CRISPR is king. That’s what Cooperative Extension Professor in Animal Genomics and Biotechnology at UC Davis, Alison Van Eenennaam, and her post-doc, Alba Ledesma, found out when the European Food Safety Authority asked them to do a review of the global research in genome editing of livestock for food and agricultural production.

“About 80% of all of the edits detailed in peer-reviewed research publications were being done using the CRISPR/Cas9 system. Other editing technologies such as zinc-finger nucleases and TALENs, predated CRISPR/Cas9, and comparatively they're more complicated and expensive to use. They do the same thing, make a double-stranded break in the DNA at a targeted location in the genome, but they're more expensive and complicated to use,” Van Eenennaam says. “That's part of the attractiveness and the democratization of genome editing is that with CRISR/Cas9, you just need to order a different CRISPR guide and you can target the Cas9 to cut at a different region in the genome.”

Last week during the 100th Annual USDA Agricultural Outlook Forum, Van Eenennaam shared the data compiled from the 195 peer-reviewed publications, as well as an update on the work Genus plc has done to develop a new generation of CRISPR-edited pigs that are resistant to porcine reproductive and respiratory virus syndrome.

Of the reviewed papers, approximately 30% of them detailed editing in mammalian somatic cell lines, followed by somatic cell nuclear transfer (SCNT) cloning to produce live edited animals. About 61% of the publications outlined approaches where the editing was done in developing embryos. Seventy-five percent of the applications involved gene inactivation, or as Van Eenennaam refers to it, “gene knockouts targeting a gene that when knocked out results in a particular phenotype or characteristic you want.”

Those specific targeted traits included heat stress tolerance, disease resistance, color alterations, hypoallergenic milk, meat or eggs, reproductive, meat quality and animal welfare traits, such as having cows that don’t grow horns or boars that don’t progress through to sexual maturity to avoid the development of boar taint.

“As a breeder, a lot of these are traits are in the normal breeding objective, using conventional selection,” Van Eenennaam says. “So, we haven't radically changed what we're after. We just have a new way to actually introduce some of these characteristics in a very targeted way.”

Sixty percent of the gene editing work has been conducted in mammals with pigs leading the effort. Van Eenennaam says this is partly due to pigs being a litter bearing species, where you get many offspring in each production cycle.

As for countries leading the research efforts in gene editing, China has published the most and in all animal categories: ruminants, monogastric, aquatics, avian and insects. The U.S. is second, with Japan following right behind.

However, when it comes to the regulatory landscape for getting these gene-edited animals to market, the country policies vary from region to region. For the United States, if an animal is gene edited, irrespective of the nature of that edit or any pathway to risk, the edit will be treated as a new animal drug and will require FDA approval in order to commercialize it.

Long road to FDA approval

The Genus PRRS resistant pig is the first gene edited food animal application that's been publicly announced to be going through full FDA investigational new animal drug (INAD) regulatory review. Van Eenennaam applauds the lengthy efforts Genus plc, the parent company of PIC (Pig Improvement Company), has undergone to get to this point.

“It's one thing to edit one cow, but if you are going to introduce this in the same way in plant breeding, you're going to have to introduce it into all of the founder lines that make up the four-way cross that is common in many breeding programs,” she says. “You can't just edit the Duroc in this case, you have to edit the Duroc and get that edited in the homozygous condition, and similarly the Landrace, and the Large White, and the Synthetic grandparent stock.”

The work involves going through a “rainbow of pigs” Van Eenennaam says to figure out which ones are non-mosaic homozygous or heterozygous knocked out, and which ones are undesired mosaics with multiple edited genotypes. And because all of these animals carry edits that are considered unapproved animal drugs, none of these pigs can enter commerce or the food supply.

“So, whoever's developing this has to pay the cost … of not allowing them to go in the food supply similar as what the salmon people had to do while they were waiting for their FDA approval,” Van Eenennaam says. “This is not something that a university's going to do. This is going to be a large multinational company that can afford this.”

In order to accomplish this, the company determined it needed 10 to 20 high genetic merit animals in each of the four parent lines, or around 40-80 homozygous non-mosaic pigs. Once those are approved, they can then go forward and breed them into the commercial population.

Last week, Genus was able to share the details of that work in a new report published in The CRISPR Journal titled, “Generation of a Commercial-Scale Founder Population of Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs Using CRISPR-Cas.” 

“They’ve accomplished this, which is a pretty impressive effort, and so they now have to get those animals to go through the regulatory approval and show that these pigs are safe to eat and for the environment, and that the gene edit does in fact make the pigs resistant to the PRRS virus, with performance otherwise comparable to non-edited lines. Once they get regulatory approval, then they'll start breeding it into their population,” Van Eenennaam says. “That’s an example of what it would take in this case to get new animal drug approval in the United States.”

Last year the Colombian government issued a favorable regulatory determination that these pigs do not contain “foreign DNA.” This means Colombia will treat the PRRS-resistant pigs the same as conventionally bred pigs rather than as “GMOs.”