Toward a CRISPR-Cas9-Based Gene Drive in the Diamondback Moth Plutella xylostella.

Promising to provide powerful genetic control tools, gene drives have been constructed in multiple dipteran insects, yeast, and mice for the purposes of population elimination or modification. However, it remains unclear whether these techniques can be applied to lepidopterans. Here, we used endogenous regulatory elements to drive Cas9 and single guide RNA (sgRNA) expression in the diamondback moth (DBM), Plutella xylostella, and test the first split gene drive system in a lepidopteran. The DBM is an economically important global agriculture pest of cruciferous crops and has developed severe resistance to various insecticides, making it a prime candidate for such novel control strategy development. A very high level of somatic editing was observed in Cas9/sgRNA transheterozygotes, although no significant homing was revealed in the subsequent generation. Although heritable Cas9-medated germline cleavage as well as maternal and paternal Cas9 deposition were observed, rates were far lower than for somatic cleavage events, indicating robust somatic but limited germline activity of Cas9/sgRNA under the control of selected regulatory elements. Our results provide valuable experience, paving the way for future construction of gene drives or other Cas9-based genetic control strategies in DBM and other lepidopterans.

LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization from coronavirus disease 2019 (COVID-19) when administered early. However, SARS-CoV-2 variants of concern (VOCs) have negatively affected therapeutic use of some authorized mAbs. Using a high-throughput B cell screening pipeline, we isolated LY-CoV1404 (bebtelovimab), a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody. LY-CoV1404 potently neutralizes authentic SARS-CoV-2, B.1.1.7, B.1.351, and B.1.617.2. In pseudovirus neutralization studies, LY-CoV1404 potently neutralizes variants, including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved, except for N439 and N501. The binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The broad and potent neutralization activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants.

LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants.

SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization when administered early during COVID-19 disease. However, the emergence of variants of concern has negatively impacted the therapeutic use of some authorized mAbs. Using a high throughput B-cell screening pipeline, we isolated a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody called LY-CoV1404 (also known as bebtelovimab). LY-CoV1404 potently neutralizes authentic SARS-CoV-2 virus, including the prototype, B.1.1.7, B.1.351 and B.1.617.2). In pseudovirus neutralization studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant and retains binding to spike proteins with a variety of underlying RBD mutations including K417N, L452R, E484K, and N501Y. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved with the exception of N439 and N501. Notably, the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The breadth of reactivity to amino acid substitutions present among current VOC together with broad and potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants causing COVID-19. In Brief: LY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated. Highlights: LY-CoV1404 potently neutralizes SARS-CoV-2 authentic virus and known variants of concern including the B.1.1.529 (Omicron), the BA.2 Omicron subvariant, and B.1.617.2 (Delta) variantsNo loss of potency against currently circulating variantsBinding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID databaseBreadth of neutralizing activity and potency supports clinical development.

Engineered expression of the invertebrate-specific scorpion toxin AaHIT reduces adult longevity and female fecundity in the diamondback moth Plutella xylostella.

BACKGROUND: Previous genetic pest management (GPM) systems in diamondback moth (DBM) have relied on expressing lethal proteins ('effectors') that are 'cell-autonomous', that is, they do not leave the cell in which they are expressed. To increase the flexibility of future GPM systems in DBM, we aimed to assess the use of a non-cell-autonomous, invertebrate-specific, neurotoxic effector - the scorpion toxin AaHIT. This AaHIT effector was designed to be secreted by expressing cells, potentially leading to effects on distant cells, specifically neuromuscular junctions. RESULTS: Expression of AaHIT caused a 'shaking/quivering' phenotype that could be repressed by provision of an antidote (tetracycline): a phenotype consistent with the AaHIT mode-of-action. This effect was more pronounced when AaHIT expression was driven by the Hr5/ie1 promoter (82.44% of males, 65.14% of females) rather than Op/ie2 (57.35% of males, 48.39% of females). Contrary to expectations, the shaking phenotype and observed fitness costs were limited to adults in which they caused severe reductions in mean longevity (-81%) and median female fecundity (-93%). Quantitative polymerase chain reactions of AaHIT expression patterns and analysis of piggyBac-mediated transgene insertion sites suggest that restriction of the observed effects to the adult stages may be due to the influence of the local genomic environment on the tetO-AaHIT transgene. CONCLUSION: We demonstrated the feasibility of using non-cell-autonomous effectors within a GPM context for the first time in Lepidoptera, one of the most economically damaging orders of insects. These findings provide a framework for extending this system to other pest Lepidoptera and to other secreted effectors. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Engineered action at a distance: Blood-meal-inducible paralysis in Aedes aegypti.

BACKGROUND: Population suppression through mass-release of Aedes aegypti males carrying dominant-lethal transgenes has been demonstrated in the field. Where population dynamics show negative density-dependence, suppression can be enhanced if lethality occurs after the density-dependent (i.e. larval) stage. Existing molecular tools have limited current examples of such Genetic Pest Management (GPM) systems to achieving this through engineering 'cell-autonomous effectors' i.e. where the expressed deleterious protein is restricted to the cells in which it is expressed-usually under the control of the regulatory elements (e.g. promoter regions) used to build the system. This limits the flexibility of these technologies as regulatory regions with useful spatial, temporal or sex-specific expression patterns may only be employed if the cells they direct expression in are simultaneously sensitive to existing effectors, and also precludes the targeting of extracellular regions such as cell-surface receptors. Expanding the toolset to 'non-cell autonomous' effectors would significantly reduce these limitations. METHODOLOGY/PRINCIPAL FINDINGS: We sought to engineer female-specific, late-acting lethality through employing the Ae. aegypti VitellogeninA1 promoter to drive blood-meal-inducible, fat-body specific expression of tTAV. Initial attempts using pro-apoptotic effectors gave no evident phenotype, potentially due to the lower sensitivity of terminally-differentiated fat-body cells to programmed-death signals. Subsequently, we dissociated the temporal and spatial expression of this system by engineering a novel synthetic effector (Scorpion neurotoxin-TetO-gp67.AaHIT) designed to be secreted out of the tissue in which it was expressed (fat-body) and then affect cells elsewhere (neuro-muscular junctions). This resulted in a striking, temporary-paralysis phenotype after blood-feeding. CONCLUSIONS/SIGNIFICANCE: These results are significant in demonstrating for the first time an engineered 'action at a distance' phenotype in a non-model pest insect. The potential to dissociate temporal and spatial expression patterns of useful endogenous regulatory elements will extend to a variety of other pest insects and effectors.

Advising patients about herbs and nutraceuticals: tips for primary care providers.

Herbs and nutraceuticals are commonly used in many households in the United States. This article discusses supplement use patterns and offers resources for primary care providers wishing to counsel patients more effectively about dietary supplements. Supplement safety issues, types of herbal formulations, and a summary of the uses, doses, side effects, and evidence ratings for 10 commonly used herbs and 11 commonly used nutraceuticals are provided.