CRISPR-based gene drives for pest control
Invasive pests impact the environment, economy and society. Current control methods are costly and largely inadequate, and they often lead to unwanted suffering in target and non-target species. Gene drives that enable super-mendelian inheritance of a transgene may offer a more cost-effective, humane and species-specific alternative to current methods. By harnessing gene drives to distort the sex-ratio of the breeding population it may be possible to control a population’s reproductive success. Using CRISPR-Cas gene editing technology, this PhD project aimed to design, model and engineer a safeguard gene drive, known as a split gene drive, in mice that could spread female infertility through a laboratory-contained mouse population. Three gene drive strategies were designed and in silico modelled in wild mouse populations. Reagents were generated to engineer two of the three split gene drive strategies using mouse embryonic stem cell technology. Both these approaches aim to disrupt an essential female fertility gene (OOEP) to confer a recessive female-infertility phenotype. Split gene drive harbouring mouse embryonic stem cells were engineered using plasmid donor-DNA and a combination of SpCas9 ribonucleoprotein or plasmid-based AsCas12a endonuclease. Engineered cells were screened through a pipeline, which included analyses by PCR, droplet digital PCR, Sanger sequencing and functional testing of the integrated transgenic systems. These validated split gene drive embryonic stem cells and the corresponding regulatory approval for animal testing now allows for two split gene drive mouse models to be generated by blastocyst injection of the engineered cells. It is hoped that the findings from this PhD project will help guide the future development of safe gene drive systems for vertebrate pest management.