Genomic insertion locus and Cas9 expression in the germline affect CRISPR/Cas9-based gene drive performance in the yellow fever mosquito Aedes aegypti

The yellow fever mosquito Aedes aegypti is a major vector of arthropod-borne viruses, including dengue, chikungunya, and Zika. A novel approach to mitigate arboviral infections is to generate mosquitoes refractory to infection by overexpressing antiviral effector molecules. Such an approach requires a mechanism to spread these antiviral effectors through a population, for example, by using CRISPR/Cas9-based gene drive systems. Here we report an autonomous single-component gene drive system in Ae. aegypti that is designed for persistent population replacement. Critical to the design of a single-locus autonomous gene drive is that the selected genomic locus be amenable to both gene drive and the appropriate expression of the antiviral effector. In our study, we took a reverse engineering approach to target two genomic loci ideal for the expression of antiviral effectors and further investigated the use of three promoters for Cas9 expression (nanos, β2-tubulin, or zpg) for the gene drive. We found that both promoter selection and genomic target site strongly influenced the efficiency of the drive, resulting in 100% inheritance in some crosses. We also observed the formation of inheritable gene drive blocking indels (GDBI) in the genomic locus with the highest levels of gene drive. Overall, our drive system forms a platform for the further testing of driving antipathogen effector genes through Ae. aegypti populations.

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Gene Drives Across Engineered Fitness Valleys: Modeling a Design to Prevent Drive Spillover

10.1101/2020.10.29.360404 ◽

2020 ◽

Author(s):

Frederik J.H. de Haas ◽

Sarah P. Otto

Keyword(s):

Local Population ◽

Target Population ◽

Drive System ◽

High Threshold ◽

Gene Drive ◽

Time Model ◽

Population Replacement ◽

Low Threshold ◽

Drive Systems ◽

Gene Drives

1AbstractEngineered gene drive techniques for population replacement and/or suppression have potential for tackling complex challenges, including reducing the spread of diseases and invasive species. Unfortunately, the self-propelled behavior of drives can lead to the spread of transgenic elements beyond the target population, which is concerning. Gene drive systems with a low threshold frequency for invasion, such as homing-based gene drive systems, require initially few transgenic individuals to spread and are therefore easy to implement. However their ease of spread presents a double-edged sword; their low threshold makes these drives much more susceptible to spread outside of the target population (spillover). We model a proposed drive system that transitions in time from a low threshold drive system (homing-based gene drive) to a high threshold drive system (underdominance) using daisy chain technology. This combination leads to a spatially restricted drive strategy, while maintaining an attainable release threshold. We develop and analyze a discrete-time model as proof of concept and find that this technique effectively generates stable local population suppression, while preventing the spread of transgenic elements beyond the target population under biologically realistic parameters.

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Development and testing of a novel killer–rescue self-limiting gene drive system in Drosophila melanogaster

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2019.2994 ◽

2020 ◽

Vol 287 (1925) ◽

pp. 20192994 ◽

Cited By ~ 5

Author(s):

Sophia H. Webster ◽

Michael R. Vella ◽

Maxwell J. Scott

Keyword(s):

Drosophila Melanogaster ◽

Aedes Aegypti ◽

Drive System ◽

R Gene ◽

Agricultural Pest ◽

Gene Drive ◽

Wild Type ◽

Drosophila Suzukii ◽

Population Replacement ◽

Population Suppression

Here we report the development and testing of a novel self-limiting gene drive system, Killer–Rescue (K–R), in Drosophila melanogaster . This system is composed of an autoregulated Gal4 Killer (K) and a Gal4-activated Gal80 Rescue (R). Overexpression of Gal4 is lethal, but in the presence of R activation of Gal80 leads to much lower levels of Gal4 and rescue of lethality. We demonstrate that with a single 2 : 1 engineered to wild-type release, K drives R through the population and after nine generations, more than 98% of the population carry R and less than 2% of the population are wild-type flies. We discuss how this simple K–R gene drive system may be readily adapted for population replacement in a human health pest, Aedes aegypti , or for population suppression in an agricultural pest, Drosophila suzukii .

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