Use of Gene Editing to Introduce the Polled Trait into Elite Germplasm

Physical dehorning of dairy cattle is a standard practice to protect both human dairy workers and other animals from injury. However, it is not only costly for producers, but also painful and stressful for the animals. As a result, dehorning is currently facing increased public scrutiny as an animal welfare issue. Despite these factors, 94% of U.S. dairy cattle producers report routine dehorning.

Horns are inherited as an autosomal recessive trait, meaning that horned cattle have two copies (pp) of a recessive allele that results in horns. Naturally-occurring dominant (P) alleles of the POLLED gene locus (specific position on the chromosome) are prevalent in beef cattle breeds such as Angus, and also exist at a low frequency in some dairy breeds. Inheriting a single copy of this P allele results in a hornless or polled animal. However, dairy animals carrying the dominant P polled allele(s) tend to have lower genetic merit (lifetime net merit (NM$)). Horns do not have a cause and effect relationship with dairy genetic merit; rather they happened to come along as genetic hitchhikers when selecting for elite dairy genetics.

The American Veterinary Medical Association (AVMA) has proposed using polled genetics as an alternative to dehorning. However, there are few polled dairy sires with high genetic merit for important economic indexes, so this approach has not been widely adopted. Figure 1 (see page 5) shows that animals carrying the P allele tend to have a lower NM$, meaning that daughters of polled sires will earn less over their lifetimes.

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Figure 1. The average NM$ of the top 50% of homozygous polled (PP), heterozygous (Pp), and horned (pp) Jersey (brown bars) and Holstein bulls (black and white bars) registered with the National Association of Animal Breeders (NAAB) in March 2018. (Mueller et al., unpublished)

Dr. John Cole from the USDA proposed adding the economic value of polled ($40) to selection indices but showed that this is not an effective method for increasing the frequency of polled animals in the population. The frequency of the P allele is very low in U.S dairy cattle (< 0.01), so carriers are unlikely to be among the top ranked bulls based on NM$. Therefore, only adding the economic value of polled to the NM$ index does not effectively increase the frequency of the P allele (Cole, 2015).

Gene editing has the potential to resolve these economic concerns by producing high-genetic merit polled bulls, thereby eliminating the need for dehorning (Carlson et al., 2016). Gene editing refers to a category of new tools that can be used to precisely edit or change the genetic code. It enables useful alleles to be introduced into elite germplasm without traditional crossbreeding. This often brings in a lot of undesired genetic information, known as “linkage drag,” and refers to all of the unwanted traits that come along with the desired allele when practicing traditional crossbreeding. Breeders then must spend several generations breeding out the unwanted genetics while retaining the desired allele.

As the name “gene editing” suggests, these technologies enable researchers to add, delete, or replace letters in the genetic code. In the same way that spell check identifies and corrects single letter errors in a word or grammar errors in a sentence, gene editing can be used to identify and change the letters that make up the genetic code (i.e. DNA) within an individual.

The current available set of gene editors, known by acronyms ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), and the trendy CRISPR (clustered regulatory interspersed short palindromic repeat)-Cas9 associated system, are effectively precise molecular scissors. They can be targeted to the POLLED locus that is responsible for horn development and used to replace 10 base pairs of the dairy “p” allele with 212 base pairs of the naturally-occurring “polled” P allele. This P allele by sequence introduced gene editing is exactly the same allele that is found in beef breeds, and when inherited it results in the polled or hornless phenotype in the resulting calves, making them genetically dehorned. The edits can take place at the single cell stage of embryogenesis (i.e. just after fertilization), or in cell culture lines which can then be cloned following confirmation that the intended edits have been successfully written into the genetic code.

Given the extensive use of artificial insemination (AI) in the dairy industry, even if only a small proportion (1%) of elite AI sires were gene edited to be homozygous PP, the P allele could be rapidly disseminated to the dairy population while maintaining the rate of genetic gain. This would be superior to using existing polled genetics. Recent simulation studies in both Holstein and Jersey populations found that if existing homozygous polled sires were used exclusively, it would both slow the rate of genetic gain and dramatically increase inbreeding in both the Holstein and Jersey breeds (Mueller et al., 2018a,b).

While there are a lot of possibilities for gene editing in animal breeding, the regulatory status of animals carrying intentional gene edits, such as the P allele discussed above, is unclear. A 2017 draft FDA guidance 187, Regulation of Intentionally Altered Genomic DNA in Animals (FDA, 2017) proposes that intentional genomic alterations, such as those introduced by gene editing, but not those introduced by selective breeding and random mutagenesis, will be subject to mandatory, multigenerational premarket “new animal drug” evaluation.

The FDA draft Guidance specifies that additional “new animal drug” regulatory oversight will be triggered by intentional nucleotide insertions, substitutions, or deletions introduced by gene editing. It further specified that in general, each specific genomic alteration will be considered a separate “drug” subject to new animal drug approval requirements, irrespective of the novelty of the alteration or the existence of any hazards in the resulting product (Van Eenennaam, 2018).

This is diametrically opposed to the approach announced by the USDA on regulating gene edited plants. In a March 28, 2018 press release, U.S. Secretary of Agriculture, Sonny Perdue, clarified that the USDA does not have any plans to additionally “regulate plants that could otherwise have been developed through traditional breeding techniques.” Historically, neither plant nor animal breeding have been formally regulated. Rather, US law prohibits the commercial sale of unsafe food, irrespective of production method.

 
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