CRISPR/Cas12a mediated knock-in of the Polled Celtic variant to produce a polled genotype in dairy cattle.

In modern livestock farming horned cattle pose an increased risk of injury for each other as well as for the farmers. Dehorning without anesthesia is associated with stress and pain for the calves and raises concerns regarding animal welfare. Naturally occurring structural variants causing polledness are known for most beef cattle but are rare within the dairy cattle population. The most common structural variant in beef cattle consists of a 202 base pair insertion-deletion (Polled Celtic variant). For the generation of polled offspring from a horned Holstein-Friesian bull, we isolated the Polled Celtic variant from the genome of an Angus cow and integrated it into the genome of fibroblasts taken from the horned bull using the CRISPR/Cas12a system (formerly Cpf1). Modified fibroblasts served as donor cells for somatic cell nuclear transfer and reconstructed embryos were transferred into synchronized recipients. One resulting pregnancy was terminated on day 90 of gestation for the examination of the fetus. Macroscopic and histological analyses proved a polled phenotype. The remaining pregnancy was carried to term and delivered one calf with a polled phenotype which died shortly after birth. In conclusion, we successfully demonstrated the practical application of CRISPR/Cas12a in farm animal breeding and husbandry.