<i>CD46</i> Gene Editing Confers Ex Vivo BVDV Resistance in Fibroblasts from Cloned Angus Calves
2025
Aspen M. Workman | Michael P. Heaton | Brian L. Vander Ley
A previous study demonstrated that a 19-nucleotide edit, encoding a six amino acid substitution in the bovine <i>CD46</i> gene, dramatically reduced bovine viral diarrhea virus (BVDV) susceptibility in a cloned Gir (<i>Bos indicus</i>) heifer. The present study aimed to replicate this result in American Angus (<i>Bos taurus</i>) using genetically matched controls and larger sample sizes. CRISPR/Cas9-mediated homology-directed repair introduced the identical <i>CD46</i> edit, encoding the A<sub>82</sub>LPTFS amino acid sequence, into exon 2 of <i>CD46</i> in primary Angus fibroblasts. Thirty-three cloned embryos (22 <i>CD46</i>-edited and 11 unedited) were transferred to recipient cows. However, all pregnancies resulted in pre- and perinatal losses due to cloning-related abnormalities, preventing in vivo BVDV challenge. Consequently, ex vivo BVDV susceptibility assays were performed on primary fibroblast cell lines rescued from deceased cloned Angus calves. Infection studies revealed significantly reduced susceptibility in the edited lines, comparable to the resistance previously observed from the edited Gir heifer. These studies extend the applicability of this finding from Gir to the most common US beef breed, Angus, suggesting the potential for broad application of <i>CD46</i> editing in BVDV control. Continued advancements in cloning technology will enhance the potential of gene-editing for producing disease-resistant livestock.
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