CD46 gene-editing confers ex vivo BVDV resistance in fibroblasts from cloned Angus calves

A 2023 proof-of-concept study demonstrated that a 19-nucleotide precision edit, encoding a six-amino acid substitution in the bovine CD46 gene, dramatically reduced bovine viral diarrhea virus (BVDV) susceptibility in a cloned Gir breed heifer. The present study aimed to replicate this result in a popular taurine beef breed (American Angus), while enhancing the design with genetically matched controls and expanded sample sizes. CRISPR/Cas9-mediated homology-directed repair was used to introduce a19-nucleotide edit encoding the A82LPTFS amino acid sequence into exon 2 of the CD46 gene of a primary skin fibroblast line from a male Angus calf (Banner). Cloned Banner embryos were produced via somatic cell nuclear transfer (SCNT) and transferred into recipient cows. Primary skin fibroblasts isolated from the unedited and CD46-edited cloned calves were used to measure ex vivo BVDV susceptibility. Thirty-three cloned embryos (22 CD46-edited and 11 unedited) were transferred to 33 recipient cows. Although six fetuses (three edited, three unedited) reached 200 days gestation, all pregnancies resulted in pre- and perinatal losses due to cloning-related abnormalities. Consequently, no live calves were available for a natural BVDV challenge. Therefore, primary skin fibroblast lines were established from four cloned Banner calves: two each CD46-edited and unedited controls. The CD46-edited calf fibroblasts showed significantly reduced ex vivo BVDV susceptibility compared to unedited controls and had resistance levels comparable to those from the CD46-edited Gir heifer. Ex vivo infection studies in Angus fibroblast lines confirm the observation that the CD46 gene-edit strongly inhibits BVDV infection by blocking viral entry. These studies extend the applicability of this finding from the Gir breed to the most common US beef breed, Angus, suggesting the potential for broad application of CD46 editing in BVDV control. Continued advancements in cloning technology will further enhance the potential of gene-editing for producing disease-resistant livestock.