Location: Animal Disease Research
Project Number: 2090-32000-043-004-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2024
End Date: Jul 31, 2025
Objective:
Devise functional genomics strategies, including gene editing, for developing traits in ruminant livestock to enhance disease resistance, reproductive efficiency, and improve resiliency to ticks, tick-borne diseases and other diseases made more prevalent through changing environmental conditions.
Approach:
1. Develop CRISPR-Cas9 gene editing strategies in cattle and sheep to confer resistance or reduced susceptibility to infectious disease. This work will focus on traits that confer resistance to two high-impact pathogens: Mannheimia hemolytica and Morbillivirus caprinae. Leukotoxin produced by M. hemolytica plays a major role in morbidity and mortality due to bovine respiratory disease (BRD). M. caprinae causes Peste des Petits Ruminants (PPR), a transboundary disease that causes high mortality in sheep and goats, and thus threatens food security in many parts of the world.
To reduce the impact of BRD, CRISPR-Cas9 based gene editing will be used to modify the CD18 allele in cattle, initially discovered at WSU as a target to reduce susceptibility to M. haemolytica leukotoxin. The immune cells with this altered allele are postulated to have resistance to bacterial leukotoxin binding, which in turn will reduce cell death, inflammation and thus disease severity caused by the M. hemolytica component of BRD. Prototype CD18 gene edited animals will be produced for testing resistance.
To reduce the impact of PPR, an ovine organoid platform with genome-wide, CRISPR- Cas9 editing will be screened to identify genes that confer resistance to PPRV. As an initial surrogate for PPRV, vesicular stomatitis virus pseudotypes bearing PPRV glycoproteins will be used to identify all host cell receptors, and other factors such as proteases, that play a role in entry of PPRV. Knockout of known receptors used for PPRV entry, including CD46, CD150, and Nectin-4 will help validate the screen. Sheep embryos with CRISPR-Cas9 precision edits in identified candidate PPR resistance genes will be produced and cryopreserved for future transfer to generate prototype animals.
2. Develop novel technologies for precision gene editing of the male germline and dissemination of the resulting disease resistance traits via natural mating. This work will include the development and refinement of methods required for in vitro propagation of sheep and cattle sperm-producing stem cells, including isolation of stem cells from testicular tissue, engineering novel feeder cells lines to support stem cell propagation, and optimizing media formulations. Additionally, methods for clonal expansion of stem cells will be developed. The efficiency of CRISPR-Cas9 gene editing of sheep and cattle sperm stem cells will be improved by optimizing delivery of the gene editing components required to generate precision DNA modifications.
Finally, to enable dissemination of disease resistance traits via natural mating, Surrogate Sires breeding strategies for sheep and cattle will be developed and refined. Nucleus herds of breeding animals carrying NANOS2 knockout alleles will be produced via CRISPR-Cas9 gene editing. Sperm development in these animals will be inhibited thus allowing for transplantation of in vitro propagated and precision gene edited sperm stem cells in the testes of the knockout bulls and rams. These techniques will be optimized to achieve natural fertility.
