Generation of wolf epidermal keratinocyte cell lines to test for effects of the CBD103 pigmentation-associated variant on gray wolf viral response

In North American gray wolves, black coat color is dominantly inherited, via a three base pair coding deletion in the canine beta defensin 3 (CBD103) gene. We generated an immortalized epidermal keratinocyte cell line from a North American gray wolf homozygous for the wildtype, non-deletion allele of CBD103 (genotype Kyy). We then used CRISPR/Cas9 to generate, from the wildtype line, a cell line that is heterozygous for the 3 base pair deletion in CBD103 (genotype KyB). Whole genome sequencing was performed on the two cell lines to confirm the expected CBD103 sequence and to identify potential off-target mutations introduced during the targeted 3 base pair deletion.