Novel synthetic 3'-untranslated regions for controlling transgene expression in transgenic Aedes aegypti mosquitoes.

Transgenic technology for mosquitoes is now more than two decades old, and a wide array of control sequences have been described for regulating gene expression in various life stages or specific tissues. Despite this, comparatively little attention has been paid to the development and validation of other transgene-regulating elements, especially 3'-untranslated regions (3'UTRs). As a consequence, the same regulatory sequences are often used multiple times in a single transgene array, potentially leading to instability of transgenic effector genes. To increase the repertoire of characterized 3'UTRs available for genetics-based mosquito control, we generated fifteen synthetic sequences based on the base composition of the widely used SV40 3'UTR sequence, and tested their ability to contribute to the expression of reporter genes EGFP or luciferase. Transient transfection in mosquito cells identified nine candidate 3'UTRs that conferred moderate to strong gene expression. Two of these were engineered into the mosquito genome through CRISPR/Cas9-mediated site-specific insertion and compared to the original SV40 3'UTR. Both synthetic 3'UTRs were shown to successfully promote transgene expression in all mosquito life stages (larva, pupa and adults), similar to the SV40 3'UTR, albeit with differences in intensity. Thus, the synthetic 3'UTR elements described here are suitable for regulating transgene expression in Ae. aegypti, and provide valuable alternatives in the design of multi-gene cassettes. Additionally, the synthetic-scramble approach we validate here could be used to generate additional functional 3'UTR elements in this or other organisms.

CRISPR-based gene editing of non-homologous end joining factors biases DNA repair pathway choice toward single-strand annealing in Aedes aegypti.

To maintain genome stability, eukaryotic cells orchestrate DNA repair pathways to process DNA double-strand breaks (DSBs) that result from diverse developmental or environmental stimuli. Bias in the selection of DSB repair pathways, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), is also critical for efficient gene editing and for homing-based gene drive approaches developed for the control of disease-transmitting vector mosquitoes. However, little is understood about DNA repair homeostasis in the mosquito genome. Here, we utilized CRISPR/Cas9 to generate indel mutant strains for core NHEJ factors ku80, DNA ligase IV (lig4), and DNA-PKcs in the mosquito Aedes aegypti and evaluated the corresponding effects on DNA repair. In a plasmid-based assay, disruption of ku80 or lig4, but not DNA-PKcs, reduced both NHEJ and SSA. However, a transgenic reporter strain-based test revealed that those mutations significantly biased DNA repair events toward SSA. Interestingly, ku80 mutation also significantly increased the end joining rate by a yet-characterized mechanism in males. Our study provides evidence that the core NHEJ factors have an antagonistic effect on SSA-based DSB repair of the Ae. aegypti genome. Down-modulating the NHEJ pathway can enhance the efficiency of nuclease-based genetic control approaches, as most of those operate by homology-based repair processes along with extensive DNA end resection that is antagonized by NHEJ.

Engineering a self-eliminating transgene in the yellow fever mosquito, Aedes aegypti.

Promising genetics-based approaches are being developed to reduce or prevent the transmission of mosquito-vectored diseases. Less clear is how such transgenes can be removed from the environment, a concern that is particularly relevant for highly invasive gene drive transgenes. Here, we lay the groundwork for a transgene removal system based on single-strand annealing (SSA), a eukaryotic DNA repair mechanism. An SSA-based rescuer strain (kmoRG ) was engineered to have direct repeat sequences (DRs) in the Aedes aegypti kynurenine 3-monooxygenase (kmo) gene flanking the intervening transgenic cargo genes, DsRED and EGFP. Targeted induction of DNA double-strand breaks (DSBs) in the DsRED transgene successfully triggered complete elimination of the entire cargo from the kmoRG strain, restoring the wild-type kmo gene, and thereby, normal eye pigmentation. Our work establishes the framework for strategies to remove transgene sequences during the evaluation and testing of modified strains for genetics-based mosquito control.

A knockout screen of genes expressed specifically in Ae. aegypti pupae reveals a critical role for stretchin in mosquito flight.

Aedes aegypti is a critical vector for transmitting Zika, dengue, chikungunya, and yellow fever viruses to humans. Genetic strategies to limit mosquito survival based upon sex distortion or disruption of development may be valuable new tools to control Ae. aegypti populations. We identified six genes with expression limited to pupal development; osi8 and osi11 (Osiris protein family), CPRs and CPF (cuticle protein family), and stretchin (a muscle protein). Heritable CRISPR/Cas9-mediated gene knockout of these genes did not reveal any defects in pupal development. However, stretchin-null mutations (strnΔ35/Δ41) resulted in flightless mosquitoes with an abnormal open wing posture. The inability of adult strnΔ35/Δ41 mosquitoes to fly restricted their escape from aquatic rearing media following eclosion, and substantially reduced adult survival rates. Transgenic strains which contain the EGFP marker gene under the control of strn regulatory regions (0.8 kb, 1.4 kb, and 2.2 kb upstream, respectively), revealed the gene expression pattern of strn in muscle-like tissues in the thorax during late morphogenesis from L4 larvae to young adults. We demonstrated that Ae. aegypti pupae-specific strn is critical for adult mosquito flight capability and a key late-acting lethal target for mosquito-borne disease control.