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1.
ACS Sens ; 6(11): 3957-3966, 2021 11 26.
Article in English | MEDLINE | ID: mdl-34714054

ABSTRACT

The development of an extensive toolkit for potential point-of-care diagnostics that is expeditiously adaptable to new emerging pathogens is of critical public health importance. Recently, a number of novel CRISPR-based diagnostics have been developed to detect SARS-CoV-2. Herein, we outline the development of an alternative CRISPR nucleic acid diagnostic utilizing a Cas13d ribonuclease derived from Ruminococcus flavefaciens XPD3002 (CasRx) to detect SARS-CoV-2, an approach we term SENSR (sensitive enzymatic nucleic acid sequence reporter) that can detect attomolar concentrations of SARS-CoV-2. We demonstrate 100% sensitivity in patient-derived samples by lateral flow and fluorescence readout with a detection limit of 45 copy/µL. This technology expands the available nucleic acid diagnostic toolkit, which can be adapted to combat future pandemics.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Nucleic Acid Amplification Techniques , RNA, Viral , Ruminococcus
2.
Nat Commun ; 12(1): 4388, 2021 07 19.
Article in English | MEDLINE | ID: mdl-34282149

ABSTRACT

Mosquito-borne diseases, such as dengue and malaria, pose significant global health burdens. Unfortunately, current control methods based on insecticides and environmental maintenance have fallen short of eliminating the disease burden. Scalable, deployable, genetic-based solutions are sought to reduce the transmission risk of these diseases. Pathogen-blocking Wolbachia bacteria, or genome engineering-based mosquito control strategies including gene drives have been developed to address these problems, both requiring the release of modified mosquitoes into the environment. Here, we review the latest developments, notable similarities, and critical distinctions between these promising technologies and discuss their future applications for mosquito-borne disease control.


Subject(s)
Insecticides , Mosquito Control/methods , Nucleic Acid Amplification Techniques/methods , Vector Borne Diseases/genetics , Animals , Animals, Genetically Modified , CRISPR-Cas Systems , Female , Humans , Malaria/prevention & control , Malaria/transmission , Male , Mosquito Vectors , Pest Control, Biological , Wolbachia/genetics
3.
medRxiv ; 2020 Oct 20.
Article in English | MEDLINE | ID: mdl-33106816

ABSTRACT

Since its first emergence from China in late 2019, the SARS-CoV-2 virus has spread globally despite unprecedented containment efforts, resulting in a catastrophic worldwide pandemic. Successful identification and isolation of infected individuals can drastically curtail virus spread and limit outbreaks. However, during the early stages of global transmission, point-of-care diagnostics were largely unavailable and continue to remain difficult to procure, greatly inhibiting public health efforts to mitigate spread. Furthermore, the most prevalent testing kits rely on reagent- and time-intensive protocols to detect viral RNA, preventing rapid and cost-effective diagnosis. Therefore the development of an extensive toolkit for point-of-care diagnostics that is expeditiously adaptable to new emerging pathogens is of critical public health importance. Recently, a number of novel CRISPR-based diagnostics have been developed to detect COVID-19. Herein, we outline the development of a CRISPR-based nucleic acid molecular diagnostic utilizing a Cas13d ribonuclease derived from Ruminococcus flavefaciens (CasRx) to detect SARS-CoV-2, an approach we term SENSR (Sensitive Enzymatic Nucleic-acid Sequence Reporter). We demonstrate SENSR robustly detects SARS-CoV-2 sequences in both synthetic and patient-derived samples by lateral flow and fluorescence, thus expanding the available point-of-care diagnostics to combat current and future pandemics.

5.
J Vis Exp ; (159)2020 05 24.
Article in English | MEDLINE | ID: mdl-32510506

ABSTRACT

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.


Subject(s)
Culex/embryology , Culex/genetics , Gene Editing , Microinjections/methods , Mosquito Vectors/genetics , West Nile Fever/veterinary , West Nile virus/pathogenicity , Animals , Culex/virology , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Embryo, Nonmammalian/virology , Female , Mutagenesis, Site-Directed , West Nile Fever/immunology , West Nile Fever/virology
6.
J Exp Biol ; 223(Pt Suppl 1)2020 02 07.
Article in English | MEDLINE | ID: mdl-32034041

ABSTRACT

Vector-borne diseases, such as dengue, Zika and malaria, are a major cause of morbidity and mortality worldwide. These diseases have proven difficult to control and currently available management tools are insufficient to eliminate them in many regions. Gene drives have the potential to revolutionize vector-borne disease control. This suite of technologies has advanced rapidly in recent years as a result of the availability of new, more efficient gene editing technologies. Gene drives can favorably bias the inheritance of a linked disease-refractory gene, which could possibly be exploited (i) to generate a vector population incapable of transmitting disease or (ii) to disrupt an essential gene for viability or fertility, which could eventually eliminate a population. Importantly, gene drives vary in characteristics such as their transmission efficiency, confinability and reversibility, and their potential to develop resistance to the drive mechanism. Here, we discuss recent advancements in the gene drive field, and contrast the benefits and limitations of a variety of technologies, as well as approaches to overcome these limitations. We also discuss the current state of each gene drive technology and the technical considerations that need to be addressed on the pathway to field implementation. While there are still many obstacles to overcome, recent progress has brought us closer than ever before to genetic-based vector modification as a tool to support vector-borne disease elimination efforts worldwide.


