Engineering of Dengue Virus Refractoriness in Aedes aegypti and Development of an Underdominant Gene Drive System in Drosophila melanogaster

Vector-borne diseases have a profound impact on world health. The two most well-known and costly diseases are dengue fever and malaria, both spread by mosquito vectors. In the last decade, many new solutions to halting the spread of these diseases have been sought, including vector-mediated disease suppression. The work presented here seeks to generate alleles to effect this suppression, and engineer a drive system to replace the native population. Additional work on systems to keep engineered organisms genetically isolated from native populations has also been carried out. Initial studies in C. elegans investigated use of the transitive nature of RNAi in this species to genetically isolate one population from another. This type of speciation could be used in plant populations to limit gene flow of engineered crops into local environments. The next series of studies details work on engineering of refractoriness alleles. Dengue virus has several enzymatic activities that are essential for its replicative cycle, including an RNA-dependednt RNA polymerase (RdRp) responsible for synthesizing both the sense and antisense viral genomes and a protease responsible for several essential cleavages of the viral polyprotein. Artificial substrates for these proteins were created to act as sensors, triggering an apoptotic response when viral infection occurs. Several generations of constructs were tested, but so far no completely functional sensor has been generated. Lastly, a series of underdominant gene drive architectures were built and tested in Drosophila melanogaster. Initial systems utilized a Drosophila cell death protein, Hid, as toxin, and engineered microRNAs designed to target the Hid proteins as antidote. Two toxin-antidote pairs were mismatched and positioned on separate chromosomes so that an organism carrying both chromosomes survives, but an organism carrying only a single chromosome is unviable. Construction of a proof-of-principle in the eye was successful, but work in essential tissues is ongoing. Systems using engineered microRNAs as toxins and resupply of the native protein as antidote were tested in essential tissues. Testing of many components has contributed to the development of these systems, but a complete system has not yet been constructed.</p

iRMCE in <i>Ae</i>. <i>aegypti</i>.

<p>(a) Schematic diagram of iRMCE in <i>Ae</i>. <i>aegypti</i> and (b) corresponding fluorescent phenotypes in fourth instar <i>Ae</i>. <i>aegypti</i> larvae following OX4714 injections. Black arrows indicate engineered <i>FRT</i> and <i>loxP</i> sites present in donor and acceptor constructs; AmpR represents the plasmid backbone sequences, which includes the ampicillin-resistance gene; grey arrows indicate <i>piggyBac</i> ends. The donor plasmid’s 3xP3-DsRed2 cassette is exchanged for the docking construct’s 3xP3-Amcyan. Larvae are shown under (i) white light, (ii) cyan, and (iii) red excitation light and filters. Images show complete exchange of the 3xP3-AmCyan cassette (OX4476), by integration of 3xP3-DsRed2 (seen in OX4476[OX4714]), and excision of the 3xP3-AmCyan marker (‘excised’ larva). There was reduced expression of the docking cassette’s 3xP3-AmCyan marker following ΦC31-<i>att</i> integration (white arrows: panel (ii), OX4476[OX4714]). Images (i-iii) were taken under the same magnification. Scale bar represents 0.5 mm. White dashed lines outline larvae.</p

iRMCE in <i>P</i>. <i>xylostella</i>.

<p>(a) Schematic diagram of iRMCE in <i>P</i>. <i>xylostella</i> and (b) corresponding fluorescent phenotypes in <i>P</i>. <i>xylostella</i> pupae. Black arrows indicate engineered <i>FRT</i> and <i>loxP</i> sites present in donor and acceptor constructs; AmpR represents the plasmid backbone sequences, which includes the ampicillin-resistance gene; grey arrows indicate <i>piggyBac</i> ends. Pupae are shown under (i) white light, (ii) green excitation light and filters, and (iii) red excitation light and filters. Images (i–iii) were taken under the same magnification. Scale bar repersents 1 mm.</p

Summary of <i>Ae</i>. <i>aegypti</i> OX4312 injections for ΦC31-RMCE.

<p>*All G1 transformants from OX4312 injections into OX4372A resulted in incomplete cassette exchange due to only a single pair of attachment sites recombining. Embryos of this partially recombined line, called ‘OX4372A[OX4312]-incomplete’ were injected with additional ΦC31 recombinase mRNA resulting in a second recombination step in G1 progeny (Efficiency = 18.5%).</p><p>Recombination events (R) are defined as the number of transgenic pools. Efficiency = minimum calculated integration efficiency.</p><p>Summary of <i>Ae</i>. <i>aegypti</i> OX4312 injections for ΦC31-RMCE.</p

Genomic flanking sequences of OX4372 and OX4476 in <i>Ae</i>. <i>aegypti</i>.

<p>The duplicated TTAA <i>piggyBac-</i>insertion site was present in all 5’ and 3’ insertion boundaries (underlined).</p><p>Genomic flanking sequences of OX4372 and OX4476 in <i>Ae</i>. <i>aegypti</i>.</p

Summary of <i>Ae</i>. <i>aegypti</i> and <i>P</i>. <i>xylostella</i> injections for ΦC31-<i>att</i> integration.

<p>Integration events (I) are defined as the number of transgenic pools. Efficiency = minimum calculated integration efficiency.</p><p>Summary of <i>Ae</i>. <i>aegypti</i> and <i>P</i>. <i>xylostella</i> injections for ΦC31-<i>att</i> integration.</p

Summary of <i>Ae</i>. <i>aegypti</i> and <i>P</i>. <i>xylostella</i> injections for Cre and FLP injections.

<p><i>Ae</i>. <i>aegypti</i> (‘<i>Aae</i>’) strains were injected with Cre and FLP in parallel for direct comparison. The number of <i>Ae</i>. <i>aegypti</i> pools with progeny apparently lacking the AmCyan marker (due to weak fluorescence) is shown (‘marker loss’); this was not a problem with the <i>P</i>. <i>xylostella</i> (‘<i>Pxy</i>’) strain so values in this column reflect true marker loss. Excision events (E) were confirmed by PCR and sequencing, and the minimum calculated excision frequencies (Efficiency) are shown as a percentage.</p><p>Summary of <i>Ae</i>. <i>aegypti</i> and <i>P</i>. <i>xylostella</i> injections for Cre and FLP injections.</p