C_LI

molecular biology↗

bioRxiv2025

A transcriptional control model for doublesex-dependent sex differentiation in Nasonia wasps

The transcription factor Doublesex (DSX) orchestrates insect sex differentiation. DSX affects Drosophila melanogaster male and female transcriptome, yet how DSX regulates gene expression in other species is poorly understood. We investigated sex-biased gene expression during the development of the parasitoid wasp Nasonia vitripennis, finding that more than three-quarters of its genes are sex-biased in at least one developmental point. Next, we transiently knocked down dsx expression to infer its role in sex-specific transcriptome regulation, revealing thousands of affected genes in males and a more subtle effect in females. Finally, we performed an in vitro DNA-protein interaction assay to identify DSX binding sites on the genome and primary DSX target genes. By integrating these three datasets, we defined DSXs regulatory function for all genes in N. vitripennis, revealing that DSX acts mainly in males as both an activator and a repressor. This male-centric model for DSX-mediated regulation is likely to apply to many other insect species.

molecular biology↗

bioRxiv2023

A Library of Electrophysiological Responses in Plants - A Model of Transversal Education and Open Science

Electrophysiology in plants is understudied, and, moreover, an ideal model for student inclusion at all levels of education. Here we report on an investigation in "open science", whereby scientists worked with students and faculty from Chile, Germany, Serbia, South Korea, and the USA. The students recorded the electrophysiological signals of >15 plants in response to a flame or tactile stimulus applied to the leaves. We observed that approximately 60% of the plants studied showed an electrophysiological response with a delay of [~]3-6 seconds after stimulus presentation. In preliminary conduction velocity experiments, we verified that observed signals are indeed biological in origin, with information transmission speeds of [~]2-9 mm/s. Such easily replicable experiments can serve to include more investigators and students in contributing to our understanding of plant electrophysiology.

plant biology↗

bioRxiv2021

Mitochondria dysfunction in Charcot Marie Tooth 2B Peripheral Sensory Neuropathy

Recent evidence has uncovered an important role of Rab7 in regulating mitochondrial morphology and function. Missense mutation(s) of Rab7 underlies the pathogenesis of Charcot Marie Tooth 2B (CMT2B) peripheral neuropathy. Herein, we investigated how mitochondrial morphology and function were impacted by the CMT2B associated Rab7V162M mutation in fibroblasts from human CMT2B patients as well as in a knockin mouse model. In contrast to recently published results from studies of using heterologous overexpression systems, our results have demonstrated significant mitochondrial fragmentation in fibroblasts of both human CMT2B patients and CMT2B mouse embryonic fibroblasts (MEFs). Furthermore, we have shown that mitochondria were fragmented and axonal mitochondrial movement was dysregulated in primary cultured E18 dorsal root ganglion (DRG) sensory neurons, but not in E18 hippocampal and cortical primary neurons. We also show that inhibitors to either the mitochondrial fission protein Drp1 or to the nucleotide binding to Rab7 normalized the mitochondrial deficits in both MEFs and E18 cultured DRG neurons. Our study has revealed, for the first time, that expression of CMT2B Rab7 mutation at physiological level enhances Drp1 activity to promote mitochondrial fission, that may potentially underlie selective vulnerability of peripheral sensory neurons in CMT2B pathogenesis.

neuroscience↗