Subject(s)
Gene Drive Technology , Malaria , Zika Virus Infection , Zika Virus , CRISPR-Cas Systems , Gene Editing , Humans , Malaria/prevention & control , Population Control
7.
Front Genet ; 10: 1072, 2019.
Article in English | MEDLINE | ID: mdl-31737050

ABSTRACT

While efforts to control malaria with available tools have stagnated, and arbovirus outbreaks persist around the globe, the advent of clustered regularly interspaced short palindromic repeat (CRISPR)-based gene editing has provided exciting new opportunities for genetics-based strategies to control these diseases. In one such strategy, called "population replacement", mosquitoes, and other disease vectors are engineered with effector genes that render them unable to transmit pathogens. These effector genes can be linked to "gene drive" systems that can bias inheritance in their favor, providing novel opportunities to replace disease-susceptible vector populations with disease-refractory ones over the course of several generations. While promising for the control of vector-borne diseases on a wide scale, this sets up an evolutionary tug-of-war between the introduced effector genes and the pathogen. Here, we review the disease-refractory genes designed to date to target Plasmodium falciparum malaria transmitted by Anopheles gambiae, and arboviruses transmitted by Aedes aegypti, including dengue serotypes 2 and 3, chikungunya, and Zika viruses. We discuss resistance concerns for these effector genes, and genetic approaches to prevent parasite and viral escape variants. One general approach is to increase the evolutionary hurdle required for the pathogen to evolve resistance by attacking it at multiple sites in its genome and/or multiple stages of development. Another is to reduce the size of the pathogen population by other means, such as with vector control and antimalarial drugs. We discuss lessons learned from the evolution of resistance to antimalarial and antiviral drugs and implications for the management of resistance after its emergence. Finally, we discuss the target product profile for population replacement strategies for vector-borne disease control. This differs between early phase field trials and wide-scale disease control. In the latter case, the demands on effector gene efficacy are great; however, with new possibilities ushered in by CRISPR-based gene editing, and when combined with surveillance, monitoring, and rapid management of pathogen resistance, the odds are increasingly favoring effector genes in the upcoming evolutionary tug-of-war.

8.
PLoS Negl Trop Dis ; 8(5): e2833, 2014 May.
Article in English | MEDLINE | ID: mdl-24810399

ABSTRACT

In 2006, we reported a mariner (Mos1)-transformed Aedes aegypti line, Carb77, which was highly resistant to dengue-2 virus (DENV2). Carb77 mosquitoes expressed a DENV2-specific inverted-repeat (IR) RNA in midgut epithelial cells after ingesting an infectious bloodmeal. The IR-RNA formed double-stranded DENV2-derived RNA, initiating an intracellular antiviral RNA interference (RNAi) response. However, Carb77 mosquitoes stopped expressing the IR-RNA after 17 generations in culture and lost their DENV2-refractory phenotype. In the current study, we generated new transgenic lines having the identical transgene as Carb77. One of these lines, Carb109M, has been genetically stable and refractory to DENV2 for >33 generations. Southern blot analysis identified two transgene integration sites in Carb109M. Northern blot analysis detected abundant, transient expression of the IR-RNA 24 h after a bloodmeal. Carb109M mosquitoes were refractory to different DENV2 genotypes but not to other DENV serotypes. To further test fitness and stability, we introgressed the Carb109M transgene into a genetically diverse laboratory strain (GDLS) by backcrossing for five generations and selecting individuals expressing the transgene's EGFP marker in each generation. Comparison of transgene stability in replicate backcross 5 (BC5) lines versus BC1 control lines demonstrated that backcrossing dramatically increased transgene stability. We subjected six BC5 lines to five generations of selection based on EGFP marker expression to increase the frequency of the transgene prior to final family selection. Comparison of the observed transgene frequencies in the six replicate lines relative to expectations from Fisher's selection model demonstrated lingering fitness costs associated with either the transgene or linked deleterious genes. Although minimal fitness loss (relative to GDLS) was manifest in the final family selection stage, we were able to select homozygotes for the transgene in one family, Carb109M/GDLS.BC5.HZ. This family has been genetically stable and DENV2 refractory for multiple generations. Carb109M/GDLS.BC5.HZ represents an important line for testing proof-of-principle vector population replacement.


Subject(s)
Aedes/genetics , Aedes/virology , Dengue Virus/immunology , Insect Vectors/genetics , Insect Vectors/virology , Aedes/immunology , Animals , Base Sequence , Gene Frequency , Genetic Fitness , Genomic Instability , Insect Vectors/immunology , Molecular Biology , Molecular Sequence Data , Phenotype , RNA Interference , Transgenes
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