Abstract
Dehorning is a common practice in the dairy industry, but raises animal welfare concerns. A naturally occurring genetic mutation (PC allele) comprised of a 212 bp duplicated DNA sequence replacing a 10-bp sequence at the polled locus is associated with the hornless phenotype (polled) in cattle. To test the hypothesis that the 10 bp deletion alone is sufficient to result in polled, a CRISPR-Cas9 dual guide RNA approach was optimized to delete a 133 bp region including the 10 bp sequence. Timing of ribonucleoprotein complex injections at various hours post insemination (hpi) (6, 8, and 18 hpi) as well as in vitro transcribed (IVT) vs synthetic gRNAs were compared. Embryos injected 6 hpi had a significantly higher deletion rate (53%) compared to those injected 8 (12%) and 18 hpi (7%), and synthetic gRNAs had a significantly higher deletion rate (84%) compared to IVT gRNAs (53%). Embryo transfers were performed, and bovine fetuses were harvested between 3 and 5 months of gestation. All fetuses had mutations at the target site, with two of the seven having biallelic deletions, and yet they displayed horn bud development indicating that the 10 bp deletion alone is not sufficient to result in the polled phenotype.
Highlights
Dehorning is a common practice in the dairy industry, but raises animal welfare concerns
The findings from this study demonstrated that CRISPR-Cas[9] dual guide RNAs microinjected as RNPs 6 hpi in bovine embryos is an efficient method to obtain biallelic deletion animals, the 133 bp deletion, including the[10] bp deletion found in the P C allele, is not sufficient to result in the polled phenotype
We designed guide RNA (gRNA) targeting the 5′ and 3′ regions surrounding the targeted 10 bp in genotypically horned embryos that resulted in high rates of mutation and, when co-injected, resulted in the predicted 133 bp deletion, the smallest possible deletion containing the 10 bp we could achieve based on gRNA design and mutation efficiency in embryos
Summary
Dehorning is a common practice in the dairy industry, but raises animal welfare concerns. Dehorning and disbudding are common practices used in the beef and dairy industry to physically remove an animal’s horns These procedures are done as preventative measures to protect both animals and handlers, they are costly to the producer and painful to the animal. Carlson et al demonstrated that recapitulating the deletion and duplication of the PC allele is sufficient to result in the polled phenotype. This was done by substituting the P C allele for the p allele at the polled locus in a cell lined derived from a horned dairy bull, followed by somatic cell nuclear transfer (SCNT) cloning to produce two polled dairy bull c alves[3].
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