Toward an hymenoptera epigenetic molecular model

This project will develop the first insect epigenetic model by creating jewel wasp, Nasonia vitripennis lines that have altered levels of methylation at single loci. The study of epigenetics has been hampered by a lack of the ability to easily alter epigenetic molecular markers (e.g. methylation). This project will provide this ability in a tractable insect model.

The study of epigenetics has for the most part been correlational. Epigenetics is defined as the heritable change in expression of a gene without any change in the DNA sequence1. The methylation of the fifth position of cytosine’s ring is one of the most widespread epigenetic markers2. A common correlational research strategy is to examine the methylation differences between two phenotypes and hypothesise that the differences found are the causes of the phenotype. What is rarer is a strategy analogous to reverse genetics, whereby the methylation is changed and the resultant phenotype studied to confirm methylation’s role. Until recently, reverse epigenetics has been of a general, crude sort. For example, early studies of the role of methylation in mammalian development, simply knocked out DNMT3, the enzyme responsible for the production of new methylation marks3. This reduced methylation throughout the genome and the resultant phenotype was measured. A number of labs have very recently developed altered CRISPR Cas9 systems to change the DNA methylation status of a given loci in mice4-5. Liu et al.5 fused Tet1 (decreased methylation) or Dnmt3a (increased methylation) with a catalytically inactive Cas9 (dCas9) to enable targeted DNA methylation editing.

This proposal will develop the same system which allow targeted DNA methylation editing in an insect model. The wasp Nasonia vitripennis is a prime contender as an insect model for epigenetics. The fruit fly, Drosophila melanogaster, has long been the predominant insect model for genetics. However Drosophila, for the most part, lacks CpG methylation6. Nasonia, like all hymenoptera, has a functional methylation system7 and replicates many of the abilities of the Drosophila model. Recently, CRISPR/Cas9 technology have been used to induce site specific mutations on the cinnabar gene in N. vitripennis adding a new powerful molecular tool for reverse genetics for this insect8. The proposed project will use CRISPR/Cas9 mediated precise homology-directed repair (HDR), cloning, methylomic assays and phenotypic analysis to generate transgenic Nasonia lines in order to establish Nasonia as a functional epigenetic molecular model system. The transgenes lines will express either dCas9-Dnmt3a or dCas9-Tet1CD and specific guide RNA (sgRNA) for target specificity. The targets methylation and expression level will be measured using a variety of methods including qPCR of bisulphite converted DNA and exon specific qPCR. The progeny changes in phenotype will also be correlated to the site specific methylation changes.

The project will involve molecular biology and bioinformatics coupled with behavioural analysis and would suit a student with a strong genetics/molecular biology background, but with a keen interest in whole animal biology, behaviour and development. Students with a zoology/organismal biology background but who can demonstrate strong molecular biology knowledge and skills may also be considered. A first or good 2:1 degree is essential.