Using CRISPR-interference to identify new drug targets to treat inflammatory disease.

Inflammatory diseases such as emphysema, asthma, heart disease and arthritis cause much illness in the developed world. We have little understanding of how the severe inflammation associated with something like pneumonia can completely resolve, while other sorts of inflammation persist with associated tissue damage. In fact, we know little of the processes that cause resolution of inflammation in any setting. The primary inflammatory leukocyte, the neutrophil, is key to mounting an inflammatory response, however the removal of these cells is critical for successful inflammation resolution.

In our lab we are interested in neutrophil removal from inflammatory sites. It is increasingly recognised that neutrophils are able to migrate away from inflammatory sites once they have completed their role. Our laboratory has published in high impact journals our findings that neutrophil reverse migration is tightly regulated in the zebrafish inflammation model. This process can be manipulated both genetically and pharmacologically to influence the outcome of inflammation. Neutrophil recruitment to inflammatory sites is regulated in large part by interaction of chemokines and chemokine receptors. Chemokine receptors are part of the family of G-protein coupled receptors (GPCRs) and are expressed in these cells and play important roles in sensing the presence of chemoattractants, transducing signals that lead to the production of inflammatory cytokines and regulation of intracellular and intercellular communications. In addition to recruiting neutrophils, we have indirect evidence that neutrophils are held at inflammatory sites by signalling through GPCRs but don’t know which ones are important for this process.

The aim of this project is to generate a transgenic zebrafish system to rapidly screen through all candidate GPCRs by knocking out their expression specifically in neutrophils to study their roles in retention signalling during tail fin injury. The hypothesis is that signalling through GPCRs leads to neutrophil retention at sites of inflammation. Blocking this signalling could lead to inflammation resolution, defining a new type of treatment for inflammatory disease. Using new CRISPR interference (CRISPRi) technology, we will manipulate inflammatory genes in the zebrafish. CRISPRi allows for sequence-specific repression or activation of gene expression. This technique allows for cell specific expression of dCas9, meaning that we can knock out genes quickly and easily in neutrophils. This approach will allow us to identify new phenotypes, and will lead to new understanding of clinically important biological questions. This project will investigate specific candidate GPCRs, to identify essential genes involved in the regulation of inflammation resolution, and elucidate targets for translatable drug therapies.

Ours is a lively, fun and exciting lab to work in. There are lots of post-docs around to help. We strongly encourage students attendance international conferences, and we operate a publication-focussed lab culture that allows students to publish manuscripts at an early stage.