Advances in genetic biocontrol: heritable vertebrate sex reversal and efficient base editors

Genetic biocontrol is a method of pest control where genetically modified organisms are released into the environment to reduce the harm caused by a pest population. The modifications are typically intended to reduce the population’s reproductive capacity, but they could also spread disease refractoriness, sensitivity to pesticides, or other alleles that reduce pest damage. Transgenic expression of genome modifying enzymes can enhance the spread of alleles for population suppression or modification. Owing to recent advances in genome engineering, enzymes can now be programmed to efficiently edit specific genomic loci. This thesis documents using CRISPR/Cas9 and cytosine base editors (CBEs) to create genetic biocontrol systems which distort sex ratios towards males.

One of the most powerful methods for biasing the sex ratio of a population towards males is heritable sex reversal. As well as being used as a genetic biocontrol, female-to-male sex reversal could also facilitate generating all-male cultures for the commercially important Nile tilapia. In the model species, zebrafish, Danio rerio, we employed CRISPR/Cas9 to inactivate fancl, which conferred the super-Mendelian inheritance of alleles that cause sex reversal. To determine the extent of the sex reversal we compared the male bias in the transgenic offspring to the nontransgenic. In the F2 generation there was a clear bias towards males, a bias that was also seen in the earlier F1 generation. Genotyping of both the F1 generation and their F2 progeny showed that there was moderate germline editing, suggesting that extensive germline editing isn’t required for this method of sex reversal.

In a separate study, we characterised highly efficient CBEs through genetically modifying the fruit fly, Drosophila melanogaster, to express six types of CBE. We used two promoters, a germline-specific nos promoter, and a ubiquitous actin promoter to each drive the expression of three types of cytidine deaminase. We assessed base editing efficiency by targeting the ebony gene, where germline transmission of a nonsense mutation would produce F2 ebony progeny. To optimise editing conditions, we compared both maternal and paternal transmission of the base editor at the F0 stage, along with maternal and paternal transmission at the F1 stage. The F2 offspring for each condition were assessed phenotypically and genotyped by Amp-seq.

For all three actin-expressed base editors there was > 99% editing, with lower efficiency seen for nos-expressed base editors. The highest efficiency was seen for the actin-expressed AID deaminase, where > 99.5% editing efficiency was seen for all different pathways of transmission. Using this base editor, we attempted to create dominant female sterile mutations in the Easter gene, however we didn’t observe female sterility.