The identification of novel therapeutic targets for human and animal tuberculosis using CRISPR/dCas9 genome interference technology

Non RVC Co-supervisors Dr Shan Goh Department of Biological & Environmental Sciences The University of Hertfordshire

The respiratory disease tuberculosis (TB) presents a burden to both human and animal populations. TB in humans, caused by Mycobacterium tuberculosis, is one of the top 10 causes of death world-wide with 1.5 million people dying each year. TB in domestic species, caused by Mycobacterium bovis, represents a large economic burden and potential zoonotic risk. In the UK, the annual cost of the disease control strategy for bovine tuberculosis is estimated to be approximately £100 million. Current control strategies through vaccination and chemotherapy (for humans) need to be improved and novel therapeutic targets identified.

Whole genome screening methods such as TraDIS (Transposon Directed Insertion Seqeuncing) are useful for predicting genes that are essential for the survival of pathogenic mycobacteria, and these genes represent potential therapeutic targets. However, single gene level investigations are required for verification and evaluation of the therapeutic potential of a novel target. Deletion of genes can be useful for verification, but can only be used providing candidates are dispensable for growth in vitro. Additionally, current methods for gene deletion are slow, time-consuming and not always effective.

The recent discovery of the bacterial CRISPR/Cas9 system and its application to interference has revolutionized the manipulation of animal, plant and bacterial genomes. During this project this technology will be applied to turn down the expression of genes that have been identified by whole genome screening methods to be potential therapeutic targets for tuberculosis. Quantitative PCR and western blotting will be used to verify the reduction in expression and the effect of interference on the viability of bacteria measured. The project will allow you to gain experience with cutting edge genome manipulation techniques, gene expression measurement methods, primary cell culture, molecular microbiology and fluorescent microscopy. The overall outcome will be the identification and evaluation of novel therapeutic targets for tuberculosis in both animals and humans.

The studentship will commence at the beginning of the 2017/18 academic year.