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

Sharma, Arvind; Pham, Michael N.; Reyes, Jeremiah; Chana, Randeep; Yim, Won Cheol; Heu, Chan C.; Kim, Dong-Hun; Chaverra-Rodríguez, Duverney; Rasgon, Jason L.; Harrell, Robert A.; Nuss, Andrew B.; Gulia-Nuss, Monika

doi:10.1016/j.isci.2022.103781

Cited by 56 publications

(36 citation statements)

References 71 publications

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“…Indeed, aquaporins continue to gain attention as targets of anti-tick vaccination strategies (e.g., de la Fuenta et al ., 2016, Guerrero et al ., 2017, Scoles et al ., 2022). We attempted to knock down IsAQP1 expression using RNAi for in vivo functional characterization that could give insight into potential control targets, but gene knock-down was unsuccessful (data not shown).Thanks to the recent development of new methods for targeted CRISPR genetic manipulations this species (Sharma et al ., 2022), future research could functionally test the importance of this gene in tick physiology and explore its potential for use as a tool of vector control.…”

Section: Discussionmentioning

confidence: 99%

Function and evolution of aquaporin IsAQP1 in the Lyme disease vector Ixodes scapularis

Tsujimoto

Metz

Smith

et al. 2022

Preprint

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Ticks are important vectors of pathogenic viruses, bacteria, and protozoans to humans, wildlife, and domestic animals. Due to their life cycle, ticks face significant challenges related to water homeostasis. When blood-feeding they must excrete water and ions, but when off-host (for stretches lasting several months) they must conserve water to avoid desiccation. Aquaporins (AQPs), a family of membrane-bound water channels, are key players in osmoregulation in many animals but remain poorly characterized in ticks. Here, we bioinformatically identified AQP-like genes from the deer tick Ixodes scapularis and used phylogenetic approaches to map the evolution of the aquaporin gene family in arthropods. We find that most arachnid AQP-like sequences (including those of I. scapularis) form a monophyletic group clustered within aquaglycerolporins (GLPs) from bacteria to vertebrates. This gene family is absent from insects, revealing divergent evolutionary paths for AQPs in different hematophagous arthropods. We next sequenced the full-length cDNA of I. scapularis aquaporin 1 (IsAQP1) and expressed it heterologously in Xenopus oocytes to functionally characterize its permeability to water and solutes. We additionally examined IsAQP1 expression across different life stages and adult female tissues. We found IsAQP1 to be an efficient water channel with salivary gland-specific expression, implying its function may be closely tied to osmoregulation during blood-feeding. Its functional properties were unique: unlike most GLPs, IsAQP1 has low glycerol permeability, and unlike most AQPs, it is insensitive to mercury. Together, our results suggest IsAQP1 plays an important role in tick water balance during blood feeding and may hold promise as a target for novel vector control efforts.

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Section: Discussionmentioning

confidence: 99%

Function and evolution of aquaporin IsAQP1 in the Lyme disease vector Ixodes scapularis

Tsujimoto

Metz

Smith

et al. 2022

Preprint

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“…Previous research has demonstrated the down regulation of either TRE31 or TROPSA both results in the decreased transmission of B. burgdorferi from tick to host [10,11]. Additionally, successful protocols for CRISPR/Cas9-mediated gene editing in the black-legged ticks are available [15]. Our protocol would thereby serve as the second step to determine the combined efficacy of TROPSA and TRE31 knockouts in eliminating B. burgdorferi transmission between tick and host.…”

Section: Discussionmentioning

confidence: 99%

“…Prior to commencing this protocol, TROSPA and TRE31 knockout ticks will be generated using CRISPR-Cas-9 according to Sharma et al's I. scapularis embryo injection protocol [15].…”

Section: Sourcing Tick Modelsmentioning

confidence: 99%

TROPSA and TRE31 Gene Knockouts to Prevent the Transmission of Lyme Disease from Tick to Host: A Research Protocol

et al. 2022

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Introduction: Ixodes scapularis, the blacklegged tick, is responsible for the transmission of Lyme disease. Rising temperatures and shorter winter seasons, due to climate change, is resulting in the Northward expansion of tick range. This is correlated with the increasing prevalence of Lyme disease in Canada. This research protocol aims to address this issue by genetically mutating the blacklegged tick which is primarily responsible for the transmission of Borrelia burgdorferi, the bacterium that causes Lyme disease, in North America. The proposed mutation involves two gene knockouts: TROSPA and TRE31. The blacklegged tick mutant is predicted to be unable to transmit Lyme disease to the white-footed mouse, Peromyscus leucopus. Methods: Mutated ticks will feed on the blood of Lyme positive mice and later naïve mice. The rate of Lyme disease transmission from mutated ticks will be compared to transmission rates in positive and negative wild type control groups. The statistical significance of the difference between these groups' transmission rates will be evaluated by Student's t-test with Fisher's protected least significant difference test. Results: Based on the results from literature testing each mutation independently, we predict our I. scapularis mutant, having both TROSPA and TRE31 gene knockouts, will be unable to transmit Lyme disease to the white-footed mouse. Discussion: Unsuccessful transmission of Lyme disease from mutated ticks indicates that the TROSPA and TRE31 knockouts are effective in preventing B. burgdorferi from completing its lifecycle within the tick. Based on the expected results, the combined gene-knockout model presents a novel method to hinder the transmission of Lyme disease more effectively than previously investigated single gene knockouts. Conclusion: This research protocol suggests a strategy to decrease the rate of Lyme disease amongst ticks, and thus humans. Future research could explore efficacies of knocking out other genes in combination with TROSPA or TRE31.

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“…The length of the hypostome varies depending upon sex and stage (nymph vs. adult) [ 44 ]. The adult female deer tick exhibits the largest hypostome which is ~500 μm long [ 45 ] and up to ~200 μm wide, while the males and nymphs have much smaller hypostomes [ 46 ]. Since the bacteria travel into a new host via tick saliva, the bacteria are likely deposited approximately close to the depth of the tick mouth.…”

Section: Methodsmentioning

confidence: 99%

Direct Capture and Early Detection of Lyme Disease Spirochete in Skin with a Microneedle Patch

et al. 2022

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Borrelia burgdorferi sensu lato family of spirochetes causes Lyme disease (LD) in animals and humans. As geographic territory of ticks expands across the globe, surveillance measures are needed to measure transmission rates and provide early risk testing of suspected bites. The current standard testing of LD uses an indirect two-step serological assay that detects host immune reactivity. Early detection remains a challenge because the host antibody response develops several weeks after infection. A microneedle (MN) device was developed to sample interstitial fluid (ISF) and capture spirochetes directly from skin. After sampling, the MN patch is easily dissolved in water or TE buffer, and the presence of spirochete DNA is detected by PCR. Performance was tested by spiking porcine ear skin with inactivated Borrelia burgdorferi, and showed an approximate recovery of 80% of spirochetes. With further development, this simple direct PCR method could be a transformative approach for early detection of the causative agent of Lyme disease and enable rapid treatment to patients when infection is early, and numbers of systemic spirochetes are low.

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

Cited by 56 publications

References 71 publications

Function and evolution of aquaporin IsAQP1 in the Lyme disease vector Ixodes scapularis

Function and evolution of aquaporin IsAQP1 in the Lyme disease vector Ixodes scapularis

TROPSA and TRE31 Gene Knockouts to Prevent the Transmission of Lyme Disease from Tick to Host: A Research Protocol

Direct Capture and Early Detection of Lyme Disease Spirochete in Skin with a Microneedle Patch

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