Gene-editing tool "not as precise as expected", say researchers in new study. Report: Claire Robinson

CRISPR gene editing in rice varieties caused a wide range of undesirable and unintended on-target and off-target mutations, according to an important new study authored by a Chinese and Australian team of scientists and published in the Journal of Genetics and Genomics.[1]

The researchers were trying to improve the yield of already high-performing varieties of rice by disrupting the function of a "green revolution" semi-dwarfing gene (SD1). They used a stable transformation method that ensured that the CRISPR editing tool remained active in the plants over four generations, so that they could examine the effects over time.

In order to disrupt or knock-out the SD1 gene, they designed the CRISPR gene-editing tool to produce small indels (insertions and deletions of bases in the genome). However, what they got was quite different. In many cases they found large insertions, deletions, and rearrangements of DNA, raising the possibility that the function of genes other than just the targeted SD1 could have been altered. In addition, and surprisingly, the range of on-target mutations (unintended mutations at the intended editing site) varied, depending on the rice variety.

The study confirms that the wide range of undesirable and unintended on-target and off-target mutations seen in gene-edited animal and human cells (see examples here) also occur in plants.

London-based molecular geneticist Dr Michael Antoniou commented, "Given these findings, the likelihood of unpredictable changes in multiple gene functions leading to altered biochemistry in gene-edited food plants, with consequent health risks (toxicity, allergenicity) is very real."

As for the hoped-for increased yield, the opposite was found: Yield was reduced in the CRISPR-induced mutants, along with the desired reduction in plant height. Therefore, the researchers conclude that "the application of CRISPR/Cas9 for successful breeding" may be a "long way" off.

CRISPR "not as precise as expected"

The authors of the new paper warn that CRISPR "may be not as precise as expected in rice”. They further say, "early and accurate molecular characterization and screening must be carried out for generations before transitioning of CRISPR/Cas9 system from lab to field". They add, “Understanding of uncertainties and risks regarding genome editing is necessary and critical before a new global policy for the new biotechnology is established".

EFSA: Wrong again

The new study comes at a time when the European Food Safety Authority (EFSA) has just concluded its public consultation on how to regulate the so-called SDN-1 (gene disruption) and SDN-2 (gene alteration) techniques of gene editing.[2]

In its draft opinion, EFSA downplays the potential for SDN-1 and SDN-2 gene-editing techniques to cause unintended effects that are different from those caused by techniques used in conventional breeding, such as random mutagenesis. EFSA appears to believe that SDN-1 and SDN-2 only produce small changes to the organism’s DNA and that risks that normally relate to genetically modified organisms only apply to gene editing when it is used to add foreign (exogenous) DNA – a type of gene editing known as SDN-3.

However, the new paper exposes EFSA's thinking as completely out of touch with the science. This latest study graphically illustrates that SDN-1 can frequently produce large indels and DNA rearrangements at the intended editing site, which can interfere with multiple gene functions. Furthermore, while the scientists intended to make only SDN-1-type gene disruptions, they ended up with SDN-3-type incorporations of exogenous DNA in a massive 25 out of 31 second-generation CRISPR-edited rice lines. The foreign DNA in this case consisted of elements from the genetic construct – the plasmid – that was used to carry the gene-editing tool into the plant cells.

This finding of exogenous DNA (an outcome supposedly restricted to SDN-3) from an SDN-1 editing event makes a nonsense of EFSA's attempt to divide up gene-editing technologies into SDN-1, -2, and -3. It shows that the agency's assumption that the first two are somehow safer than the last is wrong.

Does EFSA’s opinion matter?

EFSA’s opinion matters because it was requested by the European Commission – and some members of the Commission will use EFSA’s opinion to back their desire to promote gene editing in line with the GMO industry’s wish. That could entail exempting gene editing from the GMO regulations, meaning that gene-edited products will be subjected to little or no safety checking and no labelling.

Tissue culture-induced mutations

Placing the new study in context, a previous study published in 2018 separated out the various stages of developing CRISPR-edited rice plants and examined how many off-target and on-target mutations were caused by each different stage. The stages examined were:
* Tissue culture only
* Agrobacterium infection (used to carry the CRISPR gene-editing tool into the plant cells) only, and
* Introducing the different CRISPR gene editing tools (Cas9 backbone and Cpf1 backbone) via Agrobacterium infection, but without including the guide RNA molecules that direct the editing tools to the desired edit site in the DNA.

They found few off-target effects from the CRISPR editing tools – only 2 out of 49 plants had them.

However, they found a large number of mutations from the tissue culture (200 per rice plant). In the authors' words, "Our results clearly show that most mutations in edited plants are created by the tissue culture process, which causes approximately 102 to 148 single nucleotide variations (SNVs) and approximately 32 to 83 insertions/deletions (indels) per plant."

The results also showed Agrobacterium infection increased the number of mutations over and above the number caused by tissue culture.

In contrast, seed saved from wild type non-GM, naturally bred rice plants had only 30 to 50 spontaneous mutations per plant.[3]

The authors of the new paper on CRISPR-edited rice state that the off-target mutations they observed could similarly have resulted from the tissue culture phase of development, which they did not investigate in their study. But as tissue culture is an obligatory part of making gene-edited (or older-style transgenic) plants, there doesn't appear to be a realistic chance of avoiding the mutations induced by this procedure. Regulators would do well to bear this in mind.

Quality as well as quantity important

It's not only the quantity of mutations that is important, but also the quality – what the mutations do. This question has not been answered because developers are not doing the detailed molecular characterization requested by the authors of the new paper on CRISPR-edited rice. They have also not carried out animal feeding studies to see the biological effects of consuming gene-edited plants over time.

Developers must not be allowed to self-regulate

Tight regulation of gene editing in plants is clearly needed – and it needs to start with an understanding of the technique used to generate the plant ("process-based regulation"). We cannot rely on developers to "regulate" themselves, as illustrated by the case of the gene-edited cattle that turned out to contain antibiotic resistance genes. The developer had denied that any off-target effects were present in the cattle and these were only found by scientists at the US Food and Drug Administration (FDA). FDA scientists are using this episode to argue for strong regulation of gene-edited animals. They state, "It is paramount... that as we move forward, we maintain standards of safety and effectiveness."

We agree with the conclusions of the authors of the new paper and the FDA scientists. EFSA – and the Brexited UK – should not be pushing for the de-regulation of gene editing. This push, driven by economic interests, flies in the face of what the science is telling us and puts public safety at risk.
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Notes

1. Biswas S, Tian J, Li R, et al. Investigation of CRISPR/Cas9-induced SD1 rice mutants highlights the importance of molecular characterization in plant molecular breeding. Journal of Genetics and Genomics. Published online May 21, 2020. doi:10.1016/j.jgg.2020.04.004. https://www.sciencedirect.com/science/article/pii/S1673852720300916

2. The SDN-1 form of gene editing (sometimes written without the hyphen, as SDN1) should not be confused with the SDN1 semi-dwarfing gene referred to in this article – they are two separate things.

3. Tang X et al. A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 (Cas12a) nucleases in rice. Genome Biology 19, Article number: 84 (2018). https://genomebiology.biomedcentral.com/articles/10.1186/s13059-018-1458-5