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CRISPR gene editing: GM or not?

The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs).

18 Jan 2017

CRISPR is the new genome editing tool that could transform the fields of biology, medicine, agriscience, and biotechnology, among others but remains contentious. Progress with CRISPR gene editing technology and its possible impact on the breeding of new crop varieties was recently discussed by the SCI Agrisciences Group committee. Dr Derek Hollomon has reviewed how the technology has been evolving and the issues surrounding its commercial use.



From yoghurt to molecular scissors

A Danish dairy company might seem an unlikely place to begin a story about gene editing and a novel technique to generate genetically modified organisms (GMOs). However, Dr Rudolphe Barranguo was exploring ways to prevent bacteria used in yoghurt production from being contaminated with bacterial viruses (phages) when he sequenced the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) elements from many Streptococcus thermophilus strains. Surprisingly, elements contained sequence segments homologous with phages. Bacterial strains with the same CRISPR profiles were resistant to the same phage. Further experiments confirmed that CRISPR elements, together with Cas endonucleases (enzymes that cleave DNA at certain points) and a guide protein, deleted homologous phage sequences and prevented infection.

Barranguo had discovered a bacterial immune system and published a paper in 2009. Further research was quickly followed up in a joint project by Jennifer Doudna at Berkeley and Emmanuelle Charpentier at Umea University in Sweden (and later at the Max Plank Immunology Institute, Berlin). Surprisingly, the bacterial immune system also worked in eukaryotes, allowing development of a molecular scissors system by linking CRISPR, an improved endonuclease (Cas9) that deleted both DNA strands, and an engineered guide RNA rather than a protein. This system precisely, selectively, and affordably cuts DNA in a programmed manner, but allows for repair by including a desired DNA segment, which may be inserted.

The CRISPR Cas9 system offered advantages over established genetic manipulation techniques. It was available in kit form and could predictably modify any part of a known genome sequence by insertions, deletions, and even single base changes. Few errors occurred and targeted changes reduced the need to screen many transformants. Significantly, selectable markers were not required, so foreign DNA need not be involved. Gene editing in plants is achieved by transfecting protoplasts with kit components and regenerating transformants, first as callus and then as whole plants.

Patent disputes

CRISPR Cas9 gene editing has been actively taken up by researchers in many fields, including crop and food production. Several potential products have been developed, such as herbicide tolerant oilseed rape, waxy maize, mildew resistant wheat, low phytate maize, and non-browning mushrooms. As yet, none have been commercialised, in part because of ongoing patent disputes. Doudna and Charpentier published their work in May 2012 and, through their company, Caribou (linked with the leading seed company DuPont Pioneer) filed a patent the same month. Feng Zhang at the Broad Institute, MIT, had done similar work with CRISPR Cas9 to gene edit human and mouse cells. He filed a similar patent in December 2012, published his work in early 2013, and was awarded the patent in April 2013, having opted for a faster review process. At this time in the USA, patents were awarded to the inventor and so Doudna and Charpentier challenged this on the grounds that they were the inventors of the technology. The dispute has not been resolved.

In Europe, nine companies involved in CRISPR Cas9 research have challenged Zhang’s patent, but the European Patent Office is not expected to make a decision until at least the latter part of 2017. One impact of these patent disputes has been to delay further research on the use of CRISPR Cas9.

GM or not?

Because products generated by the CRISPR Cas9 technique contain no foreign DNA, the US food regulators (FDA) considers they should not be regulated as GMOs. Similar decisions have been taken in Germany, Sweden and Argentina. But this is a contentious issue. In the US, there are concerns about labelling food products and the potential impact on biodiversity.

In Europe, Greenpeace and Friends of the Earth are adamant that organisms produced by CRISPR Cas9 are GMOs. While this technique might not involve foreign DNA, the proteins generated, especially by insertion or deletion, could be proteins with a novel function. EU regulators have delayed a decision for more than a year. Until this dispute is resolved, products generated using this gene editing technique are unlikely to be used in crop and animal production systems, at least in the EU.

The Nuffield Council on Bioethics has recently (September 2016) published a report on the issues around gene editing and the ethics of applying the technology. This can be downloaded here.

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