Development of a gene drive system for locally confined and reversible population replacement in Aedes aegypti using chromosomal translocations
Final Report Abstract
The aim of my project was to generate chromosomal translocations to be used as a tool for population replacement. We were able to increase the frequency with which we can generate translocations compared to first generation constructs by switching to a CRISPR/Cas9 based approach. Unfortunately, the created translocation bearing stocks had a substantial fitness cost and failed to replace a population in drive experiments. At this point during my stay, we decided to focus on other projects. We build two versions of suppression homing endonuclease gene drives (HEG) using CRISPR/Cas9 with multiplexed guide RNAs (gRNA). We could show that multiplexing of guides effectively prevents the creation of resistant alleles, a problem that all previous single gRNA HEG systems had. However, we also found that the homing process itself is error prone, resulting in a large number of incomplete homing events. Furthermore, females conferred a high fitness load to all offspring coming from maternal deposition of active Cas9/gRNA complexes. In drive experiments, we showed that the HEGs failed to suppress a population. Building upon the lessons learned in the HEG project, we designed a novel toxin-antidote type of replacement gene drive system named Cleave and Rescue (CleaveR). The simple two component system consists of Cas9 targeting an essential gene (the cleaver) and a recoded rescue copy of the essential gene, resistant to Cas9 induced cleavage (the rescue). When individuals carrying CleaveR mate with non-CleaveR bearing individuals, Cas9 (the “toxin”) can inactivate the wildtype copies of the essential target gene. This will result in the death of offspring that no longer carries a functional copy of the essential gene. CleaveR individuals, however, are not affected because they carry the recoded rescue copy that is resistant to Cas9 cleavage (the “antidote”). We confirmed this predicted behaviour in several drive experiments, where CleaveR rapidly spread to fixation.
Publications
- “Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs.” PNAS
Oberhofer G, Ivy T, Hay BA
(See online at https://doi.org/10.1073/pnas.1805278115)
