First gene-edited calf with reduced susceptibility to a major viral pathogen

Authors: Workman, Aspen; Heaton, Michael - Mike; VANDER LEY, BRIAN - University Of Nebraska; WEBSTER, DENNIS - Recombinetics, Inc; SHERRY, LUKE - Recombinetics, Inc; LARSON, SABREENA - Acceligen Inc; KALBFLEISCH, THEODORE - University Of Kentucky; Harhay, Gregory; JOBMAN, ERIN - University Of Nebraska; CARLSON, DANIEL - Recombinetics, Inc; SONSTEGARD, TAD - Acceligen Inc

Submitted to: bioRxiv
Publication Type: Pre-print Publication
Publication Acceptance Date: 12/8/2022
Publication Date: 12/8/2022
Citation: Workman, A.M., Heaton, M.P., Vander Ley, B.L., Webster, D.A., Sherry, L., Larson, S., Kalbfleisch, T.S., Harhay, G.P., Jobman, E.E., Carlson, D.F., Sonstegard, T.S. 2022. First gene-edited calf with reduced susceptibility to a major viral pathogen. bioRxiv. Article 519336. https://doi.org/10.1101/2022.12.08.519336.
DOI: https://doi.org/10.1101/2022.12.08.519336

Interpretive Summary: Bovine viral diarrhea virus (BVDV) is a widespread and economically important viral infection in cattle, causing respiratory and gastrointestinal diseases, and reproductive failure. While vaccines for BVDV have been available for more than 50 years, BVDV remains a significant global pathogen. Modifying the bovine receptor for BVDV via CRISPR/Cas9 gene-editing technology offers a novel way to potentially control infections in cattle. The main bovine receptor for BVDV is CD46, which we modified by replacing six amino acids in the domain that binds to the virus. This six amino acid substitution dramatically reduced susceptibility to BVDV infection when tested in the laboratory. The same CD46-edit was then made in skin cells taken from an adult Gir breed cow and used to produce reproductively cloned, gene-edited animals for study. At 100-days gestation, primary cells were isolated from multiple organs of a gene-edited fetus and compared those from an unedited fetal clone. When infected in the laboratory, cells from all organs of the gene-edited fetus had a significant reduction in BVDV susceptibility. In 2021, a normal and healthy Gir heifer calf was born with the CD46 edit and has continued to thrive with no observed adverse effects from the CD46 edit in the first year of life. To test whether the gene-edited Gir calf could resist BVDV infection, she was co-housed with a Holstein calf that was naturally and persistently infected with BVDV. Upon this natural exposure BVDV challenge, the CD46-edited Gir calf had minimal signs of infection and her white blood cells were not infected. Together, these results provide proof-of-concept for using intentional genome alterations in CD46 to reduce the burden of BVDV infections in cattle. This represents the first demonstration of editing a bovine gene to reduce susceptibility to a viral pathogen.

Technical Abstract: Bovine viral diarrhea virus (BVDV) is one of the most important viruses affecting the health and well-being of bovine species throughout the world. Here we used CRISPR-mediated homology-directed repair and somatic cell nuclear transfer to produce a live calf with a six amino acid substitution in the BVDV binding domain of bovine CD46. The result was a gene-edited calf with dramatically reduced susceptibility to infection as measured by clinical signs and the lack of viral infection in white blood cells. The edited calf has no off-target edits and appears normal and healthy at 16 months of age without obvious adverse effects from the on-target edit. This precision bred, proof-of-concept animal provides the first evidence that intentional genome alterations in CD46 may reduce the burden of BVDV-associated diseases in cattle, and is consistent with our stepwise, in vitro and ex vivo experiments with cell lines and matched fetal clones.