2016: a gene-edited button mushroom falls outside US GMO rules

Three pages, signed and dated by hand

Case 15-321-01 at the United States Department of Agriculture closed on 13 April 2016, in three typed pages. Michael J. Firko, APHIS Deputy Administrator for Biotechnology Regulatory Services, told Yinong Yang of Penn State that the agency did not consider his CRISPR/Cas9-edited white button mushroom, Agaricus bisporus, to be regulated under 7 CFR part 340. The subject line set the question out exactly: a request for confirmation that a transgene-free, CRISPR-edited mushroom is not a regulated article. That case number appears nowhere on the letter itself — it belongs to the agency’s file and to the address the document is served from — and the date is handwritten above the ruled line marked Date on the signature page, where the scan reads 4/13/2016. It is absent from the file’s text layer, which is why the reply is sometimes dated from press coverage instead. The signed letter is public, with a scan of the original inquiry bound in behind it.

The outcome turned on how the rule had been drafted. Part 340, made under the Plant Protection Act, reached organisms engineered using a donor, recipient, vector or vector agent listed in section 340.2 that meets the definition of a plant pest; organisms unclassified or of unknown status; and anything the Administrator determined to be, or had reason to believe was, a plant pest. The finished mushroom answered to none of those descriptions. APHIS added that the white button mushroom is not a federal noxious weed under 7 CFR part 360, and that it saw no reason to think an anti-browning trait would make it weedier. Genome editing as a technique is nowhere assessed in the letter; it appears only in the account of what was done to the mycelium.

The laboratory account, as its author gave it

Yang’s letter of inquiry, dated 30 October 2015 and preceded by a presentation at APHIS in Riverdale on 7 October, reads like a methods section. The delivery construct was a pUC19-based plasmid carrying a codon-optimised Cas9 nuclease from Streptococcus pyogenes, U6 and gpd promoters taken from the mushroom itself, a synthetic guide RNA and a BsaI cloning site. It entered protoplasts by polyethylene-glycol-mediated transformation and was meant to be expressed only transiently. Mycelia were regenerated from those protoplasts without antibiotic selection, and the strains taken forward were, the letter states, those in which PCR and Southern blot analyses found no integrated foreign DNA. The edit itself amounts to deletions of between one and fourteen base pairs in a specific polyphenol oxidase gene.

The parts list also includes sequences from Escherichia coli and from Agrobacterium tumefaciens. None of them, the applicant argued, ended up in the mushroom genome, and that, together with the button mushroom being neither a plant pest nor a noxious weed, was the whole of the case he put.

The verb “states” is doing heavy lifting there, and it does the same work throughout the reply. APHIS tested nothing and repeated no experiment. It recites what the applicant told it, reaches its conclusion on the basis of the information cited in that letter, and obliges Yang to notify the agency in writing the moment he becomes aware of anything that might affect that conclusion. This was a desk review of a representation rather than a laboratory verification, and it is the distinction most often lost when the episode is retold.

What it did not decide

It was not an approval, a marketing authorisation or a safety assessment, and the letter concedes as much in its own register: the variety described may still fall to other regulators, and it names the FDA and the EPA. Nor did the mushroom leave USDA oversight, a claim the story has attracted ever since. APHIS is part of USDA. Importing these mushrooms, like any other mushrooms, remains subject to Plant Protection and Quarantine permit and quarantine requirements, and any line from the same transformation that retained inserted genetic material would still be regulated under part 340, needing a notification or a permit for import, interstate movement or environmental release.

It was not even the first ruling of its kind. The agency wrote that it was answering consistently with previous responses to similar letters of inquiry; what was new was the subject, not the gesture. The phrase that carried the story around the world — the first CRISPR-edited organism to receive a green light from the US government — belongs to Emily Waltz, writing for Nature, and not to APHIS. It cannot be checked against the agency’s own records either, because the present confirmation-letters table covers only the SECURE-era confirmations and holds no entry for 15-321-01. Two other familiar figures come from that same report, published the next day on 14 April 2016, rather than from the document: one of six PPO genes knocked out, and enzyme activity reduced by 30 per cent.

The browning itself

The file concerns regulatory status alone, so none of it should be read as saying that such a mushroom reached a shop, in the United States or anywhere else. It says nothing about European Union or Polish law, and an American determination does not travel to either; this entry does not attempt to answer how the same case would be treated in Europe. Neither the letter nor the report asks whether a slower-browning mushroom tastes better, keeps more of anything or is in any way safer, and nor does this entry. The case made for the trait was stated plainly and was about something else entirely: appearance, shelf life, and automated mechanical harvesting. That is a grower’s argument.

The chemistry, though, is open to anyone with a knife. Polyphenol oxidase and its substrates sit in separate compartments of intact tissue; damage brings them together, oxygen does the rest, and the melanin that forms darkens the exposed surface. That is why a bruised or sliced mushroom discolours along the injury while an untouched cap does not. One caution while watching it happen: the deep chocolate-brown of a mature cap’s gills is spore colour, not browning at all. The surface to keep an eye on is the pale flesh the knife has just laid open.