Preparing for disease control by gene editing for a more sustainable livestock production

One of the most difficult traits to improve by traditional breeding is disease resistance. New gene editing technologies enables high-throughput CRISPR screening to identify which genes are important for different pathogens in livestock. The overall idea of a genome-wide CRISPR screen is that every gene in the genome is targeted for knock-out by guideRNAs (gRNAs). By adding a pathogen to the targeted cells, it will be possible to find candidate genes for resistance as we can sequence cells surviving the pathogen to find which gRNA knock-outs contributed to resistance. WP1: Protocols have been established for relevant cell lines in pigs and cattle. Good cell cultures are crucial as a high-throughput CRISPR screen for all the genes in a genome requires a lot of cells. In pigs, IPEC-J2 intestinal cells stably expressing Cas9 have been created. In cattle, turbinate (BT) nasal cells expressing Cas9 is being made. Because of the low MOI (multiplicity of infection) required to have single gRNAs transfected in cells, there is not enough Cas9 in a single lentivirus for successful transfection, and Cas9 expressing cell lines overcome this problem. WP2: The aim of this work package is to design genome-wide gRNA libraries. Human and mice have commercially available libraries, but this is not available for any other species. In-house designed libraries therefore needed to be made. General features of the libraries were set. To ensure that each gene was successfully knocked out, 4-5 guides were chosen per coding gene. The cut site should be between 5 and 65% of the coding gene to make sure that the knock-out would destroy the corresponding protein properly. We wanted no matches to other sites in the genome as off-target cutting would give rise to unintended mutations. Moreover, we included 3-4 guides per non-coding gene, such as long non-coding RNAs, which often have regulatory functions. Non-targeting guides were included as controls. Two different efficiency scores were implemented for optimal cutting efficiency of the guides (Moreno-Mateos Score and Fusi/doench Score). The pig library has been designed and delivered to us as oligos, whereas the bovine library is ready to be ordered. WP3: In this work package we will conduct the genome wide screening with E.coli pathogens to identify molecular targets important for diarrhea in pigs. The easiest way to have a screenable assay with pathogens is if the pathogen kills cells that are not resistant. For E.coli, we tested five different pathogenic strains from pig. Two of the strains killed the cells and sequencing of the strains showed four different hemolysin genes in common for these two strains. Hemolysin is pore forming toxin that inserts itself into the membrane of host cells and causes calcium influx, potassium efflux, activation of apoptotic pathways, osmotic swelling and lysis. Supernatants of the two strains causing cell death were checked for hemolysin activity and with optimization of hemolysin production, the supernatants maintained activity at different temperatures. WP4: The aim of this work package is to conduct genome-wide screening with P. multocida and genome-wide association analyses to identify molecular targets important for bovine respiratory disease in cattle. This work has not started yet. WP5: In this work package, we aim to verify top hits from the screening in WP3 and 4. The work will start once the screens are complete.

Livestock meat industries are aiming for a more sustainable production and better food safety through focus on animal welfare, disease resistance, mortality and antibiotic use. Animal welfare and production efficiency can be severely affected by the presence of diseases, however, breeding for disease resistance is a difficult task as it requires high quality phenotypes on the disease status of animals in the population. This is hard to get because diseases are often caused by different pathogens and influenced by environmental factors. Gene editing technology offers new opportunities in breeding for disease resistance through identification and editing of important genes. The objective of this project is incorporating CRISPR screening tools in pig and cattle, to help solve the challenge of breeding for disease resistance. Main activities include screening of whole genome CRISPR edited cells with pathogens causing animal welfare and production losses due to diarrhea and lung infections in Norwegian livestock. Whole genome CRISPR screens have been successfully used to identify functional genes in several infectious diseases in human, however, these tools have not been developed in livestock previously. In Norway, diarrhea is the most important disease leading to piglet death, whereas respiratory infection is the most common disease in calves. Antibiotics are often used to treat both piglet diarrhea and bovine respiratory disease, however, antibiotics resistance is a concern. The identification of functional genes involved in host-pathogen interactions can not only be used for future gene editing to obtain resistant animals but also for identifying genetic markers to be used in traditional breeding today. Better animal welfare, less production loss and reduced carbon footprint contributes to further developed competitiveness of Norwegian livestock in a world that increasingly request sustainably produced food.