Intractable pests such as bovine viral diarrhoea virus (BVDV) represent a significant barrier to transforming livestock production to meet welfare and sustainability targets for the industry. BVDV, present in approximately 2% of the Australian cattle herd, is implicated in several disease complexes, including bovine respiratory disease, the leading cause of mortality in Queensland feedlots. Despite widespread global efforts to vaccinate and eradicate BVDV, persistent infection with non-cytopathic strains of the virus have enabled vertical transmission and dissemination of this pathogen throughout cattle production systems. Advances in gene editing technologies such as CRISPR-Cas9 offers an alternative approach to preventing the spread of BVDV by targeting host susceptibility factors. Therefore, the aim of this project is to validate genetic modification of bovine cell lines to block virus entry and replication. Specifically, Madin Darby Bovine Kidney (MDBK) cells containing an inducible Cas9 transgene will be used for high throughput screening of optimum genotypes which can inhibit BVDV infection whilst maintaining native cellular function. Preliminary investigations of BVDV resistance phenotypes using Oxford Nanopore long read sequencing have revealed potential multigenic modifications of both the CD46 and ADAM17 locus may be required to develop immunity to BVDV. Furthermore, prevention of secondary cell-to-cell transmission of BVDV may require targeting innate immune factors in addition to blocking viral entry pathways. Construction of transgenic MDBKs has indicated over one hundred novel genomic safe harbours may exist in the bovine genome, suggesting broad gene editing potential within cattle beyond disease resilience. Optimisation of transgenic cell lines to streamline selection of superior genotypes for BVDV resistance will form a valuable basis for future applications of gene editing technology in livestock production.