BBSRC EASTBIO DTP - A genome editing approach to identify genes involved in enhanced resistance or susceptibility to bovine TB

Infectious diseases of livestock remain a major barrier to increased production despite current control measures in the UK, EU and world-wide. Improved efficacy and availability of livestock vaccines and breeding for enhanced disease resistance are crucial. However this requires understanding of specific host-pathogen interactions as species differences makes extrapolating from mouse or human models to livestock problematic. Macrophages are of central importance in pathogen defence as well as inflammation and its resolution. There are several endemic pathogens with major impacts on the health and welfare of UK cattle and consequently the livestock economy which are not well controlled. These include the zoonotic pathogen Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB). This intracellular pathogen is tropic for bovine macrophages where it is able to survive within an intracellular niche enabling long term survival and persistence leading to chronic infection. However, exposure to M. bovis does not necessarily lead to disease. Host genetic differences have been shown to contribute to resistance to bTB (Heredity, 2014, 112, 543–551) and recently, these have been exploited by the commercial release of estimated breeding values of bulls which define bTB risk.

The genome editor revolution has allowed researchers unprecedented opportunities to precisely modify the genome of their target species. At their simplest, genome editors are used to create a double strand break at a specific locus in the genome. In the absence of a suitable template for homology directed repair, mammalian cells utilise the non-homologous end joining pathway to re-join the broken ends. This pathway is error-prone, resulting in small insertions or deletions at the target site, the outcome of which is often disruption of the targeted gene sequence and loss of function of the encoded protein.

Three forms of genome editor currently dominate the research landscape; zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. Unlike ZFNs and TALENs, the CRISPR/Cas9 system lends itself very well to application in genome-wide cellular knockout (GECKO) as demonstrated by Shalem et al., 2014 (Science 343(6166):84-7). Using this approach it is possible to knock out every gene in a mammalian genome then interrogate pooled cells for modifications of a specific trait. We currently have a BBSRC-funded grant to develop a GECKO library targeting the 22000 genes of the bovine genome. The focus of this studentship will be to apply this library to bovine macrophages and identify genes associated with either increased susceptibility or resistance to infection by M. bovis, enhancing our understanding of genetic resistance and informing future breeding strategies.

Through this project the student should acquire a solid understanding of the varied applications of genome editors, initially as mediators of genetic cell modification then latterly as tools for more subtle transcriptome manipulation. He/she will become proficient in a wide variety of cell and molecular biology techniques including culture and manipulation of macrophages, production and application of lentivirus libraries, immunochemistry and immunoblotting, gene expression analyses by next-generation sequencing and qPCR. The project brings together the complementary expertise of the supervisors Dr Jayne Hope (host-pathogen interactions/M. bovis biology) and co-supervisors Professor Bruce Whitelaw and Dr Simon Lillico (genetically engineered livestock including gene editing) in a new collaboration, where the student will have access to state of the art facilities and expertise in genome manipulation and immune biology.