Cas9-mediated gene editing in the black-legged tick, Ixodes scapularis, by embryo injection and ReMOT Control.

Despite their capacity to acquire and pass on an array of debilitating pathogens, research on ticks has lagged behind other arthropod vectors, such as mosquitoes, largely because of challenges in applying available genetic and molecular tools. CRISPR-Cas9 is transforming non-model organism research; however, successful gene editing has not yet been reported in ticks. Technical challenges for injecting tick embryos to attempt gene editing have further slowed research progress. Currently, no embryo injection protocol exists for any chelicerate species, including ticks. Herein, we report a successful embryo injection protocol for the black-legged tick, Ixodes scapularis, and the use of this protocol for genome editing with CRISPR-Cas9. We also demonstrate that the ReMOT Control technique could be successfully used to generate genome mutations outside Insecta. Our results provide innovative tools to the tick research community that are essential for advancing our understanding of the molecular mechanisms governing pathogen transmission by tick vectors and the underlying biology of host-vector-pathogen interactions.

Exploiting a Y chromosome-linked Cas9 for sex selection and gene drive.

CRISPR-based genetic engineering tools aimed to bias sex ratios, or drive effector genes into animal populations, often integrate the transgenes into autosomal chromosomes. However, in species with heterogametic sex chromsomes (e.g. XY, ZW), sex linkage of endonucleases could be beneficial to drive the expression in a sex-specific manner to produce genetic sexing systems, sex ratio distorters, or even sex-specific gene drives, for example. To explore this possibility, here we develop a transgenic line of Drosophila melanogaster expressing Cas9 from the Y chromosome. We functionally characterize the utility of this strain for both sex selection and gene drive finding it to be quite effective. To explore its utility for population control, we built mathematical models illustrating its dynamics as compared to other state-of-the-art systems designed for both population modification and suppression. Taken together, our results contribute to the development of current CRISPR genetic control tools and demonstrate the utility of using sex-linked Cas9 strains for genetic control of animals.

Development of a Rapid and Sensitive CasRx-Based Diagnostic Assay for SARS-CoV-2.

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.

Silencing the alarm: an insect salivary enzyme closes plant stomata and inhibits volatile release.

Herbivore-induced plant volatiles (HIPVs) are widely recognized as an ecologically important defensive response of plants against herbivory. Although the induction of this 'cry for help' has been well documented, only a few studies have investigated the inhibition of HIPVs by herbivores and little is known about whether herbivores have evolved mechanisms to inhibit the release of HIPVs. To examine the role of herbivore effectors in modulating HIPVs and stomatal dynamics, we conducted series of experiments combining pharmacological, surgical, genetic (CRISPR-Cas9) and chemical (GC-MS analysis) approaches. We show that the salivary enzyme, glucose oxidase (GOX), secreted by the caterpillar Helicoverpa zea on leaves, causes stomatal closure in tomato (Solanum lycopersicum) within 5 min, and in both tomato and soybean (Glycine max) for at least 48 h. GOX also inhibits the emission of several HIPVs during feeding by H. zea, including (Z)-3-hexenol, (Z)-jasmone and (Z)-3-hexenyl acetate, which are important airborne signals in plant defenses. Our findings highlight a potential adaptive strategy where an insect herbivore inhibits plant airborne defenses during feeding by exploiting the association between stomatal dynamics and HIPV emission.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis.

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

A Sensitive, Rapid, and Portable CasRx-based Diagnostic Assay for SARS-CoV-2.

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.

Cas9-Mediated Gene-Editing in the Malaria Mosquito Anopheles stephensi by ReMOT Control.

Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by Anopheles mosquitoes. CRISPR/Cas9-based gene disruption is a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially Anopheles) are difficult and inefficient, particularly for non-specialist laboratories. Here, we adapted the ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector Anopheles stephensi without injecting embryos. In Anopheles, ReMOT Control gene editing was as efficient as standard embryo injections. The application of ReMOT Control to Anopheles opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species.

Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing.

Cas9-mediated gene editing is a powerful tool for addressing research questions in arthropods. Current approaches rely upon delivering Cas9 ribonucleoprotein (RNP) complex by embryonic microinjection, which is challenging, is limited to a small number of species, and is inefficient even in optimized taxa. Here we develop a technology termed Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) to deliver Cas9 RNP to the arthropod germline by injection into adult female mosquitoes. We identify a peptide (P2C) that mediates transduction of Cas9 RNP from the female hemolymph to the developing mosquito oocytes, resulting in heritable gene editing of the offspring with efficiency as high as 0.3 mutants per injected mosquito. We demonstrate that P2C functions in six mosquito species. Identification of taxa-specific ovary-specific ligand-receptor pairs may further extend the use of ReMOT Control for gene editing in novel species.

Geometric morphometrics of nine field isolates of Aedes aegypti with different resistance levels to lambda-cyhalothrin and relative fitness of one artificially selected for resistance.

Aedes aegypti, a mosquito closely associated with humans, is the principal vector of dengue virus which currently infects about 400 million people worldwide. Because there is no way to prevent infection, public health policies focus on vector control; but insecticide-resistance threatens them. However, most insecticide-resistant mosquito populations exhibit fitness costs in absence of insecticides, although these costs vary. Research on components of fitness that vary with insecticide-resistance can help to develop policies for effective integrated management and control. We investigated the relationships in wing size, wing shape, and natural resistance levels to lambda-cyhalothrin of nine field isolates. Also we chose one of these isolates to select in lab for resistance to the insecticide. The main life-traits parameters were assessed to investigate the possible fitness cost and its association with wing size and shape. We found that wing shape, more than wing size, was strongly correlated with resistance levels to lambda-cyhalothrin in field isolates, but founder effects of culture in the laboratory seem to change wing shape (and also wing size) more easily than artificial selection for resistance to that insecticide. Moreover, significant fitness costs were observed in response to insecticide-resistance as proved by the diminished fecundity and survival of females in the selected line and the reversion to susceptibility in 20 generations of the non-selected line. As a practical consequence, we think, mosquito control programs could benefit from this knowledge in implementing efficient strategies to prevent the evolution of resistance. In particular, the knowledge of reversion to susceptibility is important because it can help in planning better strategies of insecticide use to keep useful the few insecticide-molecules currently available.