Targeted genome editing in Aedes aegypti using TALENs

Aryan, Azadeh; Myles, K.M.; Adelman, Zach N.

doi:10.1016/j.ymeth.2014.02.008

Cited by 39 publications

(32 citation statements)

References 43 publications

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“…TALEN plasmids targeting the Ae. aegypti kmo gene were as previously described (46). TALEN expression plasmids targeting dcr2, ago2, and lig4 genes were generated by the University of Utah Mutation Generation and Detection (MGD) Core using the TALEN Golden Gate kit (11), with modifications as previously described (36,47).…”

Section: Methodsmentioning

confidence: 99%

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Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis inAedes aegypti

Basu

Aryan

Overcash

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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141

129

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Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24-90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2-3%, similar to traditional transposon-or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.

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Section: Methodsmentioning

confidence: 99%

“…All photographs of larvae and adult mosquitoes were obtained using a Leica MZ16FL microscope with a Canon Powershot S3 IS digital camera. Embryonic injections were performed as described previously (46). For TALEN-based editing experiments, injection mixes contained 0.3 μg/μL of each TALEN expression construct.…”

Section: Methodsmentioning

confidence: 99%

Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis inAedes aegypti

Basu

Aryan

Overcash

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

141

129

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“…Embryonic collection and microinjections were largely performed following previously established procedures [70,80] . Injected embryos were hatched in deoxygenated water and surviving adults were placed into cages.…”

Section: Construct Assemblymentioning

confidence: 99%

Live calcium imaging ofAedes aegyptineuronal tissues reveals differential importance of chemosensory systems for life-history-specific foraging strategies

Bui

Shyong²,

Lutz

et al. 2018

Preprint

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Aedes aegypti have a wide variety of sensory pathways that have supported success as a species as well as a highly competent vector of numerous debilitating infectious pathogens. Investigations into mosquito sensory systems and their effects on behavior are valuable resources for the advancement of mosquito control strategies. Numerous studies have elucidated key 1 . CC-BY-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/345389 doi: bioRxiv preprint first posted online Jun. 12, 2018; aspects of mosquito sensory systems, however there remains critical gaps within the field. In particular, compared to that of the adult form, there has been a lack of studies directed towards the immature life stages. Additionally, although numerous studies have pinpointed specific sensory receptors as well as relevant response behaviors, there has been a lack of studies able to monitor both concurrently. To begin filling aforementioned gaps, here we engineered Ae. aegypti to ubiquitously express a genetically encoded calcium indicator, GCaMP6s. Using this strain, combined with advanced confocal microscopy, we were able to simultaneously measure live stimulus-evoked calcium responses in both neuronal and muscle cells with a wide spatial range and resolution. Moreover, by coupling in vivo calcium imaging with behavioral assays we were able to gain functional insights into how stimulus-evoked neural and muscle activities are represented, modulated, and transformed in mosquito larvae enabling us to elucidate mosquito sensorimotor properties important for life-history-specific foraging strategies. Significance StatementUnderstanding mosquito sensory systems and resulting behavior has been a major factor in the advancement of mosquito control innovations. Aedes aegypti larvae offer an effective life stage for further elucidating information on mosquito sensory systems. Due to their relatively simplified nervous system, mosquito larvae are ideal for studying neural signal transduction, coding, and behavior. Moreover, a better understanding of the larval sensory system may enable the development of novel control methodologies able to target mosquitoes before they reach a vector-competent stage. Here we engineer Ae. aegypti to ubiquitously express a genetically encoded calcium indicator, GCaMP6s and use this tool to observe links between sensorimotor responses and behavior by exploiting live calcium imaging as well as live tracking based behavioral assays. Main Text IntroductionThe yellow fever mosquito, Aedes aegypti , is a global vector of numerous debilitating arboviruses including Chikungunya, Dengue, Yellow Fever, and Zika [1] . Due to its ability to transmit copious pathogens, adaptability to diverse climates, flexibility in oviposition sites, and desiccation-tolerant eggs, Ae. aegypti are significant worldwide epidemiological...

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“…Further, a detailed procedure for target selection (kmo gene), assessing the activity of TALEN, embryonic microinjection and detection of target site mutations in Ae. aegypti genome was described by the same group in the following year (47). In another reverse genetics study, Smidler et al reported the targeted disruption of the thioestercontaining protein1 (TEP1) gene using TALEN in Anopheles gambiae mosquitoes which spreads malaria.…”

Section: Application Of Talen In Genome Editing Of Vector Mosquitoesmentioning

confidence: 99%

Current status of genome editing in vector mosquitoes: A review

Reegan¹,

Ceasar

Paulraj³

et al. 2016

BST

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Targeted genome editing in Aedes aegypti using TALENs

Cited by 39 publications

References 43 publications

Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis inAedes aegypti

Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis inAedes aegypti

Live calcium imaging ofAedes aegyptineuronal tissues reveals differential importance of chemosensory systems for life-history-specific foraging strategies

Current status of genome editing in vector mosquitoes: A review

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